ADIPOSE STEM CELLS FOR BONE TISSUE ENGINEERING IN A HUMAN MAXILLARY SINUS FLOOR ELEVATION MODEL by Peter Vos

ADIPOSE STEM CELLS FOR BONE TISSUE ENGINEERING IN A HUMAN MAXILLARY SINUS FLOOR ELEVATION MODEL: STUDIES TOWARDS CLINICAL APPLICATIONS

Janice R. Overman

ADIPOSE STEM CELLS FOR BONE TISSUE ENGINEERING IN A HUMAN MAXILLARY SINUS FLOOR ELEVATION MODEL: STUDIES TOWARDS CLINICAL APPLICATION

Janice Ruth Overman

The studies described in this thesis were carried out at the section Oral Cell Biology of the Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, the department of Oral and Maxillofacial Surgery, VU University Medical Center/ ACTA, Amsterdam, and MOVE Research Institute Amsterdam, The Netherlands.

The printing of this thesis was kindly supported by: Nederlandse Vereniging voor Calcium- en Botstofwisseling Nederlandse vereniging voor Biomaterialen en Tissue Engineering Straumann Stichting Anna Fonds|NOREF Dent-Med Materials b.v. (Geistlich Bio-Oss® en Geistlich Bio-Gide®)

Janice Ruth Overman Fox Foto (petervos@fox-foto.nl) Gildeprint, Enschede, The Netherlands (2014) 9789461088406 Janice Ruth Overman, Amstelveen, The Netherlands, 2014

All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic of mechanical, including photocopying, recording, or any information storage or retrieval system, without written permission from the author.

VRIJE UNIVERSITEIT

ADIPOSE STEM CELLS FOR BONE TISSUE ENGINEERING IN A HUMAN MAXILLARY SINUS FLOOR ELEVATION MODEL: STUDIES TOWARDS CLINICAL APPLICATION

ACADEMISCH PROEFSCHRIFT

Ter verkrijging van de graad Doctor aan de Vrije Universiteit Amsterdam, op gezag van de rector magnificus prof.dr. F.A. van der Duyn Schouten in het openbaar te verdedigen ten overstaan van de promotiecommissie van de Faculteit der Tandheelkunde op vrijdag 23 januari 2015 om 13.45 uur in de aula van de universiteit, De Boelelaan 1105

door Janice Ruth Overman geboren te Rotterdam

promotoren:

Prof.dr. J. Klein Nulend Prof.dr. E.A.J.M. Schulten

copromotoren:

Dr. M.N. Helder Prof.dr. C.M. ten Bruggenkate

- Pasensi na wan bita bòn, ma en fròktu switi f’njan - Geduld is een bittere boom, maar zijn vruchten zijn lekker om te eten -

Leden van de lees- en promotiecommissie:

Prof.dr. V. Everts Prof.dr. I.C. Heyligers Prof.dr. J.A. Jansen Dr. H-J Prins Prof.dr. G.M Raghoebar Prof.dr. M.J.P.F Ritt Prof.dr. ir. T. H. Smit

Paranimfen:

Leroy Overman Marjoleine Willems

CONTENTS CHAPTER 1

General introduction

CHAPTER 2

Human maxillary sinus floor elevation as a model for bone regeneration enabling the application of one-step surgical procedures

CHAPTER 3

Short (15 min) BMP-2 treatment stimulates osteogenic differentiation of human adipose stem cells seeded on calcium phosphate scaffolds

CHAPTER 4

Growth factor gene expression profiles of bone morphogenetic protein-2-treated human adipose stem cells seeded on calcium phosphate scaffolds in vitro

CHAPTER 5

A histomorphometrical and micro-CT study of bone regeneration in the maxillary sinus comparing biphasic calcium phosphate and deproteinized cancellous bovine bone in a human split-mouth model

CHAPTER 6

A novel approach revealing the effect of a collagenous membrane on osteoconduction in maxillary sinus floor elevation with Î˛-tricalcium phosphate

115

CHAPTER 7

Evaluation of a new biphasic calcium phosphate for maxillary sinus floor elevation: micro-CT and histomorphometrical analysis

139

CHAPTER 8

General discussion

161

GENERAL SUMMARY ALGEMENE SAMENVATTING DANKWOORD CURRICULUM VITAE

169 175 181 187

CHAPTER 1 GENERAL INTRODUCTION

Chapter 1

GENERAL INTRODUCTION The increasingly ageing population worldwide has raised the prevalence of jaw atrophy and thus the need for a sufficient treatment. Maxillary (as well as mandibular) atrophy is caused by a prolonged edentulous state after loss of natural teeth. Dental implants can be installed to restore the missing teeth, but this requires sufficient alveolar bone volume. If this requirement is not met, the alveolar bone height can be increased with a specific surgical procedure, i.e. the maxillary sinus floor elevation procedure (MSFE), which was first performed by Tatum 1, but published by Boyne 2. During the last decades, MSFE has become a standard pre-implant surgical procedure to increase alveolar bone height in the posterior maxilla. Autologous bone is still considered the golden standard as graft material for bone augmentation procedures in general and for MSFE in particular, since the graft contains osteoblasts and osteoprogenitor cells, thereby providing osteoconductive and osteoinductive properties necessary for allowing the migration and subsequent differentiation of progenitor cells 3, 4. The most common donor sites for autologous bone are the anterior iliac crest and mandibular bone. Harvesting autologous bone has several disadvantages, such as donor site morbidity and limited availability of bone 5. Alternatives for the use of autologous bone have been developed and evaluated. This resulted in the introduction and use of bone substitutes such as allograft (e.g. demineralized freeze-dried human bone, DemBone®), xenograft (e.g. demineralized bovine bone, Bio-Oss®), and purely synthetic grafting materials that are accepted and now commonly used for standard clinical dental and oral surgery procedures 6. Synthetic bone substitutes such as β-tricalcium phosphate (β-TCP, e.g. Ceros®), hydroxyapatite (HA), and biphasic calcium phosphate (BCP, mixtures of HA/β-TCP, e.g. Straumann® BoneCeramic) are interesting alternatives to use in MSFE because they are available in unlimited quantity, have an infinite half-life and may therefore be used as off-the-shelf products. The characteristics of the synthetic grafts are important for successful bone ingrowth; whereas HA is non-resorbable, β-TCP resorbs relatively fast. BCP combines these properties, and the additional high surface area and porosity of the particles facilitates attachment, proliferation and osteogenic differentiation of progenitor cells 7, 8. Using synthetic grafting materials eliminates the need for a second operation site as well as potential additional complications. Within the concept of bone tissue engineering, the sinus floor elevation model is unique by allowing histological examination of biopsies removed prior to implant insertion; we could demonstrate that synthetic bone substitutes show low bone ingrowth rates compared to autogenous bone graft due to the solely osteoconductive properties 4, 9. Therefore, additional growth factors and/or osteoblast precursor cells are required to provide the osteoinductive potential of the tissue-engineering construct. One specific growth factor is bone morphogenetic protein-2 (BMP-2), which is a potent osteoinductive molecule that, either or not combined with a carrier, has been shown to stimulate osteogenic differentiation of undifferentiated cells, and to induce healing of critical size defects in several animal studies 10-13. An in vitro study with goat adipose stem cells (ASCs) showed that the use of BMP-2 in a physiological, ng-range concentration and a short period of time can be very beneficial for tissue engineering purposes 12

General introduction

using ASCs 14. Human adipose tissue provides an easily accessible, expendable source of clinically relevant numbers of MSCs (ASCs), thereby allowing innovative one-step regenerative treatment strategies. This new concept overcomes the problems currently encountered with cellular therapies: need for in vitro expansion, high costs, and repeated surgeries. Moreover, when using non-induced, minimally manipulated cells, many regulatory hurdles can be avoided, thereby accelerating clinical introduction. The MSFE procedure is an established model to investigate oral bone tissue engineering approaches, and fits perfectly in a one-step surgical procedure 15. For an extensive review on the feasibility of this procedure we refer to chapter 1 of this thesis. This thesis concentrates on the optimization of several important steps (as illustrated in Figure 1) i.e. the use of ASCs for bone regeneration in MSFE, as well as the evaluation of the osteoconductivity of different calcium phosphate carriers. Within this research project the following scientific questions were addressed: 1. 2. 3. 4. 5.

Does the MSFE procedure using adipose stem cells fits within the one-step surgical procedure model? Does a short treatment of human ASCs with BMP-2 affect osteogenic differentiation after seeding on calcium phosphate carriers? Are the growth factor and cytokine expression profiles of human ASCs dependent on BMP-2 treatment and/or osteogenic differentiation? Is there a difference in the volume and quality of bone after MSFE with BCP 60/40 and deproteinized bovine bone? Does the ratio HA/Î˛-TCP within a BCP carrier affect the rate of new bone ingrowth after MSFE?

Figure 1. Scheme of the different steps in the one-step surgical procedure

Chapter 1

In chapter 2 a review is provided on whether the human MSFE procedure could be applied as a model for bone regeneration enabling the application of one-step surgical procedures. In chapter 3 the effect of short treatment of human ASCs with BMP-2 after seeding on a calcium phosphate carrier is evaluated. In chapter 4 the gene expression profiles of growth factors expressed by differentiating human ASCs are evaluated. In chapter 5 the gain of mineralized bone was compared between deproteinized bovine bone allograft (DBA) and biphasic calcium phosphate (BCP) after MSFE, using a split-mouth design. In chapter 6 it we explored whether a collagenous barrier membrane covering the lateral window after MSFE using Î˛-TCP affects bone formation. In chapter 7 we evaluated the performance of a new biphasic calcium phosphate (BCP 20/80) using Micro-CT and histomorphometrical analysis in a novel approach. Finally, in chapter 8 the main conclusions of this thesis are discussed and placed in a broader perspective. In addition, based on the results of our studies using calcium phosphate scaffolds and ASCs, we emphasize that our novel concept of a one-step surgical procedure for human MSFE might be applicable in other surgical disciplines as well.

General introduction

REFERENCES 1. 2. 3. 4.

5. 6.

7. 8.

10.

11.

12. 13.

14.

15.

Tatum H, Jr. Maxillary and sinus implant reconstructions. Dental clinics of North America. 1986;30(2):207-29. Boyne PJ, James RA. Grafting of the maxillary sinus floor with autogenous marrow and bone. Journal of oral surgery (American Dental Association : 1965). 1980;38(8):613-6. Browaeys H, Bouvry P, De Bruyn H. A literature review on biomaterials in sinus augmentation procedures. Clinical implant dentistry and related research. 2007;9(3):166-77. Klijn RJ, Meijer GJ, Bronkhorst EM, Jansen JA. A meta-analysis of histomorphometric results and graft healing time of various biomaterials compared to autologous bone used as sinus floor augmentation material in humans. Tissue engineering Part B, Reviews. 2010;16(5):493-507. Raghoebar GM, Louwerse C, Kalk WW, Vissink A. Morbidity of chin bone harvesting. Clinical oral implants research. 2001;12(5):503-7. Zijderveld SA, Schulten EA, Aartman IH, ten Bruggenkate CM. Long-term changes in graft height after maxillary sinus floor elevation with different grafting materials: radiographic evaluation with a minimum follow-up of 4.5 years. Clinical oral implants research. 2009;20(7):691-700. Guha AK, Singh S, Kumaresan R, Nayar S, Sinha A. Mesenchymal cell response to nanosized biphasic calcium phosphate composites. Colloids and surfaces B, Biointerfaces. 2009;73(1):146-51. Saldana L, Sanchez-Salcedo S, Izquierdo-Barba I, Bensiamar F, Munuera L, Vallet-Regi M, et al. Calcium phosphate-based particles influence osteogenic maturation of human mesenchymal stem cells. Acta biomaterialia. 2009;5(4):1294-305. Zijderveld SA, Zerbo IR, van den Bergh JP, Schulten EA, ten Bruggenkate CM. Maxillary sinus floor augmentation using a beta-tricalcium phosphate (Cerasorb) alone compared to autogenous bone grafts. The International journal of oral & maxillofacial implants. 2005;20(3):432-40. Kim CS, Choi SH, Choi BK, Chai JK, Park JB, Kim CK, et al. The effect of recombinant human bone morphogenetic protein-4 on the osteoblastic differentiation of mouse calvarial cells affected by Porphyromonas gingivalis. Journal of periodontology. 2002;73(10):1126-32. Cowan CM, Aalami OO, Shi YY, Chou YF, Mari C, Thomas R, et al. Bone morphogenetic protein 2 and retinoic acid accelerate in vivo bone formation, osteoclast recruitment, and bone turnover. Tissue engineering. 2005;11(3-4):645-58. Cowan CM, Shi YY, Aalami OO, Chou YF, Mari C, Thomas R, et al. Adipose-derived adult stromal cells heal critical-size mouse calvarial defects. Nature biotechnology. 2004;22(5):560-7. Xia L, Xu Y, Wei J, Zeng D, Ye D, Liu C, et al. Maxillary sinus floor elevation using a tissue-engineered bone with rhBMP-2-loaded porous calcium phosphate cement scaffold and bone marrow stromal cells in rabbits. Cells, tissues, organs. 2011;194(6):481-93. Knippenberg M, Helder MN, Zandieh Doulabi B, Wuisman PI, Klein-Nulend J. Osteogenesis versus chondrogenesis by BMP-2 and BMP-7 in adipose stem cells. Biochemical and biophysical research communications. 2006;342(3):902-8. Helder MN, Knippenberg M, Klein-Nulend J, Wuisman PI. Stem cells from adipose tissue allow challenging new concepts for regenerative medicine. Tissue engineering. 2007;13(8):1799-808.

CHAPTER 2 HUMAN MAXILLARY SINUS FLOOR ELEVATION AS A MODEL FOR BONE REGENERATION ENABLING THE APPLICATION OF ONE-STEP SURGICAL PROCEDURES Elisabet Farré-Guasch1*, Henk-Jan Prins1,2*, Janice R. Overman1,2, Christiaan M. ten Bruggenkate2, Engelbert A.J.M. Schulten2, Marco N. Helder3#, Jenneke Klein Nulend1#

Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, The Netherlands Department of Oral and Maxillofacial Surgery, VU University Medical Center/ACTA, MOVE Research Institute Amsterdam, The Netherlands Department of Orthopedic Surgery, VU University Medical Center Amsterdam, MOVE Research Institute Amsterdam, The Netherlands Shared first authorship, E. Farré-Guasch and H.J. Prins contributed equally to this manuscript. Shared last authorship, M.N. Helder and J. Klein-Nulend contributed equally to this manuscript.

Tissue Engineering part B 2012,19:69-82

Chapter 2

ABSTRACT Bone loss in the oral and maxillofacial region caused by trauma, tumors, congenital disorders or degenerative diseases is a health care problem worldwide. To restore (reconstruct) these bone defects human or animal bone grafts or alloplastic (synthetic) materials have been used. However, several disadvantages are associated with bone graft transplantation, such as limited bone volume, donor-site morbidity, surgical and immune rejection risks, and lack of osseointegration. Bone tissue engineering is emerging as a valid alternative to treat bone defects allowing the regeneration of lost bony tissue thereby recovering its functionality. During the last decades the increasing aged population worldwide has also raised the prevalence of maxillary atrophy. Maxillary sinus floor elevation (MSFE) has become a standard surgical procedure to overcome the reduced amount of bone, thus enabling the placement of dental implants. MSFE aims to increase bone height in the posterior maxilla, by elevating the Schneiderian membrane and placing graft material into the surgically-created space in the maxillary sinus floor. Importantly, oral bone regeneration during MSFE offers an unique human clinical model in which new cell-based bone tissue engineering applications might be investigated, since biopsies can be taken after MSFE prior to dental implant placement and analyzed at the cellular level. New approaches in oral bone regeneration are focusing on cells, growth factors, and biomaterials. Recently adipose tissue has become interesting as an abundant source of mesenchymal stem cells which might be applied immediately after isolation to the patient allowing a one-step surgical procedure, thereby avoiding expensive cell culture procedures and another surgical operation. In this new cell-based tissue engineering approach, stem cells are combined with an osteoconductive scaffold and growth factors, and applied immediately to the patient. In this review MSFE is discussed as a valid model to test bone tissue engineering approaches, such as the one-step surgical procedure. This procedure might be applied in other regenerative medicine applications as well. KEY WORDS: Adipose stem cells, oral and maxillofacial bone regeneration, one-step surgical procedure, scaffold, maxillary sinus floor elevation, stromal vascular fraction, growth factors

Human MSFE as a model for bone regeneration

INTRODUCTION The increasing aged population worldwide has raised the prevalence of maxillary and mandibular atrophy, which is mainly caused by tooth loss. Bone loss in the oral and maxillofacial region can also result from trauma, congenital disorders, ablation of tumors and enucleation of cysts, anatomical events such as pneumatization of the maxillary sinus, and infections.1 A solution to restore the lost oral function in edentulous patients is the placement of dental implants. However, a common problem for implant placement is the lack of sufficient bone height caused by excessive bone resorption, which occurs after tooth loss, particularly in the posterior maxillary bone.2 The maxillary sinus floor elevation (MSFE) procedure aims to increase bone height to allow dental implant placement and obtain initial mechanical stability.3 Since the reconstruction of (large) bone defects in the oral and maxillofacial region is a common challenge, bone tissue engineering has become a promising alternative for bone reconstruction. However, the application of bone tissue engineering approaches in the clinic is hampered by the fact that there is not a good human model available to study these concepts. MSFE could represent an excellent human model to study new bone tissue engineering modalities such as the one-step surgical procedure avoiding the use of autologous bone (Figure 1). In this one-step surgical procedure, adipose tissue is harvested and processed to obtain the stromal vascular fraction (SVF) which contains adipose stem cells (ASCs). These stem cells can be seeded on off-the-shelf scaffolds, to be applied immediately to the patient in an intraoperative concept.4, 5

Maxillary sinus floor elevation The surgical procedure of MSFE was first performed by Tatum6 and published by Boyne.7 In the procedure described, a window in the lateral wall of the maxillary sinus is made, followed by elevation of the maxillary sinus membrane (Schneiderian membrane) to create a cavity in which a bone graft can be placed (Figure 2). Nowadays, MSFE has become a standard surgical procedure to increase bone height in the posterior maxilla.2, 8 This procedure provides sufficient alveolar bone height for placement of dental implants after the appropriate healing time of the graft and increases implant success. Bone graft materials in maxillary sinus floor elevation Tatum6 introduced the use of allograft or bone from another individual of the same species, and alloplast or synthetic material, besides autograft to fill the space created when elevating the Schneiderian membrane, thereby eliminating the need for a second surgery to obtain an autologous bone graft, and thus also reducing potential complications from an additional surgery.9 The lateral window maxillary sinus floor elevation technique is commonly used nowadays, and still the best results are achieved using autologous bone.3, 10 Bone grafting material such as demineralized bone matrix is also commonly used. It induces bone formation in the human maxillary sinus by providing an osteoconductive matrix that allows migration of precursor cells from the host and differentiation into bone matrix-producing cells8 (Figure 2).

Chapter 2

Figure 2. Schematic figure of the MSFE procedure. (A) MSFE is performed via a lateral approach. After reflection of the mucoperiosteal flap, a bony window is created in the lateral wall of the maxillary sinus followed by elevation of the maxillary sinus membrane (Schneiderian membrane) to create a cavity in which the bone substitute is placed. This bone substitute provides a osteoconductive matrix that allows migration of osteoprogenitor cells from the adjacent bone, and bone formation occurs via osteoconduction. (B) The bone substitute is seeded with adipose stem cells. The addition of adipose stem cells with angiogenic and osteogenic potential stimulates osteogenesis, osteoinduction, and vascularization, which enhances bone formation throughout the whole (with bone substitute filled) maxillary sinus. Color images available online at www.liebertpub.com/teb

Human MSFE as a model for bone regeneration

Figure 1. Concept of a maxillary sinus floor elevation (MSFE) with freshly isolated adipose-derived stem cells in a one-step surgical procedure. (A) The plastic surgeon starts harvesting adipose tissue by liposuction. (B) The adipose tissue and liposuction fluid is collected in syringes. (C) The filled syringes are transferred into a Celution 800/CRS system. This device washes, digests, and centrifugates the adipose tissue to obtain the fresh stromal vascular fraction containing the adipose stem cells. After isolation of the stromal vascular fraction, cells can be shortly stimulated with the growth factors before seeding the stimulated cells onto a carrier material. (D) The freshly isolated adipose stem cells are seeded onto the calcium phosphate carrier. Unattached cells are washed off, and the calcium phosphate carrier combined with stem cells (stam= stem cells). (E) During the short attachment period of the cells (30min), the patient is prepared for the MSFE procedure via a lateral approach. After reflection of the mucoperiosteal flap, a bony window is created in the lateral wall of the maxillary sinus and carefully moved and rotated medially toward the maxillary sinus, after dissection of the maxillary sinus mucosa (trap-door technique). (F) The tissue-engineered construct is inserted immediately into the patient, and the space created is filled with the bone substitute combined with the adipose stem cells. (G) Finally, the wound is closed. Color images available online at www.liebertpub.com/teb

A variety of bone grafts are used for MSFE with different degrees of success (Table 1). The bone graft materials applied in MSFE are: 1) autologous bone graft, mostly from the iliac crest or mandibular bone, 2) allograft, 3) xenograft, from bovine or porcine origin, and 4) alloplast materials, such as β-tricalcium phosphate (β-TCP), hydroxyapatite (HA), mixtures of HA/βTCP (biphasic calcium phosphate; BCP), polymers, and bioactive glasses. Autologous bone graft is still considered as the ‘gold standard’, since it contains osteoblasts and osteoprogenitor cells, and is not only osteoconductive by allowing the migration of progenitor cells, but also osteogenic. 11-13 Since these grafts are from the same individual the tissue is recognized as “self”, and the immune system is not triggered with an immunological response.14 Although it is generally accepted that autologous bone is the ‘gold standard’ for the MSFE procedure, this may not always be the case. Abscesses, fistulas, dehiscences, and large numbers of bacteria can be found in particulated bone collected with suction devices such as bone traps,15 in addition to the disadvantage of limited bone availability and patient morbidity.14 Alternative bone graft materials frequently used are allogenic bone and bone substitutes. Allogenic bone is mostly applied in the form of demineralized freeze-dried bone matrix. The bone morphogenetic proteins (BMPs) present in the matrix of bone allograft are exposed by decalcification leading to induction of osteogenic differentiation of progenitor cells. However, The Sinus Consensus Conference held in Wellesley, MA, USA, in 1996 concluded that demineralized freeze-dried bone is not an appropriate bone substitute, since it always carries the risk of disease transmission and might cause marked bone resorption.11 Other disadvantages, such as lack of osteogenesis and osseointegration, and the risk of immune rejection at the implant site,16, 17 lead to a search for alternative graft materials. Bone substitutes are interesting alternatives because they are available in unlimited quantity and may be used as off-the-shelf products. However, bone substitutes are cell-free and require more time for bone healing than autologous bone grafts.18 It has been shown that maxillary sinus grafting with alloplast materials such as bioactive glass, HA, or β-TCP does not result in increased bone volume compared with autologous bone graft.10, 12 Several attempts 21

Chapter 2

have been made to apply a cell-based approach using stem cells combined with an osteoconductive biomaterial or scaffold, which is known as cell-based bone tissue engineering.19-23 Bone Tissue Engineering in Maxillary Sinus Floor Elevation: a Potential Model to be Used in Other Surgical Disciplines For cell-based bone tissue engineering the choice of osteoprogenitor cells and biomaterials is crucial, as well as the type of growth factor(s) that stimulate osteogenesis and improve osteogenic differentiation of the progenitor cells. Bone regeneration in the oral and maxillofacial region can be used to investigate tissue engineering approaches for later use in other surgical disciplines. Bone biopsies from patients can be taken after an MSFE procedure and prior to dental implant placement. These bone biopsies can then be analyzed by micro-CT, histomorphometry, and histology24-27 and are highly valuable for evaluating new approaches for oral and maxillofacial bone tissue engineering, such as the use of different progenitor cells, growth factors, and biomaterials. Furthermore, the treatment of patients requiring an MFSE procedure at both sites of the maxilla enables the use of a bilateral split-mouth design model, allowing to study simultaneously different conditions in the same patient. Therefore, the MSFE procedure is proposed as a human model for bone regeneration to be used in a so-called one-step surgical procedure. The maxillary sinus represents an excellent model as an osteogenic chamber for bone regeneration.28 The lack of quantitative or histomorphometrical data becomes an important drawback in studies comparing different bone grafts for MSFE.29 Clinically it is difficult and time consuming to analyze a large number of samples,13 and therefore it is desirable to generate a variety of studies with similar approaches, and to combine and compare these results in a meta-analysis. A cell-based approach that uses stem cells combined with an osteoconductive scaffold may become an interesting alternative to conventional procedures, avoiding the major problems associated with bone graft transplantation. However, the paradigm of tissue engineering is to find a suitable human model to investigate the appropriate combination of scaffold and source of osteoprogenitor cells. A limitation for successful application of bone tissue engineering in MSFE is the relative absence of mechanical loading, which is a critical issue for bone regeneration. It has been shown that mechanical loading favours proliferation and osteogenic differentiation of progenitor cells while inhibiting adipogenic differentiation,30 and this should be taken into account to decide the appropriate time for implant loading. Sources of Stem/Progenitor Cells for Clinical Application In the late 60s, Friedenstein showed that the mesenchymal stroma from human bone marrow contains a population of cells that proliferate when cultured on plastic, and differentiate to cell lineages derived from the mesoderm, such as chondrocytes and osteoblasts.31 Later these precursor spindle-shape cells are referred to as mesenchymal stem cells (MSCs)32 and shown to differentiate into several lineages in vitro33 and in vivo,34, 35 making them promising candidates for regenerative medicine applications. MSCs grow when adhered to plastic and express specific surface antigens. They also possess multipotent differentiation potential and low immunogenic 22

Human MSFE as a model for bone regeneration

Table 1. (Pre)clinical in vivo studies using mesenchymal stem cells from bone marrow or adipose tissue and stromal vascular fraction from adipose tissue for oral and maxillofacial bone tissue engineering applications in human and animal models: bone volume measurements.

Calvarial defects were critical-sized. ABB, anorganic bovine bone; DBM, demineralized bone matrix; FN, fibronectin; CaP, calcium phosphate; HA, hydroxyapatite; ß-TCP, ß-Tricalcium phosphate; BCP, biphasic calcium phosphate (%HA/%ß-TCP); PLA, polylactic acid; PLGA, polylactic-coglycolide copolymers; SVF, stromal vascular fraction; ASCs, adipose stem cells; BM-MSCs, bone marrow-derived mesenchymal stem cells; BMP-2, bone morphogenetic protein-2; PRP, platelet-rich plasma; MSFE, maxillary sinus floor elevation; GBR, guided bone regeneration; n.a., not available.

Chapter 2

potential, according to the minimal criteria to define MSCs, proposed in 2006 by the International Society for Cellular Therapy.36 Their immunosuppressive properties and differentiation potential make MSCs interesting for regenerative medicine clinical applications. The main sources of MSCs for clinical studies in bone tissue engineering are bone marrow and adipose tissue.

Bone marrow-derived mesenchymal stem cells Bone marrow-derived MSCs (BM-MSCs) are widely used in bone regeneration, since they are relatively easy to harvest and show multipotent differentiation potential.33-35, 37 MSCs secrete growth factors and cytokines that inhibit apoptosis and fibrosis or scarring at the site of injury. They also stimulate angiogenesis and proliferation of progenitor cells, and exert an immunomodulatory effect in the organism. These effects, known as â&#x20AC;&#x153;the trophic activityâ&#x20AC;?,38 make them interesting candidates to improve bone regeneration in the MSFE procedure (Table 2). Addition of bone marrow-derived MSCs to bone derivative/substitute materials such as BCP particles enhances bone formation in the maxillary sinus area, with 41% mean percentage of newly formed bone observed at 3 months post-surgery.23 Another study using BCP without cells showed 27% average of bone volume at 6 months post-surgery.25 BM-MSCs applied to calcium phosphate scaffolds for MSFE promote faster bone formation and mineralization in animal models.39-42 One study in rabbits found an increase in bone formation when BM-MSCs were applied to a calcium phosphate scaffold in comparison with scaffold without cells.43 These results suggest that BM-MSCs may improve new bone regeneration in the maxillary sinus. The use of BM-MSCs also has disadvantages, such as pain associated with the harvest procedure and low cell numbers upon harvest, especially in elderly patients.44 Only one MSC per 105 adherent mononuclear cells is obtained upon harvest,33 necessitating an ex vivo expansion step to obtain therapeutic cell numbers, which is time consuming, expensive, and carries the risk of contamination. Long cell culture is associated with loss of differentiation potential and risk of cell transformation.45, 46 The number and concentration of progenitor cells with angiogenic potential in a scaffold are also important for successful bone formation in vivo, since the vascular network is crucial to ensure cell survival.47 These considerations associated with BM-MSCs have led to a search for other sources of progenitor cells for clinical application. Adipose stem cells Adipose tissue has gained interest as it contains a population of stem cells with MSC characteristics.48 Adipose tissue, as bone marrow, is derived from adult mesodermal tissue, and after tissue disaggregation, collagenase digestion and cell centrifugation, a pellet is obtained which contains a heterogeneous cell population composed of pre-adipocytes, mast cells, endothelial cells, pericytes, smooth muscle cells, fibroblasts, and multipotent progenitor cells with similar characteristics to BM-MSCs, as well as hematopoietic stem cells.48 This mixture of cells is known as the stromal vascular fraction (SVF). When the pellet is cultured, an adherent cell population is obtained which can be expanded and used in a variety of assays. A wide nomenclature is used to identify this cell population, including adipocyte precursor cells, preadipocytes, adipose-derived adult stem cells, adipose-derived stromal cells, adipose-derived adherent stromal cells, processed lipoaspirate 24

Human MSFE as a model for bone regeneration

Table 2. (Pre)clinical in vivo studies using autograft, allograft (demineralized bone matrix and mineralized cancellous bone), xenograft (from bovine or from porcine origin), inorganic materials containing calcium phosphate, polymers, and bioglass for oral and maxillofacial bone tissue engineering applications in human and animal models: bone volume measurements.

Calvarial defects were critical-sized. ABB, anorganic bovine bone; MCBA, mineralized cancellous bone allograft; DBM, demineralized bone matrix; CaCO3, calcium carbonate; CaSO4, calcium sulfate; CaP, calcium phosphate; HA, hydroxyapatite; ß-TCP, ß-Tricalcium phosphate; BCP, biphasic calcium phosphate (%HA/%ßTCP); PLA, polylactic acid; PLGA, polylactic-coglycolide copolymers; VEGF, vascular endothelial growth factor; BMP-2, bone morphogenetic protein-2; BMP-7, bone morphogenetic protein-7; PRP, platelet-rich plasma; GDF-5, growth and differentiation factor-5; MSFE, maxillary sinus floor elevation. n.a.; not available.

Chapter 2

cells, and adipose-derived stem cells.49 At the Second Annual International Fat Applied Technology Society meeting (October 3-5, 2004, Pittsburgh, PA), consensus was reached to use the term SVF for the freshly isolated stromal vascular fraction, and to refer to the stem cell-like cells within this fraction as ASCs (adipose stromal cells or adipose stem cells). Passaged cells (cells cultured to homogeneity) are also referred to as ASCs. These ASCs can differentiate into multiple mesenchymal tissue cell types, such as osteoblasts, chondrocytes, adipocytes, and myocytes.50 ASCs become an interesting alternative to BM-MSCs, since adipose tissue is easy to access, and its harvesting results in minimal patient discomfort. Furthermore, adipose tissue contains a 100 to 1000-fold higher number of stem cells than bone marrow, and ASCs proliferate faster than BM-MSCs.4, 33, 50, 51 ASCs also secrete growth factors with angiogenic and anti-apoptotic potential, which makes them interesting candidates for bone tissue engineering where blood supply is needed.51-53 These angiogenic properties allow them to survive in low oxygen environments, and make them good candidates for cell-based therapies such as maxillary reconstruction.22 ASCs also abundantly express CD34,54 in contrast with MSCs which lack CD34 expression.36, 55 Cultured ASCs show decreased CD34 expression, which is totally lost after long-term culture.49, 54 The marker CD34, which is also expressed in hematopoietic and endothelial stem cells, is related to angiogenesis and could have important implications in bone tissue engineering, where angiogenesis is crucial.52, 56 Since the amount of bone formed depends at least in part on the number of cells that survive, it is crucial to provide a high number of osteoprogenitor cells per given volume of tissue, to ensure adequate bone formation. A dose-dependent effect on bone formation has been reported for cultured ASCs applied to a calvarium rabbit bone defect.57 ASCs promote healing of critical-sized cranial defects in mice and rabbits,57-60 and have been successfully used in animal models for vertical bone regeneration,57 as well as in MSFE using the SVF containing the ASCs61 (Table 2). ASCs have been used successfully in humans to reconstruct major maxillary defects22 and traumatic calvarial defects.62 Calvarial bone is embryologically, morphologically and physiologically similar to bone in the oral and maxillofacial region. Its high ﬂat surface eases the ﬁxation of titanium osteosynthesis57 making it an interesting model to study oral bone regeneration. Adipose tissue may be an interesting source of progenitor cells for oral and maxillofacial applications, where high cell numbers may be needed for successful bone regeneration. To select the appropriate scaffold for combination with these cells is another critical issue in bone regeneration. Scaffolds for Oral and Maxillofacial Bone Tissue Engineering Not only the appropriate source of progenitor cells, but also the use of the right type of carrier scaffold with adequate pore size, porosity and composition, is a key issue in bone tissue engineering. The ideal scaffold has a high porosity and interconnected pore network, which is large enough to facilitate vascular invasion and bone development.26 Large pore sizes (>300 µm) promote neo-vascularization and favor mineralized bone ingrowth, whereas smaller pore sizes (90–120 µm) promote endochondral bone formation.63 For tissue engineering the ideal scaffold should be biocompatible with the right porosity and pore size to facilitate neo-vascularization, 26

Human MSFE as a model for bone regeneration

and an adequate surface allowing cell adhesion, proliferation, and differentiation. Furthermore the scaffold should be osteoinductive, releasing growth factors that modulate osteoblastic cell functions and enhance bone formation, and the mechanical properties should match those of living bone. Finally the scaffold degradation rate should be tuned with the growth rate of the de novo bone formation, id est the scaffold is totally degraded by the time the injury site is fully regenerated.3, 64 The size of the scaffold is also important for adequate bone regeneration. Particles with a size ranging from 250-500 μm have been shown to increase the volume of mineralized tissue in MSFE showing the presence of lamellar and woven bone.8 Absorbable scaffolds used in bone tissue engineering are divided in biodegradable polymers of synthetic origin, such as polylactic acid (PLA), polyglicolic acid (PGA), polylacticcoglycolide copolymers (PLGA), and Poly(ε-caprolactone) (PCL),65-68 natural polymers such as collagen, hyaluronic acid, fibrin, and silk fibroin,64, 69-72 bioactive glasses with a silica-based osteoinductive surface,73 inorganic materials containing calcium phosphate (ceramics), and finally composite materials, which combine for example calcium phosphates ceramics with polymers.64 Various grafts or combination of graft materials have been used successfully in the oral and maxillofacial region (Table 2). Inorganic materials or ceramics from natural origin, such as calcium carbonate, calcium phosphate, calcium sulfate, bovine-derived or coralline HA, and from synthetic origin, such as synthetic HA or ß-TCP have been widely used in MSFE due to their good biocompatibility and osteoconductivity,10, 26, 74-79 besides bone grafts,13, 16, 17, 26, 29, 74, 77, 78 polymers28, 78, 80-86 and bioactive glass.78, 87, 88 Synthetic Calcium Phosphate Scaffolds for Oral and Maxillofacial Bone Tissue Engineering HA and ß-TCP have been successfully used for MSFE procedures because of their good biocompatibility and chemical composition, which resembles the composition of natural bone matrix.10, 25, 89, 90 Advantages of using ceramics such as β-TCP is that they support cell ingrowth and promote osteogenic differentiation of osteoprogenitor cells.90, 91 However, unlike HA, β-TCP reabsorbs rapidly, but concurrent bone formation does not occur in a 1:1 ratio, and often less bone is produced as compared with the volume of β-TCP absorbed.10 BCP combines the bioactive properties of HA with the good bioresorbability of β-TCP. Due to its high surface area and 3D hierarchical porosity, it supports attachment, proliferation, and osteogenic differentiation of progenitor cells.92, 93 BCP contains a mixture of 60% HA and 40% β-TCP and has been used successfully for MSFE,25-27, 94 with an implant survival >98%27, 89 and bone formation similar to allograft and xenograft.16, 26 The macroporosity (pore diameter 300 to 600 µm) of BCP scaffold allows colonization by osteogenic cells. Osteoclasts attach to BCP surface to resorb biomaterial, stimulating osteoblast differentiation and activity through signaling molecules, while no osteoclast resorption is found on β-TCP material, which is resorbed mainly by chemical dissolution.90, 95 This suggests that BCP material may be promising for use as a scaffold for bone tissue engineering. The favorable clinical results obtained with BCP are similar as those obtained with xenografts, such as inorganic bovine bone,13, 16 or other alloplastic materials such as HA.26 However, significantly less bone formation is observed when compared with autologous bone 27

Chapter 2

grafts, indicating that autologous bone grafts still remain the ‘gold standard’, showing increased bone formation compared to alloplast material and xenografts.13, 24, 27, 77, 78, 87 In certain augmentation cases, such as maxillary sinus grafting, complete resorption of the bone substitute to be replaced by new bone is not preferable. A long-lasting active osteoconductive guiding scaffold is required to support osseointegrated dental implants without destabilization of bone.74 Therefore the stability of the graft material in the maxillary sinus and height changes of the graft material over time are important issues for successful bone regeneration in MSFE. Calcium phosphates ceramics have also drawbacks, such as lack of osteogenic properties, brittleness and low mechanical stability observed when using ß-TCP, which makes them unsuitable for reconstruction of large bone defects.65, 96 Therefore addition of osteoprogenitor cells might be required to improve the osteogenic properties of the graft, since the ideal bone graft should provide osteogenic cells as well as osteoinductive factors for bone regeneration64 (Table 1). For clinical application of bone tissue engineering, the appropriate source of stem cells together with the adequate scaffold, and the use of growth factors that speed up tissue regeneration are crucial issues for successful clinical application.97, 98 Growth Factors for Oral and Maxillofacial Bone Tissue Engineering Angiogenesis is extremely important in bone regeneration, since blood vessels carry nutrients which are crucial to ensure the survival of transplanted progenitor cells,99 and vascular development needs to be induced prior to osteogenesis.77, 100 To establish the vascular network, growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and those factors present in platelet-rich plasma (PRP), a platelet concentrate obtained from the patient’s own blood, are needed to induce and speed up angiogenesis in the regenerating tissue, and may be used in oral and maxillofacial applications to reduce the healing time and enhance bone formation53, 84, 101-105 (Table 3). These growth factors can be added to all grafting materials. There is controversy in the literature related to the use of PRP in MSFE procedures. Although some studies found an effect of PRP on bone formation in combination with bone substitutes104 and bone grafts,106 other studies did not find such an effect when PRP was combined with a xenograft,29 an autologous bone graft,12 or a synthetic bone substitute.87 Ex vivo seeded ASCs onto a scaffold secrete a high variety of angiogenic growth factors, such as VEGF and FGF-2, which makes them interesting candidates for inducing bone tissue formation.51, 107 One substantial group of growth factors for bone tissue engineering are the bone morphogenetic proteins (BMPs). BMPs have been found in the demineralized bone matrix more than 40 years ago. They belong to the TGF-ß superfamily and promote the differentiation of osteoprogenitor cells and induce osteogenesis.108 An acute increase in the secretion of bone morphogenetic protein-2 (BMP-2) and bone morphogenetic protein-7 (BMP-7), together with bone morphogenetic protein-4 (BMP-4), has been shown when applying ASCs in criticalsized mouse defects,58 which suggests a role of these growth factors in bone regeneration and remodeling. The osteoinductive properties of BMPs make them interesting candidates to promote bone formation in rehabilitation of partially or completely edentulous patients with severe 28

Human MSFE as a model for bone regeneration

maxillary or mandibular atrophy. Favorable regenerative responses to the graft materials containing BMPs in MSFE procedures have been reported.11, 15, 22 BMP-2, BMP-7, and bone morphogenetic protein-14 (BMP-14, known as growth and differentiation factor-5) have been applied in MSFE procedures75, 79, 80, 83, 109 (Table 3). Only recombinant human bone morphogenetic protein-2 (rhBMP-2) and recombinant human bone morphogenetic protein-7 (rhBMP-7) are approved by the food and drug administration (FDA) as medical device.108 Although FDA approval is limited to few orthopaedic applications, other (off-label) applications are considered safe, and may warrant the use of rhBMP-2 and rhBMP-7 clinically in MSFE procedures.

Bone morphogenetic protein-7 There are contradictory reports concerning osteoinduction of osteoprogenitor cells by BMP7. Some studies suggest that BMP-7 is osteoinductive,38, 110 while others suggest that BMP-7 stimulates chondrogenesis.5 This is in contrast to the stimulatory effect of BMP-2 on osteogenic differentiation of progenitor cells.5, 85, 111 BMP-7 applied in MSFE procedure induces bone regeneration.80, 112 However, the use of BMP-7 is associated with swelling and the presence of some granular tissue, which could indicate an inflammatory response.80 This is likely due to the putty component of the BMP-7 preparation which recruits multinucleated giant cells.113 Bone morphogenetic protein-2 BMP-2 is a potent osteoinductive molecule that has been shown to increase and speed up osteogenic differentiation, and induce healing of intrabony periodontal defects as well as critical size defects in animals.82, 86, 105, 114 BMP-2 applied to osteoprogenitor cells5, 115, 116 and/or to osteoconductive scaffolds22, 43, 76, 81-86, 105, 114 stimulates osteogenic differentiation in vitro and accelerates bone healing and new bone formation in vivo (Table 3). Carriers combined with BMP2 have also been shown to induce bone formation when applied in MSFE in sheep,85 rabbit,43, 76, 84 and pig,81 as well as in healing of critical-sized calvarial defects in rats86 and periodontal defects in dogs.82 In an in vivo study in rats using a femoral osteotomy model, a 2 mm femoral gap stabilised by an external fixator device failed to heal within 6 weeks, whereas local application of rhBMP-2 within a blood clot showed proper bone healing with bony bridging.117 This indicates that BMP-2 stimulates osteogenic differentiation of osteoprogenitor cells and might be beneficial for clinical application. The dose of growth factor is also an important issue to take into account in clinical applications. It has been shown that incubation with only 10 ng/ml BMP-2 during 15 min is enough to stimulate osteogenic differentiation of goat derived ASCs.5 This dose of the growth factor is magnitudes lower than the concentration of BMP used in in vivo and clinical studies (0.2â&#x20AC;&#x201C;2,5 mg/ml carrier), and may avoid side effects of using mg-range concentrations, such as swelling, ankylosis and calcified seromas, as observed in some studies.80-83, 114,118 It would also avoid the high costs of using high BMP-2 concentrations. BMP-2 supplementation at a low concentration (10 ng/ml) may help to maintain the viability of endothelial cells,100 as well as ASCs (Overman and FarrĂŠ-Guasch, submitted). Moreover, this rather low and short incubation with BMP-2 could easily fit within a one-step surgical procedure, where autologous stem cells are isolated, triggered with growth factors, and immediately used for bone regeneration within the 29

Chapter 2

Table 3. (Pre)clinical in vivo studies combining growth factors with mesenchymal stem cells and/or scaffolds for oral and maxillofacial bone tissue engineering applications in human and animal models: bone volume measurements.

Calvarial defects were critical-sized. ABB, anorganic bovine bone; CaP, calcium phosphate; Ă&#x;-TCP, Ă&#x;-Tricalcium phosphate; PLA, polylactic acid; PLGA, polylactic-coglycolide copolymers; BM-MSCs, bone marrow-derived mesenchymal stem cells; BMP-2, bone morphogenetic protein-2; BMP-7, bone morphogenetic protein-7; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor, PDGF, platelet-derived growth factor; PRP, platelet-rich plasma; GDF-5, growth and differentiation factor-5; FGF-2, fibroblast growth factor-2; MSFE, maxillary sinus floor elevation. n.a.; not available.

same surgical procedure (Figure 1).4, 5 One-step Surgical Procedure Approach for Maxillary Sinus Floor Elevation The stromal vascular fraction (SVF), obtained after collagenase digestion and centrifugation of adipose tissue, has potential for bone tissue engineering, since it contains ASCs with osteogenic potential, among other cell populations.62, 119, 120 For clinical application it would be advantageous to transplant the whole SVF, thereby avoiding expensive and time-consuming in vitro selection and/or expansion steps, loss of differentiation potential, and the risk of contamination. Additionally, the harvesting of human adipose tissue by liposuction is an easy and safe procedure, unlike the harvesting of human bone marrow, which is associated with donor site morbidity.44 Most studies have been performed on cultured ASCs, but before being used clinically, they must overcome 30

Human MSFE as a model for bone regeneration

prohibitively expensive good manufacturing practice production facilities.55 In addition FDA approval in the USA, or European Agency for the Evaluation of Medicinal Products approval in Europe, is required before use. These limitations hamper the use of cultured stem cells for clinical use. However, using uncultured ASCs in the form of SVF produced via CE-marked devices would overcome at least several of these restrictions.121-123 ASCs in SVF act in coordination with blood cells such as endothelial cells and hematopoietic cells, regulating their undifferentiated state as occurs in the perivascular niche.124 These cells secrete factors that increase proliferation of progenitor cells, such as ASCs, and enhance their osteogenic potential.125 When SVF was co-transplanted on calcium phosphate scaffolds subcutaneously in mice, vascular number and diameter and ectopic mineralization was increased. Therefore, vasculogenesis and osteogenesis may be improved by a synergistic action of ASCs with (pre)vascular cells present in the SVF.126 The synergistic actions of (pre) vascular cells and MSCs may provide an alternative approach for the regeneration of vascular tissues such as bone, where an adequate vascularization is pivotal in effective cell-based bone tissue engineering. For a one-step surgical procedure in MSFE an appropriate amount of adipose tissue would be obtained from the patient. Most patients have an adequate supply of adipose tissue that can be obtained by liposuction using local anesthetics, and the SVF obtained from adipose tissue is a rich source of ASCs readily available for immediate clinical application.121 The freshly isolated SVF can be shortly stimulated with growth factors such as BMP-2 before seeding the stimulated cells onto a carrier material.5 After a short attachment period,127 the tissue-engineered construct is implanted immediately in the patient.4 The feasibility of a one-step procedure relies on the ability of the ASC fraction within the SVF to attach to a scaffold material in sufficient quantities and within a short time frame, which was recently demonstrated to occur. Moreover, it was shown that multidifferentiation potential was maintained, thus likely enhancing angiogenesis and osteogenesis and final clinical outcome.127 Summary and Future Directions A cell-based bone tissue engineering approach using stem cells combined with an osteoconductive scaffold and growth factors that stimulate osteogenesis and angiogenesis may become an interesting alternative to the conventional MSFE procedure using autologous bone. Future directions in tissue engineering for MSFE should consider: 1. The need to find the appropriate scaffold for cell-based tissue engineering in MSFE. Calcium phosphate ceramic scaffolds have been widely used in MSFE and support attachment, proliferation, and promote differentiation of osteoprogenitor cells. 2. The appropriate source of stem cells as well as the cell dose to be used in MSFE and study scaffold-cell interactions. The SVF obtained from adipose tissue contains, amongst other cell types, a relatively large number of freshly isolated cells with angiogenic and osteogenic potential, required for vascularisation and osteogenesis in the tissue engineered construct. 3. The delivery method and the appropriate dose of growth factors, such as BMP-2, are critical issues to be considered to enhance bone formation in MSFE. A short (minutes) incubation with a low dose of BMP-2 of 10 ng/ml may avoid the side effects of higher concentrations seen 31

Chapter 2

in clinical studies, which are shown to be dose-dependent, and may be enough to stimulate an osteogenic response in ASCs. 4. New bone tissue engineering cellular therapeutic approaches have to be evaluated in phase I-IV clinical trials that meet the ATMP Regulation 1394/2007/EC and/or FDA regulations, as well as (local) rules and good clinical practice (GCP) guidelines. In summary, previous studies have shown the importance of scaffolds and growth factors, together with the appropriate source of stem cells, in bone tissue engineering, and have highlighted the need to find an appropriate model for clinical applications. Human MSFE may be a valid model to test bone tissue engineering approaches in a one-step surgical procedure for later use in other surgical disciplines. The comparison of large numbers of patients treated with scaffolds differing in their properties in combination with different cell types and numbers, which are either or not stimulated with different doses of growth factors, will provide critical evidence for future meta-analysis evaluating the safety, efficacy, and outcome measures of clinical trials using new bone tissue engineering approaches.

Human MSFE as a model for bone regeneration

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CHAPTER 3 SHORT (15 MINUTES) BMP-2 TREATMENT STIMULATES OSTEOGENIC DIFFERENTIATION OF HUMAN ADIPOSE STEM CELLS SEEDED ON CALCIUM PHOSPHATE SCAFFOLDS Janice R. Overman1,2*, Elisabet Farré-Guasch3,4*, Marco N. Helder5, Christiaan M. ten Bruggenkate2, Engelbert A.J.M. Schulten2, Jenneke Klein Nulend1

Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, The Netherlands Department of Oral and Maxillofacial Surgery, VU University Medical Center/ACTA, MOVE Research Institute Amsterdam, The Netherlands Department of Basic Sciences, Faculty of Medicine and Health Sciences, International University of Catalonia, Barcelona, Spain. Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, International University of Catalonia, Barcelona, Spain Department of Orthopedic Surgery, VU University Medical Center Amsterdam, MOVE Research Institute Amsterdam, The Netherlands Shared first authorship, J.R. Overman and E. Farré-Guasch contributed equally to this manuscript.

Tissue Engineering part A 2013; 19:571-581

Chapter 3

ABSTRACT A one-step concept for bone regeneration has been postulated in which human adipose tissuederived mesenchymal stem cells (hASCs) are harvested, triggered to differentiate, seeded on carriers, and implanted in the same operative procedure. Toward this goal it was investigated whether short (minutes) incubation with BMP-2 suffices to trigger osteogenic differentiation of hASCs seeded on calcium phosphate carriers. hASCs were treated with or without BMP-2 (10 ng/ml) for 15 minutes, and seeded on ß-tricalcium phosphate granules (ß-TCP; sized <0.7mm or >0.7mm) or biphasic calcium phosphate (BCP; 60%/40% or 20%/80% hydroxyapatite/ß-TCP). Attachment was determined after 10-30 minutes. Proliferation (DNA content) and osteogenic differentiation (alkaline phosphatase activity, gene expression) were analyzed up to 3 weeks of culture. hASC attachment to the different scaffolds was similar, and unaffected by BMP-2. It stimulated gene expression of the osteogenic markers CBFA1, collagen-1, osteonectin, and osteocalcin in hASCs seeded on BCP and ß-TCP. Down regulation of osteopontin expression by BMP-2 was seen in BCP-seeded cells only. BMP-2 treatment inhibited expression of the adipogenic marker PPAR-γ. In conclusion, 15 minutes BMP-2 pre-incubation of hASCs seeded on BCP/ß-TCP scaffolds had a long-lasting stimulating effect on osteogenic differentiation in vitro. These results strongly support a one-step clinical concept for bone regeneration. KEY WORDS: Mesenchymal Stem Cells, Bone, Cell Differentiation, Ceramic Scaffolds, Morphogens

Short (15 min) BMP-2 treatment stimulates osteogenic differentiation of hASCs

INTRODUCTION The use of an autologous bone graft is still the golden standard for bone reconstruction. It is the only type of bone graft supplying living bone cells, and has osteogenic, osteoinductive, as well as osteoconductive properties. This autograft does not provoke an immunological response since the tissue is retrieved from the same individual.1 There are many advantages using autologous bone, but there are also disadvantages associated with harvesting of the bone transplant, such as limited availability of bone volume, donor-site morbidity, and risk of infection. The use of allografts, xenografts, or biosynthetic substitutes eliminates these disadvantages associated with autologous bone harvesting.2 Biosynthetic substitutes such as β-tricalcium phosphate (β-TCP), hydroxyapatite (HA), and mixtures of HA/β-TCP (biphasic calcium phosphates; BCP) have been successfully used as a graft material, because of their good biocompatibility and chemical composition, which resembles the composition of the natural bone matrix.3-6 An important issue to consider regarding graft material degradation and bone ingrowth is the pore size; large pore sizes (≥ 500 µm) promote neo-vascularization and favor mineralized bone ingrowth,7 whereas smaller pore sizes (90-120 μm) primarily induce endochondral bone formation.8 Therefore in the present study β-TCP and BCP biomaterials were used with a high porosity (60-90%), and a pore size between 500 and 1400 µm for the β-TCP scaffolds and between 500 and 1000 µm for the BCP scaffolds. Nevertheless, despite these structural optimizations, the lack of osteogenic properties still results in a slower rate of new bone formation.9, 10 Autologous adult mesenchymal stem cells (MSCs) provide new and innovative tools in tissue engineering, and may be combined with synthetic scaffolds to introduce osteogenic bioactivity. These bioactive scaffolds may then be used to restore or replace tissues or organs. Bone marrow is a common source for MSCs (BM-MSCs), but this is only available in limited amounts.11 Adipose tissue has been described as an alternative source for MSCs.12 The adipose tissue-derived mesenchymal stem cells (ASCs) have similar surface marker profiles as the BM-MSCs, and also show multidifferentiation potential towards the adipogenic, chondrogenic, myogenic, neurogenic, and osteogenic lineage.13 In the field of bone tissue engineering, ASCs have been successfully used to repair critical size calvarial defects in animals 14-17 as well as in a 7 year-old girl.18 It is as yet unclear whether stimulation of ASCs with bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-β superfamily, should be performed for optimal osteogenesis, or that the local (orthotopic) micro-environment is sufficient. Earlier reports have shown beneficial effects of BMP-2 on osteogenic differentiation of stem cells/ progenitors in vitro and in vivo 19-22, and on bone formation and bone repair in vivo.23, 24 BMP-2 is available as an FDA-approved recombinant human protein 24, 25 and has been used extensively in clinical practice. Although initially only studies were published showing beneficial effects of BMP-2 treatment, recently also adverse effects such as bone overgrowth and swelling were reported.26, 27 This clearly questions the high (mg-range) dosages of BMP-2 used in clinical studies. Previously a novel clinical concept has been postulated, in which ASCs are harvested 45

Chapter 3

from adipose tissue, seeded on scaffolds, and re-implanted during the same surgical procedure.28 Also, a short ex vivo pre-incubation of the ASC preparations with osteogenic factors was envisioned. Therefore the aim of this study was to test whether a short (minutes) incubation with BMP-2 induces osteogenic differentiation of hASCs seeded on calcium phosphate carriers in vitro.

Short (15 min) BMP-2 treatment stimulates osteogenic differentiation of hASCs

MATERIALS AND METHODS Calcium phosphate scaffolds Four different calcium phosphate scaffolds were used: (1) Straumann®BoneCeramic (Straumann, Basel, Switzerland), a porous BCP with 60% HA and 40% ß-TCP (BCP 60/40), (2) Straumann®BoneCeramic, a porous BCP with 20% HA and 80% ß-TCP (BCP 20/80), (3) Ceros® TCP (Mathys, Bettlach, Switzerland), a porous ß-TCP with particle size 0.5-0.7 mm (ß-TCP, <0.7mm), and (4) Ceros® TCP, a porous ß-TCP with particle size 0.7-1.4 mm (ß-TCP, >0.7mm) (Table 1). Donors Subcutaneous adipose tissue was harvested from the abdominal wall of ten healthy women (age 23-61) undergoing elective abdominal wall correction at the Tergooiziekenhuizen Hilversum, The Netherlands. The Ethical Review Board of the VU University Medical Center, Amsterdam, The Netherlands, approved the study protocol. Informed consent was obtained from all patients. Isolation of human adipose stem cells (hASC) Human adipose tissue was obtained by resection. hASCs were isolated from the resection material as described earlier with minor modifications.29 Adipose tissue was cut into small pieces, and enzymatically digested with 0.1% collagenase A (Roche Diagnostics GmbH, Mannheim, Germany) for 45 minutes at 37ºC in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) under intermittent shaking. A single cell suspension was obtained by filtration through a 100 μm mesh filter. After thorough washing with PBS containing 1% BSA, a Ficoll density centrifugation step (1.077 g/ ml ficoll, 280 ± 15 mOsm; Lymphoprep, Axis-Shield, Oslo, Norway) was performed to remove remaining erythrocytes from the hASC-containing cell suspension called stromal vascular fraction (SVF). After centrifugation at 600xg for 10 minutes, the resulting SVF pellet containing the hASCs was resuspended in medium composed of Dulbecco’s modified Eagle’s medium (DMEM; LifeTechnologies™ Europe BV, Bleiswijk, The Netherlands) containing 10% fetal bovine serum (FBS; Hyclone Fetalclone I, Thermo Scientific, Logan, UT, USA), 500 µg/ml streptomycin sulphate (Sigma-Aldrich®, St. Louis, MO, USA), 500 µg/ml penicillin (Sigma-Aldrich®), and 2.5 µg/ml amphotericin B (Gibco®). Cell viability was assessed using the trypan blue exclusion assay. Cells were counted using a counting chamber (Burker-Turk, Marienfeld, Germany) and a light microscope at 10 x magnification. Then cells were immediately seeded and cultured on the different scaffolds, or resuspended in Cryoprotective medium (Recovery™ Cell Culture Freezing medium; LifeTechnologies™ Europe BV, Bleiswijk, The Netherlands), frozen under “controlled rate” conditions, and stored in liquid nitrogen until further use for attachment, proliferation, and differentiation studies. The latter cells are referred to as “fresh-frozen” cells below. Samples from different donors were studied individually in all experiments. Heterogeneity studies including cell characterization and multipotent differentiation potential of these cells have been reported previously by our group.29, 30 Recently, we determined that ~90% of the ASCs within the freshly isolated SVF rapidly adhere to various scaffold types31. 47

Chapter 3

Table 1. Characteristics of the different BCP and β-TCP scaffolds used.

Composition, particle size, porosity, and pore width of Straumann® BoneCeramic (60/40), Straumann® BoneCeramic (20/80), Ceros® TCP (<0.7mm), and Ceros® TCP (>0.7mm). HA, hydroxyapatite; ß-TCP, ß-tricalcium phosphate; BCP, biphasic calcium phosphate.

BMP-2 treatment and hASC attachment to BCP and TCP scaffolds Freshly isolated and “fresh-frozen” hASC-containing cell suspensions were either or not incubated for 15 minutes with 10 ng/ml BMP-2 (Peprotech®, London, UK) at room temperature or at 37°C, as previously described.20 Then the cells were washed with PBS, centrifuged, and resuspended in DMEM without supplements. Cell suspensions were seeded at 1x105 cells per 25-35 mg of scaffold in 2 ml tubes (Eppendorf Biopur®, Hamburg, Germany). Cells were allowed to attach for 30 minutes. Then hASC-seeded scaffolds were washed with PBS, lysis buffer was added, and the DNA content (as a measure for cell number) was determined using the Cyquant Cell Proliferation Assay Kit (Molecular Probes/Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Absorption was read at 480 nm excitation and 520 nm emission in a microplate reader (BioRad Laboratories GmbH, Munich, Germany). Culture of human adipose stem cells Cryopreserved hASCs were thawed and seeded at 2.5x105 cells per 25-35 mg of BCP scaffold (Straumann®BoneCeramic, 60/40, and Straumann®BoneCeramic, 20/80) and β-TCP scaffold (Ceros®TCP, <0.5mm, and Ceros®TCP, >0.5mm). After osteogenic induction with BMP-2 as described in paragraph 2.4, the hASC-seeded scaffolds were cultured up to 21 days in 12 well plates with Costar® Transwell® containers (Corning Life Sciences, Lowell, MA, USA) containing expansion medium (DMEM) supplemented with 10% FBS, antibiotics, and 50 µM ascorbic acid (Merck, Darmstadt, Germany). hASCs seeded on tissue culture plastic (control) were cultured in expansion medium in the presence of 10 mM β-glycerol phosphate (Sigma) to provide a phosphate donor. The hASC-seeded scaffolds were incubated at 37°C under 5% CO2 in a humidified atmosphere, and medium was changed three times per week. hASC proliferation on BCP and β-TCP scaffolds hASC proliferation was assessed by determination of the DNA content of hASC cultures. Cells were seeded on BCP and β-TCP scaffolds, allowed to attach, and cultured as described above in the paragraphs “BMP-2 treatment and hASC attachment to BCP and TCP scaffolds” and “Culture of human adipose stem cells”. After 4, 14, and 21 days of culture, the hASC-seeded scaffolds were washed with PBS, and transferred to Eppendorf tubes. Cyquant® lysis buffer was added and the DNA content per tube was determined using the CyQuant® Cell Proliferation Assay 48

Short (15 min) BMP-2 treatment stimulates osteogenic differentiation of hASCs

Kit as described above in paragraph 2.4. Osteogenic differentiation of hASCs on BCP and β-TCP scaffolds ALP activity was measured to assess the osteoblastic phenotype of hASC cultures. Cells were seeded on BCP and β-TCP scaffolds, allowed to attach, and cultured as described above in the paragraphs “BMP-2 treatment and hASC attachment to BCP and TCP scaffolds” and “Culture of human adipose stem cells”. After 4, 14, and 21 days of culture, the cells were lysed with CyQuant® lysis buffer as described above in paragraph 2.6 to determine ALP activity and protein content. P-nitrophenyl-phosphate (Merck) at pH 10.3 was used as substrate for ALP as described earlier.19 The absorbance was read at 410 nm. ALP activity was expressed as µMole/ µg cellular protein. The amount of protein was determined by using a BCA Protein Assay reagent Kit (PierceTM, Rockford, Ill, USA), and the absorbance was read at 540 nm with a microplate reader (BioRad Laboratories). Donors were also used in the study by Jurgens et al.31 In this study, expression of mature osteogenic differentiation markers was demonstrated both at the gene expression level as well as at the protein level. In our study alkaline phosphatase enzyme activity was quantitatively determined in addition to the mRNA expression profiles to verify the osteogenic differentiation at the protein level. Colony-forming unit fibroblasts (CFU-f) assay and CFU-f depletion assay CFU-f assays were performed to assess if the colony forming capacity of hASCs within the isolated SVF was affected by BMP-2 treatment, as described elsewhere.32 A total of 1x103 or 1x104 cells were seeded in 6-well plates (Greiner Bio-OneTM, Alphen a/d Rijn, The Netherlands). After 14 days of culture the cells were fixed in 4% formaldehyde and stained with a 0.2% toluidine blue in borax buffer (pH 12) for 1 minute. A colony was defined as a group of cells consisting of ≥ 10 clustered cells. The number of colonies was counted using a light microscope at 100x magnification. The percentage of CFU-f per total number of hASCs seeded was calculated. The “CFU-f depletion” assay was used as an indirect measurement to determine hASC attachment to the scaffolds31. The scaffold washing steps were collected to obtain the non-adhered cells, which were pelleted and used for CFU-f frequency determination as described above. After 14 days of culture the percentage of retrieved colonies was divided by the percentage of colonies obtained from the plastic-seeded hASCs, as an indirect measurement of hASC attachment and viability. Analysis of gene expression Total RNA was extracted from hASCs of eight donors cultured on tissue culture plastic and biphasic calcium phosphate (BCP 60/40 and BCP 20/80) and β-tricalcium phosphate (β-TCP, <0.7mm, and β-TCP, >0.7mm) scaffolds for 4, 14, and 21 days, using TRIzol® reagent (LifeTechnologies™) according to the manufacturer’s instructions, and stored at –80ºC prior to assay. The cDNA synthesis was performed in a thermocycler GeneAmp® PCR System 9700 PE (Applied Biosystems, Foster City, CA, USA), using SuperScript® VILO™ cDNA Synthesis Kit (LifeTechnologies™) with 0.1 µg total RNA in a 20 µl reaction mix containing VILO™ Reaction Mix and SuperScript® Enzyme Mix. cDNA was stored at –20ºC prior to real-time PCR analysis. 49

Chapter 3

Real-time PCR reactions were performed using 2.5 μl cDNA and SYBR® Green Supermix (Roche Laboratories, IN, USA) according to the manufacturer’s instructions in a LightCycler® (Roche Diagnostics). The target and reference genes were amplified in separate wells. All reactions were performed in triplicate. In each run the reaction mixture without cDNA was used as negative control. All primers used for real-time PCR were from LifeTechnologies™. For quantitative realtime PCR, the values of relative target gene expression were normalized for relative YWHAZ housekeeping gene expression. Real-time PCR was used to assess expression of the following genes: cbfa1/runx2 (CBFA1), collagen 1 (COL1), alkaline phosphatase (ALP), osteonectin (ON), osteopontin (OPN), osteocalcin (OC), and peroxisome proliferator-activated receptor gamma (PPARγ). In each assay for osteogenic markers, mRNA preparations of osteoblasts were used as a reference and internal control for the primer sets to pick up the specific mRNA of interest. Human primary osteoblasts were used as positive control. Gene expression was compared between cells seeded on BCP and β-TCP scaffolds with or without BMP-2 treatment. Statistical analysis Data were obtained from hASCs of ten donors in total. All data were expressed as mean ± SEM. The effect of BMP-2 treatment compared to non-treated cells was tested with the Student’s t-test for single group mean, or with the Wilcoxon Signed Rank test for single group median. Differences in DNA content, ALP activity, and gene expression between groups were tested with Student’s paired two-tailed t-test. ANOVA two-way analysis of variance was used to compare attachment data between BCP and β-TCP scaffolds, and to compare DNA content and ALP activity between the different time points (day 4, 14, and 21). To determine a timedependent increase of osteogenic gene expression levels, the linear regression coefficiency was determined. Differences were considered significant if p<0.05. Statistical analysis was performed using SPSS 17.0 (SPSS Inc., Chicago, Ill, USA), Kyplot v2.0 beta 15, Japan, and GraphPad Prism® 5.01 (GraphPad Software Inc., La Jolla, CA, USA).

Short (15 min) BMP-2 treatment stimulates osteogenic differentiation of hASCs

RESULTS Does BMP-2 pre-stimulation affect human ASCs similar to goat ASCs on tissue culture plastic? BMP-2 increased the colony forming unit frequency of hASCs seeded on tissue culture plastic. The number of CFU-f was counted 14 days after seeding of freshly isolated hASC preparations which were pre-stimulated with or without BMP-2 for 15 minutes. Cells were cultured on plastic during this 14-day culture period (n=6). BMP-2 treatment significantly increased the percentage of CFU-f by 1.8-fold (p<0.0001; Fig. 1).

Figure 1. Effect of short stimulation with BMP-2 on colony forming unit formation from hASCs cultured on tissue culture plastic. A 15 minutes BMP-2 pre-treatment increased the colony forming potential of hASCs cultured on tissue culture plastic for two weeks (p=5.5x10-7), reflecting the number of viable hASCs in adipose tissue. Values are mean ± SEM (n=6). ***Significant effect of BMP-2, p<0.001. CFU-f, colonyforming unit fibroblasts; BMP-2, bone morphogenetic protein-2; plastic, tissue culture plastic.

Human ASCs, pre-stimulated for 15 minutes with BMP-2, did not show upregulation of osteogenic gene expression, nor downregulation of PPAR-γ expression, when seeded on tissue culture plastic. BMP-2 did not significantly upregulate the expression levels of CBFA1, COL1, and ON in hASC cultured on tissue culture plastic compared to control cultures (Fig. 2A-C). BMP-2 also did not significantly inhibit PPAR-γ expression (Fig. 2D). Interestingly osteogenic gene expression, especially COL1 and ON gene expression, increased in hASCs during three weeks of culture. To confirm a correlation between the osteogenic gene expression levels and the culture time, the correlation coefficient was calculated. These calculations indicated that culture time positively affected the increase in gene expression of CBFA1, COL1, and ON in BMP-2–treated cells (Table 2). The correlation coefficient between culture time and the decreasing PPAR-γ gene expression levels was also calculated, and a positive correlation in BMP-2–treated cells was found. 51

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Figure 2. Effect of short stimulation with BMP-2 on osteogenic gene expression and PPAR-γ gene expression in hASCs cultured on tissue culture plastic. A 15 minutes BMP-2 pre-treatment did not increase the expression levels of CBFA1, COL1, or ON after 14 or 21 days of culture. BMP-2 also did not inhibit PPAR- γ expression levels after 21 days (p=0.08). Values are mean ± SEM (n=2-6), and relative to the control value at time point 0. CBFA1, core binding factor A1 (or Runx-2); COL1, collagen type1; ON, osteonectin; PPAR-γ, peroxisome proliferator-activated receptor gamma.

Table 2. Correlation between osteogenic and PPAR-γ gene expression and culture time of hASCs cultured on tissue culture plastic.

Regression coefficient (R2) and their p-values (p) calculated from the gene expression levels of CBFA1, COL1, ON, and PPAR-γ of hASCs cultured on tissue culture plastic. CBFA1, core binding factor alpha 1; COL1, collagen type 1; ON, osteonectin; PPAR-γ, peroxisome proliferator-activated receptorgamma. *Significant correlation, p<0.05.

Short (15 min) BMP-2 treatment stimulates osteogenic differentiation of hASCs

How do human ASCs behave on calcium phosphate scaffolds? BMP-2 did not affect cell attachment on different scaffolds and increased hASC number. Before starting the analyses of proliferation and differentiation, the optimal conditions for the hASCs to be handled in vitro were investigated, thereby keeping in mind the conditions preferred for a one-step surgical procedure. Freshly isolated hASCs as well as ”fresh-frozen” hASCs were seeded on BCP and β-TCP scaffolds (without any pre-treatment for 10, 20, and 30 minutes, and at room temperature or 37°C). After removal of the non-attached cells, the DNA content was determined as a measure of cell number. The results indicated that the maximum number of cells did attach to the carriers within 10 minutes (data not shown; data are in line with previously reported data.31 No marked difference was observed between attachment at room temperature and at 37°C. BMP-2 did not affect cell attachment to the BCP and/or β-TCP scaffolds (Fig. 3A). Remarkably, it seemed that a higher number of cells was attached to both types of BCP scaffolds used, whether hASCs had been BMP-2 treated or not, compared to TCP scaffolds. The number of CFU-f cultured from the BCP and β-TCP washings was less than 0.03% of the total number of CFU-f (Fig. 3B). This indicated that at least the hASCs within the heterologous primary hASC isolates did adhere to the scaffolds, as previously described for other types of scaffolds.32 Intriguingly, the number of CFU-f of the hASCs collected from BCP 20/80 and β-TCP >0.7mm was significantly lower (p=0.05 and p<0.0001, respectively) compared to the number of CFU-f collected from the two other scaffolds (BCP 60/40 and β-TCP <0.7mm). These findings indicate that hASCs may have a higher affinity for these specific BCP and β-TCP scaffolds. The number of CFU-f cultured from the BCP and β-TCP washings was less than 0.03% of the total number of CFU-f (Fig. 3B). This indicated that at least the hASCs within the heterologous primary hASC isolates did adhere to the scaffolds, as previously described for other types of scaffolds.32 Intriguingly, the number of CFU-f of the hASCs collected from BCP 20/80 and β-TCP >0.7mm was significantly lower (p=0.05 and p<0.0001, respectively) compared to the number of CFU-f collected from the two other scaffolds (BCP 60/40 and β-TCP <0.7mm). These findings indicate that hASCs may have a higher affinity for these specific BCP and β-TCP scaffolds. BMP-2 affected hASC proliferation. BMP-2 pre-treatment increased the DNA content of hASCs seeded on the scaffolds compared to non-treated controls. On β-TCP a marginally significant increase was noted at day 14 by 2.1 ± 0.9 fold (p=0.06), and on BCP at day 21 by 2.7 ± 0.8 fold (p=0.06, Fig. 4). BMP-2 did not stimulate ALP activity in hASCs seeded on BCP or β-TCP scaffolds. ALP activity was measured after 4, 14, and 21 days of culture under control or BMP-2–prestimulated conditions. The ALP activity was expressed as fold-increase of BMP-2-treated cells versus non-treated cells. Although ALP activity increased in both BMP-2–treated and untreated hASCs during 21 days of culture, it did not reach significance at any time point measured (Fig. 5).

Chapter 3

Figure 3. Short stimulation with BMP-2 did not affect the attachment of human SVF cells to calcium phosphate scaffolds. A) A 15 minutes BMP-2 pre-treatment did not change the cell number (expressed as DNA, ng) of hASCs on different BCP and β-TCP scaffolds after allowing attachment for 30 minutes. More hASCs seemed to attach to the BCP scaffolds compared to β-TCP scaffolds. B) A 15 minutes BMP-2 pretreatment affected the percentage of CFU-f from non-attached cells dependent on the scaffold. More BMP-2–treated cells were attached to BCP 20/80 (p=0.05) and ß-TCP >0.7mm (p=2.2 x10-7). Values are calculated relative to the CFU-f proliferation assay data (Fig. 1). Values are mean ± SEM (n=6). Significant difference between scaffolds, *p<0.05, ***p<0.001. CFU-f, colony-forming unit fibroblasts; BMP-2, bone morphogenetic protein-2; BCP, biphasic calcium phosphate; β-TCP, β-tricalcium phosphate.

Figure 4. Short stimulation with BMP-2 did not affect proliferation in hASCs seeded on calcium phosphate scaffolds. hASCs were pre-treated for 15 minutes with BMP-2 and cultured on different BCP and β-TCP scaffolds for 4, 14, and 21 days. BMP-2 did not increase DNA content in hASCs seeded on BCP and β-TCP. DNA content is expressed as BMP-2-treated-over-untreated control ratio. Values are mean ± SEM (n=6). BMP-2, bone morphogenetic protein-2; BCP, biphasic calcium phosphates; β-TCP, β-tricalcium phosphate; con, control.

Figure 5. Short stimulation with BMP-2 did not increase ALP activity in hASCs cultured on calcium phosphate scaffolds. hASCs were pre-treated for 15 minutes with BMP-2 and cultured on different BCP and TCP scaffolds for 4, 14, and 21 days. BMP-2 did not increase ALP activity in hASCs seeded on BCP and β-TCP. Values are mean ± SEM (n=6-7). ALP activity is expressed as BMP-2-treated-over-untreated control ratio. ALP, alkaline phosphatase activity; BMP2, bone morphogenetic protein-2; BCP, biphasic calcium phosphate; β-TCP, β-tricalcium phosphate; con, control.

Short (15 min) BMP-2 treatment stimulates osteogenic differentiation of hASCs

BMP-2 increased osteogenic gene expression, and decreased PPAR-γ expression in hASCs seeded on calcium phosphate scaffolds. BMP-2–pre-treatment significantly increased mRNA gene expression levels of CBFA1, COL1, ON, and OC in hASCs cultured on BCP and β-TCP (Fig. 6A,B,C,D) compared to unstimulated controls. BMP-2 also upregulated ALP gene expression by hASCs cultured on BCP. On these BCP scaffolds, ALP gene expression was significantly upregulated at day 14, and decreased back to day-0-levels at day 21 (Fig. 6E). Since unstimulated controls showed a similar but delayed pattern of ALP gene expression (significant upregulation at day 21), the osteogenic induction may be primarily governed by the scaffold properties, and only facilitated by BMP-2 pre-treatment of hASCs seeded on these scaffolds. On β-TCP, a gradual but lower increase in ALP gene expression was observed, which never reached significance at any time point tested. OPN gene expression showed a decrease in time for both hASCs seeded on BCP and β-TCP. Only at day 21, and only on BCP scaffolds, the inhibition of OPN gene expression by BMP-2 was significant (Fig. 6F). BMP-2 strongly inhibited PPAR-γ gene expression by cells cultured on BCP as well as β-TCP (Fig. 6G). Furthermore a significant correlation between the expression levels of CBFA1, ALP, and OPN expression and culture time in BMP-2–treated cells cultured on β-TCP was observed, similar as in cells cultured on plastic (Table 3).

Table 3. Correlation between osteogenic and PPAR-γ gene expression and culture time of hASCs cultured on BCP and β-TCP.

Regression coefficient (R2) and their p-values (p) calculated from the gene expression levels of CBFA1, COL1, ALP, COL1, ON, OPN, OCN and PPAR-γ of ASCs cultured on tissue culture plastic. CBFA1, core binding factor alpha 1; ALP, alkaline phosphatase; COL1, collagen type 1; ON, osteonectin; OPN, osteopontin; OCN, osteocalcin, PPAR-γ, peroxisome proliferator-activated receptor-gamma. *Significant correlation, p<0.05.

Chapter 3

Short (15 min) BMP-2 treatment stimulates osteogenic differentiation of hASCs

Figure 6. Effect of short stimulation with BMP-2 on osteogenic gene expression levels and PPAR-γ expression levels in hASCs. hASCs were pre-treated for 15 minutes with BMP-2 and cultured on different BCP and β-TCP scaffolds for 4, 14, and 21 days. A) BMP-2 increased CBFA1 gene expression in hASCs seeded on BCP at day 21 (p=0.009), and in hASCs seeded on β-TCP at day 14 and 21 (p=0.004 and p=0.003 respectively). B) BMP-2 increased COL1 expression at day 14 (p=0.05) and day 21 (p=0.05) in hASCs seeded on BCP. BMP-2 also increased COL1 expression at day 14 (p=0.016) and day 21 (p=0.00005) in hASCs seeded on β-TCP. C) BMP-2 increased ON gene expression at day 14 (p=0.04) and day 21 (p=0.0005) in BCP seeded cells. BMP-2 also increased ON expression at day 14 (p=0.05) and day 21 (p=7.0x10-5) in β-TCP seeded cells. D) BMP-2 increased OC expression levels at day 21 in both BCP (p=0.04) and β-TCP (p=0.02). E) BMP-2 increased ALP gene expression in cells seeded on BCP at day 14 (p=0.04), but inhibited ALP expression at day 21 (p=0.04). A similar trend (not significant) was observed in cells seeded on β-TCP. F) BMP-2 inhibited OPN gene expression at day 21 in cells seeded on BCP (p=0.04), but did not affect OPN gene expression in ACSs seeded on β-TCP. G) BMP-2 decreased PPAR-γ expression levels at day 14 (p=0.03) and day 21 (p=0.03) in cells seeded on BCP. BMP-2 decreased PPAR- γ expression levels at day 21 (p=6.5 x10-7) in cells seeded on β-TCP. Values are mean ± SEM (n=4-8). Significant effect of BMP-2, *p<0.05, **p<0.01, ***p<0.0001. Expression levels are normalized to the housekeeping gene YWHAZ. CBFA1, core binding factor aplha1; COL1, collagen type 1; ON, osteonectin; OC, osteocalcin; ALP, alkaline phosphatase; OPN, osteopontin; PPAR-γ, peroxisome proliferator-activated receptor-gamma; BMP-2, bone morphogenetic protein-2; BCP, biphasic calcium phosphates; β-TCP, β-tricalcium phosphate.

Chapter 3

DISCUSSION The aim of this study was to investigate whether a short pre-incubation of hASCs with BMP-2 would result in a long-lasting osteogenesis-promoting effect in vitro as previously reported for goat ASCs. Furthermore we investigated whether this osteogenic induction could be enhanced by seeding of BMP-2–pre-stimulated cells on BCP and ß-TCP calcium phosphate scaffolds. The ultimate goal was to validate the BMP-2 pre-stimulation and hASC seeding steps for their feasibility to fit within the time frame of a one-step surgical procedure as described earlier by Helder et al.28 We found that (i) hASCs showed differential responses when compared to goat ASCs on tissue culture plastic after pre-stimulation of hASCs for 15 minutes with a 10 ng/ml dose of BMP-2; (ii) BMP-2 pre-stimulation significantly increased the frequency of colony forming units of hASC preparations; (iii) hASCs adhered rapidly (within 10 minutes) to the BCP and ß-TCP scaffolds, independent of BMP-2 pre-treatment; (iv) proliferation and osteogenic differentiation after seeding on the BCP/ß-TCP scaffolds were enhanced by BMP-2 pre-treatment, with concomitant downregulation of adipogenic gene expression; and (v) BCP effects appeared more pronounced and/or differentiation-accelerating compared to their ß-TCP counterparts. Our osteogenic differentiation data obtained from BMP-2–pre-stimulated hASCs cultured on plastic did not reveal a stimulation of osteogenic differentiation within 4 days, as reported by Knippenberg et al. using goat ASCs.20 A plausible explanation for this discrepancy could be the origin of the cells used, since species-determined differences may exist. For example, rat and human mesenchymal stem cells differ in their properties regarding metabolism and proliferation.33 An alternative explanation of this discrepancy may be that hASCs respond stronger to the co-factors ascorbic acid and ß-glycerol phosphate present in the culture medium; the cells already showed upregulation of differentiation markers without BMP-2 pre-treatment, which was observed to a much lower degree for goat ASCs. The increase in colony forming unit frequency upon BMP-2 pre-stimulation is surprising. Increased proliferation rates (see below) may be part of the explanation, since this will enhance the chance of colonies growing fast enough to reach the threshold size (i.e. ≥10 clustered hASCs). Alternatively, BMP-2 pre-treatment may enhance hASC attachment rates on tissue culture plastic and/or efficiency of the hASCs within the fresh isolates which increases the number of colonies. We intend to address this issue in more detail in follow-up studies. We have not specifically tested to what extent BMP-2 is washed off from the cell surface. It may well be that at least part of the BMP-2 is still sequestered by the BMP-2 receptors on the cell surface. However, even if some aspecific sticking of BMP-2 occurs, this will never exceed the administered concentration of 10 ng/ml, as also used in the study of Knippenberg et al.20 The rapid attachment of the hASCs to the scaffolds was in line with previous studies of our group for other classes of scaffolds, consisting of polymeric and collagenous biomaterials.31 Interestingly, our data set indicates a slightly higher attachment rate of hASCs (as determined by measuring DNA content) on BCP scaffolds versus their ß-TCP counterparts. We speculate, but did not study in detail, that this may be due to different scaffold surface characteristics caused by surface topography and/or material compositional differences (either or not HA-containing), 58

Short (15 min) BMP-2 treatment stimulates osteogenic differentiation of hASCs

which were shown previously to have major differences on osteoconductivity, osteoinductivity, and cell behaviour.34, 35 The enhancement of proliferation and osteogenic differentiation of the hASCs on calcium phosphate scaffolds by BMP-2 pre-treatment resembles the outcome of a study where rat ASCs were cultured on a β-TCP scaffold.36 However, an obvious difference between that study and our study is that the BMP-2 in the study with rat ASCs was continuously present in the culture medium at 100 ng/ml concentration, whereas our study employed only a 15 minutes exposure of 10 ng/ml BMP-2 followed by culture in “plain” expansion medium for the full culture period. Nevertheless the study using rat ASCs confirms the clear difference in differentiation efficiencies on tissue culture plastic vs. calcium phosphate scaffolds. The efficacy of 15 minutes stimulation has been shown earlier to be similar for both BMP-2 and BMP-7 (OP-1).20 Interestingly, the responses to both growth factors were divergent; BMP-2 induced an osteogenic response, while BMP-7 resulted in a chondrogenic phenotype of ASCs. In conjunction with the observed osteogenic differentiation, the differentiation towards adipogenesis was strongly down-regulated in hASCs. These findings are in line with data presented in the literature and support the view that stimulation into the osteogenic direction simultaneously inhibits differentiation along the adipogenic pathway. The level of PPAR-γ gene expression is an important parameter for adipogenesis; previous research showed that activation of the PPAR-γ receptor induces adipogenesis in bone marrow stromal cells37, whereas PPAR-γ haploinsufficiency stimulates osteoblastogenesis in mouse stem cells.38 More recently it has been shown that two signaling cascades promote osteoblastic differentiation from MSCs through two distinct modes of PPAR-γ transrepression39 and that BMPs might interfere with the adipogenic differentiation of MSCs dependent on the type of BMP-receptor involved.40 Thus the positive effect of BMP-2 on hASC differentiation is also confirmed by the concomitant downregulation of PPAR-γ expression. The observation that osteogenic differentiation may be enhanced and/or accelerated in hASCs seeded on BCP versus ß-TCP scaffolds may be explained by surface topography and/ or material compositional differences as described above. However, another consideration may be that the HA component in the BCP may provide additional stiffness to the scaffold, which favors bone differentiation as highlighted in recent reports.41-44 This study clearly shows that the interaction with the calcium phosphate (CaP) scaffolds markedly enhanced the osteogenic phenotype of the cells compared with culture plates. We hypothesize that this rapid attachment to the stiff bone-like surface may contribute positively to the osteogenic differentiation process, and may possibly at least in part overrule possible diverting signals such as microenvironmental cytokines, growth factors, and BMP antagonists which may affect in vivo outcome.43, 45-47.

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CONCLUSIONS In conclusion, this study revealed that a 15 minutes incubation with a low dose of BMP-2 is sufficient to stimulate hASCs to gain an osteogenic phenotype in vitro after culturing on either BCP or β-TCP. Our findings indicate that this short pre-treatment is a very promising tool for its use in a clinical one-step surgical procedure. These results will be extrapolated and applied in further development of the one-step surgical concept 28 in a clinical maxillary sinus floor elevation model. Whether the differences in osteogenic gene expression by hASCs seeded on the different scaffold types will influence bone formation in this clinical setting needs yet to be established. ACKNOWLEDGEMENTS The work of J.R. Overman was supported by the Research School of the Academic Centre for Dentistry Amsterdam (ACTA), The Netherlands. The work of E. Farré Guasch was partly supported by a travel grant from AGAUR, Spain, grant number 2009 BE2 00114 and a fellowship from the International University of Catalonia. The authors wish to extend their thanks to the surgical staff of Tergooiziekenhuizen Hilversum Hospital, The Netherlands, for kindly providing the human adipose tissue for this study. They also thank J. Hogervorst for excellent technical assistance in the isolation of ASCs, and V. Everts and A.D. Bakker for critical scientific feedback on the manuscript.

Short (15 min) BMP-2 treatment stimulates osteogenic differentiation of hASCs

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CHAPTER 4 GROWTH FACTOR GENE EXPRESSION PROFILES OF BONE MORPHOGENETIC PROTEIN-2â&#x20AC;&#x201C;TREATED HUMAN ADIPOSE STEM CELLS SEEDED ON CALCIUM PHOSPHATE SCAFFOLDS IN VITRO Janice R. Overman1,2, Marco .N. Helder3, Christiaan M. ten Bruggenkate2, Engelbert A.J.M. Schulten2, Jenneke Klein Nulend1, Astrid D. Bakker1

Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute, Amsterdam, The Netherlands Department of Oral and Maxillofacial Surgery, Academic Centre for Dentistry Amsterdam/ VU University Medical Center, MOVE Research Institute Amsterdam, Amsterdam, The Netherlands Department of Orthopedic Surgery, VU University Medical Center Amsterdam, MOVE Research Institute Amsterdam, The Netherlands

BIOCHIMIE, Special Secretome Issue 2013; 95: 2304-2313

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ABSTRACT The secretome of stem cells strongly determines the outcome of tissue engineering strategies. We investigated how the secretome of human adipose stem cells (hASCs) is affected by substrate, BMP-2 treatment, and degree of differentiation. We hypothesized that as differentiation progresses, hASCs produce increasingly more factors associated with processes such as angiogenesis and bone remodeling. Human ASCs were treated for 15 min with BMP-2 (10 ng/ml) to enhance osteogenic differentiation, or with vehicle. Subsequently, hASCs were seeded on plastic or on biphasic calcium phosphate (BCP) consisting of 60% hydroxyapatite and 40% β-tricalcium phosphate. A PCR array for ~150 trophic factors and differentiation-related genes was performed at day 21 of culture. A limited set of factors was quantified by qPCR at days 0, 4, 14 and 21, and/or ELISA at day 21. Compared to plastic, BCP-cultured hASCs showed ≥2-fold higher expression of ~20 factors, e.g. cytokines such as IL-6, growth factors such as FGF7 and adhesion molecules such as VCAM1. Expression of another ~50 genes was decreased ≥2-fold on BCP vs. plastic, even though hASCs differentiate better on BCP than on plastic. BMP-2-treatment increased the expression of ~30 factors by hASCs seeded on BCP, while it decreased the expression of only PGF, PPARG and PTN. Substrate affected hASC secretion of Activin A and seemed to affect P1NP release. No clear association between hASC osteogenic differentiation and growth factor expression pattern was observed. Considering our observed lack of association between the degree of differentiation and the production of factors associated with angiogenesis and bone remodeling by hASCs, future bone regeneration studies should focus more on systematically orchestrating the secretome of stem cells, rather than on inducing osteogenic differentiation of stem cells only. Short incubation with BMP-2 may be a promising treatment to enhance both osteogenic differentiation and environmental modulation. KEY WORDS: Adipose stem cells; trophic factors; secretome; BMP-2; osteogenic differentiation; calcium phosphate. ABBREVIATIONS: (h)ASC, (human) adipose stem cell BCP, biphasic calcium phosphate BMP-2, bone morphogenetic protein-2 GAPDH, glyceraldehyde 3-phosphate dehydrogenase PCR, polymerase chain reaction YWHAZ, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide

Growth factor gene expression profiles of BMP-2 treated hASCs

INTRODUCTION Three components are considered to play a key role when developing a tissue engineered construct to repair large bone defects: mesenchymal stem cells (MSCs), an osteoconductive matrix, and inductive factors1, 2. These components are supposed to create a bio-active construct that, once implanted, contributes to the repair of bone. In the past decade, a vast amount of in vitro research has been performed on the proper combination of stem cells, substrate and inductive factors that induces osteogenic differentiation of stem cells, and enhances bone matrix formation by these cells 3-6. These investigations have led to valuable insights into the biological processes leading to stem cell differentiation. However, translation of these basic research data towards clinical application has proven difficult, partly because tissue engineering strategies that are interesting from an academic point of view often lack clinical applicability, and partly because a true understanding of the nature of the multitude of factors driving bone healing is still lacking. Adipose tissue is a rich source of MSCs, from which adipose-derived mesenchymal stem cells (ASCs) can be obtained in clinically relevant quantities and seeded on carriers within a single operative procedure with minimal patient discomfort. This so called â&#x20AC;&#x153;one-step surgical conceptâ&#x20AC;? has been preclinically tested in a goat spinal fusion model 7, 8, and hASCs are currently being applied to enhance jaw bone height using maxillary sinus floor elevation in humans, using calcium phosphate as a carrier 1. MSCs not only differentiate, but also have been shown to have a modulatory and chemo-attractive effect on their microenvironment 9, 10, which strongly contributes to the regeneration of tissues such as bone 11-13. MSCs produce numerous trophic factors that play a major role in the onset of fracture repair, inflammation modulation, and concomitant events such as angiogenesis and bone remodeling 14-17. ASCs have been shown to secrete a plethora of trophic factors. For example, ASCs secrete VEGF, and thus potentially stimulate the start of the vascularization process when implanted in vivo 17. VEGF promotes the growth of vascular endothelial cells in vitro and induces vascular leakage in vivo, which is important in inflammation control and de novo blood vessel formation and sprouting 18. ASCs also produce members of the fibroblast growth factor and transforming growth factor-Ă&#x; family 19. Thus, ASCs produce factors that potentially support bone regeneration in vivo. Bone regeneration is a complex event in which intercellular communication between implanted cells and their surrounding cell types is extensive and crucial in orchestrating the repair process 20-23. To date, little is known about the set of secreted trophic factors produced by ASCs that influence the aforementioned regeneration processes. It is also largely unknown how substrate and growth factor stimulation affect the secretome, and how the secretome of ASCs changes during their osteogenic differentiation. Therefore we investigated the dynamic changes in the secretome of hASCs cultured on two different substrates, while stimulating osteogenic differentiation by treatment with BMP-2. We hypothesized that as differentiation progresses, hASCs produce increasingly more factors associated with processes such as angiogenesis and bone remodeling. PCR array was performed to compare the gene expression levels of trophic factors produced by hASCs that were pre-treated with BMP-2 or with vehicle, seeded on plastic or on 67

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biphasic calcium phosphate (BCP), and cultured for 21 days. Previously we showed that a short BMP-2 treatment (15 min, 10 ng/ml) of hASCs induces osteogenic differentiation after 14 and 21 days of culture. The lowest amount of differentiation was observed when hASCs were seeded on tissue culture plastic, and the most was seen when cells were seeded on calcium phosphate carriers 21, 22. In the current study we found that substrate and BMP-2 treatment, but not the degree of osteogenic differentiation exerted a strong modulatory effect on trophic factor production by hASCs.

Growth factor gene expression profiles of BMP-2 treated hASCs

MATERIALS AND METHODS Donors Adipose tissue was harvested from residues of abdominal wall resections of 3 donors, aged 28, 44, and 58, who underwent elective abdominal wall correction at the Tergooiziekenhuizen Hilversum, The Netherlands. The Ethical Review Board of the VU Medical Center, Amsterdam, The Netherlands, approved the protocol. Informed consent was obtained from all patients. Calcium phosphate scaffold For this study we used Straumann® BoneCeramic (Straumann, Basel, Switzerland), a porous biphasic calcium phosphate (BCP) scaffold with 60% hydroxyapatite and 40% ß-tricalcium phosphate. hASC isolation hASCs were isolated from the resection material as described earlier with minor modifications 24. Adipose tissue was cut into small pieces, and enzymatically digested with 0.1% collagenase A (Roche® Diagnostics GmbH, Mannheim, Germany) for 45 min at 37ºC in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA; Sigma-Aldrich®, St. Louis, MO, USA) under intermittent shaking. A single cell suspension was obtained by filtration through a 100 μm mesh filter. After thorough washing with PBS containing 1% BSA, a Ficoll® density centrifugation step (1.077 g/ml Ficoll®, 280 ± 15 mOsm; Lymphoprep®, Axis-Shield, Oslo, Norway) was performed to remove remaining erythrocytes from the stromal vascular fraction (SVF). After centrifugation at 600xg for 10 min, the resulting SVF pellet containing the hASCs was resuspended in expansion medium composed of Dulbecco’s modified Eagle’s medium (DMEM; Gibco®, Paisley, UK) containing 10% fetal bovine serum (Gibco®) supplemented with 500 µg/ml streptomycin sulphate (Sigma-Aldrich®, St. Louis, MO, USA), 500 µg/ml penicillin (Sigma-Aldrich®), and 2.5 µg/ml amphotericin B (Gibco®). Cell viability was assessed using the trypan blue exclusion assay. Cells were counted using light microscopy and immediately seeded as indicated below, or cells were resuspended in Cryoprotective medium (Freezing Medium, BioWhittaker®, Cambrex, Verviers, Belgium), frozen under “controlled rate” conditions, and stored in liquid nitrogen until further use. hASC seeding, attachment and culture Fresh-frozen SVF was, after thawing, directly used for the experiments, i.e. without a preculture step. SVF was seeded at 2.5x105 cells/well in 12-well plates and left to attach for 3 hours, after which non-adherent cells were removed by washing with PBS. The resulting culture contains a high percentage ASCs. Alternatively, SVF was either or not incubated for 15 min with 10 ng/ml BMP-2 in PBS (Peprotech®, London, UK) at 37°C after which SVF cell suspensions were seeded at 2.5x105 cells per 25-35 mg of BCP scaffold in 2 ml tubes (Eppendorf Biopur®, Hamburg, Germany). Attachment was allowed for 30 min, after which cell-seeded scaffolds were washed with PBS. Cell seeded BCP scaffolds were transferred to 12-well plates in Costar®Transwell® plates (Corning Life Sciences®, Lowell, MA, USA). All well plates received expansion medium 69

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(DMEM) supplemented with 10% fetal bovine serum and antibiotics, with different additives depending on the scaffold: Cells seeded on BCP were supplemented with 50 mg/ml ascorbic acid (Merck®, Darmstadt, Germany), whereas their counterparts seeded on tissue culture plastic were supplemented with ascorbic acid and b-glycerol phosphate (10 mmol/l) as phosphate source. Culture was performed for up to 21 days in 5% CO2 at 37°C in a humidified atmosphere, and medium was changed three times a week. PCR analysis of trophic factor expression by hASCs At day 0, 4, 14 and 21 of culture, cell-seeded BCP scaffolds and trypsin-EDTA dissociated ASCs from the tissue culture plastic wells were transferred to 2 ml tubes (Eppendorf Biopur®, Hamburg, Germany), and washed with PBS. RNA was extracted using Trizol® reagent (Invitrogen®, Carlsbad, USA) according to manufacturer’s instructions, and stored at -80°C prior to further use. For PCR Array, experiments were performed with cells from 2 donors. RNA samples from both donors, obtained at day 21, were separately converted to cDNA. cDNA was made with 1 mg total RNA, using the RT2 First Strand Kit (QIAGEN®, SABiosciences, Frederick, USA) in a thermocycler GeneAmp PCR System 9700 PE (Applied Biosystems®, Foster City, CA). Subsequently, the PCR Array kits ‘Cytokines’ and ‘Mesenchymal Stem Cells’ (QIAGEN®) were employed according to the manufacturer’s guidelines. Fifteen genes were present in both kits. Within each experiment we averaged the expression levels of those genes. The PCR Array reactions were performed using the LightCycler® 480 (Roche® Diagnostics). Gene expression levels were displayed relative to the mean expression level of 5 housekeeping genes (B2M, HPRT1, RPL13A, GADDH and ACTB). For qPCR, RNA samples obtained at day 0, 4, 14 or 21 from cultures performed with cells from 3 donors were converted to cDNA. If cell quantities allowed, experiments were repeated on a separate occasion. cDNA was prepared using the SuperScript® VILO™ cDNA Synthesis Kit (LifeTechnologies™), with 0.1 mg total RNA in 20 mL reaction mix in the thermocycler. For COL1A1, RUNX and TGFB1, qPCR was performed using 12.5 ng cDNA and 7.5 mL SYBR® Green Supermix (Roche® Laboratories, Indianapolis, IN) in a total volume of 15 ml per sample in a LightCycler® 480. Target and reference genes were amplified in separate wells, in triplicate. Expression of target genes was calculated using the relative standard curve method. Primer sequences are shown in table 3. All primers used for real-time PCR were from LifeTechnologies™. Tm was 60°C for all primers. Gene expression for BMP6, INHBA, IGF1, FGF1 and FGF2 was analyzed using TaqMan® Gene Expression assays (TaqMan®, Applied Biosystems®) in an ABI Prism® 7700 DNA sequence de- tector (Applied Biosystems®). 24 ng cDNA and 10 mL TaqMan® Master Mix were used in a total volume of 20 mL. Pre-designed, ready-made primer sets were obtained from Applied Biosystems®. For all qPCR analyses, the values of target gene expression were expressed relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene expression, except for COL1A1 and RUNX2 which were expressed relative to tyrosine 3- monooxygenase/ tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ). The relative gene expression was calculated with the ΔCt method. 70

Growth factor gene expression profiles of BMP-2 treated hASCs

Protein analysis IGF-I, BMP-6 and Activin A protein levels in conditioned medium at day 21 were measured by enzyme-linked immunosorbent assays (ELISA) from RayBiotech (Norcross, GA, USA), and P1NP (Procollagen I N-Terminal Propeptide) was measured using ELISA from USCN Life Science Inc. (Houston, TX, USA). The detection limits were as follows: IGF-I: 100 pg/ml; BMP-6: 150 pg/ml; Activin A: 15 pg/ml; and P1NP 6 pg/ml. Absorbance was measured at 450 nm with a microplate reader (Bio-Rad Laboratories). After 21 days of culture, culture medium was removed and the hASCs were washed twice with PBS on ice. Cell isolates were collected in distilled water, and alkaline phosphatase activity for each isolate was quantified using a p-nitrophenyl-phosphate colorimetric assay. Alkaline phosphatase activity was calculated as mmol p-nitrophenol per mg protein, which was determined using a BCA protein assay reagent kit (Pierce, Rockford, IL, USA), the absorbance was read at 570 nm. Statistical analysis All data are expressed as meanÂąSD. Data shown in figure 3 were log transformed when not distributed normally, and tested per time point using an unpaired t-test for single group mean. Data shown in figure 4 were also log transformed and tested using 1-way ANOVA in combination with Tukey post-hoc analysis. Differences were considered significant if pâ&#x2030;¤ 0.05.

Chapter 4

RESULTS Effect of substrate on trophic factor expression by hASCs We evaluated the expression of trophic factors by hASCs cultured on plastic for 21 days, and compared this to the expression in cells cultured on BCP. Figure 1 and table 1 present the data from PCR arrays for the 21-day time point. For the sake of clarity, we did not display the error bars in figure 1. Compared to plastic, hASCs cultured on BCP showed â&#x2030;Ľ 2-fold higher expression of ~20 factors, amongst which cytokines such as IL-6, growth factors such as FGF7 and adhesion molecules such as VCAM1. Expression of another ~50 genes was decreased â&#x2030;Ľ 2-fold on BCP compared to plastic (Fig. 1A-D).

Figure 1. Effect of substrate on growth factor expression by hASCs. Relative gene expression of trophic factors by hASCs cultured for 21 days on BCP (y-axis) was plotted against expression levels of trophic factors in hASCs cultured on tissue culture plastic (x-axis). Please note the difference in axis scales between figures 1A-D. Data are displayed as mean from n =1 or 2 donors (SD not shown). The solid line represents equal expression of genes in hASCs cultured on plastic and BCP. Red symbols mark genes that are expressed at least 2-fold lower by hASCs grown on BCP compared to plastic. Green symbols mark genes that are expressed at least 2-fold higher by hASCs grown on BCP compared to plastic. Full names of genes are given in Table 1.

Growth factor gene expression profiles of BMP-2 treated hASCs

Effect of BMP-2 on trophic factor expression by BCP-seeded hASCs A short pre-treatment with BMP-2 has a strong effect on osteogenic differentiation of hASCs on BCP 21, 22. In a previous study, we demonstrated that seeding on BCP further enhanced the BMP-2 induced osteogenic differentiation 22, which was confirmed in the current study. RUNX2 and COL1A1 were highly upregulated in BMP-2 pretreated hASCs seeded on BCP at day 21 (Fig. 2, Table 1). Figure 2 shows that BMP-2 enhances the expression of ~30 factors by hASCs seeded on BCP (Fig 2A-D). Treatment with BMP-2 decreased the expression of just 3 factors by more than 2-fold, i.e. PGF (5.7-fold), PPARG (2.4-fold) and PTN (3.3-fold). There were 27 factors that showed differential expression depending on substrate type and an effect of BMP-2 (Table 2). Strikingly, amongst these 27 factors there were 6 members of the fibroblast growth factor family, i.e. FGF1, FGF2, FGF5, FGF9, FGF14, FGF19, but only 1 bone morphogenetic protein, i.e. BMP-6.

Figure 2. Effect of BMP-2 on trophic factor expression by hASCs cultured on a biphasic calcium phosphate scaffold after 21 days. Relative gene expression of trophic factors by BMP-2-pretreated hASCs (y-axis) was plotted against the expression levels untreated hASCs (control, x-axis). Please note the difference in axis scales between figures 2A-D. Data are displayed as mean from n = 2 donors (SD not shown). The solid line represents equal expression of genes in hASCs treated with BMP-2 and cultured on BCP and untreated controls grown on BCP. Red symbols mark genes that are expressed at least 2-fold lower by hASCs grown on BCP compared to plastic. Green symbols mark genes that are expressed at least 2-fold higher by hASCs grown on BCP compared to plastic. Full names of genes are given in Table 1.

Chapter 4

qPCR analysis Based on the data in figures 1 and 2, we performed qPCR analysis of the following osteogenic differentiation markers and growth factors (Fig. 3): COL1A1, RUNX2, TGFB1, BMP6, INHBA, IGF1, FGF1, and FGF2. We found that short treatment of hASCs with BMP-2 enhanced COL1A1 expression equally at days 14 and 21 (4-fold; fig 3A), and enhanced RUNX2 expression by 4-fold at days 14 and by 6-fold at day 21 (Fig 3B). No convincing stimulating effect of BMP-2 treatment on TGFB1 expression in hASCs from the 3 different donors was found at any of the time points measured, including day 21 (Fig. 3C). BMP-6 expression seemed to decrease over time, with the lowest expression at day 14. As expected from the PCR array, at day 21 BMP-2-treated hASCs expressed significantly more BMP-6 than control hASCs (Fig 3D). Expression of INHBA, another member of the TGFÎ˛ family, gradually increased over time, reaching the highest expression at day 21. INHBA expression was significantly higher in BMP-2 treated hASCs than in the vehicletreaded cells (Fig 3E). BMP-2 treatment also seemed to enhance IGF-1 expression by hASCs at day 21, but the effect of BMP-2 was only significant at day 14. At this time point IGF-1 expression was very low compared to the expression at day 0 (Fig 3F). FGF1 expression increased over time. BMP-2 tended to increase FGF-1 expression in hASCs, but significance was not reached due to the low number of donors (Fig 3C). No clear effect of time or BMP-2 treatment was observed with regard to FGF-2 expression by hASCs (Fig 3H).â&#x20AC;&#x192; Protein analysis IGF1 and BMP-6 spent by hASCs in the culture medium at day 21 remained below the detection limits of 100 pg/ml and 150 pg/ml, respectively. The homodimer of INHBA is Activin A. In the absence of BMP-2, hASCs grown on BCP for 21 days produced 5.2-fold less Activin A compared to hASCs grown on plastic. BMP-2 seemed to increase the Activin A secretion by 1.5-fold in hASCs cultured on BCP, but this effect was not significant (Fig 4A). ASCs grown on plastic showed a clear trend towards higher P1NP levels compared to both the BCP only and the BMP2 treated cells (p=0.1 and p=0.13 respectively). Compared to both the BCP only and the BMP- 2 treated hASCs, cells grown on plastic had a lower ALP activity (p < 0.01 for both).

Growth factor gene expression profiles of BMP-2 treated hASCs

Figure 3. Relation between osteogenic differentiation and expression of trophic factors by ASCs. Relative expression levels of genes were measured after 14 or 21 days of culturing ASCs on BCP without stimulation of osteogenic differentiation or after stimulation of osteogenic differentiation (BMP2 pre-treatment). A) COL1A1, B) RUNX2, C) TGFB1, D) BMP6, E) INHBA, F) IGF1, G) FGF1, and H) FGF2. Values are meanÂą SD from n=2-6 separate experiments. * Significant effect of BMP-2 p<0.05; ** Significant effect of BMP-2, p<0.01. BCP, biphasic calcium phosphate; BMP-2, bone morphogenetic protein

Chapter 4

Figure 4. Effect of BMP-2 or substrate on Activin A and P1NP secretion, and on ALP activity in hASCs cultured on a biphasic calcium phosphate scaffold for 21 days. A) Culture of ASCs on plastic enhanced Activin A production compared to cells cultured on BCP. BMP2 treatment did not significantly enhance Activin A production by hASCSs cultured on BCP, although a trend towards an increase was visible. B) P1NP protein levels, a measure for newly produced collagen type I, seemed increased in cells cultured on tissue culture plastic, but the production was not significantly different between ASCs grown on plastic and BCP. C) Growing hASCs on BCP significantly enhanced ALP activity at day 21. Treatment of the cells with BMP-2 tended to further enhance ALP activity, but this effect was not significant. *Significant difference in protein levels, p < 0.05. **Significant difference in protein levels, p < 0.01. BCP, biphasic calcium phosphate; BMP-2, bone morphogenetic protein 2; P1NP, Procollagen Type I Intact N-Terminal Propeptide.

Growth factor gene expression profiles of BMP-2 treated hASCs

Table 1. Gene expression of trophic factors measured in hASCs after pre-treatment with BMP-2 and 21 days of culture on tissue culture plastic and biphasic calcium phosphate. All values are expressed relative to 5 housekeeping genes and multiplied by 100.

Chapter 4

Table 1 continued

Growth factor gene expression profiles of BMP-2 treated hASCs

Table 1 continued

Chapter 4

Table 1 continued

Table 2. Effect of short BMP-2 treatment on gene expression of trophic factors by hASCs cultured on BCP for 21 days. Shown are the average BMP-2 / control ratios (± SD), of those trophic factors who’s expression by ASCs is affected by both substrate type (Figure 1) and BMP-2 treatment (Figure 2) at day 21. n= 2 separate experiments with cells from 2 donors.

Growth factor gene expression profiles of BMP-2 treated hASCs

Table 3. Primer sequences for housekeeping gene GAPDH, RUNX2, COL1A1 and TGFB1 as used for gene expression analysis of human ASCs after 21 days of culture on biphasic calcium phosphate.

Chapter 4

DISCUSSION In this study we investigated how the secretome of ASCs is affected by substrate, growth factors, or degree of differentiation of stem cells. During bone healing, many distinct biological processes operate together in a coordinated fashion to facilitate the regeneration of a functional living tissue. Attraction and osteogenic differentiation of mesenchymal stem cells is a key part of this process, but is not the only important step by far 25. The environment in which the tissue regeneration takes place needs to be cleaned of damaged tissue and remodeled, inflammation needs to be allowed and suppressed at the proper stages of healing, and blood vessel formation has to keep up with the regeneration process to allow oxygen and nutrients to reach the metabolically very active bone forming osteoblasts. ASCs can both differentiate towards osteoblasts 22, and form trophic factors that influence the before mentioned processes 20 . As it would make sense that ASCs produce increasingly more trophic factors while healing propagates, we hypothesized that as the differentiation of ASCs progresses hASCs produce more factors associated with processes such as angiogenesis and bone remodeling. From previous experiments we know that ASCs in vitro show a higher differentiation potential when seeded on BCP compared to plastic, and the highest differentiation potential when treated with BMP-2 and seeded on BCP 22. Therefore we compared the growth factor expression profiles of ASCs seeded on BCP and seeded on BCP after treatment with BMP-2. Corroborating with our previous data 22, DNA analysis confirmed that the proliferation potential of cells cultured on BCP and plastic is similar (data not shown), ensuring that differences in gene expression profiles are influenced by differentiation, and not by cell number. We found no clear association between the level of osteogenic differentiation of hASCs and the pattern of trophic factor production (Figure 3). Osteogenic differentiation was higher at day 14 and 21 compared to day 0 and 4 (Fig 3A, B) 22, yet BMP6 and IGF1 had lower expression levels at day 14 and 21 compared to day 0 (Figure 3). INHBA levels increased gradually over time. In addition, based on the Array data, ASCs grown on BCP seemed to produce lower amounts of approximately 50 different factors compared to ASCs grown on tissue culture plastic, even though osteogenic differentiation of ASCs is promoted by BCP 26. Amongst these were a large fraction of cell surface molecules such as ALCAM, ANXA5, CD44, ICAM1, PDGFRB, SLC17A5 and THY1 (full names given in table 2). Signaling molecules such as EGF, FGF2 and INHBA were also lower expressed on BCP than on plastic, suggesting that those substrates most promoting osteogenic differentiation of MSCs do not automatically enhance trophic factor production as well. The results do show that trophic factor production by ASCs can be strongly affected by substrate, and this means that tissue engineering strategies may be rendered more successful once it is known which substrate properties most strongly enhance trophic factor production by ASCs promoting bone healing. As stated above, trophic factor production is mostly enhanced on plastic compared to BCP, even though differentiation of ASCs is diminished on plastic compared to BCP (Figure 1). The expression of many trophic factors by plastic adherent hASCs seems enhanced at day 21 after BMP-2 treatment (Table 1), even though short treatment with BMP-2 of hASCs grown on plastic does not significantly enhance osteogenic differentiation of those hASCs 22. The factors 82

Growth factor gene expression profiles of BMP-2 treated hASCs

produced by the ASCs thus likely do not work in a paracrine / autocrine fashion on the cultured ASCs to promote osteogenic differentiation. Alternatively, the substrate has strong direct effects on ASC differentiation, overruling the effect of trophic factors. Substrate stiffness for instance, has been shown to determine differentiation potential of MSCs via an effect on Rho/ROCK signaling 27 . However, BCP and plastic both have a high stiffness, thus it is not likely that this could explain the difference in differentiation potential. ASCs may differentiate more easily on BCP due to the higher amounts of calcium in the medium, released from the scaffold 26. For ASCs grown on tissue culture plastic we compensated for available phosphate in the culture medium, but we did not compensate for calcium. These factors may not only affect osteogenic differentiation, but also trophic factor production by MSCs. Future studies aimed to elucidate how factors such as free calcium concentration, substrate stiffness, surface roughness, or surface energy affect the secretome of MSCs are warranted. We expected that angiogenic factors, such as VEGF and FGFs 18, 28 to be highly expressed by ASCs, both on plastic and on BCP, because of the extensive angiogenic capacity of ASCs in vivo 8. We found that these factors were expressed at moderate levels when compared to other factors (Table 1). It is possible that the presence of ascorbic acid in our culture medium somewhat diminished VEGFA expression, since it has been shown that ascorbic acid-2 phosphate reduces VEGFA secretion in rat ASCs 19. Of course this does not mean that protein production is also moderate to low. In addition, the combination of angiogenic factors produced by ASCs may together still have a strong angiogenic potential. The most strongly expressed factors on mRNA level were the extracellular matrix component COL1A1 and the mesenchymal cell specific intermediate filament vimentin (VIM). In addition, the type IV collagenase MMP2 was very highly expressed by ASCs. MMP2 aides the lysis of basement membranes, thereby enhancing vascularization 29. This may partly explain the angiogenic potential of ASCs in vivo. Based on the array data, BMP-2 treatment enhanced VEGFA and VEGFC expression in ASCs grown on BCP (Fig. 2). This suggests that BMP-2 treatment may be beneficial for stimulating angiogenesis when applying ASCs for bone healing in vivo. Indeed it was found that BMP-2 treated ASCs enhance angiogenesis more strongly than ASCs alone in vivo 8, underscoring the potential of BMP-2 to promote ASC-driven bone healing. Besides stimulating the expression of angiogenic factors by ASCs, BMP-2 stimulated the expression of a panel of factors that may condition the in vivo environment to facilitate bone repair. Based on both the PCR array and qPCR analysis, BMP-2 treatment enhanced the expression of ~30 factors by hASCs seeded on BCP, amongst which IGF1, INHBA and BMP-6. Treatment with BMP-2 decreased the expression of only 3 factors, i.e. PGF, PPARG and PTN. The decrease in PPARG expression by hASCs after BMP-2 treatment is in line with earlier findings, where BMP-2 enhances the osteogenic differentiation of hASCs, while decreasing adipogenic differentiation, and thus PPARG expression 22. IGF1 regulates angiogenesis and endogenous RUNX2 activity in human endothelial cells 30-32, and promotes osteogenic differentiation of bone marrow MSCs in rats 33. BMP6 is known to enhance osteochondral bone formation 34. A homodimer of INHBA forms ActivinA, which is known to affect osteoclastogenesis as well 35. This might suggest that INHBA enhances remodeling of the repair site. Short incubation with BMP-2 may thus be a promising treatment to enhance both osteogenic differentiation of the 83

Chapter 4

stem cells and tissue remodeling. We are the first to show that a short incubation with BMP-2 of only 15 minutes can affect the secretome of ASCs up to 21 days later. It is likely that 15 minutes is more than sufficient for BMP-2 to occupy the available receptors and activate smad signaling. We speculate that smad signaling activates a positive feedback loop in the ASCs, and that this epigenetic mechanism maintains the positive effect of BMP-2 treatment for 21 days. What this positive feedback loop consist of is unknown. A clear limitation of this study is that the secretome has been evaluated primarily on the level of mRNA expression, and that additional determination of secreted factor levels succeeded for some (Activin A, P1NP), but failed in other cases (IGF-1, BMP-6). Although protein production is for 90% regulated at the level of transcriptional control, it is still possible that alterations in translation or protein activation occur. Thus, inventarization of the ASC secretome may give subtly different results when trophic factor production is studied using proteonomics approaches versus genetic screening methods. Nevertheless, a large overlap in the results is to be expected, likely giving rise to similar conclusions. We found a clear effect of substrate and growth factor treatment on the production of trophic factors by ASCs in vitro, showing that the environment strongly affects the secretome of ASCs. Once implanted, ASCs are bound to encounter a multitude of stimuli, ranging from (platelet derived) growth factors, via matrix molecules such as fibrin, to inflammatory cytokines. Factors produced by the host may trigger trophic factor production by ASCs, via reciprocal signaling between host and stem cells. The secretome of ASCs is therefore likely to be different in vivo compared to in vitro. Nevertheless, systematic in vitro investigations can shed a light on exactly which factors affect on trophic factor production by ASCs, aiding the targeted design of tissue engineering strategies.

Growth factor gene expression profiles of BMP-2 treated hASCs

CONCLUSIONS We hypothesized that as differentiation progresses, hASCs produce increasingly more factors associated with processes such as angiogenesis and bone remodeling. However, we found a lack of association between the degree of differentiation and the secretome of hASCs. Most current bone regeneration studies focus on induction of osteogenic differentiation of stem cells. Considering our findings, and considering the critical importance of cellular trophic factor production for the outcome of tissue engineering strategies, a shift in research focus towards systematically orchestrating the secretome of stem cells is indicated. Short BMP-2 treatment stimulated the expression of a panel of factors in hASCs that may play a role in the conditioning of the environment to facilitate bone repair in vivo. Short incubation with BMP-2 may thus be a promising treatment to enhance both osteogenic differentiation of stem cells as well as modulation of the wound environment. â&#x20AC;&#x192; ACKNOWLEDGEMENTS The work of J.R. Overman was supported by the Research School of the Academic Centre for Dentistry Amsterdam The authors wish to extend their thanks to the surgical staff of Tergooiziekenhuizen Hilversum Hospital, The Netherlands, for kindly providing the human adipose tissue for this study. They also thank Jolanda Hogervorst and Behrouz Zandieh Doulabi for their outstanding technical assistance in the PCR analysis.

Chapter 4

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CHAPTER 5 A HISTOMORPHOMETRICAL AND MICRO-CT STUDY OF BONE REGENERATION IN THE MAXILLARY SINUS COMPARING BIPHASIC CALCIUM PHOSPHATE AND DEPROTEINIZED CANCELLOUS BOVINE BONE IN A HUMAN SPLIT-MOUTH MODEL Janice R. Overman1*, Gert L. de Lange2*, Elisabet FarrĂŠ-Guasch1, Clara M. Korstjens1, Bastiaan Hartman1, Geerling E.J. Langenbach3, Marion A. Van Duin1, Jenneke Klein Nulend1

2 3

Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, The Netherlands Academic Center Oral Implantology Amstelveen, Amstelveen, The Netherlands Department of Functional Anatomy, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, The Netherlands

Shared first authorship, Janice R. Overman and Gert L. de Lange contributed equally to this manuscript

Oral Surg Oral Med Oral Pathol Oral Radiol. 2014; 117:8-22

Chapter 5

ABSTRACT Objective: The gain of mineralized bone was compared between deproteinized bovine bone allograft (DBA) and biphasic calcium phosphate (BCP) for dental implant placement. Study Design: Five patients with atrophic maxillae underwent bilateral sinus elevation with DBA (Bio-Oss速) and BCP (Straumann速 BoneCeramic). After 3 to 8 months, 32 Camlog implants were placed, and biopsies were retrieved. Bone and graft volume, degree of bone mineralization, and graft degradation gradient were determined using microcomputed tomography, and bone formation and resorption parameters were measured using histomorphometry. Implant functioning and peri-implant mucosa were evaluated up to 4 years. Results: Patients were prosthetically successfully restored. All but one of the implants survived, and peri-implant mucosa showed healthy appearance and stability. Bone volume, graft volume, degree of bone mineralization, and osteoclast and osteocyte numbers were similar, but BCP-grafted biopsies had relatively more osteoid than DBA-grafted biopsies. Conclusions: The BCP and DBA materials showed similar osteoconductive patterns and mineralized bone, although signs of more active bone formation and remodeling were observed in BCP- than in DBA-grafted biopsies.

A histomorphometrical and micro-CT study of bone regeneration in a human split-mouth model

INTRODUCTION Augmentation of the maxillary sinus floor with a grafting material is a well-established procedure to restore the bone height required for placing dental implants in the posterior edentulous maxillary region.1-3 Autogenous bone grafts are often used for sinus augmentation and are considered the gold standard owing to their maintenance of cellular viability and presumptive osteogenic capacity. Nevertheless, drawbacks such as the requirement for an additional surgical site, graft resorption, and increased risk of morbidity 2, 4 make bone substitutes an interesting alternative to autogenous grafts, with similar results when using some of these materials.5-7 Deproteinized bovine bone allograft (DBA) (Bio-Oss®; Geistlich AG, Wolhusen, Switzerland) is a well documented and well-established bone graft material that has been used frequently in sinus floor elevation procedures for nearly 2 decades.7-9 DBA is a calcium deficient carbonate derived from deproteinized bovine bone and is identical to human bone from a chemical and physical point of view. It performs well as a grafting material for sinus floor augmentation.10 DBA material acts as an osteoconductive scaffold, leading to the formation of lamellar bone and increased bone density.11 Osteoblasts are recruited from the adjacent preexisting bone and adhere directly to the surface of the graft particles using cell-matrix binding proteins.12 However, in a few human cases, DBA led to a foreign body reaction,13 which might have been due to residual protein.14 Both immunologic and ethical considerations have created the need for a purely synthetic material.15 The advantage of using synthetic materials is the predictable quality of production and the elimination of the risk to retain known and unknown proteins from an animal source. Biphasic calcium phosphate (BCP) (Straumann® BoneCeramic, Institut Straumann AG, Basel, Switzerland) is a new purely synthetic bone graft material consisting of a mixture of 60% hydroxyapatite (HA) and 40% β-tribasic calcium phosphate (β-TCP). HA has been found to be highly biocompatible with bone.16-18 β-TCP has also been used successfully for sinus floor elevation.19, 20 However, β-TCP degrades rather fast and has a different resorption pattern than HA.21 BCP combines the bioactive properties of HA with the good bioresorbability of β-TCP and has been successfully used for maxillary sinus floor elevation and treatment of mandibular bone defects.22-27 It has good biocompatibility and osteoconductivity, with an implant survival >90% and similar bone formation compared with allografts and xenografts such as DBA.25, 28-31 Bone volumes measured with conventional 2-dimensional (2D) histological techniques have been described to vary between 22% and 39% after grafting the sinus with BCP, with an increase in bone volume over time.22, 23, 25, 29, 32 Micro-computed tomography (micro- CT) analysis is a nondestructive radiographic procedure providing high-resolution 3-dimensional (3D) images. This technique allows the distinction between graft material and (native) mineralized bone.33 Micro-CT is useful for the investigation of hard tissue volume and bone structure after bone regeneration, which was first and independently reported in the same year by Ito34 and Chappard et al.35 Comparative studies of DBA and BCP performance in sinus floor elevation concluded that DBA and BCP produced similar amounts of newly formed bone, indicating that both materials are suitable for sinus floor augmentation to allow the placement of dental implants.23, 28 However, in these studies, most biopsies were obtained from different patients, and a comparison within 91

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one patient was not made. In the current study, a split-mouth model was used to compare BCP and DBA for their capacity to augment maxillary bone when grafted in the maxillary sinus floor. We hypothesized that BCP, which combines the bioactive properties of HA with the good bioresorbability of Î˛-TCP, may perform better in conjunction with dental implants placed in the augmented sinus floor for prosthetic rehabilitation. Five edentulous patients with thin residual sinus floors were selected for bilateral sinus floor elevation using BCP on one side and DBA on the other side in a splitmouth study design. Biopsies were retrieved at implant positions from previously augmented bony sites for histology, histomorphometry, and micro-CT analysis. The survival of implants in the augmented sites was evaluated during a 4-year follow-up period, including evaluation of the peri-implant mucosa and surrounding bone.

A histomorphometrical and micro-CT study of bone regeneration in a human split-mouth model

MATERIALS AND METHODS Patients Five healthy nonsmoking patients (4 women, 1 man; age, 64-71 years; mean age, 66 years; Table I), who had been without their maxillary dentition for many years and complained about the retention of their upper denture, were randomly selected to undergo sinus floor elevation and implant placement at 3 to 8 months after bone augmentation. All patients included had severe maxillary atrophy and were examined thoroughly. Radiographic examination included a dental panoramic tomography view and a lateral view, which revealed that the maxillary anatomy and residual sinus floor on the left and right sides were comparable. The maxillary bone height varied from 0.5 to 2 mm in the center and up to 4 mm mesially or distally, with a mean height of 2.2 mm on the left side and 2.3 mm on the right side (see Table I). All patients were informed about the necessity of sinus floor augmentation to achieve sufficient bone volume. A staged approach was used; implants were placed 3 to 8 months after bone augmentation (mean, 6 months) and were loaded after osseointegration. Early implant placement, that is, after 3 months, was considered in one patient owing to the patient’s schedule. A total treatment time of 12 months was scheduled. The protocol was reviewed by the appropriate institutional review board in compliance with the Helsinki Declaration, and ethical approval was obtained according to subcommittee CEN/ TC 258 (clinical investigation of medical devices) of the European Committee for Standardization, Central Secretariat, Brussels, Belgium. Each subject was informed of the procedures and signed a detailed informed consent form. Surgical procedures and postoperative care Patients received one preoperative antibiotic oral dose of 3 g amoxicillin. Bilateral sinus augmentation was performed during one surgical procedure. The graft material was randomly assigned to one of the sides, with DBA (Bio-Oss®; Geistlich AG) at one side and BCP (Straumann® BoneCeramic; Institut Straumann AG) at the other side (see Table I). Graft material was infused with blood. A full-thickness buccal mucosa flap was elevated, and an opening was made in the lateral sinus wall. The bony window was pushed medially to detach the schneiderian membrane from the bone. The subantral cavity created was filled with granular DBA or BCP. The window at both sides was covered with a resorbable collagenous membrane (Bio-Gide®; Geistlich AG). Complete wound closure was performed with resorbable sutures. Perforation of the schneiderian membrane did not occur. Postoperative examination was performed at the outpatient clinic. Patients were seen on a 3-week basis to check on healing. Chlorhexidine 0.2% rinse was used as an antiseptic therapy twice daily for 2 weeks. The differences in the healing period were partly due to the availability of the patients. Implant surgery Implant surgery was performed under appropriate local anesthesia. A total of 32 screw-type titanium implants (Camlog® Screw Line, Camlog Biotechnologies AG, Wimsheim, Germany) were manufactured from commercially pure titanium. The core diameter of the implants was 93

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3.8 mm, and the total length was 11 mm. Implants were sandblasted and acid-etched according to a standardized procedure (Promote速; Altatec, Wimsheim, Germany) and inserted (see Table I) with an undersized drilling technique. The implant design, with a conical shape and a selftapping screw thread, contributed to a good initial stabilization in the relatively soft regenerated bone (type D4 according to Lekholm and Zarb36). The implants were covered with mucosa, and the flap was sutured with 4-0 polyglactin 910 (Vicryl) resorbable sutures. The sutures were removed after 7 to 10 days, and the existing prosthesis was adapted with a soft material to the new situation. Patients were seen every 3 weeks to check on healing and ensure prevention of premature loading. Abutment surgery was performed after implant healing, and the soft tissues were optimized for a sufficient amount and quality of peri-implant keratinized tissue. After 1 month of soft tissue maturation, prosthetic procedures were started, either for an overdenture with bar retention (2 patients) or for fixed bridges cemented on customized titanium abutments (3 patients). Table 1. Patient and biopsy characteristics.

DBA, deproteinized bovine bone allograft (Bio-Oss速); BCP, biphasic calcium phosphate (Straumann速 Bone Ceramic).

Follow-up procedures Patients were seen every 6 months for 4 years. The peri-implant mucosa and surrounding bone were examined at 4 positions (buccal, palatal, mesial, and distal) with probe angulation as the method for pathology detection.37 Scoring was performed according to the peri-implant score of Mombelli et al.38 for (1) healthy appearance, no bleeding on gentle probing, and pocket depth <5 mm; (2) bleeding, also when the probe was angulated, with pocket depth <5 mm and radiographic bone loss <2 mm; and (3) bleeding and pus, with pocket depth >5 mm and radiographic bone loss >2 mm. The highest score dominated. A score of 1 was considered successful, whereas a score of 3 was considered unsuccessful. A score of 2 indicated the need for treatment, and when it turned out to be reversible to a healthy situation (score 1), the implant was considered successful.

A histomorphometrical and micro-CT study of bone regeneration in a human split-mouth model

Laboratory biopsy procedure and histology At 3 to 8 months after the sinus floor elevation, vertical bone biopsies of 4 patients were retrieved from the augmented sinus floor, at implant positions, during implant placement with a hollow trephine burr (3.5 mm outer diameter and 2 mm inner diameter) at approximately 12 mm depth. In total, 6 biopsies with DBA and 8 biopsies with BCP could be analyzed. One patient (No. 5) refused to have the biopsies taken. All biopsies (n = 14) were immediately fixed in 4% formaldehyde solution in 0.1M phosphate buffer, pH 7.3, at 4°C for 24 hours.39 They were then rinsed 3 times in 0.1M phosphate buffer and stored in 70% ethanol at 4°C, until ready to be embedded in low-temperature polymerizing methylmethacrylate (Merck Schuchardt OHG, Hohenbrunn, Germany) without decalcification. Three series of 6 consecutive sections with thicknesses of 5 mm were cut using a Jung K microtome (R. Jung, Heidelberg, Germany). The distance between the 3 series was 100 mm. Two sections of each set of consecutive sections were stained with the Goldner trichrome method to highlight distinct mineralized bone tissue (green) and osteoid (red). A third section of the set of consecutive sections was stained for tartrate resistant acid phosphatase (TRAP) to detect osteoclastlike cells. Three other sections served as backup. Bone histomorphometry Histomorphometric measurements were performed using a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany) connected to a computer using an electronic stage table and a Leica DC 200 digital camera. The computer software used was Leica QWin© (Leica Microsystems Image Solutions, Rijswijk, The Netherlands). The sections were digitized at x125, x250, and x400 magnification. For every biopsy, one Goldner trichrome stained section per series was analyzed, that is, 3 sections per biopsy. A demarcation line was indicated between the native (that is, original or background) alveolar bone of the residual sinus floor and the regenerated and grafted bone (Figure 1). Three consecutive areas of interest, each 625 mm2 and at a 500-mm distance, were defined in the grafted bone from caudally, at a 500 µmm distance from the sinus floor, up to the sinus bone end at the cranial side (see Figure 1). Nomenclature, symbols, and units were used as recommended by the Nomenclature Committee of the American Society for Bone and Mineral Research.40 In each area of interest, bone volume (BV) was calculated as the amount of mineralized tissue (mineralized volume, Md.V) plus the amount of osteoid tissue (osteoid volume, OV) as a percentage of the total tissue volume (TV) (thus BV/TV x100). The relative osteoid volume was calculated as the amount of osteoid tissue as a percentage of the total bone volume (OV/BV x100). The absolute graft particle volume (GV) was calculated as the amount of graft material as a percentage of the total tissue volume (GV/TV x100). The number of osteocytes (N.Ot) and the number of osteocyte lacunae (N.lac) were counted and expressed per mineralized tissue area (mm2). The number of TRAP-positive cells (osteoclasts, N.Oc) was expressed per total tissue area (mm2). Micro-CT All biopsies (n=4) were fixed in buffered formaldehyde and embedded in plastic resin before micro-CT analysis. Scanning was performed with the micro-CT equipment of Scanco Medical 95

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AG (model mCT40; Bassersdorf, Switzerland). This scanner has a tube voltage of 5 kV and a tube current of 145 mA. Scanning resolution was 15 mm. The micro-CT scanner measures the radiopacity of the material. The scanner is calibrated every week, and the calibration constants are used to convert the opacity values to mineralization degrees. The distinction between newly formed bone and graft material was made by using the highest value of the degree of mineralization in preexisting sinus floor bone as a cutoff point. The degree of mineralization, expressed in milligrams of hydroxyapatite per cubic centimeter (mgHA/cm3), was found to be 550 to 1300 mgHA/cm3. A threshold of 550 mgHA/cm3 was used to differentiate between graft bone, newly formed bone, and background bone. Values above 1300 mgHA/cm3 were assumed to be graft material. This way we were able to distinguish the graft material from the original non-grafted native bone of the residual sinus floor and newly formed bone. Volumes of interest (3 mm3) of the scanned biopsies were analyzed, starting with the sinus floor bone (caudal), and continuing in the cranial direction, every subsequent 1 mm. The 3 areas selected represented different areas of interest in the grafted sinus, whereby area of interest 1 was close to the sinus floor, in a way similar to that in the study by Cordaro28 The ratios of bone and graft volume over total volume were calculated, as well as the average degree of mineralization of bone and graft contained in each volume of interest. Data pooling and statistical analysis Implant survival was calculated by the Kaplan-Meier method as described by Hu and Lagakos.41 Bone and graft volumes, as well as the degree of mineralization of bone and graft in the volumes of interest retrieved from biopsies containing the same graft material, were pooled. The preexisting native bone from the sinus floor was used as a reference. This enabled overlay of data from biopsy sections with different lengths and eliminated possible differences in results due to different thickness of preexisting sinus floor bone, resulting in comparable areas with regard to their distance to the sinus floor to allow pooling of the data. The last millimeter of the sinus floor bone adjacent to the graft material was used as the starting point for the first volume of interest (number 1). The following volumes of interest contained the graft material in increasing distances, namely, in steps of 1 mm from the residual sinus floor. Data were expressed as mean Âą standard error of the mean (SEM). Statistical testing was performed using paired t-tests, Student independent t-test, and analysis of variance using SPSS (version 16.1; SPSS Inc, Chicago, IL, USA), KyPlot 4.0 (KyensLab Inc, Tokyo, Japan), and GraphPad PrismÂŽ 5.01 (Graph-Pad Software Inc, La Jolla, CA, USA). These methods allowed us to compare the mean bone volume, graft volume, degree of bone mineralization, and graft mineralization for the 2 graft materials, gradient of graft-degradation per millimeter in biopsy, osteoid volume, number of osteocytes, and number of TRAPpositive osteoclasts. Statistical analysis was performed on pooled data from corresponding volumes of interest obtained from at least 3 biopsies. Values of P < .05 were considered significant.

A histomorphometrical and micro-CT study of bone regeneration in a human split-mouth model

RESULTS Clinical results All patients had good postoperative healing (see Figure 1). The postoperative radiographs showed the presence of the bone substitute material in the augmented sinuses with an increase in height of 16 to 25 mm (see Figure 1). Two patients exceeded the time schedule; one (patient No. 1) owing to traveling and the other (patient No. 2) owing to a series of soft tissue augmentations in the anterior zone with connective tissue obtained from hyperplastic tuberosities, which was necessary to achieve maximal aesthetics for a fixed bridge. In one other patient (patient No. 5), the healing abutments were placed immediately after implant surgery. Unfortunately, one implant failed 6 months earlier, owing to premature loading at the BCP-grafted side. The height of the original sinus floor at the position of the failed implant (P2 ss) was only 1 mm (see Figure 1, G). The implant was removed, and a fixed bridge was made on the remaining 7 implants. All other 31 implants survived during the 4 years of follow-up. These implants resisted occlusal load and were successfully used for the prosthetic follow-up, which was 2 overdentures with bar retention (patients No. 1 and No. 3) or 3 fixed bridges (patients No. 2, No. 4, and No. 5). The mean peri-implant score for all implants was 1.2 and did not change over time. Patients with fixed prosthetics had excellent plaque control, and the mean peri-implant score of their 21 implants was 1.1. Patients with overdentures had slightly more plaque, and some of their implants had a peri-implant score of 2 as a result of bleeding on probing. With additional plaque control instructions and an intensive cleaning protocol, these patients were able to keep the peri-implant mucosa of their 10 implants supporting the overdentures healthy during the 4 years of follow-up. The mean peri-implant score did not exceed 1.4. Scores of 3, indicating severe bone loss, bleeding, or pus, were not seen. No differences were observed between the sites grafted with DBA or BCP. The bone height in the augmented sites, as observed in panoramic radiographs (see Figure 1), was maintained during the 4 years of follow-up. Histology All biopsies obtained from patients after sinus floor elevation with BCP or DBA contained mineralized bone, osteoid, and remaining graft particles of BCP or DBA (Figure 2). The native bone showed the characteristic structure of lamellar bone, with coarse bone trabeculae and marrow spaces in between. Some trabeculae were covered by an osteoid layer, but osteoclasts were also present, lining the bone surface, indicating an ongoing normal bone remodeling process of living bone. The bone trabeculae were connected to each other and ended on a thick layer of mineralized lamellar bone with a flat surface representing the (old) residual sinus floor (see Figure 2). The regenerated bone, on the other hand, had thin bone trabeculae surrounding the remaining graft material up to the sinus end. The border between the residual sinus floor and the regenerated bone was clearly visible, which enabled us to draw a demarcation line between the residual sinus floor and the regenerated (grafted) bone (see Figure 2). Histology showed direct bone deposition on graft particles, thin bone trabeculae, and woven bone with osteoid, characteristic for new bone formation, in all biopsies. Some graft material was still present in all biopsies, and both BCP and DBA grafted biopsies showed that 97

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Figure 1. Pre- and postoperative radiographs of 3 patients after bilateral sinus grafting with deproteinized bovine bone allograft (DBA, Bio-Oss速) at one side and biphasic calcium phosphate (BCP, Straumann速 BoneCeramic) at the other side. A, D, and G, Preoperative radiographs (of patients No. 1, No. 2, and No. 5) showing severe maxillary resorption and thin residual sinus floors. B, E, and H, Radiographs of the same patients after sinus floor elevation with DBA and BCP. C, F, and I, Radiographs of the same patients after implant placement. Note that both DBA and BCP graft materials are more radiopaque than bone and that the augmented sites become less radiopaque over time.

remaining graft particles were embedded in a loose cell-rich connective tissue (see Figure 2, A and B). Most BCP and DBA particles were covered with a layer of mineralized bone that hardly contained cells (see Figure 2, A and B). This mineralized bone seemed to be deposited directly on the graft material without an intermediate osteoid layer. Direct apposition/deposition of osteoid on the BCP or DBA bone graft material was occasionally observed (see Figure 2, A1 and B2). Most osteoid was located on the bone trabeculae at the marrow side (see Figure 2, A1 to A3 and B1 to B3). A gradient in bone maturation was observed from the cranial end (sinus end) down to the sinus floor; there was a decrease in BCP and DBA graft material and an increase in mineralized bone. In addition, the bone trabeculae were thicker close to the sinus floor than at the cranial end (see Figure 2, A and B). Using light microscopy, we observed in all biopsies the typical characteristics of an active bone forming process, namely, thin trabeculae of woven bone with many viable osteocytes (Figure 3, A and B). This woven bone was embedded in a loose and cell-rich connective tissue with many blood vessels, and it was surrounded with osteoid that was lined with active osteoblastic cells. Both BCP and DBA graft materials showed histologically a similar bone forming process. Signs of inflammation or foreign body reaction were not seen 98

A histomorphometrical and micro-CT study of bone regeneration in a human split-mouth model

in BCP or DBA-grafted biopsies. TRAP-positive osteoclasts were observed at the tissue-graft interface of both materials. The surface of some BCP particles clearly showed resorptive activity in relation with TRAP-positive osteoclasts (see Figure 3, C) which was also the case for DBA particles (see Figure 3, D).

Figure 2. Light microscopy of nondemineralized sections of whole human biopsies after sinus floor elevation with biphasic calcium phosphate (BCP, Straumann速 BoneCeramic) and deproteinized bovine bone allograft (DBA, Bio-Oss速). A and B, Overviews of the whole biopsies of the augmented sinuses with BCP (A) and with DBA (B). The alveolar side (caudal) includes native mineralized bone (green) of the original sinus floor with coarse bone trabeculae ending in a dense layer of lamellar bone. The amount of mineralized bone in the augmented sinus decreases from caudal to cranial. Three areas of interest are located in the grafted bone, which are aligned at a 500 mm distance from each other and used for histomorphometric analysis. Both BCP and DBA graft particles are unstained (white) and surrounded by loose cell-rich connective tissue. No adverse tissue reactions are observed. The black horizontal line demarcates the native alveolar bone (bottom) and the grafted bone (top). The 3 areas of interest in the augmented sinus floor are seen with BCP (A1 to A3) and with DBA (B1 to B3). The BCP-grafted sinus shows particles with sharper angles, and their size is somewhat smaller compared with DBA. Mineralized bone (green) is deposited directly (open arrows) on the BCP graft particles (unstained, G). Osteoid tissue (red) borders mineralized bone, indicating new bone formation, and is located mainly at the marrow side (black arrows). Direct deposition of mineralized bone on the majority of the DBA particles is visible (B1 to B3; hollow arrows). The bone trabeculae are thicker in area 1 than in the other areas, and some contain woven bone (B1). Most osteoid is located at the marrow side (black arrows). Goldner trichrome stained nondemineralized sections. Scale bar, 100 mm. Mineralized bone (green), osteoid tissue (red), cell nuclei (black).

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Figure 3. Light microscopy of nondemineralized sections of whole human biopsies after sinus floor elevation with biphasic calcium phosphate (BCP, Straumann速 BoneCeramic) (A and C) and deproteinized bovine bone allograft (DBA, Bio-Oss速) (B and D). In A and B, typical structures of a bone forming process are seen, with many blood vessels, thin bone trabeculae of woven bone containing numerous bone cells (black arrows), a mineralization front with osteoid, and osteoblasts embedded in a loose and cellrich connective tissue. In C and D, tartrate-resistant acid phosphatase (TRAP) positive cells (red) indicate osteoclast-like cells. TRAP-positive cells, bordering the BCP granules, seem to dissolve the material at the surface (black arrows). The DBA granules are also surrounded by TRAP-positive cells (black arrows), of which some invade the granule pores. Goldner trichrome stained sections. Scale bar, 100 mm.

Histomorphometry Bone volume (BV/TV x 100) in native bone was similar in all biopsies. Mineralized bone volume (Md.V/TV x 100) ranged between 12.6% and 11.7% close to the sinus floor (area 1) for both BCP and DBA grafted biopsies, and it decreased cranially (area 3) to about 7% (Figure 4, A). There was no difference in mineralized bone volume between the 2 grafting materials (see Figure 4, A). However, the bone volume (BV/TV x 100) (including osteoid tissue and bone) in area 2 was 1.4-fold higher (p=0.034) in BCP-grafted biopsies in comparison with DBA-grafted biopsies (see Figure 4, B). Whereas the bone volume (BV/TV x 100) increased with decreasing distance from the native bone, the graft volume (see Figure 4, C) increased significantly by 1.6-fold from area 2 to area 3 in the BCP-grafted biopsies (P =0.036) and by 1.4-fold from area 1 to area 2 in the DBA-grafted biopsies (P =0.05). The relative osteoid volume (OV/BV x 100) increased with increasing distance from the native sinus floor, especially in the BCP-grafted biopsies, indicating active bone formation (see Figure 4, D). The number of osteocytes per mineralized tissue area (mm2) was slightly (but not significantly) higher in area 3 than in area 1 in BCP-grafted biopsies, but not in DBA-grafted biopsies (see Figure 4, E). TRAP-positive cells were sparsely observed in the BCP and DBA-grafted biopsies. The number of TRAP-positive cells was slightly (but not significantly) higher in DBA-grafted biopsies than in BCP-grafted biopsies (see Figure 4, F).

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Figure 4. Bone formation and bone resorption parameters measured in biopsies from biphasic calcium phosphate (BCP) grafted and deproteinized bovine bone allograft (DBA) grafted maxillary sinuses. A, Percentage of mineralized bone (Md.V) per total bone volume (TV) of the 3 areas of interest after sinus floor augmentation with BCP and DBA. The percentage of mineralized bone volume is highest in the area closest to the native sinus floor in both BCP- and DBA-grafted biopsies. B, Percentage of total bone including osteoid (BV) per TV of the 3 areas of interest after sinus floor augmentation with BCP or DBA. The amount of bone formation in area 2 was significantly higher in BCP-grafted biopsies than in DBA-grafted biopsies. C, Percentage of graft material (GV) per total tissue volume. The amount of graft material was decreasing from cranially to the sinus floor in BCPand DBA grafted biopsies (P < .05). D, Percentage of osteoid volume (OV) per BV in areas 1 to 3. E, Number of osteocytes per mineralized tissue area (mm2) in the 3 consecutive areas, 1 to 3. F, Number of tartrate-resistant acid phosphatase (TRAP) positive cells per mineralized tissue area (mm2). Data from the 3 areas were pooled because the cell numbers were very low.

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Bone volume and remaining graft volume Reconstruction by micro-CT showed mineralized bone and DBA graft material in the biopsies (Figure 5). The residual bone showed coarse trabeculae and abundant intermediate space, whereas the augmented part of the biopsy showed thin bone trabeculae with graft material, which filled up almost the entire remaining intermediate spaces, causing a dense appearance of the augmented bone (see Figure 5, A). The transition between residual bone and the augmented bone was clearly visible (see Figure 5, B). Graft material was seen throughout the entire length of the biopsies. The volume of graft material was slightly less than that of the newly formed mineralized bone (see Figure 5, B and C). The micro-CT reconstruction of the BCP-containing biopsies also showed a difference between the trabecular structure of the residual bone and the newly formed mineralized bone in the grafted area, similar to the DBA-containing biopsies (Figure 6). The newly formed bone and the remaining BCP graft (see Figure 6, B and C) displayed the same pattern in the cranial direction as was observed with DBA. The volume of the newly formed bone in DBA grafted biopsies was highest in the first 2 mm of the biopsy and decreased slowly in the cranial direction, whereas the graft material increased in volume more cranially (Figure 7, A). The volume of newly formed mineralized bone in BCP-grafted biopsies was highest in the first 2 mm of the biopsy, similar to DBA-grafted biopsies (see Figure 7, B), but this volume was 5% less in BCP-grafted biopsies than in DBAgrafted biopsies. BCP graft material was seen over the entire length of the biopsies, and the volume of the BCP graft material was similar to the volume of newly mineralized bone. More graft material than bone was found cranially. When comparing the total volume of mineralized tissue (the sum of bone and graft volumes) between DBA and BCP-grafted sinuses, the DBA graft material was consistently more prevalent than the BCP material, except in the most cranial area of the biopsies. No significant differences between BCP- and DBA-grafted biopsies were found for either bone or graft volumes in volumes of interest 2 to 7 (see Figure 7).

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Figure 5. Three-dimensional reconstruction by micro-CT of a vertical biopsy taken from previously augmented sites during implant surgery at implant locations after sinus floor elevation with deproteinized bovine bone allograft (DBA, Bio-Oss速). A, Mineralized bone only. B, Mineralized bone and DBA graft material. Bone (transparent gray), DBA graft (blue). C, DBA graft material only. D, Longitudinal section through the center of the biopsy showing bone and DBA graft. Bone (transparent gray), DBA graft (blue). VOI, volume of interest; sample regions from caudal to cranial were separately analyzed for bone and graft volumes.

Figure 6. Three-dimensional reconstruction by micro-CT of a vertical biopsy taken from previously augmented sites during implant surgery at implant locations after sinus floor elevation with biphasic calcium phosphate (BCP, Straumann速 BoneCeramic). A, Mineralized bone only. B, Bone and BCP graft material. Bone (transparent gray), BCP graft (blue). C, BCP graft only. D, Longitudinal section through the center of the biopsy showing bone and BCP graft. Bone (transparent gray), BCP graft (blue). VOI, volume of interest; sample regions from caudal to cranial were separately analyzed for bone and graft volumes.

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Figure 7. Bone volume per total volume (BV/TV) and graft volume per total volume (GV/TV) assessed by micro-CT analysis in biopsies retrieved after bilateral sinus floor elevation with (A) deproteinized bovine bone allograft (DBA, Bio-Oss®) and (B) biphasic calcium phosphate (BCP, Straumann® BoneCeramic). Bone volume (white) and graft volume (black) were expressed per total tissue volume and assessed for each volume of interest containing DBA or BCP graft material. Volume of interest 1 represents the caudal most millimeter of the biopsy (containing part of the original maxillary sinus floor and thus native bone), whereas volume of interest 8 represents the cranial-most millimeter of the biopsy.

Degree of mineralization Micro-CT analysis of the degree of mineralization (expressed as mgHA/cm3) of the newly formed bone showed the same mean degree of mineralization in the 7 consecutive volumes of interest for both DBA and BCP grafting materials (Figure 8). The degree of mineralization of the DBA graft material itself was significantly higher than that of BCP graft material. However, the degree of mineralization of the newly formed bone showed very little variation throughout the whole length of the biopsies for both DBA and BCP (see Figure 8) (DBA, 878 ± 48; BCP, 847 ± 51; mean ± SEM). A gradient in mineralization could not be detected. Gradient All biopsies showed a gradient of less bone volume and more graft material volume in the consecutive volumes of interest from residual sinus floor to cranial direction. Comparison of DBA and BCP graft materials found that the bone volume close to the residual sinus floor was 28% for DBA and 20% for BCP, and slowly decreased to 26% for DBA and 19% for BCP more cranially (Figure 9, A). The volume of DBA and BCP graft materials showed the opposite (see Figure 9, B); graft material increased from 12% for DBA and 15% for BCP close to the residual sinus floor to 19% for DBA and 22% for BCP more cranially.

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Figure 8. Degree of mineralization of bone and graft material as assessed by micro-CT analysis in biopsies retrieved after bilateral sinus floor elevation with (A) deproteinized bovine bone allograft (DBA, Bio-Oss速) and (B) biphasic calcium phosphate (BCP, Straumann速 BoneCeramic). Bone mineralization and graft mineralization were expressed as mgHA/cm3 and calculated for each volume of interest containing DBA or BCP graft material. Volume of interest 1 represents the caudal-most millimeter at the end of the biopsy (containing part of the maxillary sinus floor and thus native bone), whereas volume of interest 8 represents the cranial most millimeter of the biopsy.

Figure 9. Comparison of the gradient of (A) bone volume per total volume (BV/TV) and (B) graft volume per total volume (GV/TV) as assessed by micro-CT analysis in biopsies retrieved after bilateral sinus floor elevation with deproteinized bovine bone allograft (DBA, Bio-Oss速; white dots) and biphasic calcium phosphate (BCP, Straumann速 BoneCeramic; black dots). Bone volume and graft volume were expressed per total tissue volume, and assessed for each volume of interest containing DBA or BCP graft material. Volume of interest 2 represents the caudal-most millimeter of the biopsy that contains graft material, whereas volume of interest 7 represents the last area where >3 measurements in different biopsies could be made. BV/TV, bone volume per total volume; GV/ TV, graft volume per total volume.

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DISCUSSION This study found that both DBA and BCP facilitate the formation of new bone when placed in the maxillary sinus. Similar results were obtained for the volume of newly formed bone, the remaining graft volume, the gradient of graft consolidation, and the degree of mineralization of the newly formed bone in DBA- and BCP-grafted biopsies using histomorphometry and microCT. Traditionally, autologous bone is used for sinus augmentation and has proven its efficacy in the formation of new bone, as seen in histologic sections of biopsies taken from the grafted sites.42, 43 However, many patients as well as clinicians want to avoid the use of autologous bone, given that it is accompanied by donor site morbidity and that time is spent on the donor site surgery and hospitalization.4 As previously explained, we selected 2 promising bone substitute materials, DBA and BCP, the latter of which is a mixture of 60% HA and 40% β-TCP. HA is often used because of its high biocompatibility and low solubility 16, 44, 45 and because it can serve as a scaffold for osteoblasts.18 β-TCP is also a biocompatible calcium phosphate and has been used successfully for sinus floor elevation.19, 20 However, β-TCP degrades rather fast and has a different resorption pattern than HA has.21, 46, 47 β-TCP also has a relatively late-occurring remodeling phase.20 Osteogenic cells infiltrate around and into the pores of the β-TCP particles, and degradation of these particles mainly occurs through chemical dissolution of the material rather than by osteoclastic resorption.48 Mixtures of HA/β-TCP in different ratios have been studied for their osteoconductive properties. These mixtures were osteoconductive in animal models, with 60:40 and 80:20 mixtures of HA/β-TCP having similar degradation and bone formation rates as DBA.24 DBA is also widely used for sinus floor augmentation. Sometimes DBA is combined with autologous bone.49 Both DBA and BCP are osteoconductive and therefore need more time for bone regeneration compared with autologous bone. Some patients fear protein transmission when using DBA, although this has never been detected so far 50 and is very unlikely to occur because the donor bone is subjected to heat preparation before transplantation.51 Most studies comparing bone substitutes in sinus floor elevation use biopsies obtained from unilateral sinus floor elevation and different patients. Because the sinus anatomy of these patients varies, reliable results are obtained only when the study includes a high number of patients. To reduce the number of patients while maintaining statistical reliability, we used a splitmouth design for graft comparison and obtained biopsies from 4 patients with similar anatomy and maxillary atrophy on the left and right sides. A split-mouth design is a strong design, allowing within-subject comparison of different bone grafts, thereby removing interindividual intrinsic variation in healing time, physiology, general health, and oral health20 and reducing the number of patients needed to reach statistical significance. This design is an excellent way to compare the performance of BCP and DBA, especially when it concerns edentulous patients with an atrophic maxilla, comparable large sinus spaces, and thin sinus floors.11, 42 It would be interesting to use the split-mouth design in studies comparing different time points in bone healing as well as implant healing for bone regeneration in sinus floor elevation.

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Clinical results All treatments were considered successful, because all patients experienced uneventful treatment, and the peri-implant tissues around all implants were healthy during the 4 years of follow-up. Plaque control was only a minor issue and was easily solved with standard procedures. Variations in bone height, as measured on radiographs, were not observed, which is in agreement with a previous report.52 The 32 Camlog screw-type implants performed well in all patients, and a relatively high initial stability was experienced despite the softness of the grafted bone after 6 months of healing. In one patient, 4-mm-long healing abutments were placed immediately on the 8 implants. These implants were loaded by the removable denture, which was not part of the protocol for unloaded implant healing. Unfortunately, one implant failed in this patient at the site where the original bone height was only 0.5 mm. We consider this failure to be due to premature loading rather than to an insufficient performance of the BCP in conjunction with dental implants. Including this failure, the implant survival rates were 100% at the DBA-grafted site and 94.1% at the BCP-grafted site. The mean implant survival for all implants placed in the augmented sinus was 97.1%. One study reported an implant survival of 97.3% when the sinus floor is thin (â&#x2030;¤4 mm) and an implant survival decreasing to 90.2% with increasing intermaxillary distance.53 All patients in our study had a sinus floor height of <4 mm, and one patient had a large intermaxillary distance. Most clinicians still prefer to use autologous bone for sinus floor elevation and use DBA or BCP only when the residual sinus floor height is more than 4 or 5 mm. This provides sufficient implant stability also when there is limited newly formed bone.2 Our results indicate that both DBA and BCP grafting materials performed clinically well in relation to the limited sinus floor height and implant survival. This suggests that it is not necessary to use autologous bone as long as the bone and implant are allowed sufficient time to heal. Histology and histomorphometry No differences in bone volume were observed between DBA and BCP materials. We measured a bone volume between 5% and 14%, which was less than was found in other studies, which reported bone volumes between 20% and 37% after 5 to 8 months of healing.11, 22, 23, 28 In our study, the healing time was only 3 to 8 months, and longer healing times may lead to increased bone volume.11, 42 Another factor that might have contributed to less bone volume in our biopsies is the maxillary bone anatomy. The patients included in this study were edentulous for many years. They were classified according to the Cawood classification with scores from VI to VII, because they had severe maxillary atrophy, with an initial sinus floor height ranging from 0.5 to 4 mm. These values were small but not a contraindication for inclusion in the present study. A thin original sinus floor (<5 mm) is related to low implant survival.53 From a biologic view, atrophic maxillary bone and a thin sinus floor are also unfavorable, because the recruitment of bone cells from the surrounding bone will be easier if the patient has thick bone walls and a thick residual sinus floor with a high amount of vital bone. Thus the bone regeneration process in our patients is not the result of spontaneous bone repair but rather is dependent on the bone graft material properties. The BCP-grafted sinuses had a higher amount of osteoid in the area between the native bone end and the sinus bone end of the biopsies. When osteoid volume was related 107

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to the bone volume, BCP-grafted biopsies showed relatively more osteoid in all 3 areas of interest in comparison with DBA-grafted biopsies, suggesting more active bone formation in BCP-grafted sinuses than in DBA-grafted sinuses. High amounts of osteoid in vertical biopsies 6 months after grafting with BCP for sinus floor elevation have been reported by others as well.22 We conclude that the bone was vital in all areas of interest in both BCP- and DBA-grafted biopsies, and no foreign body reaction or signs of inflammation were noticed. Micro-CT results: bone volume and remaining graft volume Micro-CT analysis of biopsies has several advantages over traditional histology: it provides 3D information about the tissue sample without cutting the material; it delivers radiographic images of mineralized material that can be 3D-reconstructed; and if the biopsy is properly fixed and embedded in plastic, then histologic sections can be obtained afterward. There is, however, also a disadvantage. The micro-CT reconstruction algorithm does not produce sharp transitions between the graft material and the other materials but rather produces gradual transitions over several voxels. The consequence is that all graft material appears to be surrounded by a thin layer of material with a lower mineral density. During the evaluations, this layer was identified as mineralized bone. This is clearly visible in Figure 5, D, where the graft material is covered by a thin white layer at all transitions from graft material to unmineralized tissue. In this study, the volume of all these layers (which is not negligible) was erroneously added to the mineralized bone volume. Because we mainly looked at gradients in the cranial direction and because the volume of the graft material was almost constant in the cranial direction, we are convinced that this artifact did not influence our conclusions. We found that both DBA and BCP are osteoconductive when used as a sinus augmentation material in patients with an estimated linear growth speed between 0.5 and 1.0 mm per month. BCP-containing biopsies showed a more evenly distributed bone growth, whereas bone formation in DBA-containing biopsies seemed to decline in areas with increasing distance from the preexisting sinus floor bone. This result could be due to a higher rate of bone formation in BCP-grafted sinuses and to a faster rate of degradation of the material compared with DBA. We did not find a significant difference in the degree of mineralization of the newly formed bone when using DBA and BCP. Interestingly, our micro-CT data indicate the existence of a certain volume relation between graft and regenerated bone. As in most patient studies, the newly formed maxillary bone plus remaining graft material composed about half of the total volume. When the graft material decreased, the bone volume increased and replaced the graft material over time. The total volume of graft plus bone was maintained (approximately 50%), and the other half was composed of soft tissue. Valentini et al.54 found a similar shift in bone volume and graft volume between 6 and 12 months after grafting. Biopsies retrieved from native maxillary bone at the molar region also showed 45% to 50% bone volume.42 Higher percentages are seldom seen. In maxillary sinus grafting, a fast resorption of the bone substitute to be replaced by new bone would not be preferable. A long-lasting active osteoconductive guiding scaffold is needed to support osseointegrated implants without bone destabilization.52 Therefore the stability of the graft material in the maxillary sinus and height changes of the graft material over time are important issues to consider for a successful bone regeneration in 108

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maxillary sinus floor elevation procedures. A bone substitute that gradually degrades may be desirable for these bone augmentation procedures. BCP is purely synthetic and is a mixture of HA and β-TCP in a 60:40 ratio. The resorption rate of BCP has been found to be dependent on the HA/β-TCP ratio and proportional to the amount of β-TCP present; HA/β- TCP 20:80 resorbs more rapidly than does HA/β-TCP 60:40, whereas DBA does not degrade within 52 weeks.24 Another study found that resorption of BCP graft occurs faster than resorption of DBA after 1 year of functional loading (0.43 mm vs 0.29 mm mean graft resorption).30 Cordaro28 found less graft substitute in BCP-grafted sinuses than in DBA-grafted sinuses, a finding similar to ours. Degree of mineralization For micro-CT analysis, we have used the highest degree of mineralization value of residual bone (1300 mgHA/ cm3) as a maximum standard and 550 mgHA/cm3 as a minimum standard for mineralized bone. It is possible that the amount of newly formed and less mineralized bone is slightly underestimated, because new bone can have a lower degree of mineralization. This also applies to uncalcified bone. Graft material and bone could be easily distinguished from each other, because both DBA and BCP graft materials contained more than 1300 mgHA/cm3, and shape and contrast differed from mineralized bone. BCP particles had sharp edges, whereas DBA particles were more rounded, and both BCP and DBA particles differed in shape from bone trabeculae. When colors were used instead of grayscale, the images closely mimicked those obtained by traditional histology of undecalcified (ground) sections. Micro-CT analysis and histology of biopsies obtained after sinus grafting with b-TCP have been reported after pseudocoloring of the graft material.55 However, the gray value for bone or graft material was not provided in this study, and a comparison between micro-CT and histologic analysis was not made.55 The main information of bone volume and bone growth after bone regeneration in the maxillary sinus stems from traditional 2D histology and histomorphometry. These reports show much variation between patients, which is caused by anatomic and biologic differences as well as different healing times. This makes a comparison between 2 materials difficult. The reported mean volumes of newly formed bone after sinus grafting using various materials are as follows: for β-TCP, 19%20 and 27%55; for DBA, 20%,28 22%,23 23%,11 25%,29 28%,23,56 and 33%25; and for BCP, 22%,28 27%,22 28%,23 30%,29 31%,25 34%,27 and 39%.26 A histologic comparison between DBA and BCP has been described in an elegant study reporting similar mean volumes of newly formed bone of 19.8% for DBA and 21.6% for BCP,28 indicating that both materials produced similar amounts of newly formed bone. DBA was significantly more retained than BCP, and a higher amount of mineralized tissue in biopsies containing DBA was observed.28 The latter was consistent with our observation of a higher mineralization of DBA graft material, but not the retaining of DBA. Gradient Using histology, we observed that with increasing bone volume, the graft material was decreasing, with a mean volume of 36.8% for BCP and 41.7% for DBA. A similar shift in the gradient between bone volume and graft material was reported when using DBA in maxillary sinus floor elevation.11,28 Using micro-CT, we also observed a gradient of decreasing bone volume and increasing graft volume from residual sinus floor to cranial direction, 109

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suggesting osteoconduction. Such a gradient has also been observed in histologic sections of biopsies from mini pigs.57 In our study, we performed histomorphometry and micro-CT analysis separately. The correlation between micro-CT analysis and traditional histology is still unclear. Obviously, the 2D data from histomorphometry provide an incomplete picture of what is actually a 3D specimen. A discrepancy for bone volume of about 8% to 10% has been reported when histomorphometry and micro-CT data were compared.58, 59 There was still a strong correlation (r = 0.93) of bone volumes found in 2D slices of specimens analyzed by microCT and histomorphometry.59

CONCLUSION Our findings suggest that both DBA and BCP graft materials were effective for regaining adequate maxillary bone height for implant placement and prosthetic rehabilitation after sinus floor elevation in patients with severe maxillary atrophy. Both materials had similar osteoconductive patterns and similar volumes of mineralized bone. Although our split-mouth study found more osteoid in BCP-grafted biopsies than in DBA-grafted biopsies, indicating more active new bone formation and remodeling, we may not conclude that BCP performed better in conjunction with dental implants.

ACKNOWLEDGEMENTS The authors thank Dr L. van Ruijven for excellent support in performing micro-CT analysis and Dr P. Randelzhofer for clinical support.

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CHAPTER 6 A NOVEL APPROACH REVEALING THE EFFECT OF A COLLAGENOUS MEMBRANE ON OSTEOCONDUCTION IN MAXILLARY SINUS FLOOR ELEVATION WITH Î˛-TRICALCIUM PHOSPHATE Engelbert A.J.M. Schulten1*, Henk-Jan Prins1,2*, J.R. Overman1,2, Marco N. Helder3, Christiaan M. ten Bruggenkate1,4, Jenneke Klein Nulend2

Department of Oral and Maxillofacial Surgery, Academic Centre for Dentistry Amsterdam (ACTA)/VU University Medical Center, MOVE Research Institute Amsterdam, The Netherlands Department of Oral Cell Biology, ACTA, University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, The Netherlands Department of Orthopedic Surgery, VU University Medical Center Amsterdam, MOVE Research Institute Amsterdam, The Netherlands. Department of Oral and Maxillofacial Surgery, Rijnland Hospital, Leiderdorp, The Netherlands Shared first authorship, Engelbert A.J.M. Schulten and Henk-Jan Prins contributed equally to this manuscript.

European Cells and Materials 2013;25: 215-28

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ABSTRACT Calcium phosphates are used in maxillary sinus floor elevation (MSFE) procedures to increase bone height prior to dental implant placement. Whether a collagenous barrier membrane coverage of the lateral window affects bone formation within a bone substitute augmentation is currently an important matter of debate, since its benefit has not been irrefutably proven. Therefore, in this clinical study twelve patients underwent an MSFE procedure with a β-tricalciumphosphate (β-TCP). The lateral window was either left uncovered, or covered with a resorbable collagenous barrier membrane. After a 6-months healing period, bone biopsies were retrieved during implant placement. Consecutive 1 mm regions of interest of these biopsies were assessed for bone formation, resorption parameters, as well as bone architecture using histology, histomorphometry, and micro-computed tomography (micro-CT). Comparable outcomes between the groups with and without membrane were observed regarding osteoconduction rate, bone and graft volume, osteoclast number, and structural parameters of newly formed bone per region of interest. However, osteoid volume in grafted maxillary sinus floors without membrane was significantly higher than with membrane. In conclusion, our results obtained with a novel method employed using 1 mm regions of interest, demonstrate that the clinical application of a bioresorbable collagenous barrier membrane covering the lateral window after an MSFE procedure with β-TCP was not beneficial for bone regeneration, and even decreased osteoid production which might lead to diminished bone formation in the long run.

KEY WORDS: Maxillary sinus floor elevation, Collagenous barrier membrane, Histomorphometry, Microcomputed tomography, Calcium phosphate, Osteoconduction, Bone formation Bone regeneration, Bone architecture, Bone resorption

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INTRODUCTION Maxillary sinus floor elevation (MSFE) is a widely accepted and routinely used pre-implant surgical procedure to increase bone height in the posterior maxilla 1, 2. MSFE enables dental implant placement and provides a stable basis for these dental implants. For bone reconstruction in the oral and maxillofacial region autologous bone is still the “gold standard” as grafting material. These bone grafts can be harvested from the iliac crest 3, 4, calvarium 5, tibia 6, rib 7, or intraoral donor sites such as the maxillary tuberosity, the retromolar area of the mandible 4, 8, or chin 7. Autologous bone grafts have excellent osteoinductive, osteoconductive, and osteogenic properties 9. However, the use of autologous bone has also disadvantages such as limited graft availability 10, risk of infection 11, the chance of morbidity at the donor site 12, 13, including pelvic instability 14, and sensitivity disturbances 15-17. Finally, the autologous bone grafts have an unpredictable resorption rate 18. To overcome these disadvantages and to improve the overall patient’s comfort, there is a continuous search for alternative treatments. A variety of allogenic, xenogenic, and alloplastic bone grafting materials or combinations have been used as an alternative for autologous bone grafts in MSFE procedures 19. Recently a meta-analysis demonstrated that β-TCP is the best alternative for autologous bone with regard to osteogenic potential 20. The major advantages of the use of synthetic grafting materials are the reproducible production in unlimited quantities enabling the use as off-the-shelf products, and the absence of disease transmission risk. Calcium phosphate ceramics are very similar to the inorganic components of natural bone and therefore highly biocompatible. The use of a membrane covering the lateral window of the maxillary sinus was suggested to be considered for all MSFE procedures 21. Moreover, a systematic review concluded that the use of a barrier membrane increases the survival rate of endosseous dental implants in the grafted maxillary sinus 22. A resorbable collagenous barrier membrane has a bilayer structure with a porous surface (facing the bone) allowing the ingrowth of bone forming cells, and a dense surface (facing the soft tissue) preventing the ingrowth of fibrous soft tissue into the graft-filled area of the sinus. The use of barrier membranes for guided bone regeneration is not limited to the MSFE procedure, but is also used in restoration of large bone defects 23. The collagen is resorbed within 4 months in bone cavities in animals 24. The use of β-TCP in the MSFE procedure has been reported 25-28. However, the effect of a collagenous barrier membrane to cover the lateral window on bone formation after an MSFE procedure with β-TCP has not been reported. Moreover, the reported percentages of bone volume in the grafted maxillary sinus floor are highly variable, since the residual maxillary sinus floor height and the spatial distribution of the newly formed bone throughout the grafted maxillary sinus were not taken into account. Evidently, a higher percentage of residual bone volume strongly influences the total bone volume percentage, which then does not represent an accurate measurement of the amount of newly formed bone as a result of the grafting. In this study, we attempted to tackle this problem by dividing the biopsies in consecutive so-called regions of interest (ROI) of 1 mm length. Therefore, the purpose of this study was to evaluate the effect of using a collagenous membrane covering the lateral window in MSFE with β-TCP on bone formation and resorption parameters as well as bone structure by combining clinical data, radiological data, and 119

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histomorphometrical and micro-CT data obtained using our novel approach for determination of osteoconduction in bone regeneration.

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MATERIALS AND METHODS Patient selection Twelve patients who were partially edentulous in the posterior maxilla and requiring dental implant(s) for dental rehabilitation, were included in this study. Since all patients had insufficient maxillary bone height, MSFE procedures were performed as described previously 1. The vertical alveolar bone height before MSFE was ≤ 7 mm in the posterior maxilla, but at least 4 mm at the dental implant positions. The mean age of the patients was 57 years, ranging from 36 to 73 years (without membrane: 57±15 years; with membrane: 57±8 years) (Table 1). This study was conducted with the approval of the local medical ethical committee, and all patients signed a written informed-consent before participation in the study. All patients were nonsmokers or smoked <10 cigarettes per day. Patients with systemic diseases, drug abuse, and/or pregnancy were excluded from participation in this study. Patients who required horizontal bone augmentation were also excluded. Maxillary sinus floor elevation surgery In all patients a pre-operative panoramic radiograph was made, which was carefully examined for contour lines of the maxillary sinus to determine the bone height at each planned implant position. After the MSFE procedure using the lateral top hinge swing door technique according to Tatum 1, wound healing and ingrowth of bone in the grafted area was allowed for approximately 6 months. Dental implants were then placed as described previously 29. The open space created within the maxillary sinus between the top-hinge trap door and the maxillary sinus floor was filled with Ceros® β-TCP granules (β-TCP) with 60% porosity and a grain size of 0.7–1.4 mm (Thommen Medical AG, Waldenburg, Switzerland) (Figure 1a). In six patients a resorbable collagenous Biogide® 25 x 25 mm membrane (Geistlich, Wolhusen, Switzerland) was used to cover the lateral window of the maxillary sinus (Figure 1b; Table 1). Thereafter, the wound was closed using Gore-Tex sutures (W.L. Gore and Associates, Newark, DE, USA), which were removed 10-14 days postoperatively. All patients received antibiotic prophylaxis, consisting of 500 mg amoxicillin 3-times daily, starting one day pre-operatively and continuing one week post-operatively. After a healing period of 6 months, prior to the dental placement a panoramic radiograph was made. Both panoramic radiographs, made prior to MSFE and dental implant placement, were used for morphometric measurements to calculate the vertical increase of bone height in the grafted area. Calculations were performed with the use of a conversion factor that adjusted for magnification (1.2x magnification) of the panoramic radiograph. Dental implant surgery was performed under local anesthesia. A crestal incision was made with mesial and distal buccal vertical release incisions. A full-thickness mucoperiosteal flap was raised to expose the underlying alveolar ridge, which was inspected for sufficient bone volume to allow dental implant placement. Then implant preparations were made and biopsies were taken at the planned dental implant positions using a hollow trephine drill with an outer diameter of 3.5 mm, and an inner diameter of 2.5 mm (Straumann trephine drill, Institute Straumann AG, Basel, Switzerland), using sterile saline for copious irrigation. Regular neck Straumann dental implants with a diameter of 4.1 mm, a length of 10 or 12 mm, and a SLA 121

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(sand-blasted, large grit, acid-etched) surface were placed in the augmented maxillary sinus. The dental implants were placed in a single-stage surgical procedure, mounted with healing caps and sutured as described previously 30. Sutures were removed 10-14 days post-operatively. Patients were instructed to avoid loading of the dental implants during integration time postimplant surgery. After a 3 months dental implant integration period the superstructures were manufactured and placed in an outpatient clinic. Table 1. Data of patients treated with or without collagenous barrier membrane. Twelve patients (gender, age in years) who required a maxillary sinus floor elevation (MSFE) were treated either with β-TCP Ceros®>0.7 mm only, or with β-TCP Ceros®>0.7 mm in combination with a collagenous barrier membrane covering the lateral window. The Fédération Dentaire Internationale (FDI) system was used for the dental implant position.

Figure 1. Clinical photographs of a maxillary sinus floor filled with β-TCP with or without a collagenous barrier membrane covering the lateral window. (a) Clinical photograph showing the open space created within the maxillary sinus between the top-hinge trap door and the maxillary sinus floor, that is filled with 100% Ceros® β-TCP granules. The lateral window was left uncovered. (b) Maxillary sinus floor filled with Ceros® β-TCP granules and a resorbable collagenous barrier membrane covering the lateral window. Scalebars represent 1 cm.

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Biopsy analysis The bone biopsies, taken during dental implant surgery using a trephine burr, were fixated in 4% phosphate-buffered formaldehyde (Klinipath BV, Duiven, The Netherlands). Biopsies were removed from the burrs, transferred to 70% ethanol, and stored until used for micro-CT analysis and histomorphometry as described below. A pre-selection on the retrieved biopsies was done to obtain comparable samples. To this end, broken biopsies and biopsies taken from nongrafted areas were excluded. Intact biopsies from implant positions in the graft-filled area of the maxillary sinus were selected, based on blinded radiographic selection, for histomorphometrical and micro-CT analyses. Micro-computed tomography analysis Bone biopsies were kept in 70% ethanol, and three-dimensional (3D) reconstructions of the biopsies were obtained using a high-resolution micro-CT system (µCT 40, Scanco Medical AG, Bassersdorf, Switzerland). To this end, biopsies were fixed in synthetic foam and placed vertically in a polyetherimide holder and scanned at a 10 µm isotropic voxel size, 70 kV source voltage, and 113 µA current. Grey values, depending on radiopacity of the scanned material, were converted into corresponding values of degree of mineralization by the analysis software (Scanco Medical AG). The distinction between newly formed bone and graft material was made using the highest value of the degree of mineralization in the pre-existing sinus floor bone as threshold value. Thereby a distinction could be made between the original non-grafted native bone of the residual sinus floor and the graft material, since the mineralization degree of the graft material was significantly higher than the mineralization degree of bone. A low threshold of 650 mg HA/cm3 to distinguish bone tissue from connective tissue and bone marrow, and a high threshold of 270 ‰ was defined to distinguish graft material from bone tissue. These two thresholds were calculated by averaging the thresholds determined in 3 slices of three bone biopsies by two independent observers. Using this simple thresholding resulted initially in a thin layer of bone covering the graft material throughout the grafted area of the sinus. Therefore, a new and so called “onion-peeling” algorithm (Scanco Medical AG) was used to discriminate between the newly formed bone deposited on the graft material and the graft material itself. This method peels off voxels from the thin layer of bone (as measured with the simple thresholding), and removes this layer when β-TCP was detected within a predefined extent of space. The digital images of the scanned biopsies were analyzed, starting from the caudal side of the biopsy, and continuing towards the cranial side (Figure 2a-c). ROI of 1 mm thickness were defined. The bone volumes obtained from the ROI in the residual native bone part were pooled. ROI, numbered in a consecutive sequence starting from the residual sinus floor (ROI 1) up to the most cranial part of the biopsy, were analyzed for bone volume and graft volume. The following microstructural parameters of the bone were determined: trabecular connectivity density (Conn.D) per mm3, trabecular number (Tb.N) per mm, trabecular thickness (Tb.Th) expressed in mm, trabecular spacing/separation (Tb.Sp) expressed in mm, and bone mineral density (BMD) expressed in mg HA per cm3.

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Histology and histomorphometrical analysis After micro-CT scanning and dehydration in ascending alcohol series, the bone specimens were embedded without prior decalcification in low temperature polymerizing methylmethacrylate (MMA, Merck Schuchardt OHG, Hohenbrunn, Germany) as previously described 31. Longitudinal sections of 5 µm thickness were prepared using a Jung K microtome (R. Jung, Heidelberg, Germany). Midsagittal histological sections of each biopsy were stained with Goldner’s Trichome, in order to distinct mineralized bone tissue (green) and unmineralized osteoid (red) 32. The histological sections were divided in ROI of 1 mm2 for blinded histomorphometrical analysis. Depending on the length of the biopsy, the number of ROI ranged from 9-15 (Figure 2b,c). A consecutive section was immuno-stained for tartrate-resistant acid phosphatase (TRAP) and counterstained with light green to detect any osteoclast-like cells. TRAP staining was carried out according to the method described by Van de Wijngaert and Burger (1986)33. For each separate ROI, the histomorphometrical measurements were performed with a computer using an electronic stage table and a Leica DC 200 digital camera. The computer software used was Leica QWin© (Leica Microsystems Image Solutions, Rijswijk, The Netherlands). Digital images of the sections were acquired at 100x magnification. A demarcation line was indicated between the “residual native bone” floor and the regenerated “grafted sinus floor” bone. Consecutive ROI of 1 mm2 each were defined and numbered throughout the whole biopsy. Data from the residual native bone part of the biopsy were pooled. Each ROI from the sinus floor towards the cranial side of the biopsy was analyzed separately (Figure 2b,c). Using this new method we were able to compare similar ROI for all biopsies (with and without membrane) with respect to the bone regeneration in the augmented maxillary sinus as indicated by the amount of osteoid and bone formed, the presence of TRAP-positive multinucleated osteoclasts, and the volume of remaining graft material. In each ROI, the mineralized tissue volume (Md.V), graft volume (GV),

Figure 2. Micro-CT and histomorphometrical analysis of bone biopsies: methods. (a) Micro-CT analysis showing bone volume and graft volume in a whole biopsy. For micro-CT analysis biopsies were divided in consecutive ROI of 1 mm thickness, and bone volume as well as graft volume were determined in each ROI. New bone formation in the grafted maxillary sinus floor was determined in the ROI from the sinus floor towards the cranial side of the biopsies, whereby section 1 indicates the first ROI where substantial graft material (>1% graft volume per total volume) was observed when analyzing from the caudal to the cranial side of the biopsy. (b) Overview of a midsagittal section of a whole biopsy stained with Goldner’s Trichome. For histomorphometrical analysis, biopsies were divided in consecutive ROI of 1 mm2. The maxillary sinus floor indicates the border between the residual native bone and the grafted maxillary sinus floor. New bone formation (red arrows) in the grafted sinus floor can be seen around the TCP remnants (*). Bone (B) ingrowth, osteoconduction, is determined from the maxillary sinus floor towards the cranial side of the biopsies. (c) Schematic diagram showing the alignment of the biopsies. Three different biopsies differing in size, and residual maxillary sinus floor vertical bone height, are shown. The biopsies are positioned at a 90º angle, with the caudal side at the left and the cranial side at the right. All biopsies were divided and analyzed in 1 mm volumes of interest (dotted lines). Data from the residual native bone were pooled. The maxillary sinus floor shows the border (solid line) between the residual native bone and the grafted part of the biopsy with newly formed bone. Each numbered ROI represents the measurements in the grafted sinus floor region of the biopsy. Scalebars represent 1 mm.

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and osteoid volume (OV) were calculated as a percentage of the total tissue volume (TV) as previously described 34. The number of TRAP-positive cells (osteoclasts, N.Oc) was expressed per total tissue area (mm2). Statistical analysis Data are presented as mean Âą standard deviation (SD). Statistical analysis was performed using SPSS version 15.0 software. The Mann-Whitney test was performed to compare results obtained from the different volumes of interest between the biopsies with and without a membrane. Statistical significance was considered when p<0.05. 125

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RESULTS Clinical evaluation and implant survival All MSFE procedures using β-TCP as a bone grafting material, with or without a collagenous barrier membrane covering the lateral window, were performed without any Schneiderian membrane (antral mucosa) perforation and showed uneventful healing. No wound dehiscences were observed, and no prematurely membrane exposure occurred. Primary stability was achieved with all dental implants, which demonstrated excellent osseointegration after a healing period of 3 months. No clinical signs of inflammation were observed during follow-up. No implant failures were observed during a follow-up of at least one year in the patient group with a collagenous barrier membrane, nor in the group without a membrane, resulting in a 100% dental implant survival (Table 2). Table 2. Histomorphometrical analysis of complete bone biopsies with or without collagenous barrier membrane.

The complete bone biopsies were evaluated using histomorphometry for the mean percentage of bone, osteoid, graft material and connective tissue/marrow. Data on ≥1-year implant survival indicated that no implants failed in the groups with or without a collagenous barrier membrane. Data are presented as mean ± SD. a Significant effect of membrane, p<0.05.

Radiological evaluation Two panoramic radiographs were made of each patient, id est one radiograph was made prior to MSFE (Figure 3a), and another one prior to dental implant placement (Figure 3b). The mean gain in height of the maxillary sinus floor at the implant positions was similar for patients without a membrane (Figure 3c) and patients with a collagenous barrier membrane (Figure 3d) (without membrane: 7.8 ± 1.9 mm; with membrane: 7.2 ± 1.5 mm; mean ± SD). Quantitative histomorphometric evaluation To assess osteoconduction and new bone formation in the grafted area, bone biopsies of the corresponding implant positions were embedded, cut into sections, and stained using Goldner’s Trichome. Newly formed mineralized bone tissue, containing lacunae with live osteocytes, unmineralized osteoid areas, and connective tissue, were observed around the β-TCP particles cranial to the native residual bone. Close contact between newly formed bone and bone substitute was observed. Histomorphometric evaluation of complete biopsies without membrane (n=6) and biopsies with a collagenous barrier membrane (n=6) revealed that the OV/TV was significantly (p=0.026) higher in biopsies without a membrane then with a membrane (Table 2; without membrane: 0.6 ± 0.4%; with membrane: 0.3 ± 0.1%). No differences between biopsies 126

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Figure 3. Radiographic evaluation of the bone height prior to MSFE and prior to dental implant placement. (a) Bone height in the posterior maxilla on panoramic radiographs at the planned dental implant position prior to MSFE, and (b) 6 months after MSFE (prior to dental implant placement). (c) Evaluation of the bone height (in mm) prior to MSFE and prior to dental implant placement for patients augmented with β-TCP Ceros® >0.7 mm (n=6 patients), and (d) for patients augmented with β-TCP Ceros® >0.7 mm in combination with a collagenous barrier membrane (n=6 patients). Mean increase in bone height ± SD was 7.8 ± 1.9 mm without membrane, and 7.2 ± 1.5 mm with a collagenous barrier membrane. Scalebars represent 1 cm.

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without and with a membrane were observed with regard to the following histomorphometric parameters: mean percentage of mineralized volume (Table 2; without membrane: 24 ± 8%; with membrane: 19 ± 4%), mean percentage of graft volume (Table 2; without membrane: 19 ± 10%; with membrane:19 ± 7%), mean percentage of connective tissue and marrow volume (Table 2; without membrane: 56 ± 4%; with membrane: 62 ± 5%), and mean number of osteoclasts (Table 2; without membrane: 1.8 ± 1.8, with membrane: 0.6 ± 0.5). To gain insight in whether the distribution of the values of the histomorphometric parameters was homogeneous or not throughout the residual bone and the newly formed bone, biopsies were divided in consecutive ROI, which were then analyzed separately (Figure 2b). The bone ingrowth from the native residual alveolar bone towards the cranial side of the biopsy was similar in both patient groups without and with a membrane (without membrane: 3 ± 0.9 mm; with membrane: 2.8 ± 1.2 mm) (Figure 4a). This indicates that the rate of osteoconduction was 0.5 mm per month for both groups. The residual native bone was predominantly composed of lamellar bone, containing little osteoid, and some multinucleated TRAP-positive osteoclasts. In all biopsies (without and with membrane) the newly formed bone volume decreased substantially from the residual native bone towards the cranial side of the biopsies (ROI 1-7; Figure 4b). No graft was observed in the residual native bone. The graft volume increased from the residual native bone towards the more apical side of the biopsies (ROI 1-3; Figure 4c), and was ~40% at the most apical ROI of the bone biopsies (ROI 4-7; Figure 4c). In all biopsies (without and with membrane) the unmineralized osteoid volume was clearly more prominent in the newly formed bone area (Figure 4d). The osteoid volume in all ROI of the grafted area in the biopsies without a membrane was higher than in the biopsies with a membrane, with a significant difference between the groups in ROI 3 and 4 (Figure 4d). Multinucleated TRAP-positive osteoclasts were observed predominantly close to the residual native bone in biopsies without and with membrane (ROI 1-3; Figure 5a), indicating that active bone remodeling was taking place. TRAP-positive osteoclasts were absent in ROI with little or no newly formed bone (ROI 4-7; Figure 5b). No significant differences were observed in the mean number of osteoclasts between biopsies without or with a collagenous barrier membrane. Micro-computed tomography evaluation For 3D analysis and evaluation, bone biopsies were scanned using micro-CT. The biopsies were divided in ROI of 1 mm volumes of interest and micro-CT analysis was performed on each consecutive ROI (Figure 2a). Using this method a clear distinction could be made between graft and bone. The bone ingrowth into the graft-filled area could be clearly visualized (Figure 6a-c), and quantified (Figure 6d,e). The mineralized bone volume decreased substantially from the residual native bone towards the cranial side of the biopsies (ROI 1-5; Figure 6d). This pattern corresponded to our observations using histomorphometry (Figure 4b). As expected no graft was observed in the residual native bone, and percentages of graft volume were increasing from the ROI close to the sinus floor towards those at the apical side of the biopsies (ROI 1-3; Figure 6e). The graft volume remained rather constant (~20%) throughout the more apical side of the biopsies (ROI 4-7; Figure 6e). 128

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Figure 4. Histomorphometrical analysis of bone biopsies with and without collagenous barrier membrane. Bone ingrowth (osteoconduction) in mm was determined from the maxillary sinus floor towards the cranial side of the biopsies. Data are presented as mean ± SD, and were similar for both patient groups; without membrane: 3 ± 0.9 mm; with membrane: 2.8 ± 1.2 mm. (a) Osteoconduction rate for both groups was 0.5 mm per month. Histomorphometrical analysis of (b) the mineralized bone volume (Md.V), (c) graft volume (GV), and (d) osteoid volume (OV) as a percentage of the total tissue volume (TV) per area for the group without (–) membrane (black bars), and the group with (+) a collagenous barrier membrane (white bars). Data are presented as mean ± SD. For both groups (with and without membrane), 6 biopsies were analyzed, and only ROI with data from at least 3 biopsies are shown (n≥3). a Significantly different, p<0.05.

Figure 5. Osteoclast activity in bone biopsies with and without a collagenous barrier membrane. (a) To visualize osteoclasts within the bone biopsies, consecutive sections were stained for tartrateresistant acid phosphatase (TRAP; red stain). An example of a large TRAP-positive, multinuclear osteoclast laying on a piece of bone is depicted by a red arrow. (b) The number of TRAP-positive osteoclasts was counted in each separate ROI in each biopsy. Measurements from the native bone part, without graft, were pooled. Each numbered ROI represents the number of osteoclasts in the grafted part of the biopsy. Data are presented as mean ± standard deviation. For both groups, without (-) membrane (black bars) and with (+) a collagenous barrier membrane (white bars), 6 biopsies were analyzed, and only ROI with data from at least 3 biopsies are shown (n≥3). Scalebar represent 25 µm.

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Figure 6. Micro-CT evaluation of bone biopsies with and without collagenous barrier membrane: bone and graft volume. Micro-CT was used to create 3D reconstructions of the biopsies. (a) Total bone volume (yellow), (b) total graft volume (white), and (c) the combination of bone (yellow) and graft (white) are shown. The red line indicates the position where the transverse sections were made. For the combination of bone and graft a midsagittal section is shown in C. To evaluate bone and graft volumes, biopsies were divided in consecutive ROI of 1 mm (see Figure 2A). The residual native bone part of the biopsies contained less than 0.2% graft. (d) Bone volume (BV) and (e) graft volume (GV) are shown as a percentage of the total tissue volume (TV). Data are presented as mean Âą SD. For both groups, without (-) membrane (black bars) and with (+) collagenous barrier membrane (white bars), 6 biopsies were analyzed, and only ROI with data from at least 3 biopsies are shown (nâ&#x2030;Ľ3). Scalebars represent 500 Âľm.

To shed more light on the microarchitectural parameters of the newly formed bone in the graft-filled area, and to compare these parameters with those in the residual native bone, micro-CT was used to analyze bone mineral density, trabecular number, trabecular thickness, trabecular spacing, and trabecular connectivity density. The latter was highest close to the border between the residual native bone and the grafted sinus floor (ROI 1-2; Figure 7a). In these ROI a slightly higher number of trabeculae was observed (Figure 7b) as well as a lower trabecular thickness (Figure 7c), and less space between the trabeculae (Figure 7d). The ROI at the apical side of the biopsies (ROI 3-5) demonstrated a decreased number of trabeculae. These trabeculae were also thinner, and consequently there was more space between the trabeculae, when compared to the ROI at the caudal side of the biopsies (residual native bone and ROI 1-2; Figure 7b-d). The 3D reconstructions (Figure 6a-c) clearly represented these findings on trabecular structural parameters. The bone mineral density of the newly formed bone in the graft-filled area was lower than the bone mineral density of the native bone (Figure 7e). No significant differences in the micro-architectural parameters were observed between the biopsies without and with collagenous barrier membrane.

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Figure 7. Micro-CT evaluation of bone biopsies with and without a collagenous barrier membrane: micro-architectural parameters. Micro-CT was used to evaluate the structural parameters of the newly formed bone. Biopsies were divided in consecutive ROI of 1 mm length (Figure 2a), and in each slice the following parameters were determined: (a) the trabecular connectivity density (Conn.D) per mm3, (b) the trabecular number (Tb.N) per mm, (c) trabecular thickness (Tb.Th) expressed in mm, (d) trabecular spacing/separation (Tb.Sp) expressed in mm, and (e) bone mineral density (BMD) expressed in mg HA per cm3. Measurements from the native bone part, without graft, were pooled. Each numbered ROI represents the measurements in the grafted part of the biopsy. Data are presented as mean Âą standard deviation. For both groups, without (-) membrane (black bars) and with (+) a collagenous barrier membrane (white bars), 6 biopsies were analyzed, and only ROI with data from at least 3 biopsies are shown (nâ&#x2030;Ľ3).

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DISCUSSION The aim of this study was to assess whether covering the lateral window with a resorbable collagenous barrier membrane during augmentation of the maxillary sinus floor with β-TCP is beneficial for bone regeneration. Evaluation was performed by multiparameter analysis, id est by combining clinical, radiological, histological, histomorphometrical, and micro-CT data. The latter two data sets were analyzed using a novel approach, in which consecutive 1 mm ROI were defined, thus allowing identification of new bone formation in a highly detailed manner. Using this novel approach it was shown that after a healing period of 6 months, β-TCP is an effective bone substitute for pre-implant vertical bone augmentation in the human maxillary sinus with high survival rates of dental implants. Moreover, it was demonstrated that the use of a resorbable collagenous barrier membrane covering the lateral window during MSFE procedures did not affect mineralized bone volume (24% without vs. 19% with membrane), but decreased the amount of osteoid, which might indicate either less active osteoblasts and/or an inhibition of the ingrowth of active osteoblasts in these bone biopsies. Our novel approach to obtain highly detailed information on new bone formation in the grafted area provides solutions for both the residual native bone height in the posterior maxilla (which hampers correct statements about the extent of new bone formation), and allows to assess the spatial distribution of the newly formed bone throughout the grafted maxillary sinus. First, the biopsies are divided in consecutive 1 mm ROI. Subsequently, bone biopsies are aligned at the remaining maxillary sinus floor bone height, allowing separation of bone volume data in the residual bone part, and those of the newly formed bone in the graft-filled area of the augmented maxillary sinus. Finally, histomorphometrical data are determined in each ROI, thus allowing identification of new bone formation in a highly detailed manner. The histomorphometrical data were compared with the results of the micro-CT analysis of the biopsies, using similar ROI. By using a unique “onion-peeling” algorithm and specific threshold settings, we were able to distinguish between native bone and grafted ROI, and to gain insight into the 3D structure of the bone grown into the grafted maxillary sinus floor. Minor differences between histomorphometrical results and micro-CT results could be attributed to small differences in 2D and 3D measurements. Lower percentages of graft volume were observed by micro-CT analysis compared to histomorphometry throughout all grafted ROI in biopsies with or without membrane. This might be explained by the fact that graft volumes are massive when analyzed by histomorphometry but not by micro-CT, although the structure of the β-TCP granules used was porous. The percentage of graft volume was approximately twofold lower when analyzed by micro-CT compared to histomorphometry, which is in line with the porosity of 60% of the β-TCP granules. These results indicate that micro-CT is highly useful for 3D evaluation of bone regeneration in MSFE. Using our novel approach, we demonstrate that osteoconduction exclusively occurred upwards from the maxillary sinus floor in cranial direction, independently of the placement of a collagenous barrier membrane, and that no bone ingrowth had taken place from the lateral sides of the biopsies. This indicates that close contact of the graft material with a bony environment was required for new bone formation in the grafted area, and that the osteoprogenitor cells 133

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that repopulate the grafted maxillary sinus floor were mainly derived from cells residing in the residual maxillary alveolar ridge. No differences were observed between bone formation volumes between both patient groups. This is in concordance with a recent meta-analysis by Klijn et al.20, showing no significant effect of the use of a (non-)resorbable membrane over the lateral window on the amount of total bone volume of various biomaterials as sinus floor augmentation materials. Remarkably, even a decrease in the amount of osteoid formed and an increase in the percentage of connective/fibrous tissue was observed in the grafted area of the biopsies in membrane-covered compared to uncovered MFSEs, thereby also potentially affecting bone regeneration at the position where the dental implants were placed. Our data are in contrast with observations by Wallace et al.35, who concluded that vital bone formation in MSFE procedures using deproteinized bovine bone mineral (Bio-Oss®) as grafting material is improved when a barrier membrane, either the same bioresorbable Bio-Gide® membrane as used in our study, or a non-resorbable Gore-Tex® membrane was placed over the lateral window. However, in the Wallace study the beneficial effect appeared to be primarily caused by rather low vital bone percentage (12.1% vs. 17.6% in the membrane groups), whereas other studies found substantial higher bone percentages by using Bio-Oss® without a membrane 36-38. Differences in degradation of deproteinized bovine bone xenograft, such as Bio-Oss®, and calcium phosphate ceramics, such as β-TCP, might explain different results obtained when using these two materials in MSFE. A pre-clinical dog study shows that β-TCP particles are completely resorbed 24 months after implantation, whereas no significant resorption of inorganic xenograft is observed beyond 6 months 39. Bio-Oss degrades very slowly 40; no clinical signs of resorption are seen up to 6 years 41. Graft materials with a very low resorption rate will not remodel and thus not functionally adapt to surrounding bone, which might result in negative mechanical cues. Slow or even a lack of resorption of graft material prevents its replacement by new bone, which may hamper proper and timely bone to dental implant interface formation, thus possibly resulting in lower implant survival. However, the clinical relevance of these differences in degradation rate and/or whether the effect of a collagenous barrier membrane is similar in combination with other types, shapes and sizes of graft materials, such as injectable cements, and any other application needs further (pre-)clinical investigation, especially on the long term changes. Last but not least: the collagenous barrier membrane is composed of collagen extracted from animals, id est veterinary certified pigs, and the use of and exposure to animal-derived materials in clinical settings should be prevented as much as possible. The benefits of using a xenogenic collagenous barrier membrane in combination with any graft material should be considered very carefully, and only be used when benefits have been irrefutably proven.

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CONCLUSION The MSFE procedure represents a unique human model, since after this procedure, clinical bone biopsies can be taken and studied 19. In the current study, using an innovative multiparametric analysis in combination with a novel “onion-peeling” algorithm and a unique 1 mm ROI evaluation strategy, we optimized MSFE biopsy evaluation by establishing a novel strategy that allows identification and spatial distribution of new bone formation in a highly detailed manner, and simultaneously provides a solution for the amount of residual native bone height. Using this novel approach, we demonstrated that the use of a resorbable collagenous barrier membrane covering the lateral window during MSFE procedures using β-TCP as a grafting material, did not have any beneficial effects on the bone ingrowth into the grafted area, the prevention of ingrowth of fibrous tissue into the grafted area, and the survival of dental implants. The collagenous barrier membrane even decreased the amount of osteoid, which might lead to an inhibition of bone formation in the long run. The value of the MSFE procedure reaches far beyond the maxillary reconstruction per se, since it allows detailed and microscopic clinical evaluation of all types of novel therapies, such as stem cell-seeded bone substitutes for bone regeneration. Our novel approach may thus represent a pivotal prerequisite for adequate evaluation of new innovative bone therapeutic modalities, and may be used to measure safety and efficacy in clinical trials.

ACKNOWLEDGEMENTS This research was supported by ZonMw - The Netherlands Organization for Health Research and Development (project number 116001009). The authors thank Marion van Duin and Peter Brugman (Departments of Oral Cell Biology and Functional Anatomy , ACTA-UvA and VU University Amsterdam) for excellent technical assistance, Elisabeth Farré-Guasch (Department of Oral and Maxillofacial Surgery, International University of Catalonia, Spain) for help with histomorpometrical analysis, and Britt van den Berg, Leo van Ruiven, and Geerling Langenbach (Departments of Oral Cell Biology and Functional Anatomy, ACTA-UvA and VU University Amsterdam) for help with micro-CT analysis.

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CHAPTER 7 EVALUATION OF A NEW BIPHASIC CALCIUM PHOSPHATE FOR MAXILLARY SINUS FLOOR ELEVATION: MICRO-CT AND HISTOMORPHOMETRICAL ANALYSIS Janice R. Overman1, Marco N. Helder2, Henk-Jan Prins1, Mardi D. Kwehandjaja1, Christiaan M. ten Bruggenkate3, Jenneke Klein Nulend1, Engelbert A.J.M. Schulten3

In preparation

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ABSTRACT Maxillary sinus floor elevation (MSFE) is a frequently performed pre-implant surgical procedure to restore insufficient jaw bone height allowing dental implant placement in the lateral maxilla. Synthetic calcium phosphate scaffolds are commonly used as substitutes for autologous bone. Despite the successful use of biphasic calcium phosphate with a hydroxyapatite/tricalcium phosphate (HA/TCP) ratio of 60/40, the high percentage of HA may hamper efficient scaffold remodeling. We hypothesize that the use of BCP 20/80 in a MSFE procedure will result in a higher quantity of bone and/or better bone quality in the grafted maxillary sinus compared to BCP 60/40. A comparative study between these two types of calcium phosphate scaffold has not been performed before in a human model. Two groups of 11 patients were included in this study based on strict inclusion criteria. One group received BCP 60/40, the other group received BCP 20/80 during the MSFE procedure. After six months the implants were placed, with concomitant harvesting of biopsies using trephine drills for evaluation by a novel approach for micro-ct and histomorphometrical analysis. Although not significant, there is a clear trend in both the Âľ-CT and the histomorphometrical analyses towards more bone ingrowth in the 20/80 versus the 60/40 BCP variant. Osteoid volumes were comparable between both groups, while osteoclastic activity was significantly higher in the 60/40 group, indicating more balance towards bone formation in the BCP 20/80-treated patients. We conclude that the novel BCP 20/80 scaffold in MSFE performs at least equal, but most likely better in bone augmentation when compared to the BCP 60/40 standard.

KEY WORDS Maxillary sinus floor elevation, Biphasic calcium phosphate, HA/TCP ratio, Micro-CT analysis, Histomorphometry

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INTRODUCTION Maxillary sinus floor elevation (MSFE) is a common pre-implant surgical procedure for restoring insufficient jaw bone height in the lateral maxilla, enabling the placement of dental implants 1, 2 . This procedure was first described by Boyne and James 3 and performed for the first time by Tatum 4. MSFE is an internal vertical augmentation of the maxillary sinus floor, which is approached via the lateral wall while keeping the Schneiderian membrane intact 5. Autologous bone as grafting material for MSFE has been the golden standard, since it does not show adverse reactions and yields a high bone volume when the dental implants are placed 6, 7 . Furthermore, autologous bone has both osteoconductive and osteoinductive properties, meaning that it is capable of influencing its surroundings in the maxillary sinus floor to attract cells that are involved in bone regeneration 8-10. Because of disadvantages of using autologous bone, such as limited availability of bone transplants and morbidity at the donor site 11 several types of synthetic bone substitutes have been proposed and studied; xenografts as well as allografts have been used in different clinical fields 5. The choice of a bone substitute is dependent on the type of tissue and the size of the defect that needs to be engineered. The properties of the graft need to match the needs of the tissue it will be applied to. A recent study described the importance of the substitute choice, since substitute properties such as pore width and particle size can be of major influence on the outcome of the regeneration process. For MSFE and other dental and maxillofacial procedures, a variety of synthetic bone grafting materials is available to substitute autologous bone. Grafting materials containing hydroxyapatite (HA), β-tricalcium phosphate (β-TCP) or a combination of HA and β-TCP, also known as biphasic calcium phosphate (BCP), are most commonly used for these procedures 12-17. Both HA and β-TCP are part of the inorganic component of bone and are highly biocompatible with natural bone. Where HA is rigid, brittle and hardly resorbed after application in MSFE, β-TCP degrades faster and has a different resorption pattern 18, 19. For proper bone scaffolding, there should be a proper balance between the resorption time of the scaffold and the timing of new bone formation. The 60/40 variant represents the slowest resorbing variant of BCP currently used in the clinic. Due to its slow resorption rate, BCP 60/40 hampers rapid bone remodeling while the 100% β-TCP has the shortest resorption time and may lose its scaffolding properties too early. Several studies have evaluated and compared bone substitutes with different HA/ β-TCP ratios against autologous bone or each other 7, 15, 20. Up to now two animal studies have been published where BCP 60/40, BCP 20/80 and other bone substitutes were compared to autologous bone in an animal model (21, 22). The minipig study concluded that the results of BCP 20/80 application were almost similar to those obtained when autologous bone was applied 21, 22 . In another animal model (sheep) it has been demonstrated that BCP 50/50 versus BCP 30/70 did not differ regarding bone formation, mineral dissolution from the biomaterial scaffolds, and active cell-mediated resorption 18. The combination of 60% HA and 40% β-TCP (BCP 60/40) has proven to be a good formula as a bone substitute and is already widely used within dental and oral surgery practices 10, 23, 24. To our knowledge the 20/80 variant has not been tested before in a clinical human model. We 143

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hypothesized that the use of BCP 20/80 in human MSFE results in a higher bone volume and/or improved bone structure compared to the widely used BCP 60/40. We compared two groups of patients who underwent a MSFE. We used a new method of micro-CT and histomorphometrical analysis which provided excellent insight in the bone structure and cellular components within the lateral sinus cavity. Six months after the MSFE we evaluated the biopsies taken from the maxilla prior to implant placement using this new method of micro-CT and histomorphometry. Both evaluation methods complemented each other, and provided a complete view of the new bone regeneration process. In order to eliminate influences of material processing on the outcome of our study, the BCP 20/80 variation was provided by the same manufacturer that provided the BCP 60/40.

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MATERIALS AND METHODS Patients In total 22 partially edentulous patients were included in this study. Six males (aged 53 ± 10.1) and five females (aged 57 ± 18.8) received BCP 60/40 during MSFE. Their mean residual alveolar bone height was 6.4 ± 2.6 mm. In the group that received BCP 20/80, three males (65 ± 9.0) and eight females (48 ± 10.6) were included, with a total mean alveolar bone height of 5.7 ± 1.4 mm.Both patient groups were comparable with regard to age, alveolar jaw bone height, and pre-implant bone height. Average ages were comparable between both groups; even though a significant difference between males and females in the BCP 20/80 group was observed (Table 1). We did not stratify for this parameter, for we do not expect this a major influencing factor on outcome parameters. Prior to participation in the study, all patients signed a written informed consent on the procedures and materials used. All procedures were performed by one oral surgeon either at the Rijnland Hospital in Leiderdorp, or at the VU University Medical Center in Amsterdam, The Netherlands. The patients included in the study were either non-smokers or moderate smokers (less than 10 cigarettes per day; Table 1). Patients who required horizontal bone augmentation, as well as patients with systemic diseases, drug abuse, heavy smokers, other semi-invasive dental treatments and/or pregnancy were excluded from participation in this study. Table 1. Age, alveolar bone height, pre-implant bone height, and bone increase after MSFE surgery in patients treated with BCP 60/40 or BCP 20/80.

Data are mean±SD. Groups were compared with an unpaired t-test. Statistical significant difference when p≤0.05. BCP, biphasic calcium phosphate. Moderate smoking, 10 or less cigarettes/day.

Calcium phosphate scaffolds Two types of calcium phosphate scaffolds from the same manufacturer were used: eleven patients received porous BCP with 60% HA and 40% ß-TCP (BCP 60/40; Straumann®BoneCeramic (Straumann, Basel, Switzerland)), and eleven patients received porous BCP with 20% HA and 80% ß-TCP (BCP 20/80; Straumann, Basel, Switzerland). Both BCP 60/40 and BCP 20/80 scaffolds had a porosity of 90%, and a particle size and pore width ranging from 500 to 1000 µm. 145

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Maxillary sinus floor elevation procedure All 22 patients underwent a unilateral maxillary sinus floor procedure. In this procedure the sinus floor was elevated according to Tatumâ&#x20AC;&#x2122;s protocol 4. The surgical technique consisted of making a top-hinge trap door in the lateral maxillary sinus wall (Fig. 1). A horizontal incision on the top of the maxilla was made in combination with one or two vertical incisions. After elevation of the buccal flap the bony trapdoor preparation was performed with a large round diamond burr. This bone preparation followed the outer contour of the maxillary sinus, leaving the maxillary sinus membrane intact. The buccal trapdoor was carefully pushed inward. Then the fragile Schneiderian membrane needed to be prepared from the inner aspect of the sinus, leaving this membrane intact. After mobilization and elevation of the sinus membrane and the bony trapdoor a new sinus floor was created. The cavity underneath the trap door was filled with the graft material. Six male and 5 female patients received BCP 60/40. Three male and 8 female patients received BCP 20/80. The wound was closed using Gore-Tex sutures (W.L. Gore and Associates, Newark, DE, USA), which were removed 10-14 days postoperatively. All patients received antibiotic prophylaxis, consisting of 500 mg amoxicillin 4 times daily, starting one day pre-operatively and continuing one week post-operatively.

Figure 1. MFSE procedure, implant placement, and radiography. Clinical and radiographic images of the MSFE procedure and implant placement six months later. (A) pre-operative image, (B) sinus filled with graft material, (C) implants were installed after six months, and (D) end result after abutments were installed. Radiographic evaluation of the sinus and the alveolar bone height occurred during all stages of MSFE implant placement.

Implant placement and biopsy retrieval Six months after the MSFE procedure, dental implant surgery was performed under local anesthesia. A crestal incision was made with mesial and distal buccal vertical release incisions. A full-thickness mucoperiosteal flap was raised to expose the underlying alveolar ridge. Then implant preparations were made and simultaneously biopsies were taken at the planned dental implant positions using a trephine drill with an outer diameter of 3.5 mm, and an inner diameter 146

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of 2.5 mm (Straumann® Trephine Drill, Institute Straumann AG, Basel, Switzerland), using sterile saline for copious irrigation. Regular neck Straumann® dental implants with a diameter of 4.1 mm, a length of 10 or 12 mm, and a SLA (Sand-blasted, Large grit, Acid-etched) surface were placed in the augmented maxillary sinus. The implants were mounted with healing abutments to heal in a non-submerged fashion as described previously 25. The sutures were removed 1014 days postoperatively. Patients were instructed to avoid loading of the dental implants during integration time post-implant surgery. After a 3 months integration period the superstructures were manufactured and placed in an outpatient clinic. Pre- and postoperative panoramic radiographic images were made to determine the size and contour of the maxillary sinuses, and the height of the augmented sinus floor fill. (Fig. 1B). The calculations were performed using a conversion factor that adjusted for the magnification (1.25 x) of the panoramic radiograph. The retrieved biopsies were first fixated in a standard formalin solution for at least 24 hours. They were then transferred to containers with a 70% ethanol solution prior to further evaluation. For each patient one biopsy was selected. Micro-computed tomography analysis Micro-CT analysis was performed on selected biopsies that were transferred to a cylindricalshaped container made of porous synthetic foam soaked in alcohol 70%. This cylinder was custom made for the polyetherimide holder inserted into the micro-CT scanner. The micro-CT device was a Scanco Medical AG, model PX5-925EA (Basel, Switzerland). This scanner had a tube voltage of 55kV and a tube current of 145mA (70 kV source voltage, and 113 μA current). The scanning resolution was 10 micron. The images of the biopsies were reconstructed by measuring the radiolucency of the object with sensors and rotating x-ray beams from different angles, and subsequent calculation of the three-dimensional structure. The software of the micro CT-scans (Scanco Medical AG) was able to convert gray scale values into corresponding values of degree of mineralization. The distinction between bone and graft material was made using the highest value of the degree of mineralization in the pre-existing sinus floor bone as threshold value. Therefore we could distinguish between the patient’s native alveolar bone and the graft material, since the mineralization degree of the graft material was significantly higher than the mineralization degree of bone. The degree of mineralization was expressed in milligrams of hydroxyapatite per cubic centimeter (mgHA/cm3). For bone we initially determined the ultimate thresholds as the values between 650 and 1300 mgHA/cm3, and for graft material we measured values between 1300 and 2500 mgHA/cm3. This thresholding method resulted initially in a thin layer of computing-generated bone covering the graft material throughout the grafted area of the sinus. Therefore, a new and so-called “onion-peeling” algorithm (Scanco Medical AG, evaluation program no. 12) was used to discriminate between the newly formed bone deposited on the graft material and the graft material itself. This method peels off voxels from the thin layer of bone (as measured with the previous tresholding), and removes this layer when the graft was detected within a predefined extent of space. The digital images of the scanned biopsies were analyzed, starting from the caudal side of the biopsy, and continuing towards the cranial side (Fig. 2A). Volumes of interest of 1 mm thickness were defined, and numbered in a consecutive sequence starting from the residual sinus floor (volume of interest area #1) up to the most 147

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cranial part of the biopsy. In each volume of interest the following micro structural parameters of the bone were determined: bone volume and graft volume over total tissue volume (BV/TV and GV/TV respectively). The bone volumes obtained from the areas of interest in the alveolar native bone were pooled. The borders between the graft area and the native bone were aligned (Fig. 2B). The biopsies sometimes differed in length (due to breakage), and therefore statistical analysis was not always possible on the results obtained from the most cranially situated areas of the bone biopsies, due to the low number of biopsies available for measurement in that particular area.

Figure 2. Novel methods of micro-CT and histomorphometrical analysis. Each biopsy was first analyzed by micro-CT, where the biopsies were virtually divided into 1 mm long areas for determination of bone volume and graft volume. After embedding in methylmethacrylate (MMA) followed by Goldnerâ&#x20AC;&#x2122;s trichrome-staining the sections were divided into 1 mm2 areas for determination of mineralized bone volume, unmineralized bone volume (osteoid), and graft volume (A). The collected data per area were aligned and pooled according to the scheme to ensure a proper evaluation of new bone ingrowth (B).

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Histology and histomorphometrical analysis After micro-CT scanning and dehydration in increasing concentrations of alcohol solutions, the bone specimens were embedded without prior decalcification in low temperature polymerizing methylmethacrylate (MMA, Merck Schuchardt OHG, Hohenbrunn, Germany). Longitudinal sections of 5 μm thickness were prepared using a Jung K microtome (R. Jung, Heidelberg, Germany). Midsagittal histological sections of each biopsy were stained with Goldner’s Trichrome, in order to distinct mineralized bone tissue (green) and unmineralized osteoid. The histological sections were divided in areas of interest of 1 mm2 for blinded histomorphometrical analysis. Depending on the length of the biopsy, the total number of areas ranged from 9 up to 15 (Fig. 2A). For each separate area of interest, the histomorphometrical measurements were executed using a computer with an electronic stage table and a Leica DC 200 digital camera. The computer software used was Leica QWin© (Leica Microsystems Image Solutions, Rijswijk, The Netherlands). The sections were digitized at 100x magnification. A demarcation line was indicated between the “residual native bone” floor and the newly formed bone. The definitive criteria for locating the demarcation were based on multiple factors; we considered the clinically determined native bone height derived from the radiographic images. We also considered the histological changes that occurred in the transition area from native bone to new bone; native bone contains bone marrow, defined by the presence of adipose tissue, while the area where new bone is formed contains more blood vessels, and a specific pattern of the (woven) bone formation around calcium phosphate particles. Consecutive areas of interest of 1 were defined and pooled in the same manner as described under ” micro-CT analysis” (page 13, Fig. 2B). We compared similar areas of interest for all biopsies with respect to bone regeneration in the augmented maxillary sinus as indicated by the amount of osteoid and bone formed, and the volume of remaining graft material. In each area of interest, the mineralized tissue volume (Md.V), graft volume (GV), and osteoid volume (OV) were calculated as a percentage of the total tissue volume (TV) as previously described 26. Tartrate-resistant acid phosphatase (TRAcP) staining was used to visualize bone resorbing multinuclear cells (osteoclasts) within the biopsy sections. These sections were selected adjacent to biopsy sections that were stained with Goldner’s Trichrome method. TRAcP staining was performed according to a standardized protocol 27. The number of TRAcP osteoclasts in each section was measured at 200x magnification with the same computer software and microscope as used for quantification of bone volume and osteoid volume in the Goldner-stained sections. Each tissue section used for TRAcP staining of osteoclasts was divided in consecutive optical areas of 1 mm2, overlapping with the optical areas in the Goldner-stained sections as closely as possible. Within each area all red-coloured multinuclear cells were identified as TRAcP-positive osteoclasts, and for each section the total number of TRAcP-positive osteoclasts was calculated. The data were pooled, and compared between groups as described under ”Micro-CT analysis”. Statistical analysis Statistical analysis was performed using KyPlot version 2.0 beta (32 bit) and GraphPad Prism®5.0 (2007). In Fig. 3, 5, and 6, the data are presented as mean ± standard deviation (SD), with a minimum of three biopsies per area of interest for valid statistical analysis. The first four 149

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areas of interest contained 11 measurements, the consecutive two areas contained between 6-9 measurements. To be able to draw conclusions as reliable as possible, we only performed statistical analysis on these first four areas. We do show the results of all six consecutive areas of interest in the grafted part of the biopsies. The Mann-Whitney U-test was performed to compare bone volume, mineralized volume, osteoid volume, and the number of TRAcP+ cells between the groups of biopsies containing BCP 60/40 and BCP 20/80. After pooling the data, that MannWhitney-U test was also used for comparison of means. Data were considered significantly different when p â&#x2030;¤ 0.05.

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RESULTS During and after the MSFE procedure no adverse events and/or complications were reported. The augmentation height increased after the MSFE procedure, and was similar in all patients (Table 1). Wound healing went uneventful, and no implants were lost two years after implantation. Micro-computed tomography analysis 3D evaluation of the bone structure within the biopsies. Bone and graft could be clearly distinguished based on differences in grey-scale values as well as obvious visible differences in structure. New bone volume (BV) and graft volume (GV) could be determined up to 10 mm from the border with native bone. In the BCP 60/40 biopsies, bone ingrowth was observed up to 6 mm from the border with native bone, with volumes ranging from 27 % ± 11% in area 1, to 5.4 ± 7% in area 6 (Fig. 3A). In the BCP 20/80 biopsies, bone ingrowth was also measured up to 6 mm from the native bone. BV ranged from 36.8 ± 10% in area 1, to 2.8 ± 4.5% in area 6. A clear trend towards more bone formation in the 20/80 group was observed. To obtain information on BV in two larger areas of the biopsies, we pooled BV data of areas 1-4, and 5-6. In addition, we pooled the BV data obtained from all areas of interest, which provides information on the mean BV in the whole biopsy. The pooled BV data also showed that BCP 20/80 showed a trend towards more bone formation than BCP 60/40, although the difference between BCP 20/80 and BCP 60/40 did not reach significance (BV area 1-4: 14.4 ± 4.7% versus 21.7 ± 6.1%, p=0.94; BV total biopsy: 12.3 ± 8% versus 16.0 ± 13%, p=0.82; Fig. 3B). The alveolar native bone height in both BCP 20/80 and BCP 60/40 groups was similar (Bone height: BCP 60/40: 30.3 ± 10.3%; BCP 20/80: 31.5 ± 11.4%,.

Figure 3. Micro-CT analysis of bone and graft volumes measured in biopsies taken after MSFE with BCP 60/40 or BCP 20/80. (A) Comparison of the gradient of bone volume per total volume (BV/TV) as assessed by microCT analysis in biopsies retrieved after bilateral sinus floor elevation with BCP. Bone volume was expressed per total tissue volume, and assessed for each area of interest containing BCP 60/40 and BCP 20/80 graft material. Area #1 represents the most caudal-millimeter of the biopsy that contains graft material, whereas area # 6 represents the cranial side of the biopsy, which is the area furthest from the native bone. (B) Pooled data of measurements in areas 1-4 (p=0.94), areas 5-6 , and all areas together (total, p=0.82). Values are mean ± SD. Statistical significance when p≤0.05. For a more clear display in (A), the BCP 20/80 data set has been moved to the right along the x-axis by 20%. MSFE, maxillary sinus floor elevation, BV/TV, bone volume per total volume. BCP 60/40, BCP with 60% HA and 40% β-TCP. BCP 20/80, BCP with 20% HA and 80% β-TCP.

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GV of BCP 60/40 and BCP 20/80 granules ranged between 10-20% throughout the biopsies. No differences were observed between GV of both types of graft material measured after MSFE (data not shown). Histology and histomorphometrical analysis Mineralized volume (Md.V) was determined in Goldner’s trichrome-stained sections up to 6 mm from the native bone (Fig. 4A). Md.V/TV showed a similar pattern when measured in the consecutive areas followed a similar pattern as in the micro-CT assessments (Fig. 3A). Increased Md.V/TV was observed in most areas of interest in the BCP 20/80 biopsies compared to BCP 60/40 biopsies. Md.V/TV ranged from 25.6 ± 11% to 0.15 ± 0.4% using BCP 60/40, and from 31.6 ± 15% to 2.2 ± 7% using BCP 20/80. The pooled Md.V/TV data (Fig. 4B) revealed a similar trend towards higher Md.V/TV in BCP 20/80 compared to BCP 60/40 groups, similar as the trends observed the pooled data from area 1-4 and area 5-6 (Fig. 4A). The mean BV of the whole biopsies was also similar for both BCP scaffolds (BCP 60/40: 11.7 ± 8%; BCP 20/80: 15.7 ± 13%, p=0.69). Osteoid volume was similar between BCP 20/80 and BCP 60/40 biopsies (Fig. 4C,D).

Figure 4. Histomorphometrical analysis of bone biopsies after MSFE with BCP 60/40 or BCP 20/80. Mineralized volume (Md.V), and osteoid volume (OV) as a percentage of total tissue volume (TV) measured in each area of interest in biopsies obtained from patients treated with BCP 60/40 or BCP 20/80 (A, C). Data are also pooled for areas 1-4 (p=0.49), areas 5-6, as well as for the whole biopsy (total, p=0.69) (B, D). Area #1 represents the most caudal-millimeter of the biopsy that contains graft material, whereas area # 6 represents the cranial side of the biopsy, which is the area furthest from the native bone. Values are mean ± SD. Statistical significance when p≤0.05. For a more clear display in (A, C), the BCP 20/80 data set has been moved to the right along the x-axis by 20%. MSFE, maxillary sinus floor elevation. BCP 60/40, BCP with 60% HA and 40% β-TCP. BCP 20/80, BCP with 20% HA and 80% β-TCP.

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Histology revealed that in both patient groups the newly formed bone was deposited against the BCP particles in a similar way (Fig. 5A,B). In most biopsies the demarcation between native bone and newly formed bone could be easily determined using criteria as described in the materials and methods section. A rather sharp transition from the native bone area to the grafted area can be seen (Fig. 5B). The fatty aspect of bone marrow as part of the patient’s native bone (black arrows) is in contrast with woven bone surrounding the synthetic granules (stars) and blood vessels (bv) on the right side. Osteoid (red) is being deposited against the granules and the blood vessels. The histology, histomorphometry, together with the structural images obtained by micro-CT and the radiographic images, revealed that in some biopsies the newly formed woven bone had already been remodeled into lamellar bone indicating adaptation to mechanical loads, which resembled the lamellar structure of the native bone. TRAcP-positive stained multinucleated cells (osteoclasts were mostly found clustered together against the bone, i.e. against both newly formed woven bone as well as native alveolar bone (Fig. 5C,D).

Figure 5. Histological evaluation of bone and graft volumes in biopsies taken after MSFE with BCP 60/40 or BCP 20/80. Goldner’s trichrome-stained sections of biopsies from (A) BCP 60/40, and (B) BCP 20/80 treated patients, showing mineralized bone (green), unmineralized bone (red, short black arrows), bone marrow tissue within the native bone area characterized by clustered adipose tissue cells (triangle), and the spaces where the particles (*) used to be. Both images show the transition area from native alveolar bone to the grafted area right next to the native bone. Image 5B clearly shows the transition from native bone to grafted area; adipose fat cells (triangle) are a marker of bone marrow within mature native bone. Images 5C and 5D show the TRAcP-stained sections (orange colored multinuclear cells, black long arrows) of biopsies from group BCP 60/40 and BCP 20/80 respectively. In both groups the cells were mostly found clustered and/or lined against the bone (green). BCP 60/40, BCP with 60% HA and 40% β-TCP. BCP 20/80, BCP with 20% HA and 80% β-TCP. TRAcP, tartrate-resistant acid phosphatase; BV, blood vessel.

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Osteoclasts The number of TRAcP-positive osteoclasts in the alveolar native bone and in the grafted areas of the biopsy sections was quite variable between patients in both BCP 20/80 and BCP 60/40 grafted groups (Fig. 6A). After pooling the data on osteoclast number, there was a significant difference (p=0.01) in the number of TRAcP-positive osteoclasts between the BCP 20/80 and BCP 60/40 groups; more osteoclasts were observed in the BCP 60/40 group than in the BCP 20/80 group, indicating more active bone remodeling with BCP 60/40 compared to BCP 20/80 (Fig. 6B).

Figure 6. Number of TRAcP-positive osteoclasts in biopsies taken after MSFE with BCP 60/40 or BCP 20/80. The number of TRAcP-positive osteoclasts was counted in each separate area of interest in the grafted sinus (A). Pooled data show that the number of osteoclasts in BCP 60/40 biopsies was significantly higher than in BCP 20/80 biopsies, p=0.009 (B). Values are mean ± SD. Statistical significance when p≤0.05. TRAcP, tartrate-resistant acid phosphatase; MSFE, maxillary sinus floor elevation; BCP 60/40, BCP with 60% HA and 40% β-TCP. BCP 20/80, BCP with 20% HA and 80% β-TCP.

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DISCUSSION This study aimed to compare the performance of BCP 20/80 with BCP 60/40 in a human maxillary sinus floor elevation (MFSE) model. The patient data were evaluated using clinical parameters (radiography), micro-CT analysis, and extended histomorphometrical analysis. We found that BV seemed higher after MSFE with BCP 20/80 than with BCP 60/40, especially in the areas of interest closest to the native residual bone. Although this difference was not statistically significant, this observation suggests that the presence of BCP 20/80 in general might facilitate osteoconduction better than BCP 60/40, since the residual native bone volume was similar in both BCP 20/80 and BCP 60/40 groups. In addition, the percentage of osteoid was also similar in both patient groups, showing that active bone deposition was taking place at a similar rate in both study groups. Strikingly, the number of TRAcP-positive osteoclasts present in the BCP 60/40 biopsies was significantly higher than in the BCP 20/80 biopsies. It is well known that calcium phosphate attracts osteoclasts 7. This implies that bone remodeling in BCP 60/40 biopsies may advance faster compared to the BCP 20/80 grafted biopsies. Regarding the measured remaining graft volumes we did not observe any significant differences between BCP 60/40 and BCP 20/80. Remarkably, we observed a discrepancy between the graft volumes measured with micro-CT compared to measurements done by histomorphometry, which is not uncommon; a similar discrepancy was found in our previous study 12. This discrepancy can be explained by the fact that micro-CT data analysis comprises 3D volumetric measurements, whereas bone and graft volumes are calculated as a percentage of the whole measured volume of interest, which also includes the soft tissue and air within the porous particles. However, during histological preparation, the grafted particles are dissolved due to the acidity of the dyes used for the staining process. Therefore the total surface of the space that is left by the particles is measured as graft, giving the false impression that the granules are solid while they are porous. Corrections for air are automatically done when measuring graft volume. It must also be kept in mind that the histomorphometrical analysis extrapolates data derived from 2D measurements to 3D data. Therefore, even though the tissue sections of the biopsies are representative for the whole biopsy, the outcome of histomorphometrical measurements can differ from micro-CT measurements in a particular area. Despite the differences in data obtained using micro-CT and histomorphometry, these methods are complementing each other very well; micro-CT analysis is based on differences between structures and mineralized compounds, while histomorphometry adds cellular and soft tissue information that is required to answer the question whether and how new bone ingrowth takes place. We defined a new method to determine the exact position of the demarcation between native bone and newly formed bone in the grafted area, by considering the presence of bone marrow adipose tissue as a marker of residual native bone. Especially this parameter that can be derived from the histological images added significantly to our new insights on the position of this demarcation line. Without using this new method, false demarcations would have been assigned 1 to 2 mm more cranial than the original border. This method also allowed the otherwise overlooked observation that after 6 months, newly formed bone has been remodeled 155

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from a woven structure to a lamellar structure. To our knowledge our new method to determine the demarcation line needed for proper histological evaluation of biopsies has not been described before. This method may also be applicable in other studies where native bone needs to be distinguished from newly formed bone such as in studies on osteoconduction. Based on the difference in composition we would expect a higher resorption rate in BCP 20/80-grafted biopsies due to lower percentage of HA, thus allowing more space for osteoconduction. Our results revealing differences in bone formation and bone resorption parameters might be explained by the difference in properties between both bone substitutes; the higher bone volume using BCP 20/80 compared to BCP 60/40 could be caused by a higher percentage of Ă&#x;-TCP leading to a larger bone resorption surface, thus leaving more room for new bone ingrowth to take place. Next to surface properties also the porosity of a bone substitute is highly important for the osteoconduction after MSFE. In this study we aimed to rule out differences in graft performance due to differences in scaffold porosity by having BCP 60/40 and BCP 20/80 manufactured by the same company, excluding possible differences as a result of differences in manufacturing, which might affect the macro- and microstructure of a calcium phosphate, which is crucial for osteoconduction. In addition, this macro- and microstructure determines cell and vessel movement inside the pores, which are related to the degree of interconnection of the pores and to the pore size 28, 29. Previous studies have shown the effectiveness of biphasic calcium phosphate in human MSFE procedures. Several studies have also compared the performance of different synthetic bone substitutes 14, 24, 30-34. Several studies have also compared the performance of different synthetic bone substitutes. However, there are no studies comparing BCPs with different HA/Ă&#x;TCP ratios in a human model of bone augmentation such as the MSFE model. In addition we refined our method to determine the demarcation between native bone and newly formed bone by considering the presence of bone marrow adipose tissue as a marker of residual native bone.

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CONCLUSIONS From the results we can conclude that the ingrowth of newly formed bone tissue was observed using both BCP 20/80 and 60/40 in a human MSFE procedure. With both BCP 20/80 and BCP 60/40, exhibiting different HA/Î˛-TCP ratios, it was possible to provide a stable environment for implant placement after 6 months. Strikingly a clear trend of more bone formation with BCP 20/80 than BCP 60/40 after 6 months was observed, while the number of bone resorbing osteoclasts was significantly lower in BCP 20/80 than in BCP 60/40 biopsies. This might be due to either a faster bone remodeling rate, or an earlier start of bone remodeling in BCP 20/80 treated patients. This also suggests that, even though the volume of newly formed bone was not statistically different, BCP 20/80 might perform better than BCP 60/40 as a scaffold in the MSFE model during a so-called one-step surgical procedure.

ACKNOWLEDGEMENTS The work of J.R. Overman was supported by the Research School of the Academic Centre for Dentistry Amsterdam (ACTA), The Netherlands. The authors also thank Marion A. van Duin and Leo R. van Ruijven for their excellent technical assistance.

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CHAPTER 8 GENERAL DISCUSSION

Chapter 8

GENERAL DISCUSSION The developing fields of bone tissue engineering and regenerative medicine have identified skeletal defects, such as insufficient jaw bone for oral implant placement, as attractive translational targets. Bone tissue engineering comprises cells, biomaterials (preferably biodegradable), environmental stimuli and biologics to trigger differentiation of stem cells. The ultimate goal of this thesis is to achieve regeneration of functional bone tissue during and after sinus floor elevation, and therefore an evaluation of the applied stem cells is needed, as well as an evaluation of the properties of calcium phosphate scaffolds involved. This thesis describes the functionality of ASCs for bone tissue engineering and proposes a novel concept of a one-step surgical procedure for human sinus floor elevation using calcium phosphate scaffolds. In previous studies we evaluated goat and human ASCs for use in spinal interbody fusion; it was shown that goat ASCs could be applied in a one-step procedure as part of a spinal fusion model 1. In line with these studies the feasibility of their human ASC counterparts for their use in a one-step surgical procedure for human maxillary sinus floor elevation was evaluated, addressing attachment time to scaffolds, and optimal treatment with differentiationenhancing growth factors. Next, the effects of the secretome of osteogenically stimulated ASC after seeding on calcium phosphate carriers were also evaluated. Furthermore the known methods for evaluating bone ingrowth within a biopsy were refined, which provided better insights in the processes of new bone formation. This improved method allowed us to compare new bone formation when different bone graft materials were used in sinus floor elevation. The before mentioned validations occurred in different phases and on different levels throughout this study, and together provide the framework for successful implantation strategies. In chapter 2 the feasibility of the human MSFE model for bone tissue engineering studies was discussed. In chapter 3, the short treatment of hASCs with BMP-2 in a low dose was evaluated. This was prompted by two considerations: (1) induction times should be really short in order to fit within the one-step surgical concept; and (2) the use of BMP-2 has been extensively debated recently, since it could be damaging to cells and tissues when administered in higher dosages in vitro as well as locally in vivo 2, 3. Our ex vivo stimulation and physiological dosaging in the ng-range ensure patient safety in our MSFE model by reducing the body exposure to BMP-2 to a minimum. As a start, the properties of hASCs, and the culturing conditions for hASC attachment to several calcium phosphate carriers were thoroughly investigated and optimized. Aspects like cell attachment time, influence of temperature on cell attachment, time of BMP-2 treatment of hASCs, and a surface (topography, chemistry) effect on proliferation and osteogenic differentiation of ASCs were evaluated. The authors found that hASCs attached rapidly to the surface of different calcium phosphate carriers, independent of BMP-2 pre-treatment. Interestingly it was concluded that subjection of hASCs to a low dose of BMP-2 for 15 minutes was sufficient to stimulate the expression of osteogenic genes and to inhibit expression of an adipogenic marker in vitro for at least 21 days. The exact mechanism for such a prolonged effect is yet unknown. 164

General discussion

When hASCs were seeded on biphasic calcium phosphate (BCP) and Î˛-tricalcium phosphates (TCP) scaffolds, a slight difference in osteogenic gene expression levels was observed on the two scaffold types. However, the most striking finding was the strong osteogenesispromoting effect of the surface topography of the biphasic calcium phosphate (BCP) and Î˛-tricalcium phosphates (TCP) scaffolds compared to cells cultured on plastic. The strongest osteogenic differentiation was found when combining BMP-2 induction followed by seeding on a calcium phosphate scaffold. combining BMP-2 induction followed by seeding on a calcium phosphate scaffold. To evaluate whether the secretome of osteogenically stimulated hASCs seeded on BCP and TCP scaffolds may affect the wound repair process in parallel with their osteogenic differentiation, in vitro studies of the dynamic changes of the ASC secretome were performed. Therefore, in chapter 4, the effects of hASCs after seeding in vitro on BCP and TCP were tested; the dynamic changes of the angiogenic secretome of the hASCs during the osteogenic differentiation process were investigated. Surprisingly, no correlation was found between the degree of differentiation and the secreted growth factor expression pattern of hASCs. This suggests that the growth factors produced by hASCs do not seem to work in an autocrine/paracrine fashion to stimulate osteogenic differentiation in vitro, yet the substrate still does have a strong effect on the differentiation of hASCs. An alternative explanation may be that our in vitro study conditions lack interaction with environmental factors present in vivo (e.g. iterative interactions with local cells). Previous research also showed that MSCs and/or their secretome in particular could be applied in a therapeutic way; they are able to migrate to injured tissues and, in some cases, even able to replace damaged tissue 4-10. In the next three chapters the effect of bone substitutes, and peri-operative techniques during sinus floor elevation, on the ingrowth of new bone was investigated. Firstly in chapter 5 two different scaffolds, biphasic calcium phosphate (BCP) and deproteinized bovine bone (DBA) were compared within a split-mouth model for MSFE. The authors found that after six months there were no differences regarding bone and graft volume, however BCP showed a more active bone remodeling process. In this study experiments with the histological evaluation technique as well as the micro-Ct analysis were executed. In the following studies these techniques were applied in conjunction with other study parameters. In chapter 6 it was evaluated whether a collagenous membrane, placed over the maxillary sinus cavity after the elevation procedure, would result in enhanced bone ingrowth. The authors showed that the use of a resorbable collagen barrier did not increase mineralized bone volume, and actually decreased the amount of osteoid, indicating a lower activity of osteoblasts, or a lesser amount of active osteoblasts in the sites. This difference in results might be explained by our extensive and multifactorial methods (1mm-regions of interests, micro-CT, histomorphometry and clinical x-ray data), yet more human studies with a larger number of patients need to be done to confirm our data. In chapter 7 the performance of a new biphasic calcium phosphate (BCP 20/80) Was evaluated In contrast to previous bone biopsy evaluating studies, in which parameters were averaged over the whole biopsy or over two regions only (native bone and graft area) 11, 12, our refined techniques resulted in a better and more detailed insight in the processes involved in 165

Chapter 8

bone (re)generation in MSFE. Although not significant, there was a clear trend in both the Âľ-CT and the histomorphometrical analyses towards more bone ingrowth in the 20/80 versus the 60/40 BCP variant. Osteoid volumes were comparable between both groups, while osteoclastic activity was significantly higher in the 60/40 group, indicating more balance towards bone formation in the BCP 20/80-treated patients. The authors concluded that the novel BCP 20/80 scaffold in MSFE performs at least equal, but most likely better in bone augmentation when compared to the BCP 6/40 standard.

SUMMARY AND PERSPECTIVE In summary, the authors proposed and performed preparatory studies to implement and optimize a novel bone tissue engineering approach for maxillary sinus floor elevation performed in patients with insufficient jaw bone height to allow dental implant placement. In this thesis, we provide strong indications that ASCs may contribute to the (re)generation of bone tissue after a MSFE procedure using bone substitutes, and that their application may well be performed in the one-step surgical concept postulated by our group. In fact, in a parallel project, a phase I clinical trial has been performed which confirmed this statement; it was found that the one-step surgical concept was feasible, safe, and that there was an additive effect of the stem cells (significant on some parameters; clear trend on other parameters) with respect to new bone formation when compared to BCP/TCP scaffolds alone. Our experimental data suggest that the efficacy of ASC-mediated bone formation may be increased by performing a short (15 min) BMP-2 stimulation of the freshly isolated ASCs prior to in vivo implantation. However, the latter approach needs further validation in in vivo bone formation studies, e.g. in a calvaria defect model and/or goat large bone defect models. We postulate that the inclusion of the BMP-stimulation step may be a next-generation approach to further increase adipose stem cell-mediated bone formation efficacy. The BMP-2 stimulation may also compensate for the likely suboptimal (too low) loading mechanical loading conditions in our current MSFE model. This aspect of non-optimal loading condition, which likely results in an underestimation of the full potential of the ASCsupplementation since mechanical stimuli are an important stimulatory factor for ASC bone formation-promoting actions, should be given more attention. In this respect, an earlier dental implant placement than the currently used 6-month time frame may have beneficial effects since it is expected that the dental implants will transduce the loading much more efficiently to the bone augmentation area. The authors conclude that the use of freshly isolated ASCs in a one-step surgical procedure is a feasible and innovative cellular basis for bone tissue engineering, and that the MSFE model is well-suited for monitoring the ingrowth of new bone and understanding the mechanisms behind the bone remodeling process. The outcome of these and future studies will have pivotal implications for bone tissue engineering models in other fields such as orthopaedic surgery; predictions can be made for outcomes regarding spinal fusion surgeries and knee 166

General discussion

defect reconstructions 13. Also for craniofacial surgeries the MFSE model can be useful; for frontal sinus reconstructions, cranioplastic reconstructions, mandibular reconstructions and nasal septum corrections, the process of culturing ASCs will not be necessary and the outcome can be predicted without taking biopsies after surgery 14. Still additional clinical research needs to be done to evaluate the outcome of a one-step surgical model in larger defects, as well as defects that are not completely surrounded by a natural bone cavity.

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REFERENCES 1. 2.

4. 5.

8. 9. 10. 11.

12. 13.

14.

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Helder MN, Knippenberg M, Klein-Nulend J, Wuisman PI. Stem cells from adipose tissue allow challenging new concepts for regenerative medicine. Tissue engineering. 2007;13(8):1799-808. Carragee EJ, Hurwitz EL, Weiner BK. A critical review of recombinant human bone morphogenetic protein-2 trials in spinal surgery: emerging safety concerns and lessons learned. The spine journal : official journal of the North American Spine Society. 2011;11(6):471-91. Zuk P, Chou YF, Mussano F, Benhaim P, Wu BM. Adipose-derived stem cells and BMP2: part 2. BMP2 may not influence the osteogenic fate of human adipose-derived stem cells. Connective tissue research. 2011;52(2):119-32. Otto WR, Wright NA. Mesenchymal stem cells: from experiment to clinic. Fibrogenesis & tissue repair. 2011;4:20. Mirza A, Hyvelin JM, Rochefort GY, Lermusiaux P, Antier D, Awede B, et al. Undifferentiated mesenchymal stem cells seeded on a vascular prosthesis contribute to the restoration of a physiologic vascular wall. Journal of vascular surgery. 2008;47(6):1313-21. Elman JS, Li M, Wang F, Gimble JM, Parekkadan B. A comparison of adipose and bone marrowderived mesenchymal stromal cell secreted factors in the treatment of systemic inflammation. Journal of inflammation (London, England). 2014;11(1):1. Akram KM, Samad S, Spiteri MA, Forsyth NR. Mesenchymal stem cells promote alveolar epithelial cell wound repair in vitro through distinct migratory and paracrine mechanisms. Respiratory research. 2013;14:9. Frazier TP, Gimble JM, Kheterpal I, Rowan BG. Impact of low oxygen on the secretome of human adipose-derived stromal/stem cell primary cultures. Biochimie. 2013;95(12):2286-96. Teven CM, Liu X, Hu N, Tang N, Kim SH, Huang E, et al. Epigenetic regulation of mesenchymal stem cells: a focus on osteogenic and adipogenic differentiation. Stem cells international. 2011;2011:201371. Patel DM, Shah J, Srivastava AS. Therapeutic potential of mesenchymal stem cells in regenerative medicine. Stem cells international. 2013;2013:496218. Tadjoedin ES, de Lange GL, Bronckers AL, Lyaruu DM, Burger EH. Deproteinized cancellous bovine bone (Bio-Oss) as bone substitute for sinus floor elevation. A retrospective, histomorphometrical study of five cases. Journal of clinical periodontology. 2003;30(3):261-70. Xavier SP, Dias RR, Sehn FP, Kahn A, Chaushu L, Chaushu G. Maxillary sinus grafting with autograft vs. fresh frozen allograft: a split-mouth histomorphometric study. Clinical oral implants research. 2014. Jurgens WJ, Kroeze RJ, Zandieh-Doulabi B, van Dijk A, Renders GA, Smit TH, et al. One-step surgical procedure for the treatment of osteochondral defects with adipose-derived stem cells in a caprine knee defect: a pilot study. BioResearch open access. 2013;2(4):315-25. Sandor GK, Numminen J, Wolff J, Thesleff T, Miettinen A, Tuovinen VJ, et al. Adipose stem cells used to reconstruct 13 cases with cranio-maxillofacial hard-tissue defects. Stem cells translational medicine. 2014;3(4):530-40.â&#x20AC;&#x192;

GENERAL SUMMARY ADIPOSE STEM CELLS FOR BONE TISSUE ENGINEERING IN A HUMAN MAXILLARY SINUS FLOOR ELEVATION MODEL: STUDIES TOWARDS CLINICAL APPLICATION

GENERAL SUMMARY The increasingly ageing population worldwide has raised the prevalence of jaw atrophy and thus the need for a sufficient treatment. Maxillary atrophy is caused by a prolonged edentulous state after loss of natural teeth. To restore the missing maxillary teeth with prosthetic devices, dental implants can be installed if the alveolar bone volume is sufficient. If this requirement is not met, the maxillary alveolar bone height can be increased by means of a specific surgical procedure, i.e. the maxillary sinus floor elevation procedure (MSFE). For decades MSFE has been a standard pre-implant surgical procedure to increase alveolar bone height in the posterior maxilla. Autologous bone is still considered the golden standard as graft material for bone augmentation procedures in general and for MSFE in particular, since the graft contains osteoblasts and osteoprogenitor cells, thereby providing osteoconductive and osteoinductive properties necessary for allowing the migration and subsequent differentiation of progenitor cells. Harvesting autologous bone (from the anterior iliac crest or mandible) has several disadvantages, such as donor site morbidity and limited availability of bone. Alternatives for the use of autologous bone have been developed and evaluated. This resulted in the introduction and use of bone substitutes such as allografts, xenografts and purely synthetic grafting materials that are accepted and now commonly used for standard clinical dental and oral surgery procedures. Synthetic bone substitutes such as β-tricalcium phosphate (β-TCP, e.g. Ceros®), hydroxyapatite (HA), and biphasic calcium phosphate (BCP, mixtures of HA/βTCP, e.g. Straumann BoneCeramic®) are interesting alternatives to use in MSFE because they are available in unlimited quantity, have an infinite half-life and may therefore be used as offthe-shelf products. Using synthetic grafting materials eliminates the need for a second surgical procedure as well as potential additional complications. Growth factors and/or osteoblast precursor cells are required to provide the osteoinductive potential of the tissue-engineering construct. One specific growth factor is bone morphogenetic protein-2 (BMP-2), which is a potent osteoinductive molecule that has been shown to stimulate osteogenic differentiation of undifferentiated cells. An in vitro study with goat adipose stem cells (ASCs) showed that the use of BMP-2 in a physiological, nanogram-range concentration and a short period of time can be very beneficial for tissue engineering purposes using ASCs. In this thesis progenitor cells were used isolated from human adipose tissue. This tissue provides an easily accessible, expendable source of clinically relevant numbers of mesenchymal stem cells, thereby allowing innovative one-step regenerative treatment strategies. This new concept overcomes the problems currently encountered with cellular therapies: need for in vitro expansion, high costs, and repeated surgeries. Moreover, when using non-induced, minimally manipulated cells, many regulatory hurdles can be avoided, thereby accelerating clinical introduction. Within the concept of bone tissue engineering, the maxillary sinus floor elevation model is unique by allowing histological examination of biopsies that have been retrieved prior to implant insertion. Therefore, this thesis evaluates and describes the functionality of human ASCs 172

General summary

for bone tissue engineering and proposes a novel concept of a one-step surgical procedure for human maxillary sinus floor elevation using calcium phosphate scaffolds. In chapter 2 the authors reviewed whether the human MSFE procedure could be applied as a model for bone regeneration enabling the application of one-step surgical procedures. It was concluded that the use of freshly isolated ASCs in a one-step surgical procedure is a feasible and innovative cellular basis for bone tissue engineering, and that the MSFE model is well-suited for monitoring the ingrowth of new bone and understanding the mechanisms behind the bone remodeling process. In chapter 3 the effect of short treatment of human ASCs with BMP-2 after seeding on a calcium phosphate carrier was evaluated. It was investigated whether short (15 minutes) incubation with BMP-2 suffices to trigger osteogenic differentiation of hASCs seeded on calcium phosphate carriers. The authors found that hASC attachment to the different scaffolds was similar, and unaffected by BMP-2. BMP-2 stimulated gene expression of the osteogenic markers CBFA1, collagen-1, osteonectin, and osteocalcin in hASCs seeded on BCP and ß-TCP. Down regulation of osteopontin expression by BMP-2 was seen in BCP-seeded cells only. BMP2 treatment inhibited expression of the adipogenic marker PPAR-γ. Thus, 15 minutes BMP-2 pre-incubation of hASCs seeded on BCP/ß-TCP scaffolds had a long-lasting stimulating effect on osteogenic differentiation in vitro. The secretome of stem cells strongly determines the outcome of tissue engineering strategies. It was investigated how the secretome of human adipose stem cells (hASCs) is affected by substrate, BMP-2 treatment, and degree of differentiation. Therefore, in chapter 4 it was hypothesized that as differentiation progresses, hASCs produce increasingly more factors associated with processes such as angiogenesis and bone remodeling. The authors found that, compared to plastic, hASCs cultured on BCP showed ≥ 2-fold higher expression of ~20 factors, amongst which cytokines such as IL-6, growth factors such as FGF7 and adhesion molecules such as VCAM1. However, expression of another ~50 genes was decreased ≥ 2-fold on BCP compared to plastic, even though hASCs differentiate better on BCP than on plastic. BMP-2treatment increased the expression of ~30 factors by hASCs seeded on BCP, while it decreased the expression of only PGF, PPARG and PTN. No clear association between the degree of osteogenic differentiation of hASCs and the pattern of trophic factor production was observed. Considering the observed lack of association between the degree of differentiation and the production of factors associated with angiogenesis and bone remodeling by hASCs, future bone regeneration studies should focus more on systematically orchestrating the secretome of stem cells, rather than on inducing osteogenic differentiation of stem cells only. Short incubation with BMP-2 may be a promising treatment to enhance both osteogenic differentiation and environmental modulation. In chapter 5 the gain of mineralized bone in human biopsies was compared between deproteinized bovine bone allograft (DBA) and BCP after MSFE, using a split-mouth design. The authors found that patients were prosthetically successfully restored. All but one of the implants survived, and peri-implant mucosa showed healthy appearance and stability. Bone volume, graft volume, degree of bone mineralization, and osteoclast and osteocyte numbers were similar, but BCP-grafted biopsies had relatively more osteoid than DBA-grafted biopsies. 173

The authors concluded that the BCP and DBA materials showed similar osteoconductive patterns and mineralized bone, although signs of more active bone formation and remodeling were observed in BCP- than in DBA-grafted biopsies. In chapter 6 the effect of a collagenous barrier membrane covering the lateral window in MSFE procedures on the bone formation in β-TCP was described. For decades this has been an important topic of debate, since the benefit of a collagenous barrier membrane has not been irrefutably proven. The authors found that comparable outcomes between the groups with and without a membrane were observed regarding osteoconduction rate, bone and graft volume, osteoclast number, and structural parameters of newly formed bone per region of interest. However, osteoid volume in grafted maxillary sinus floors without a membrane was significantly higher than with membrane. In conclusion, these findings demonstrate that the clinical application of a bioresorbable collagenous barrier membrane covering the lateral window in MSFE procedures using β-TCP was not beneficial for bone regeneration, and even decreased osteoid production, which might lead to diminished bone formation in the long run. Further research needs to be done to confirm these data. Despite the successful use of biphasic calcium phosphate with a hydroxyapatite/ tricalcium phosphate (HA/TCP) ratio of 60/40, the high percentage of HA may hamper efficient scaffold remodeling. Therefore, in chapter 7, it was hypothesized that the use of BCP 20/80 in a MSFE procedure will result in a higher quantity of bone and/or better bone quality in the grafted maxillary sinus compared to BCP 60/40. A comparative study between these two types of calcium phosphate bone substitutes has not previously been performed in a human model. The authors found that, although not significant, there is a clear tendency in the µ-CT and the histomorphometrical analysis towards more bone ingrowth in the 20/80 versus the 60/40 BCP variant. Osteoid volumes were comparable between both groups, while osteoclastic activity was significantly higher in the 60/40 group, indicating more balance towards bone formation in the BCP 20/80-treated patients. The authors concluded that the novel BCP 20/80 bone substitute performs at least equal, but most likely better in MSFE procedures compared to the BCP 60/40. Finally, in chapter 8 the main conclusions of the studies described in this thesis are discussed as well as the future implications of the findings. In short, the authors concluded that the use of freshly isolated and BMP-2-pretreated human ASCs in a one-step surgical procedure is a feasible and innovative cellular basis for bone tissue engineering, and that the MSFE model is well-suited for monitoring the ingrowth of new bone and understanding the mechanisms behind the bone remodeling process. The outcome of these and future studies will have pivotal implications for bone tissue engineering models in other fields, such as orthopedic surgery.

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ALGEMENE SAMENVATTING HUMANE VETSTAMCELLEN VOOR BOTREGENERATIE IN EEN SINUSBODEMELEVATIE MODEL: STUDIES VOOR KLINISCHE TOEPASSING

ALGEMENE SAMENVATTING Met de wereldwijde vergrijzing nemen ook de prevalentie van kaakatrofie en de daarmee geassocieerde behandelingen toe. Na verlies van eigen tanden en/of kiezen treedt doorgaans atrofie van de kaak op. In het kader van de prothetische vervanging van gebitselementen kunnen tandwortelimplantaten worden geplaatst , op voorwaarde dat het kaakbotvolume voldoende is. Indien dit laatste niet het geval is, kan onder meer in de zijdelingse delen van de bovenkaak het bot worden opgehoogd met een specifieke chirurgische procedure, namelijk de sinusbodemelevatie (SBE). Tot op heden wordt het gebruik van autoloog bot beschouwd als de gouden standaard tijdens botaugmentatieprocedures in het algemeen en de SBE in het bijzonder. Het autologe bottransplantaat bevat onder andere osteoblasten en osteogene voorlopercellen, en voorziet in osteoinductieve en osteoconductieve eigenschappen die noodzakelijk zijn voor de migratie en differentiatie van voorlopercellen. Het oogsten van autoloog bot uit de crista iliaca anterior of de mandibula heeft verschillende nadelen, zoals de met deze ingrepen gepaard gaande morbiditeit en de beperkte hoeveelheid beschikbaar bot. Daarom zijn er alternatieve materialen ontwikkeld en geëvalueerd, resulterend in de introductie en het gebruik van botsubstitutie materialen zoals allografts, xenografts, en zuiver synthetische materialen, die veelvuldig worden gebruikt tijdens tandheelkundige en kaakchirurgische ingrepen. Synthetische botsubstituten zoals β-tricalciumfosfaat (β-TCP, bijv. Ceros®), hydroxyapatiet (HA), en bi-fasisch calciumfosfaat (BCP, een combinatie van HA/β-TCP, bijv. Straumann BoneCeramic®) zijn interessante alternatieven om te gebruiken tijdens een SBE vanwege de ongelimiteerde beschikbaarheid en houdbaarheid, hetgeen ze zeer geschikt maakt als ‘off-the-shelf’ producten. Het gebruik van synthetische botsubstituten maakt de noodzaak tot additionele operaties en de daarmee geassocieerde mogelijke complicaties overbodig. Groeifactoren en/of osteoblast voorlopercellen zijn nodig voor het osteoinductieve karakter van het ‘tissue-engineering’ construct. Een specifieke groeifactor is ‘bone morphogenetic protein’ (BMP-2), een sterk osteoinductief molecuul, waarvan is bewezen dat het de osteogene differentiatie stimuleert van ongedifferentieerde cellen. Een in vitro studie waarin vetstamcellen van de geit werden toegepast liet zien dat de toepassing van BMP-2 in een fysiologische concentratie (nanogram-range), en gedurende een korte periode, zeer geschikt was voor ‘tissue-engineering’ doeleinden. In dit proefschrift werd gebruik gemaakt van voorlopercellen die werden geïsoleerd uit humaan vetweefsel. Dit weefsel is een eenvoudig toegankelijke en ongelimiteerde bron van de benodigde hoeveelheid mesenchymale cellen. Dit laatste maakt een innovatieve ‘onestep’-regeneratie strategie mogelijk. Dit nieuwe concept ondervangt de huidige problemen met celtherapieën zoals noodzakelijke celkweken, hoge kosten en meerdere chirurgische ingrepen. Met het gebruik van onbewerkte, minimaal gemanipuleerde cellen kunnen vele logistieke drempels worden omzeild, en kan daarmee de introductie in de kliniek worden versneld. Binnen het concept ’tissue engineering’ speelt de SBE een unieke rol, zodanig dat het mogelijk is om histologisch onderzoek te doen op de biopten die worden verkregen, voordat de tandwortelimplantaten worden geplaatst. In dit proefschrift wordt de toepassing van humane op 178

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vetstamcellen (hASCs) voor botregeneratie geëvalueerd en een nieuw concept geïntroduceerd, waarbij in een ‘one-step’ chirurgische procedure hASCs worden geoogst uit vetweefsel en worden uitgezaaid op een calciumfosfaatdrager (botsubstituut), waarna dit “bioactieve” materiaal tijdens een SBE wordt toegepast.. In hoofdstuk 2 wordt geconcludeerd dat het gebruik van hASCs tijdens een ‘onestep’ procedure haalbaar is, en een innovatieve basis vormt voor botweefselregeneratie. Tevens bleek het SBE model geschikt voor het meten van de ingroei van nieuw bot alsmede het verkrijgen van inzicht in de mechanismen van het regeneratieproces. In hoofdstuk 3 werd het effect van een korte behandeling met BMP-2 op hASCs onderzocht, nadat ze waren uitgezaaid en gekweekt op een calciumfosfaatdrager. Het bleek dat de aanhechting van hASCs aan verschillende botsubstituten gelijk was en niet werd beïnvloed door BMP-2. Daarnaast stimuleerde BMP-2 de genexpressie van de osteogene markers CBFA1, collagen-1, osteonectin, en osteocalcin in hASCs gekweekt op BCP en ß-TCP. Downregulatie van de osteopontine genexpressie door BMP-2 werd alleen gezien in cellen gekweekt op BCP. Behandeling met BMP-2 inhibeerde de expressie van de adipogene marker PPAR-γ. Het bleek dat een 15-minuten-durende stimulatie van BMP-2 in een lage dosis de hASCs in vitro minstens 21 dagen stimuleerde tot osteogene genexpressie en tot verminderde expressie van adipogene markers. Het secretoom van stamcellen heeft grote invloed op de resultaten van “tissue engineering” strategieën. Er werd onderzocht hoe het secretoom van hASCS wordt beïnvloed op verschillende substraten, door behandeling met BMP-2 en door de differentiatiegraad. Daarom werd in hoofdstuk 4 de hypothese geponeerd, dat gedurende differentiatie hASCs steeds meer factoren produceren die zijn betrokken bij de angiogenese en botremodelering. Er werd gevonden dat, ten opzichte van een plastic kweekbodem, hASCs die werden gekweekt op BCP, een meer dan 2-voudige expressie toonden van ongeveer 20 factoren, waaronder cytokinen zoals IL-6, groeifactoren zoals FGF7 en adhesiemoleculen zoals VCAM1. Echter, 50 genen werden meer dan 2-voudig verminderd tot expressie gebracht op BCP in vergelijking met plastic, ook al was er een betere osteogene expressie op BCP dan op plastic. Behandeling met BMP- 2 versterkte de expressie van 30 factoren door cellen gekweekt op BCP, terwijl het de expressie van PGF, PPARG en PTN verminderde. Er bleek geen duidelijk verband te zijn tussen de osteogene differentiatie van hASCs en het productiepatroon van trofische factoren. Omdat er geen associatie bleek te zijn tussen de differentiatiegraad en de productie van angiogeneseen botremodelering-gerelateerde factoren, wordt gesuggereerd dat toekomstige studies op het gebied van weefselregeneratie meer de focus zouden moeten leggen op het systematisch rangschikken van het secretoom van hASCs, en niet alleen maar op de differentiatie ervan. Een korte incubatie met BMP-2 kan een veelbelovende behandeling zijn om zowel de differentiatie als de directe invloed op de omgeving van de hASCs te versterken. In hoofdstuk 5 werd de hoeveelheid nieuw ontstaan botweefsel gemeten na SBE met gebruik van het eiwitarme boviene botgraft (DBA) vergeleken met een bifasisch calciumfosfaat (BCP) middels een ‘split-mouth design’. De gebitsrehabilitatie bleek bij alle patiënten naar tevredenheid te zijn verlopen. Eén van de geplaatste implantaten ging verloren. De periimplantaire weefsels toonden geen afwijkingen. Botvolume, graftvolume, mineralisatiegraad, en 179

aantal osteoclasten en osteocyten bleken gelijk te zijn, maar de BCP-biopten bleken meer osteoïd te bevatten dan de DBA–biopten. Het gebruik van BCP en DBA lieten dus een vergelijkbaar osteoconductief patroon en hoeveelheid gemineraliseerd bot zien, hoewel in de patiënten die waren behandeld met BCP er sprake leek te zijn van een verhoogde botremodellering. In hoofdstuk 6 werd het effect van een oplosbaar collageenmembraan op de botregeneratie beschreven, dat wordt gebruikt om de opening in de laterale wand van de kaakholte (“lateral window”) af te dekken tijdens een SBE . Het gebruik van het genoemde membraan is al langere tijd een onderwerp van discussie, omdat het nooit onomstotelijk is bewezen of het gebruik ervan voordelig is. Er werden vergelijkbare resultaten gevonden tussen de groepen met en zonder collageenmembraan wat betreft de snelheid van osteoconductie, bot- en graftvolume, aantal osteoclasten en de structurele parameters van nieuw gevormd bot per aandachtsgebied (“region of interest”). Echter de hoeveelheid osteoïd in de geopereerde kaakholte zonder membraan was significant hoger dan in de groep waar wel een membraan was gebruikt. Deze bevindingen laten zien dat de toepassing van een collageenmembraan over de opening in de laterale wand van de kaakholte tijdens een SBE met β-TCP niet bijdraagt aan de botregeneratie en zelfs de osteoïdvorming vermindert, hetgeen op lange termijn zou kunnen leiden tot verminderde botvorming. Ondanks het succesvolle klinische gebruik van bifasische calciumfosfaten (BCP) met een hydroxyapatiet/tricalciumfosfaat (HA/TCP) ratio van 60/40, zou het hoge percentage hydroxyapatiet het botremodeleringsproces mogelijk kunnen vertragen. Daarom werd in hoofdstuk 7 de hypothese geponeerd, dat het gebruik van BCP 20/80 tijdens een SBE uiteindelijk een grotere hoeveelheid bot, dan wel botweefsel van een beter kwaliteit oplevert ten opzichte van BCP 60/40. Een vergelijkende studie tussen deze twee typen botsubstituut was nog niet eerder in een humaan model uitgevoerd. De groep met BCP 20/80 bleek minstens even goede resultaten te laten zien als de groep met BCP 60/40. Ondanks het ontbreken van significantie is er een duidelijke tendens zichtbaar in de micro-computertomografie en histomorfometrische analyses, die duidt op een betere ingroei van bot in de ‘20/80’ groep. De osteoïdvolumes van beide groepen waren vergelijkbaar, terwijl in de ‘60/40’ groep de osteoclastenactiviteit significant hoger was; dit duidt op een betere balans in de botremodelering in de ‘20/80’ groep. Er werd geconcludeerd dat het nieuwe botsubstituut BCP 20/80 tenminste even goed, maar mogelijk zelfs beter presteert in vergelijking met BCP 60/40. Tenslotte, in het afsluitende hoofdstuk 8 worden de belangrijkste conclusies van de hierboven beschreven studies nader beschouwd, alsmede een uiteenzetting gegeven van de implicaties van de resultaten voor de toekomst. Samenvattend kan worden gesteld dat het gebruik van direct geïsoleerde en met BMP-2 voorbehandelde humane vetstamcellen (hASCs) binnen een ‘one-step’ procedure een haalbare en innovatieve basis vormt voor de regeneratie van botweefsel. Daarnaast is het SBE model zeer geschikt voor het meten van ingroei van nieuw gevormd bot en draagt het bij aan een beter begrip van het botremodeleringsproces. De resultaten van deze studie vormen een belangrijke basis voor toepassing van botweefselregeneratie technieken in andere vakgebieden, zoals de orthopedische chirurgie.

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DANKWOORD Met het verdedigen van dit proefschrift eindigt een fase waarin in veel heb geleerd over wetenschap, over geduld, en over het leven. Een fase die veel langer heeft geduurd dan ik had voorzien, maar zoals het gezegde ongeveer luidt: ‘het gaat niet om bestemming van de reis, maar om de reis naar de bestemming’ En dat is maar al te waar. Met de opgedane ervaringen tijdens deze reis zal ik sterker terugkomen in mijn carrière en de rest van mijn levensweg. Uiteraard kan ik niet anders dan even stilstaan om iedereen te bedanken die heeft bijgedragen aan de totstandkoming van dit proefschrift en/of aan mijn welzijn gedurende deze periode. Ik begin bij mijn promotoren, Prof.dr. Klein Nulend en Prof.dr. Schulten; Beste Jenneke, bedankt voor jouw enthousiasme en tomeloze inzet. Ik kan me de lange dagen nog herinneren die het hebben gekost om, met jouw kennis van de wetenschap, presentaties en manuscripten afgerond te krijgen. Jij er hebt altijd werk van gemaakt om letterlijk en figuurlijk de puntjes op de i te zetten; geen komma stond verkeerd en de lay-out van een manuscript was altijd netjes verzorgd. Niets was jou teveel in jouw doel om AIO’s op te leiden tot echte wetenschappers! Beste Bert, bedankt voor de goede samenwerking. Ik waardeer jouw betrokkenheid, jouw pragmatische instelling en jouw bereidheid om altijd kritische vragen te blijven stellen en zaken vanuit een klinisch oogpunt te belichten. Mijn co-promotoren, Dr. Helder en Prof.dr. ten Bruggenkate; Beste Marco, jouw kalmte, jouw nuchtere karakter en jouw perspectieven hebben mij zeker geholpen om door de bomen het bos te blijven zien, en de manuscripten naar een hoger niveau te tillen. Bedankt voor jouw inzet en de gezellige tijd in Quebec! Beste Chris, ondanks jouw drukke schema en werk in Leiderdorp heb je altijd tijd vrij weten te maken om net als Bert tijdens besprekingen met een kritische en klinische blik naar ons project te kijken. Zeer veel dank voor jouw inzet, enthousiasme en betrokkenheid. Een onderzoeksafdeling staat of valt met een goed geolied team van analisten en onderzoekers. Orale Celbiologie mag zich wat dat betreft zeker in de handjes wrijven! Beste Vincent, als hoofd van de afdeling wist jij feilloos de wetenschap te combineren met jouw scherpe humor. Bedankt voor jouw wetenschappelijke en kritische input in combinatie met de grote glimlach die ik van jou gewend ben. Beste Astrid, het is altijd een verademing om te zien met hoeveel plezier jij aan jouw projecten werkt. Daarom was ik ook erg blij om met jou samen te werken aan ons artikel over groeifactoren. Je bent vindingrijk en je probeert een idee altijd van alle kanten te belichten, ook al zijn zij ‘ out of the box’. Bedankt voor al jouw hulp en voor de gezelligheid tijdens congressen, borrels en meetings. Beste Gert, Clara en Elisabet, jullie bijdrage aan het ‘Geistlich’ artikel was werkelijk onmisbaar! Zeer veel dank voor jullie hulp en inzet. Henk-Jan, bedankt voor de prettige samenwerking gedurende de periode dat jij op OCB werkte; jouw energieke aanwezigheid en je inzet hebben hun vruchten afgeworpen. 184

Dankwoord

Jolanda, zoals altijd duidelijk aanwezig dankzij jouw vrolijke persoonlijkheid in stereo! En altijd erg punctueel met het bijhouden van de verjaardagskalender, want stel je voor dat er onnodig een dag zonder traktatie voorbijgaat....bedankt voor jou hulp op het lab en voor jouw bijdrage als onderdeel van het ‘stamcelteam’. Cor, jij hebt mij aan het begin van mijn contract wegwijs gemaakt in het lab en de belangrijkste praktische zaken bijgebracht. Ik waardeer jouw vrolijkheid en opgeruimde karakter en jouw bereidheid om altijd een handje te helpen. Marion, jij bent het ultieme bewijs dat stille wateren een onverwacht kurkdroge humor kunnen hebben! Ik ben ontzettend blij met jouw bijdragen aan de histomorfometrische analyses, jouw werk was zoals altijd uitstekend. Dirk-Jan, bedankt voor jouw hulp bij het uitboren van de bovenkaaksbiopten en het verzorgen van de bestellingen van alle spullen die ik nodig had. Behrouz en Ton S, bedankt voor jullie hulp met de PCR apparatuur en de genexpressie analyses. Ton B, Teun en Don, bedankt voor jullie onderwijs, alle hulp op het lab en de gezellige gesprekken. Mardi en Bastiaan en Britt, ook jullie bedankt voor jullie bijdrage aan de publicaties tijdens jullie studie. Ook buiten de afdeling Orale Celbiologie ben ik mensen mijn dank verschuldigd. De microCT analyses had ik niet kunnen uitvoeren zonder medewerking van de afdeling Functionele Anatomie; Peter, Hans, Geerling en Leo, bedankt voor jullie bijdrage, geduld en gezelligheid. Ook medewerkers van de afdelingen Orthopedie en Plastische Chirurgie van het VU Medisch Centrum hebben mij geholpen tijdens de vetstamcel isolaties en de aanlevering van de stamcellen voor mijn experimenten. Robert-Jan, Wouter en Benno, bedankt. Vanuit de kliniek trad ik binnen in de wereld van het ‘AIO-bestaan’. Een wereld van verschil, waarin ik mij vaak als een vis in het verkeerde water voelde. Gelukkig had ik een verzameling van prettige mede-AIO’s om mij heen die mij wegwijs maakten in deze tijdelijke bestaansvorm. De pieken waren vrolijker en de dalen minder ondraaglijk, en dat zal ik nooit vergeten. Marjoleine, jij was de eerste die mij wegwijs maakte over het reilen en zeilen op OCB. Bedankt voor de gezellige tijden en ik waardeer het dat jij mijn paranimf bent. Ook Petra, Nina, Greetje, Heinze, Anna, Sreeda, Janak, Alejandra, Jenny, Rishikesh, Ana Paula, Veerle, en alle andere AIO’s die ik heb verzuimd te noemen, bedankt voor al die gezellige lunches, etentjes, feestjes, dagjes uit, PhTea’s, tennis partijtjes, besprekingen, congressen en borrels gevuld met lachbuien, vrijdagmiddag-humor, af en toe een traan, zang, dans, dubbele regenbogen, Duitse techno muziek, filosofische momenten en een speeddate. De dames en heren van het Promovendi Overleg verdienen eveneens lof voor hun enthousiasme en inzet tijdens mijn jaren als algemeen lid en voorzitter. Mijn ‘her-intrede’ in de kliniek vond plaats in Amersfoort. Collega’s van de afdeling Cardiologie van het Meander Medisch Centrum, ik heb het afgelopen jaar bij jullie veel geleerd en genoten van de gezellige momenten. Zeer veel dank en misschien tot ziens!

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Als laatste, maar zeker niet in het minste, wil ik mijn dank uitspreken naar mijn familie en vrienden. Ik ben niet iemand die het hart op de tong draagt, juist daarom wil ik benadrukken hoeveel steun ik aan jullie heb gehad, ook al hadden jullie het niet altijd door. Ik bedank mijn beide ouders, en vooral mijn moeder; yu gi mi alla fu yu srèfi dat mèki mi na mi. Fu dati mi è taigi yu Gran tangi. U heeft de basis gelegd voor alles wat ik heb willen doen en dat zal ik nooit vergeten. Mijn broer en zus, Gwen en Leroy, ik beschouw ons drieën als de ‘musketiers’. Wij delen een taal en een humor die alleen wij begrijpen en dat zal altijd zo blijven. Gwen, jij bent een ontzettende inspiratie voor mij. Ik heb de afgelopen jaren gezien hoe jij jouw ongekende creatieve talenten steeds meer tot expressie laat komen in jouw kunstwerken. Ik ben trots op jou! Leroy, mijn paranimf, zelden hebben wij een gesprek waarin jij mij niet plaagt of mij ervan probeer te overtuigen dat jouw mening eigenlijk gewoon de waarheid is. Het is jouw manier is om te zeggen dat je mij toch best wel aardig vindt. Jij bent een coole kerel en ik had mij geen betere broer kunnen wensen. Beiden bedankt voor jullie steun en relativerende gesprekken wanneer ik het nodig had.  Oom Humphrey, u bent als een tweede vader voor mij. Ik waardeer uw onvoorwaardelijke steun, de vele muzikale intermezzo’s en uw open hart. Gran tangi.  Vincent Pino, your guitar lessons have ignited the spark to explore my creative mind once again. I cannot thank you enough for your inspiration and patience.  My friends from overseas; Raymond, Deidre and Indu, thank you for your outstanding hospitality. I had a great time in Virginia! Hopefully we meet again sometime.  Beryl en Ona, jullie raken geïnspireerd door positiviteit en daarom zijn jullie een inspiratie voor mij. Bedankt voor de gezelligheid en de goede gesprekken tijdens het hardlopen en de overige uitjes. Laura, bedankt voor jouw adviezen, de gezellige uitjes in Rotterdam en de leuke reisverhalen over Suriname en andere landen! Aan alle overige familieleden en vrienden die ik niet heb genoemd, maar die wel veel voor mij betekenen, heel erg bedankt voor jullie liefde en steun.

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CURRICULUM VITAE

CURRICULUM VITAE Personal data Last name Overman Given names Janice Ruth Date and place of birth January 27, 1979, Rotterdam, NL Nationality Dutch Email jroverman79@yahoo.com Scientific education 2000-2007 Medicine at Erasmus University Rotterdam, NL. Graduate research on Multiple Organ Failure at department of Intensive Care and Traumatology, Erasmus University Rotterdam, NL. Internship at department of General Surgery, Diakonessenhuis Paramaribo, Surinam, South America. 2007 MD degree 1998-2000

Pharmacy at University of Utrecht, NL. Bachelorâ&#x20AC;&#x2122;s degree.

1991-1997

Erasmiaans Gymnasium, Rotterdam, NL

Professional experience 2008-2012: PhD student at department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, NL, in collaboration with department of Oral and Maxillofacial Surgery, VU University Medical Center/ ACTA, Amsterdam, NL. Title thesis Adipose Stem Cells for Bone Tissue Engineering in a Human Maxillary Sinus Floor Elevation Model: studies towards clinical applications. 2008

Ward doctor at departments of Internal Medicine, Nephrology, Pulmonology, and Orthopedics, Vlietland Hospital, Schiedam, NL.

2007-2008 Ward doctor at department of Neurosurgery, Slotervaart Hospital, Amsterdam, NL. 2007 Ward doctor at department of Cardiology, IJsselland Hospital, Capelle a/d Ijssel, NL.

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PhD courses • Course Regenerative Medicine Module II, Dec 6-9, 2011, Rotterdam, NL. • Course Successful Grant Writing, March 1 and 8, 2011, Amsterdam, NL. • Course ‘Dentistry for non-dentists’, Oct 6-8, 2010, Amsterdam, NL. • Scientific writing in English for publication, Feb 4 - Apr 15, 2010, Amsterdam, NL. • Course Oral Biology, Nov 15 - Dec 3, 2009, Amsterdam, NL. • Laboratory Animal Science, June 8-19, 2009, Amsterdam, NL. • Course Statistics, Dec 11, 2008 - Feb 19, 2009, Amsterdam, NL. Memberships professional societies 2008-2012 Dutch Society for Calcium and Bone Metabolism. 2008-2012 Netherlands Society for Biomaterials and Tissue Engineering. Scientific meetings 2012 Overman JR, Farré-Guasch E, Helder MN, ten Bruggenkate CM, Schulten EAJM, Klein-Nulend J. Short (15 minutes) treatment stimulates osteogenic differentiation of human adipose stem cells seeded on calcium phosphate scaffolds in vitro. 34th American Society for Bone and Mineral Research 2012 Annual Meeting, Minneapolis, MN, USA, October 12-15, 2012 (poster presentation). Overman JR, Farré-Guasch E, Helder MN, ten Bruggenkate CM, Schulten EAJM, Klein-Nulend J. Short (15 minutes) treatment stimulates osteogenic differentiation of human adipose stem cells seeded on calcium phosphate scaffolds in vitro. 10th Anniversary Meeting of the International Federation for Adipose Therapeutics and Science, Quebec-City, Canada, October 5-7, 2012 (oral presentation). Overman JR, Farré-Guasch E, Helder MN, ten Bruggenkate CM, Schulten EAJM, Klein-Nulend J. Short (15 minutes) treatment stimulates osteogenic differentiation of human adipose stem cells seeded on calcium phosphate scaffolds in vitro. 20th Annual Meeting of the European Orthopedic Research Society, Amsterdam, NL, September 26-28, 2012 (oral presentation). Overman JR, Farré-Guasch E, Helder MN, ten Bruggenkate CM, Schulten EAJM, Klein-Nulend J. Osteogenic differentiation of BMP-2 induced human adipose stem cells seeded on biphasic calcium phosphate and β-tricalcium phosphate scaffolds. IOT Dental Research Meeting 2012, Lunteren, NL, February 2-3, 2012 (oral presentation).

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2011 Overman JR, Helder MN, ten Bruggenkate CM, Schulten EAJM, Klein-Nulend J: Osteogenic differentiation of BMP-2-induced human adipose stem cells seeded on biphasic calcium phosphate and β-tricalcium phosphate scaffolds. 21th Annual Meeting of the Dutch Society for Calcium and Bone Metabolism, Zeist, NL, November 10-11, 2011 (oral presentation). Overman JR, Prins H, Helder MN, ten Bruggenkate CM, Schulten EAJM, Klein-Nulend J: Bone augmentation with adipose stem cells and calcium phosphate carriers for human maxillary sinus floor elevation: an ongoing phase I clinical trial. 3th Annual MOVE Research meeting, Amsterdam, NL, September 28, 2011 (oral presentation). Overman JR, Helder MN, ten Bruggenkate CM, Schulten EAJM, Klein-Nulend J: Osteogenic differentiation of BMP-2-induced human adipose stem cells seeded on biphasic calcium phosphate and β–tricalcium phosphate carriers. 5th VUMC Day of Science and Technology, Amsterdam, NL, March 11, 2011 (poster presentation). 2010 Overman JR, Helder MN, ten Bruggenkate CM, Schulten EAJM, Klein-Nulend J: Osteogenic gene expression of BMP-2-induced human adipose stem cells seeded on biphasic calcium phosphate carriers. 19th Annual Meeting of the Netherlands Society for Biomaterials and Tissue Engineering, Lunteren, The Netherlands, December 2-3, 2010 (oral presentation). Overman JR, Helder MN, ten Bruggenkate CM, Schulten EAJM, Klein-Nulend J: Osteogenic gene expression of BMP-2-induced human adipose stem cells seeded on biphasic calcium phosphate carriers. 20th Annual Meeting of the Dutch Society for Calcium and Bone Metabolism, Zeist, The Netherlands, November 11-12, 2010 (oral presentation). Overman JR, Helder MN, ten Bruggenkate CM, Schulten EAJM, Klein-Nulend J: Osteogenic differentiation of BMP-2-induced human adipose stem cells seeded on biphasic calcium phosphate and β–tricalcium phosphate carriers. 2nd MOVE Annual Research Meeting, Amsterdam, NL, October 28, 2010 (poster presentation). Overman JR, Helder MN, ten Bruggenkate CM, Schulten EAJM, Klein-Nulend J: Osteogenic differentiation of BMP-2-induced human adipose stem cells seeded on biphasic calcium phosphate and β–tricalcium phosphate carriers. Gordon Research Conference on Musculoskeletal Biology & Bioengineering, Andover, NH, USA, August 1-6, 2010 (poster presentation).

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2009 Overman JR, Helder MN, ten Bruggenkate CM, Schulten EAJM, Klein-Nulend J: Osteogenic differentiation of BMP-2-induced human adipose stem cells seeded on biphasic calcium phosphate and β–tricalcium phosphate carriers. 18th Annual Meeting of the Netherlands Society for Biomaterials and Tissue Engineering, Lunteren, NL, December 14-15, 2009 (oral presentation). Overman JR, Helder MN, ten Bruggenkate CM, Schulten EAJM, Klein-Nulend J: Osteogenic differentiation of BMP-2-induced human adipose stem cells seeded on biphasic calcium phosphate and β–tricalcium phosphate carriers. 19th Annual Meeting of the Dutch Society for Calcium and Bone Metabolism, Zeist, NL, November 12-13, 2009 (oral presentation).

Publications Overman JR, Farré-Guasch E, Helder MN, ten Bruggenkate CM, Schulten EAJM, Klein-Nulend J. Short (15 minutes) Treatment Stimulates Osteogenic Differentiation of Human Adipose Stem Cells Seeded on Calcium Phosphate Scaffolds in Vitro. Tissue Engineering Part A, 2013; 19:571-581. Farré-Guasch E, Prins H, Overman JR, ten Bruggenkate CM, Schulten EAJM, Helder MN, KleinNulend J. Human Maxillary Sinus Floor Elevation as a Model for Bone Regeneration Enabling the Application of One-Step Surgical Procedures. Tissue Engineering Part B, 2012; 19:69-82. Schulten EAJM, Prins H-J, Overman JR, Helder MN, ten Bruggenkate CM, Klein-Nulend J. A Novel Approach Revealing the Effect of a Collagenous Membrane on Osteoconduction in Maxillary sinus Floor Elevation with β-tricalcium Phosphate. European Cells & Materials, 2013; 25:215-228. Overman JR, de Lange GL, Farré-Guasch E, Korstjens CM, van Ruijven LJ, van Duijn MA, Hartman RS, Klein-Nulend J. A Histomorphometrical and Micro-CT Study of Bone Regeneration in the Maxillary Sinus Comparing Biphasic Calcium Phosphate and Deproteinized Cancellous Bovine Bone in a Human Split-mouth Model. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, 2014; 117:8-22. Overman JR, Helder MN, ten Bruggenkate CM, Schulten EAJM, Klein-Nulend J, Bakker AD. Growth Factor Gene Expression Profiles of Bone Morphogenetic Protein-2-Treated Human Adipose Stem Cells Seeded on Calcium Phosphate Scaffolds In Vitro. BIOCHIMIE, Special Secretome Issue, 2013; 95:2304-2313.

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Extracurricular activities 2009-2012 Chairman of ‘Promovendi Overleg ACTA’ (ActaPro) 2005-2006

Continuation of graduate research on Multiple Organ Failure at Erasmus Medical Center, Rotterdam, NL.

2002-2006

Nursing assistant at department of Internal Medicine, Erasmus Medical Center, Rotterdam, NL.

2004-2005

Assistant in the Clinical Skills Class at department of General Practitioner Medicine, Erasmus University, Rotterdam, NL.

2000-2004 Editorial member of faculty magazine O’dokter, Erasmus University Rotterdam, NL. Committee member of the annual ‘Parent’s Day’, Erasmus University Rotterdam, NL. General member of the Television Channel Committee at Haringvliet Student Housing Complex, Rotterdam, NL.

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