Journal of Osseointegration

jo j o u r n a l o f osseointegration

issn 2036-4121 MARch 2015 issue 1 vol. 7 www.journalofosseointegration.eu

A. Pozzuoli1*, C. Gardin2*, R. Aldegheri1, E. Bressan3, M. Isola4, J. L. Calvo-Guirado5, C. Biz1, P. Arrigoni1, L. Feroni2, B. Zavan2 1

Department of Surgical, Oncological and Gastroenterological Sciences, University of Padua, Padua, Italy Department of Biomedical Sciences, University of Padua, Padua, Italy 3 Department of Neurosciences, University of Padua, Padua, Italy 4 Department of Animal Medicine, Production and Health (MAPS) 5 Department of General Dentistry, Faculty of Medicine and Dentistry, University of Murcia, Murcia, Spain * These authors contributed equally to this work 2

Genetical stability and osteogenic ability of mesenchimal stem cells on demineralized bone matrices to cite this article Pozzuoli A, Gardin C, Aldegheri R, Bressan E, Isola M, Calvo-Guirado JL, Feroni L, Zavan B. Genetical stability and osteogenic ability of mesenchimal stem cells on demineralized bone matrices. J Osseointegr 2015;7(1):2-7.

ABSTRACT Aim Tissue engineering is a rapidly expanding field with regard to the use of biomaterials and stem cells in the orthopedic surgery.

Many experimental studies have been done to understand the best characteristics of cells, materials and laboratory methods for safe clinical applications. The aim of this study was to compare the ability of 2 different human demineralized bone matrices (DBMs), the one enriched and the other not enriched with hyaluronic acid, to stimulate in vitro the proliferation and the osteogenic differentiation of human adipose-derived stem cells (ADSCs) seeded onto an osteoconductive scaffold.

Materials and Methods ADSCs were isolated, by enzymatic digestion, from abdominal adipose tissue of 5 patients undergoing cosmetic lipoaspiration surgery. ADSCs were then seeded onto a 3D scaffold in the presence of the two different osteoinductive matrices of human demineralized bone and evaluated for proliferation and osteogenic differentiation. The safety of the methods was verified using array-Comparative Genomic Hybridization (array-CGH).

Results ADSCs were able to differentiate in osteogenic sense.

Both DBMs showed the ability to induce osteogenic differentiation of the cells.

Conclusion array-CGH showed no changes at genome level, thus confirming the safety of materials and methods.

Keywords Adipose-derived stem cells; Array-CGH; Demineralized bone matrices; Osteogenesis; Tissue engineering.

INTRODUCTION Bone regeneration is a multifactorial process in which osteoconduction, osteoinduction, and osteogenesis play

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a key role (1). Osteoconduction refers to 3D materials (scaffolds), which favour adhesion, migration and proliferation of the cells able to directly synthesize new bone (osteogenesis) (2). Osteoinductive factors, mainly bone morphogenetic proteins (BMPs), growth factors (GFs) and demineralized bone matrices (DBMs), guarantee the recruitment of osteoprogenitor cells and the differentiation of stem cells in osteoblasts. Autologous bone graft is considered the gold standard for bone repair and regeneration because of its osteogenic, osteoinductive, and osteoconductive properties related to the presence of cells from bone and bone marrow, proteins in the matrix, and a 3D structure that provides a viable scaffold for the growth of the surrounding bone (3, 4). However, the use of autologous bone graft presents several limits, mainly dependent on the low amount of harvested bone, increase in operative time, pain, infection, hematoma, and blood loss (5-11).

A recent approach able to stimulate the regeneration of fully functioning bone tissue is based on the use of mesenchymal stem cells (MSCs) derived from adipose tissue cultured on an osteoconductive scaffold in presence of DBMs (12, 13).

Ceramics, mainly hydroxyapatite (HA), are widely used as scaffolds due to their physical and chemical similarity to the mineralized cancellous bone which guarantees the osteoconductivity (14). HA has a 3D porous structure determined by a network of crystalline mineral delimiting numerous interconnected pores of various sizes. Pores smaller than 10 Âľm allow the movement of extracellular fluids, while the ones larger than 50 Âľm support the cells growth and proliferation (15-18).

Ceramics are considered an excellent vehicle for osteoinductive GFs and osteogenic cells, since they are biocompatible, not toxic, and they do not cause immunological responses (19).

DBMs are produced by removal of minerals from cadaver cortical bone and are considered allografts (20). They consist of collagen (mainly type I but also type IV and type X), non-collagenous proteins, a minimum percentage of Ca3(PO4)2 (1-6%), and importantly BMPs and GFs essential

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for bone growth and regeneration. The use of DBMs has some advantages, such as absence of rejection and foreign body reaction because antigenic surface structure of bone is destroyed during demineralization. DBMs are commercially available in various forms, including wafers, modeling paste, adhesive strips, and injectable compounds.

Moreover, they can be molded even intraoperatively, they are intraoperative washing resistant, and they allow rapid vascularization after application. Problems related to the use of DBMs consist in the potential transmissibility of viruses (which has never been reported). DBMs are very expensive and they have different repair capability among different batches of product, due to variability in the quality of donor bone (20).

Recently, the interest towards the use of human adiposederived stem cells (ADSCs) in bone repair and regeneration is increasing. ADSCs are isolated from the stromal-vascular fraction of adipose tissue obtained during abdominoplastic surgical procedures, or during subcutaneous liposuction (21). ADSCs share many properties with bone marrow stem cells (BMSCs), especially the same differentiation potential.

Notably, ADSCs possess many advantages associated to the easy harvesting, security, abundance and higher yield (high number of cells available from a single patient) (2224). Moreover, ADSCs are easily cultivated and expandable in vitro and have greater resistance in culture compared to BMSCs. ADSCs fulfill the International Society for Cellular Therapy (ISCT) criteria as they are plastic-adherent, express CD73, CD90 and CD105, lack CD34 antigens, and have a trilineage mesenchymal differentiation (25).

Many studies have shown possible stem cells involvement in the process of oncogenesis in which malignant cells have the ability to proliferate and divide indefinitely without control (26, 27). It has been hypothesized that some pathways for self-renewal, finely regulated in the healthy stem cells, are altered in neoplastic diseases (28-34). Therefore, arrayComparative Genomic Hybridization (array-CGH) analysis was performed in order to evaluate the genetic stability of ADSCs-based cultures. Array-CGH is a new technique that allows to identify copy number imbalances (gain and loss) of genetic material which are the basis of many disorders and diseases including cancer. Infact, array-CGH has many advantages with respect to classic cytogenetic methods, mainly due to higher resolution, higher reproducibility, and rapid and precise mapping of the whole genome (35).

The aim of the study was to evaluate the ability of 2 DBMs (TBM® e DBX®) to stimulate in vitro the proliferation and the osteogenic differentiation of mesenchymal stem cells isolated from adipose tissue. The safety of the methods was also investigated by means of molecular genetic tests.

MATERIALS AND METHODS Scaffold

Orthoss® Blocks 1x1x2 cm (Geistlich Pharma AG, Wolhusen, Switzerland) were used in this study.

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Orthoss® is a natural carbonated HA of bovine origin.

It is a highly osteoconductive material because of its particular structure, very similar to human cancellous bone, with interconnected macropores (100-300 μm), micropores, and nanopores (10-20 μm), resulting in a higher inner surface and excellent hydrophilic property (36).

Demineralized Bone Matrices (DBMs)

Accell TBM® (Integra LifeScience Corporation, Irvine, CA, USA) is composed of 100% lyophilized human DBM which contains a broad spectrum of natural GFs. It is obtained through the proprietary Accell® DBM processing and it is provided in a sheet or "wafer" format. Accell TBM® is sterilized by electron beam irradiation.

DBX® Putty (Synthes Inc., West Chester, PA, USA) is composed of DBM (93% by volume) in sodium hyaluronate (4%) and phosphate dibasic buffer. It consists of collagen and bone GFs (mainly TGF-β), responsible of the osteoinductive properties. DBX is completely replaced by new host bone after 4 to 6 months. DBX® is aseptically produced processing the tissues in ISO class 5 static certified clean rooms.

Isolation and expansion of human ADSCs

Adipose tissue was collected from the abdominal region of healthy patients (age, 35–58 years) who underwent liposuction cosmetic procedures. All patients gave written consent. The lipoaspirate was washed with Phosphate Buffered Saline (PBS, EuroClone, Milan, Italy) and digested using a solution of 0.075% collagenase from Clostridium histolyticum type II (Sigma-Aldrich, St. Louis, MO, USA) in Hank's Balanced Salt Solution (HBSS, Lonza S.r.l., Milano, Italy), for 3 h at room temperature and in slow agitation. At the end of the digestion, the collagenase activity was blocked with an equal volume of complete DMEM (cDMEM). cDMEM consisted of Dulbecco’s modified Eagle’s medium (DMEM, Lonza, Italy) supplemented with 10% Fetal Bovine Serum (FBS, Bidachem S.p.A., Milano, Italy) and 1% Penicillin/ Streptomycin (P/S, EuroClone). After centrifugation for 4 min at 1200 rpm, the pellet was washed in PBS and filtered with a 70 µM cell strainer (BD Biosciences, Mississauga, Ontario, Canada). The cell suspension was resuspended in cDMEM, transferred to a 25-cm2 tissue culture flask, then incubated at 37°C and 5% CO2 for 15 days. Culture medium was changed every 2 days (37).

Cell counting

The viable cells were counted using the trypan blue exclusion test (38). At the confluence point, ADSCs were detached from the flasks with a solution of 0.25% trypsin ad 0.02% EDTA (EuroClone). After the addition of cDMEM, the cells were centrifuged for 4 min at 1200 rpm. The pellet was resuspended in cDMEM, then, 20 μl of the suspension were added to 80 μl of trypan blue for each culture. Cell counting was done using a Burker’s chamber.

Cell characterization

The expression of stemness markers was evaluated by

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immunofluorescence. The cells, seeded on a glass slide, were fixed for 10 min at room temperature with 4% paraformaldehyde in PBS, pH 7.4. The slides were treated with 1% Bovine Serum Albumin (BSA, Sigma-Aldrich) for 30 min to block non-specific sites, and then incubated at 37°C for 1 h with the following primary antibodies: antihuman CD73 (Abcam, Cambridge, UK), anti-human CD90 (Abcam), anti-human CD105 (Santa Cruz Biotechnology Inc., CA, USA), anti-human fibroblasts (FU) (Abcam), and antihuman CD34-FITC (Macs, Miltenyi Biotec GmbH, Germany).

Subsequently, a second incubation was done at 37°C for 1 h with secondary antibodies: goat anti-mouse IgG DyLight 488 labeled (KPL, Gaithersburg, MD, USA) or goat anti-rabbit IgG (H+L), DyLight 549 labeled (KPL, Gaithersburg, MD, USA).

3D cell cultures

Cells were seeded at a density of 106/cm2 on the 3D Orthoss®scaffold alone (control), with TBM® (Orthoss® + TBM®) or with DBX® (Orthoss® + DBX®). Two different media were used: standard medium (cDMEM) or osteogenic differentiation medium (cDMEM supplemented with 0.1 μM dexamethasone, 200 μM L-ascorbic acid, and 10 mM β-glycerol phosphate). All 3D cultures were incubated at 37°C and 5% CO2 for 28 days, changing the medium every 2 days.

The cell cultures were prepared as represented in Figure 1.

MTT assay

The biocompatibility of the materials was assessed at day 28 verifying cell viability and proliferation by the MTT (3-4,5-dimethylthiazol-2YL-2,5-bromuro diphenyltetrazolium) assay (39). The test is based on the ability of functional mitochondria to oxidize the MTT solution, giving a blue-violet product. In detail, the supernatant was aspirated from the tissue culture plate and 1 mL of 0.5 mg/mL MTT solution in PBS was added.

After 3 h of incubation, the supernatant was aspirated, each scaffold was transferred to a microtube and 0.5 mL of 10% dimethyl sulfoxide in isopropanol was added to extract the formazan in the samples for 30 min at 37°C. For each sample, absorbance values at 570 nm were recorded in duplicate on 200 μL aliquots deposited in microwell plates using a multilabel plate reader (Victor 3 Perkin Elmer, Milano, Italy).

Real-time PCR

Total RNA was extracted on day 28 using the TRIzol® Reagent (Invitrogen, Carsbad, CA, USA). The samples were quantified using the NanoDrop spectrophotometer (NanoDrop™ 1000, Thermo Scientific). For the first-strand cDNA synthesis, 500 ng of total RNA was reverse transcribed using M-MLV RT (Moloney Murine Leukemia Virus Reverse Transcriptase, Invitrogen) according to the manufacturer's protocol. Human primers were selected for each target gene with Primer 3 software. Real-time PCRs were carried out using the designed primers at a concentration of 300 nM and FastStart SYBR Green Master (Roche Diagnostics, Mannheim, Germany) on a Rotor-Gene 3000 (Corbett Research, Sydney, Australia). Thermal cycling conditions

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ADSCs standard medium

ADSCs osteogenic differentiation medium

Orthoss®

Orthoss®

Orthoss® + TMB®

Orthoss® + TMB®

Orthoss® + DBX®

Orthoss® + DBX®

fig. 1 Schematic representation of the prepared 3D cell cultures.

were as follows: 15 min denaturation at 95°C; followed by 40 cycles of 15 s denaturation at 95°C; annealing for 30 s at 60°C; and 20 s elongation at 72°C. Values were normalized to the expression of the β-actin internal reference, whose abundance did not change under our experimental conditions. Experiments were performed with 3 different cell preparations and repeated at least 3 times.

array-CGH

In order to identify possible chromosomal aberrations, such as deletions, amplifications, and aneuploidy, array-CGH was performed using the Human Genome CGH Microarray (Agilent Technologies, Santa Clara, CA, USA) with a median probe spatial resolution of 44 Kb. array-CGH was performed following the manufacturer's protocol. Briefly, 1 µg of DNA from human ADSCs cultures (sample) and 1 µg of pooled sex-matched reference DNA (Promega, Madison, WI, USA) were digested with AluI and RsaI for 2 h at 37°C. After inactivation of the enzymes at 65°C for 20 min, each digested sample was labeled by random priming (Genomic DNA Enzymatic Labelling Kit, Agilent Technologies) for 2 h using Cy5-dUTP for sample DNA and Cy3-dUTP for reference DNA. Labeled products were then column-purified (Microcon YM-30 filters, Millipore Corporation, Billerica, MA, USA). After probe denaturation and pre-annealing with Cot-1 DNA, hybridization was performed at 65˚C with rotation for 24 h. At the end of the incubation, slides were washed and analyzed using the Agilent scanner. Data and graphics elaboration was done using the CGH Analytics software (V3.1 Agilent Technologies).

Statistical analysis

Statistical analysis was performed using the software SPSS 20.0.0. The comparison between groups was done using the Mann-Withney-U test. A p<0.05 was considered significant (*).

RESULTS Cell isolation and characterization

Human ADSCs were isolated from lipoaspirate by an enzymatic digestion, then grown as monolayer for 15 days in cDMEM at 37°C and 5% CO2. ADSCs showed

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a fibroblastic-like morphology when observed under phase contrast microscope (Fig. 2). In agreement with the ISCT25, ADSCs were phenotypically characterized by immunofluorescence at passage three. The cells were positive for anti-CD73, -CD90 and -CD105 antibodies, negative for anti-CD34 and anti-FU antibodies (Fig. 3).

Biocompatibility of the scaffolds

ADSCs were able to proliferate onto the scaffolds, both in standard medium and osteogenic differentiation medium, as demonstrated by the MTT assay performed 28 days after cell seeding (Fig. 4). These results demonstrated that the scaffolds were biocompatible and not toxic for the cell growth.

Cells seeded on Orthoss® alone were used as control. In presence of standard medium, we found a significantly higher expression of selected genes in ADSCs seeded with TBM® compared to control and ADSCs grown with DBX®.

On the contrary, there was not a significant difference in gene expression in the populations seeded with DBX® and control (Fig. 5). Osteogenic differentiation of ADSCs was, instead, similar independently of the biomaterials used (Fig. 6).

array-CGH

In order to verify the safety of both materials and methods, we performed array-CGH analyses of all the 3D ADSCs cultures after 28 days from seeding.

Real-time PCR

The gene expression level of some osteoblast markers was analyzed at day 28 by means of Real-time PCR in order to verify the osteogenic properties of the materials used in the present study. The expression of selected genes (osteopontin, osteonectin, osteocalcin, type I collagen) was evaluated in relation to the expression of the reference gene (β-actin).

fig. 3 A-E Immunofluorescence stainings of stemness markers in ADSCs at passage three. ADSCs were positive (red) for (A) CD73, (B) CD90 and (C) CD105; negative for (D) FU and (E) CD34 markers. Cell nuclei (blue) were stained with Hoechst33342.

O.D. 570 nm

2,5 2,0

Orthoss® Orthoss® + TMB®

1,5

Orthoss® + DBX®

1,0

Orthoss® Orthoss® + TMB®

0,5

Orthoss® + DBX®

0,0 ADSCs standard medium

ADSCs osteogenic differentiation medium

fig. 4 MTT assay of 3D ADSCs cultures in standard medium (left) and osteogenic differentiation medium (right). After 28 days of culture, the cells show the same ability to proliferate in presence of both TBM® and DBX®.

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2^ΔCt

fig. 2 Morphology of ADSCs isolated and cultured at 37°C with 5% CO2 at passage three. Cells show a spindle or a triangle-like morphology (20X magnification).

2,2 2,0 1,8 1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0 Osteopotin Orthoss®

Osteonectin

Osteocalcin

Orthoss® + TMB®

Collagen Type I Orthoss® + DBX®

fig. 5 Gene expression of some osteoblast markers in ADSCs cultured for 28 days in standard medium. Gene expression levels are higher in ADSCs seeded in Orthoss® + TBM® (* = p<0.05) compared to ADSCs seeded in Orthoss® alone or Orthoss® + DBX®.

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2^ΔCt

Pozzuoli A. et al.

2,2 2,0 1,8 1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0 Osteopotin Orthoss®

Osteonectin

Osteocalcin

Orthoss® + TMB®

Collagen Type I Orthoss® + DBX®

fig. 6 Gene expression of some osteoblast markers in ADSCs cultured for 28 days in osteogenic differentiation medium. Gene expression levels are similar in ADSCs seeded in Orthoss® + TBM® (* = p<0.05) compared to ADSCs seeded in Orthoss® alone or Orthoss® + DBX®.

No chromosomal aberrations were detected in the experimental sets, confirming the biosafety of the Orthoss® scaffold, of both the DBMs as well as of the cell culture method used.

DISCUSSION Bone regeneration is a complex and multifactorial process that mainly needs the presence of osteoconductive (3D scaffold), osteoinductive (BMPs, GFs, and DBM), and osteogenic (bone-forming cells) factors (1). These properties are typically found in autologous bone graft; however, the disadvantages associated to its harvesting limit its use. For this reason, an alternative approach has been extensively studied with the aim to obtain a fully functioning bone. This strategy is based on the ability of MSCs to proliferate, self-renew and differentiate. MSCs can be harvested from many tissues, among which adipose tissue represents an abundant and accessible source (40, 41). Indeed, ADSCs have similar ability to proliferate and differentiate of BMSCs, in particular in osteogenic sense (42). Moreover, ADSCs possess additional advantages compared to BMSCs, mostly related to the great availability and yield (22).

On the basis of these knowledges, in the present study we tested the ability of ADSCs to differentiate towards the osteogenic phenotype in vitro when seeded on a 3D scaffold in presence of osteogenic factors.

ADSCs were isolated, by enzymatic digestion, from the lipoaspirate of 5 healthy patients undergoing abdominal cosmetic liposuction. After an incubation period of 15 days, positivity to CD73, CD90, and CD105 stem cell markers was verified by immunofluorescence. Negativity to FU and CD44 was also assessed. Then, ADSCs were seeded on Orthoss®, an HA scaffold necessary for 3D cell culture (1). ADSCs were also 3D cultured in presence of 2 different DBMs: TBM®, entirely composed of

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demineralized bone matrix, and DBX®, adjuvanted with sodium hyaluronate, able to stimulate the osteogenic differentiation.

In order to compare the osteoinductive ability of the 2 DBMs, cells were cultured in cDMEM or in osteogenic differentiation medium for 28 days. At the end of this period, the biocompatibility of the scaffolds was tested with the MTT assay by comparing viability of the cells cultured with or without differentiation medium. Proliferation rates were higher, although not significant, in the group of cells cultured in standard medium compared to the ones treated with osteogenic factors. This result could be explained by a slower ability of the cells to differentiate in standard medium, where they maintain stemness characteristics and, therefore, a greater replicative capacity.

Moreover, we observed a lower proliferation rate of ADSCs seeded on Orthoss® + DBX® compared to Orthoss® + TBM® and also to the group only cultured on Orthoss® (control group). On day 28, the osteoinductive capacity of DBMs were evaluated by measuring the gene expression levels of osteopontin, osteonectin, osteocalcin and type I collagen. Real-time PCR results revealed the expression of all the genes analyzed, thus confirming the mature osteogenic phenotype of the cells. In detail, the results showed a significantly greater gene expression level in cells cultured with Orthoss® + TBM® compared to the control and to the Orthoss® + DBX® groups. On the contrary, there was no difference between the control and the Orthoss® + DBX® groups.

This would suggest a greater ability for TBM® to induce osteogenic differentiation in vitro.

ADSCs were able to differentiate in osteogenic sense also in the group seeded on Orthoss® alone. It has been demonstrated that Orthoss® not only has osteoconductive ability but also osteoinductive properties due to its porous structure that ensures to the cells a habitat extremely similar to that of native bone (43, 44). The main difference between TBM® and DBX® consists in the presence of hyaluronic acid in the second material. As this molecule is abundantly found in vivo in the dermis, we hypothesized that the lesser osteoinductive capacity of DBX® compared to TBM® may be due to an interaction of hyaluronic acid with its receptor (CD44), normally expressed by MSCs. This interaction could alter the normal process of MSCs osteoinduction interfering with the normal pathways of differentiation and promoting the activity of the peroxisome proliferator activated receptor γ (PPAR γ), factor required for the adipogenic differentiation (48). Finally, we evaluated the safety of the method used with array-CGH analysis. This test is particularly important for further clinical applications. array-CGH is preferentially used to test genetic alterations, index of neoplastic transformation, because of its higher resolution than other cytogenetic and molecular methods. The analysis was performed on the entire genome of all 3D cultures, after 28 days of in vitro

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culture. Results showed no alterations at the genome level. These data strongly supported the safety of both biomaterials and stem cells cultured in vitro, according to the methods used in this study.

Conclusion Based on the results of this study, it can be concluded that human ADSCs are able to adhere, proliferate and differentiate in osteoblast-like cells, also in absence of osteogenic factors. This confirms the osteoinductive ability of both the DBMs used. Moreover, the absence of genetic alterations demonstrates the safety of materials and cultures and supports their potential clinical application for bone reconstruction. The use of in vitro cultured autologous ADSCs is, therefore, promising for the clinical treatment of bone defects of various dimensions reducing the problems related to the use of non-autologous material and to the availability of great amount of materials.

ACKNOWLEDGMENTS This research was supported by funds from University of Padua, Progetto di Ateneo awarded to Barbara Zavan.

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36. Thorwarth M, Schlegel KA, Wehrhan F, Srour S, Schultze-Mosqau S. Acceleration of de novo bone formation following application of autogenous bone to particulated anorganic bovine material in vivo. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101:309316.

37. Aguiari P, Leo S, Zavan B, Vindigni V, Rimessi A, Bianchi K, Franzin C, Cortivo R, Rossato M, Vettor R, Abatangelo G, Pozzan T, Pinton P, Rizzuto R. High glucose induces adipogenic differentiation of muscle-derived stem cells. Proc Natl Acad Sci U S A 2008;105:1226-1231.

38. Strober W. Trypan blue exclusion test of cell viability. Curr Protoc Immunol 2001;Appendix 3:Appendix 3B. 39. Denizot F, Lang R. Rapid colorimetric assay for cell-growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 1986,89:271-277.

40. Asagiri M, Takayangi H. The molecular understanding of osteoclast differentiation. Bone 2007;40:251-264.

41. Cohen MM Jr. The new bone biology: pathology, molecular, and clinical correlates. Am J Med Genet 2006;140:2646-2706.

42. Landis WJ. The strength of a calcified tissue depends in part on the molecular structure and organization of its constituent mineral crystals in their organic matrix. Bone 1995;16:533544.

43. Kouropis D, Baboolal TG, Jones E, Giannoudis PV. Native multipotential stromal cell colonization and graft expander potential of a bovine natural bone scaffold. J Orthop Res 2013;31:1950-1958.

44. Gardin C, Bressan E, Ferroni L, Nalesso E, Vindigni V, Stellini E, Pinton P, Sivolella S, Zavan B. In vitro concurrent endothelial and osteogenic commitment of adipose-derived stem cells and their genomical analyses through comparative genomic hybridization array: novel strategies to increase the successful engraftment of tissue-engineered bone grafts. Stem Cells Dev 2011;21:767-777.

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M. Muñoz-Corcuera, A. Bascones-Martínez1, J. Ripollés-de Ramón Department of Oral Medicine and Surgery, School of Dentistry, Madrid Complutense University, Madrid, Spain 1 Professor at Dental School

Post-extraction application of beta-tricalcium phosphate in alveolar socket to cite this article Muñoz-Corcuera M, Bascones-Martínez A, Ripollés-de Ramón J. Post-extraction application of beta tricalcium phosphate in alveolar socket. J Osseointegr 2015;7(1):8-14.

ABSTRACT Aim The objective of this study was to assess the capacity of betatricalcium phosphate to facilitate bone formation in the socket and prevent post-extraction alveolar resorption.

Materials and methods After premolar extraction in 16 patients, the sockets were filled with beta-tricalcium phosphate.

Six months later, during the implant placement surgery, a trephine was used to harvest the bone samples which were processed for histological and histomorphometrical analyses. Data were gathered on patient, clinical, histological and histomorphometric variables at the extraction and implant placement sessions, using data collection forms and pathological reports.

Results Clinical outcomes were satisfactory, the biomaterial was radio-opaque on X-ray. Histological study showed: partial filling with alveolar bone of appropriate maturation and mineralization for the healing time, osteoblastic activity and bone lacunae containing osteocytes. The biomaterial was not completely resorbed at six months.

Conclusion Beta-tricalcium phosphate is a material capable of achieving preservation of the alveolar bone when it is positioned in the immediate post-extraction socket followed by suture; it also helps the formation of new bone in the socket. Further studies are needed comparing this technique with other available biomaterials, with growth factors and with sites where no alveolar preservation techniques are performed.

Keywords Bone graft; Calcium phosphate; Dental implant; Post-extraction alveolar socket.

INTRODUCTION In normal conditions, healthy bone is under continuous remodelling and has an effective self-repair capacity.

Bone remodelling maintains a continuous balance of

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bone formation and resorption in a dynamic process that adapts the bone to local forces (1). Above a critical defect size, however, bone cannot be repaired by its own osteogenic activity, and some type of bone grafts must be used (2).

Jaw bone defects can be caused by surgical resection, traumatic loss, ossification impairment (in the elderly), periodontal and peri-implant diseases and congenital disorders. These defects may complicate the surgical phase of implant supported rehabilitation treatment due to insufficient bone volume for an adequate implantation (3, 4). Jaw bone loss is frequently caused by post-extraction alveolar resorption, a physiological phenomenon which leads to a reduction of the original height and width of the alveolar ridge to a degree that varies among localizations and patients (5). Alveolar ridge preservation techniques have been developed to address the ensuing clinical problem, especially in aesthetic areas (5). They are conducted during or after extraction and are designed to minimize external ridge resorption and maximize bone formation inside the socket (5). Measures include autologous bone grafts, allografts, bone of animal origin (xenografts) and synthetic bone substitutes (alloplastic grafts), as well as the application of growth factors and gene therapies (3, 4, 6).

Beta-tricalcium phosphate (beta-TCP) is widely used as a biocompatible, resorbable and osteoconductive ceramic substitute to repair bone defects. Thanks to its physicochemical characteristics, it has been successfully used to fill spaces in multiple settings, including biology, veterinary medicine, human medicine and dentistry (712). It has also been proposed as a vehicle for growth factors that stimulate bone formation (12, 13). Various authors have reported on its capacity as a biomaterial for bone regeneration in animals and humans (4, 14-21).

The study is aimed at evaluating granular beta-TCP in post-extraction sockets in order to measure its bone regenerative potential and its capacity to preserve the original height and width of the alveolar bone for subsequent implant placement.

Specifically, the study objectives were the following.

1. To analyze the clinical and radiological results

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Beta-tricalcium in post-extraction sockets

obtained after placement of the biomaterial in the post-extraction socket and at the subsequent insertion of dental implants.

2. To assess the effectiveness of beta-TCP as bone filling material in the post-extraction socket.

3. To perform histological analysis of the amount and quality of bone formed in the dental socket six months after the placement of the biomaterial.

4. To determine the percentage of biomaterial particles in contact with patient bone.

fig. 1 Socket filled with a mixture of beta-TCP and patient blood.

fig. 2 Socket closed by suture using a coronally repositioned flap.

MATERIALS AND METHODS Study design

This prospective longitudinal observational clinical study complied with the principles of the Helsinki Declaration and was approved by the clinical research ethics committee of the San Carlos Clinical Hospital, Madrid (Spain).

All patients in the study were aged over 18 years and scheduled for ≥ 1 premolar extraction due to periodontal disease, caries or fractures and for subsequent replacement with dental implant(s) up to a maximum of four premolar extractions (one per quadrant) per patient.

Exclusion criteria were: failure to sign informed consent or commit to compliance with the study appointment schedule; the presence of endocrine-metabolic disease or chronic, general or local disease; the presence of disease that may be affected by the surgery or by the intraoperative or postoperative medication; alveolar socket wall defects; smoking habit of ≥10 cigarettes/ day, due to its relationship with implant failure; and treatment with bisphosphonates or antibiotics during the previous month. Patients were recruited from the School of Dentistry clinic (Complutense University of Madrid, Spain) and private clinics. A non-probabilistic sampling of consecutive cases was conducted and only patients who met the above criteria were included.

Sixteen patients were enrolled in the study between March 2008 and July 2010, with a mean age of 44.3 years (standard deviation: 10.74); seven were male (44%) with mean age of 39.7 years and nine were female (56%) with mean age of 48 years. No participant (0%) was a daily drinker of alcohol, and two (12%) were daily smokers (of 1-9 cigarettes). A total of 19 upper and 2 lower teeth were extracted (lower teeth were excluded from the analysis because of this small number).

After a baseline clinical assessment, all patients received basic periodontal therapy before the surgery and were instructed to maintain good oral hygiene throughout the study.

Surgical procedure

After applying local anaesthesia and performing fullthickness buccal and lingual flap elevation, the premolar was extracted; a full-thickness flap was elevated to

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enable a subsequent suture to keep the granules of the material in place. Any granulation tissue present in the socket was removed by surgical curettage, and the socket was filled with 0.5 g beta-TCP KeraOs® (Keramat, La Coruña, Spain) mixed with physiological saline solution or blood from the same patient (Fig. 1). The socket was then closed by suture using a coronally repositioned flap (Fig. 2). Patients were instructed to rinse daily for two weeks with 0.12% chlorhexidine digluconate. Sutures were removed at 7-10 days post-extraction.

During the implant placement surgery (about 6 months after biomaterial placement), a bone biopsy was harvested using a trephine (inner diameter of 2.2 mm, outer diameter of 3 mm), placed in a 10% buffered formalin and sent to the Ceramic Institute of Galicia (Santiago de Compostela, Spain) laboratory for processing.

Histological processing

The specimens were processed to obtain thin undecalcified sections following Donath’s method and using the EXACT system.

Briefly, specimens were fixed in buffered 10% formalin, progressively dehydrated in alcohol and then embedded in photopolymerizable methacrylate resin (Technovit 7200®, VLC-Heraus Kulzer GMBH, Werheim, Germany).

After polymerization, the specimens were cut with a diamond saw and then ground with silicon carbide papers to a width of about 70 microns. After thinning, samples were stained with Levai Laczko stain and chromotrope 2R/Harris haematoxylin. A motorized Olympus BX51 microscope with Olympus DP71 camera was used to image the specimens, with Olympus D-cell capture software and Photoshop CS3 image processing software, employing a Wacom Intuos 4 pen tablet and

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Muñoz-Corcuera M., Bascones-Martínez A. and Ripollés-de Ramón J.

Females Age (yrs) 48 Number 9 (56.25%) Smokers (1-9 cigs/day) 2 (12%) Drinkers 0 (0%)

Males Mean Standard deviation 39.7 44.3 10.74 7 (43.75%) 0 (0%) 0 (0%)

applying the Olympus MicroImage 4.0 program to obtain histomorphometric measurements.

Data were gathered on the following.

1. Patient variables, sex, age, and consumption of alcohol and cigarettes (smoker = 1-9, non-smoker = 0 cigarettes/day, to test whether a light tobacco habit affects socket healing).

2. Clinical variables, biomaterial stability within socket and primary implant stability.

3. Radiological findings.

4. Histological variables at 6 months, degree of bone neoformation in socket, amount and quality of newly formed bone, degree of contact between patient bone and beta-TCP and degree of beta-TCP resorption, all assessed by direct microscopic observation.

5. histomorphometric variables, areas of newly formed bone, immature bone, old bone, biomaterial and lamellar bone, bone-biomaterial contact index (perimeter of material in contact with bone / perimeter of whole material), remnant volume (surface of material present / [surface of material present + total bone surface]) and immature:mature bone ratio (mature bone surface / total bone surface).

Specifically designed forms were used to collect data at the following time points: tooth extraction, gathering patient variables; suture withdrawal (7-10 days postextraction), recording radiological findings; and implant placement (around 6 months post-extraction), gathering radiological findings and data on material retention in the socket and primary implant stability.

Histological data were obtained from the pathology report on samples taken at implant placement.

Microsoft Excel and SPSS were used for the statistical analyses, which included: descriptive analysis of patient, clinical and histomorphometric variables; frequency histograms for histomorphometric variables; ShapiroWilks normality tests for histomorphometric variables, age and healing time; 95% confidence intervals for histomorphometric variables; use of the Pearson correlation coefficient to analyse associations of different histomorphometric variables with each other and with healing time and age; analysis of variance (ANOVA) to determine the effect of healing time on newly formed bone area, biomaterial area and bone-biomaterial contact index; and the Student’s t test to compare newly formed bone area, biomaterial area and bone-biomaterial contact index between shorter and longer healing times (5-6 months versus 7-8 months, respectively).

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tabLE 1 Results. Variables related to the individual. Mean age 44.3 yrs (standard deviation: 10.74); 7 males (44%) with mean age 39.7 yrs and 9 females (56%) with mean age 48 yrs. No participant (0%) was a daily drinker of alcohol, and two (12%) were daily smokers of 1-9 cigarettes.

RESULTS Patient variables

One male patient abandoned the study before implant placement. Among the 15 remaining patients, 21 biopsies were obtained after a mean healing time of 6.2 months (standard deviation: ±1.05). Out of the 21 biopsies, 3 were impaired during grinding and could not be processed, and 2 were incorrectly sampled and excluded from the analyses. Hence histological and histomorphometric analyses were conducted in a final sample of 16 biopsies (Table 1).

Clinical results

None of the patients evidenced biomaterial loss at implant placement; in some cases, the most superficial area showed residual graft particles that had no effect on the surgical procedure or primary stability, which was obtained in all cases. X-ray images revealed no complications, and in all the films, high radiopacity and consequent prompt identification of the material was detected.

Histological and histomorphometric results

No biomaterial fragments or necrotic bone splinters were detected in any of the 16 biopsies analyzed. In three cases, the biomaterial was integrated in the bone and surrounded by fibrous tissue with rim of osteoblasts and osteoid matrix; in one case, the biomaterial was surrounded by lax conjunctive tissue; in five cases, it was surrounded by mature bone trabeculae with scant osteoid and osteoblastic rimming; in seven cases, modest to highly abundant immature bone trabeculae growth was observed with osteoid and osteoblasts rim. Ten of the biopsies showed the presence of medullary fibrosis, at a low level in most cases.

Evidence of vital bone growth was found in the sockets, with bone neoformation in close contact with graft particles. All samples showed residual particles of the material, with various degrees of material remodelling and resorption (Fig. 3, 4, 5).

The histological study at 6 months revealed that the degree of bone neoformation in the socket was generally moderate, that the newly formed bone was immature (consistent with the healing time) and surrounded by and in direct contact with biomaterial fragments and that the beta-TCP material showed initial signs of resorption.

Table 2 exhibits the results of the histomorphometric

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fig. 3 Newly formed bone around the biomaterial, (white arrows) with faint signs of resorption in the central area of the biopsy (red arrow). Most of the bone is newly formed, with traces of old bone at the periphery. Levai-Laczko stain. 100X.

fig. 4 Material integrated in the bone tissue; there is a predominance of lax connective tissue (red arrow), although with areas of denser connective tissue.

The biomaterial is integrated in bone trabeculae (white arrows). Levai-Laczko stain. 100X.

Variable (%) Newly formed bone area Immature bone area Old bone area Biomaterial area Lamellar bone area Bone-implant contact index Remnant volume Immature bone-mature bone relationship

Minimum value 0.30 8.34 0.43 0.33 2.02 0 0 0

variables, which were found to follow a normal distribution (Shapiro-Wilk test). The frequency histograms showed that the mean contact between bone and biomaterial was <20% in 8 out of 15 biopsies and that the newly formed bone area was >20% in most of them; the biomaterial area was <20% in most of the biopsies. Calculation of 95% confidence intervals showed significance for all variables, except for the immature bone area and lamellar bone area, for which there were measurements in only two cases (Table 3).

These two variables were excluded from analysis, using Pearsonâ&#x20AC;&#x2122;s correlation coefficient, of the relationships of histomorphometric variables with each other and with healing time and age; a positive correlation was found between remnant volume and biomaterial area (p= 0.0056) and between old bone area and the immature bone:mature bone ratio (p= 0.015).

Although the healing period was established as 6 months for this study, this time was sometimes influenced by specific patient circumstances and ranged from 5 to 8 months. The results for newly formed bone area,

fig. 5 Biomaterial surrounded by immature bone. Chromotrope 2R/Harris haematoxylin staining.100X.

Maximum value 45.33 31.80 21.03 26.25 6.11 69.70 98.85 96.07

Mean 20.15 20.07 11.98 11.40 4.06 32.31 31.98 42.62

Median 13.64 20.07 11.87 7.99 4.06 19.82 35.60 36.13

Standard deviation 15.42 16.58 7.65 8.88 2.89 24.94 25.68 36.48

tabLE 2 Results. Descriptive statistics of histomorphometric variables. Table shows the minumim and maximum values for each variable; the mean and the median are also showed for each variable.

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Muñoz-Corcuera M., Bascones-Martínez A. and Ripollés-de Ramón J.

Variable Newly formed bone area Immature bone area Old bone area Biomaterial area Lamellar bone area Bone-implant contact index Remnant volume Immature bone:mature bone ratio

Upper interval limit 29.95 169.11 19.06 18.23 30.04 46.12 46.20 63.69

Lower interval limit 10.35 -128.97 4.91 4.58 -21.92 18.49 17.76 21.56

biomaterial area and bone-biomaterial contact index were analyzed in function of healing time, finding no significant differences. Then, newly formed bone area, biomaterial area and bone-biomaterial contact index were compared between healing times of 5-6 months and 7-8 months, finding no significant differences, altough borderline significance (p=0.08) was obtained for newly formed bone area.

DISCUSSION In this study, post-extraction placement of beta-TCP in the socket did not cause any complications and achieved good clinical outcomes. There was histological evidence of bone neoformation at implant placement, with the presence of osteocytes and immature bone. The mean percentage of neoformed bone was 20.15%, in line with previous reports (22-25). The biomaterial area was less than 20% in most of the biopsies, confirming the resorbability of the biomaterial. The biomaterial was readily identifiable on X-ray, being much denser than the adjacent bone, as previously reported by Von Doernberg et al. (26). This characteristic is useful for the radiographic follow-up of healing, because the radiopacity changes as the material is resorbed and replaced by new bone.

Clinical studies on humans generally require the use of non-invasive techniques, e.g. radiology; but a biopsy study is the currently optimal method to assess the regeneration, quantity and quality of bone. A two-phase approach, inserting the graft in the first phase and the implant in the second, allows a histological sample to be obtained (20). This technique was applied in the present study.

We mixed beta-TCP with saline solution or blood from the patient, as in the study by Horowitz et al. (24), given the difficulty of managing this porous material in granular form (27).

Six months as bone healing time was selected, because most of the ceramic is resorbed, and the grafted tissue

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Statistical significance Significant Not significant Significant Significant Not significant Significant Significant Significant

tabLE 3 Results. 95% confidence intervals. Calculation of 95% confidence intervals showed significance for all variables, with the exception of immature bone area and lamellar bone area, for which there were measurements in only two cases.

can be considered sufficiently stable for functional implant loading (19, 24). A study in pigs (28) found betaTCP degradation to be slow, with 80% of the material resorbed at 28 weeks and 97% at 86 weeks; therefore, the authors recommended an interval of 5-6 months before implant placement in grafted areas, concluding that the cell response to their simultaneous placement could damage implant osseointegration. Some authors suggested lengthening this healing time in order to increase implant stability (4), and it was found that the presence of residual particles at 9 months does not compromise implant placement (23). In contrast, as reported above, Ormianer et al. achieved a 97% success rate after the immediate placement of implants in augmented areas and their immediate implant loading (22).

With regard to the mechanism of beta-TCP degradation before its substitution by bone, it was attributed by Wiltfang et al. (28) to chemical hydrolysis (halisteresis) and the activity of phagocytic cells (multinucleated giant cells). Two degradation pathways have since been described: osteoclast-mediated resorption and dissolution in interstitial fluid (23). A study in 2005 detected no osteoclastic activity in biopsies from sinuses augmented with this biomaterial, but this finding does not rule out the participation of osteoclasts although it suggested that it is limited (29). Besides these two mechanisms, it has been postulated that betaTCP resorption may also be mediated by cells other than osteoclasts (20). However, Martinez et al. (30) suggested that osteoclasts or macrophage cells may not play an important role in beta-TCP resorption, as they found in the bone-beta-TCP interface cells of the reticuloendothelial system.

Some data are available on the use of beta-TCP for alveolar preservation (22-25). Ormianer studied the use of beta-TCP alone in 338 patients, although alveolar preservation was not investigated in all of these, and the number of patients undergoing the different procedures was not specified; the mean follow-up was 19.2 months and the global implant survival rate was

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97.6%. In 2008, Brkovic reported on the use of betaTCP with collagen alone in one patient, followed up for 9 months, reporting good clinical outcomes with bone formation activity.

In 2012, Horowitz used beta-TCP with a membrane in 30 patients, followed up for a mean of 6 months, also observing good outcomes with preservation of 91% of the socket width. Finally, in the same year, Brkovic studied 20 patients in two groups, one receiving betaTCP with membrane and apically repositioned flap and the other beta-TCP alone, with a mean follow-up of 9 months, concluding that socket preservation was lower in the group without membrane. Our results are comparable to the findings of these four studies, because the implant survival was 100%, the clinical outcomes were good, bone neoformation was observed in the biopsies, and there was only a small volume of residual bone (11.98%) (Table 2).

There have also been reports on socket preservation with the use of other materials. Thus, Liasella et al. employed allografts with good results (31), while De Coster et al. (32) used biphasic ceramics but obtained poor outcomes that delayed implant placement.

After experiencing some problems in harvesting the specimens from the trephine, the protocol was modified and the samples were processed with the trephine as a block. Zerbo (33) also found it difficult to remove betaTCP biopsies in a single piece from the trephine, and Suba (20) reported that biomaterial particles frequently broke during sample preparation.

In the present study three biopsies were lost in the polishing process, due to the complexity of sample processing, and one biopsy was taken from the incorrect area, a problem that some authors have resolved by using surgical guides (25).

In the study by Horowitz 2010 (34), two cases are discussed. In the first one an identical procedure to the one here described was followed, except for the use of a resorbable membrane after the placement of the biomaterial. The clinical outcome was excellent, allowing the placement of a dental implant 6 months after extraction. The biomaterial was replaced by new vital bone, just as in our work. Their second case is that of a smoker patient; the biomaterial was placed in the socket followed by a membrane. Healing time in this case was 10 months, after which an implant was placed. The clinical, radiological and histological results are comparable to those of our study, they observed the formation of osteon and Haversian systems in the biopsy due to increased healing time.

With the limitations of this study, especially regarding the small sample size, the histological and clinical results are in agreement with reports by various authors, evidencing problem-free healing, primary stability of implants placed in the augmented area, and an adequate substitution of beta-TCP particles by newly formed bone at 6 months.

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CONCLUSION The clinical and radiographic outcomes of this procedure are satisfactory, with no associated complications. Betatricalcium phosphate seems to be a biomaterial capable of achieving preservation of the alveolar bone when it is positioned immediately in post-extraction socket followed by suture; also facilitating the formation of new bone in the socket in the first six months. This resorbable material allows predictable and reproducible bone regeneration. As advantages, it can be noted its unlimited availability, its easy handling and its great radiopacity, allowing radiographic follow-up of the area. Multiple publications have shown the suitability of this material for use in bone augmentation techniques.

Further clinical studies and randomized clinical trials are needed, comparing this technique with other available biomaterials, with growth factors and with alveoli in which no alveolar preservation techniques are performed.

ACKNOWLEDGEMENTS This study was supported by an FPU training grant for university professors from the Spanish Ministry of Education (nº AP2008-00011) and by a project of Universidad-Empresa Foundation (47/2009) signed with Keramat.

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30. Martinez A, Franco J, Saiz E, Guitian F. Maxillary sinus floor augmentation on humans: Packing simulations and 8 months histomorphometric comparative study of anorganic bone matrix and β-tricalcium phosphate particles as grafting materials. Mater Sci Eng C Mater Biol Appl 201015;30:763-9.

31. Iasella JM, Greenwell H, Miller RL, Hill M, Drisko C, Bohra AA et al. Ridge preservation with freeze-dried bone allograft and a collagen membrane compared to extraction alone for implant site development: a clinical and histologic study in humans. J Periodontol 2003;74:990-9.

32. De Coster P, Browaeys H, De Bruyn H. Healing of extraction sockets filled with BoneCeramic® prior to implant placement: preliminary histological findings. Clin Implant Dent Relat Res 2011;13:34-45.

33. Zerbo IR, Zijderveld SA, de Boer A, Bronckers AL, de Lange G, ten Bruggenkate CM et al. Histomorphometry of human sinus floor augmentation using a porous beta-tricalcium phosphate: a prospective study. Clin Oral Implants Res 2004;15:724-32.

34. Horowitz RA, Mazor Z, Foitzik C, Prasad H, Rohrer M, Palti A. b-tricalcium phosphate as bone substitute material: properties and clinical applications.

J Osseointegr 2010;2(2):61-68.

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F.S. Marchionni1-2, F. Alfonsi1, S. Santini, S. Marconcini, U. Covani, A. Barone1-2 Tuscan Stomatologic Institute, Versilia General Hospital, Lido di Camaiore, Italy 1 Department of Surgical, Medical, Molecular and of the Critical Area Pathology, University of Pisa, Italy 2 Unit of Dentistry and Oral Surgery, University-Hospital of Pisa, Italy

Maxillary sinus augmentation: collagen membrane over the osteotomy window. A pilot study to cite this article Marchionni FS, Alfonsi F, Santini S, Marconcini S, Covani U, Barone A. Maxillary sinus augmentation: collagen membrane over the osteotomy window. A pilot study. J Osseointegr 2015;7(1):15-20.

ABSTRACT Aim Implant rehabilitation has become a very reliable and safe procedure. However, in some cases, a small amount of bone could make implant surgery extremely difficult or even impossible.

Hence, a surgical technique to augment sinus floor has been developed and improved. Nevertheless, there is still controversy over the use of a membrane over the osteotomy window.

Therefore, the aim of this study was to investigate whether the use of a membrane could be beneficial in sinus floor augmentation.

Materials and methods A group of 12 patients requiring sinus floor lift were recruited. The patients were randomly allocated to either control group (membrane) or test group (no membrane) and only one sinus for patient was augmented. After 6 months, a bone biopsy was harvested from the lateral window to be processed for histological analysis.

Results The mean amount of newly formed bone in test group was 28.0±19.5%, the connective tissue accounted for a mean value of 59.2±15.6%, while 12.8±12.6% was the amount of residual graft particles. In the membrane group the newly formed bone counted for a mean value of 30.4±15.8%, the mean quantity of connective tissue was 50.3±18.9% and about residual graft particles a mean value of 18.2±20.4% was registered.

Conclusion According to our data, the use of a membrane over the lateral bone wall in sinus lift surgery does not significantly influence healing. However, the membrane could influence the residual particles resorption rate as well as soft tissue ingrowth.

Keywords Sinus; Floor; Augmentation; Lift; Membrane; Xenograft.

Introduction Following tooth extraction, alveolar ridge undergoes marked changes both in width and in height (1). This could make the prosthetic implant rehabilitation

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extremely hard for the oral surgeon, especially in posterior maxillary regions in which great care must be taken to avoid maxillary sinus lesions (2).

In order to overcome this problem, in the ‘80s some authors described different methods for bone augmentation of the sinus floor (3, 4) and since the modified Cadwell-Luc technique by Tatum (1986), several modifications have been proposed in the literature (5, 6, 7, 8).

Regarding graft materials, autologous bone and bone substitutes are the two types of available materials with the strongest literature support in sinus augmentation.

However, there is not a clear evidence on the superiority of one over the other (9, 10). In fact, autologous bone grafts have excellent osteoinductive, osteoconductive and osteogenic properties, but also some limitations such as graft availability, risk of infection, the possibility of morbidity of the donor site, sensitivity disturbances and unpredictable resorption rate (11, 12, 13). On the other hand, bone substitutes such as bovine bone and porcine bone, seem to fulfill many of the properties of autologous bone (e.g. remarkable osteoconductivity and ability to allow revascularization) without having its disadvantages (14, 32), thereby gaining clinicians’ attention. Very interesting results have been obtained also with a mix of autologous bone and bone substitutes in different ratio in sinus augmentation procedures (15). Moreover, in the literature, the need of a membrane as a barrier over the osteotomy window is still controversial and difficult to analyze due to the different protocols used in the studies. Some authors described a better implant survival rate in patients whose osteotomy windows were covered by a membrane as compared to patients whose lateral wall defects were left uncovered (16), whilst other studies did not find such evidence reporting that implant survival rate is influenced by many factors, but not by the presence of a membrane lying over the lateral window (17). In addition, according to some authors, the presence of a membrane covering the access to the maxillary sinus would guarantee a better healing, especially in terms of higher percentage of trabecular bone volume (18), higher vital bone formation (19) and prevention of soft tissue encleftation (20). Conversely, there is some evidence claiming that the use of membrane does not improve implant survival rate and mean mineralized volume, but causes a decrease in mean osteoid volume (21). Finally,

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the possible exclusion of the vascular supply from the healing area is the main criticism arisen from the use of a membrane over the lateral window in maxillary sinus augmentation procedures.

Therefore, the aim of the present study was to investigate whether a resorbable membrane could be advantageously used for a better healing after sinus floor lift procedures via a lateral approach.

MATERIALs AND METHODS Patients who could benefit from a maxillary sinus augmentation procedure, who had a residual bone height under the maxillary sinus lower than 5 mm and who were 18 years or older and able to sign an informed consent form were eligible for inclusion in the trial. Patients were not included in the study if any of the following exclusion criteria were present: systemic medical contraindications to implant surgery; history of irradiation in the head and neck area; poor oral hygiene and motivation; uncontrolled diabetes; current pregnancy and lactation; acute or chronic pathologies of the maxillary sinuses; smoking more than 20 cigarettes per day.

Ethical committee of Versilia Hospital, Lido di Camaiore (Italy) approved the study. Patients who were referred to the Versilia Hospital from April 2009 to January 2010, were asked to participate in the present study. All patients received thorough explanations and were requested to fill a written informed consent form prior to being enrolled in the trial. After the informed consent was signed, all patients underwent at least one session of oral hygiene prior to the augmentation procedures to provide an oral environment more favorable to wound healing. Each case

was accurately evaluated examining diagnostic casts to assess the inter-arch relationship; moreover, panoramic radiographs and computed tomography were taken.

Maxillary sinuses were allocated to either a control (membrane) or test (no membrane) group using a computerized random allocation process. Only one maxillary sinus was elevated for each patient.

A computer generated restricted randomization list was created. Only one (PT) of the investigators, not involved in the selection and treatment of the patients, was aware of the randomization sequence and could have access to the randomization list. The randomized codes were enclosed in sequentially numbered, identical, opaque, and sealed envelopes. All patients received prophylactic antibiotic therapy of 2 g of amoxicillin (or clindamycin 600 mg if allergic to penicillins) and 4 mg dexamethasone 1 h before the augmentation procedure and continued to take the antibiotic postoperatively, 1 g amoxicillin (or 300 mg clindamycin) twice a day for 7 days. All patients rinsed for 1 min with chlorhexidine mouthwash 0.2% prior to the surgery (and twice a day for the following 3 weeks), and were treated under local anesthesia using lidocaine with adrenaline 1 : 50,000. All surgeries were undertaken by the surgeons (A.B. and U.C.) and their surgical teams.

All the patients were treated with the same surgical technique consisting of sinus floor augmentation via a lateral approach (22).

Briefly, a mucoperiosteal flap was elevated exposing the lateral bone wall of the maxillary sinus, a modification of the conventional lateral wall approach was used to perform the osteotomy to access the sinus membrane (23) (Fig. 1, 2). A bone scraper (Safe scraperÂŽ; Meta corp.

Reggio Emilia, Italy) was used to harvest autologous cortical bone and to reduce the lateral bone thickness,

fig. 1 Direct view of the osteotomy window in a patient belonging to the test group. fig. 2 Lateral bone wall at osteotomy. fig.3 Graft procedure.

fig. 4 Grafting. fig. 5 At the end of grafting. fig. 6 After placing the resorbable collagen membrane.

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Use of membrane vs. no membrane in maxillary sinus augmentation: a pilot study

allowing an easy access to the sinus membrane with ultrasound (Piezosurgery, Mectron, Genova, Italy).

Subsequently, large flat curettes were used to raise the sinus membrane exposing the sinus bone wall up to the medial wall. Once the sinus membranes were elevated, all the sinuses were grafted with a mixture of autogenous bone, harvested from the lateral bone wall, and collagenated corticocancellous porcine bone (MP3®; Osteobiol-Tecnoss, Coazze - TO, Italy) (Fig. 3, 4) in a 1:1 ratio. After maxillary sinus grafting, the randomization envelope was opened and indicated to the blindfolded surgeons to include the sinus as a test or a control site according to the randomization list. As a result, the treatment allocation was concealed to the investigators who were involved in enrolling and treating the patients.

Sinuses in the test group did not receive any membranes over the osteotomy window (Fig. 5), while sinuses in the control group were covered with a reabsorbable collagen membrane (Evolution®; Osteobiol-Tecnoss) (Fig. 6). The mucoperiosteal flaps were sutured with 3-0 reabsorbable sutures. Patients were instructed to continue with prophylactic antibiotic therapy, and naproxen sodium 550 mg tablets were prescribed as an anti-inflammatory to be taken twice a day as long as required. Removable prosthesis, if present, was not permitted for use until they had been adjusted and refitted no sooner than 3 weeks after surgery. Patients were instructed to avoid blowing their nose and advised to administer corticosteroids, nasal drops, three times a day in both nasal cavities for 4 weeks. Patients were seen 1 week after surgery for suture removal and thereafter for regular follow-up visits. After 6 months of graft healing, radiographic examinations (orthopantomography and CT scan) were taken to evaluate the outcome of the surgical procedure. Immediately prior to the implant placement, at least one bone biopsy from each augmented maxillary sinus was harvested from the lateral window, using a trephine bur with an inner diameter of 2 mm and an outer diameter of 3 mm. Lateral window was identified by the surgeon observing the healed area in comparison with the surrounding bone. After fixation, the bone samples were forwarded to the Institute of Biomedicine, Sahlgrenska Academy Gothenburg University (Sweden) for histological examination. After the retrieval, the functional implants were inserted in the augmented maxillary sinuses.

The following outcome evaluations were considered in this study.

1.

Surgical complications during maxillary sinus augmentation procedures, in particular, hemorrhage during lateral bone wall osteotomy or perforations of the sinus membrane.

2. Dimensions of osteotomy windows to access the sinuses were evaluated such as bony window length (L), bony window height (H), and lateral bone wall thickness (T).

3. Early or late postoperative complications such as

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wound dehiscence and acute/chronic sinusitis.

4. Histomorphometric parameters such as trabecular bone volume, soft tissues, and residual graft particles percentages.

Specimen processing and analysis

Specimens were decalcified in EDTA (15%) for a period of 2 weeks. Specimens were again X-rayed to verify the decalcification procedure. After dehydratation in graded series of ethanol, the specimens were embedded in paraffin, sectioned (3–5 μm sections), and stained with hematoxyline and eosine and modified Mallory aniline blue. Examinations were performed in a Nikon Eclipse 80i microscope (Teknooptik AB, Huddinge, Sweden) equipped with an easy image 2000 system (Teknooptik AB) using X1.0 to X40 objectives for descriptive evaluation and morphometrical measurements. Histomorphometric measurements were performed in order to calculate the percentages (i.e., area fraction) of mineralized bone, residual graft materials, and soft tissue components (i.e., connective tissue and/or bone marrow) 6 months after the sinus augmentation procedure. All measurements were determined by point counting directly in the light microscope, using an optically superimposed eyepiece test square grid (distance between 6 ¥ 6 test lines ¼ 255 mm) at a magnification of 160 X. The number of points of intersection between the test lines and the outlines of mineralized bone, bone substitute particles, and non mineralized tissue were recorded.

Statistical analysis

The Mann–Whitney nonparametric test was used for comparing the differences between the two groups.

Statistical significance was set at 5%.

RESULTS A total of 15 patients were assessed for eligibility for the study; 3 patients were excluded for not having met the inclusion criteria. Going into detail, one patient was being treated with oral bisphosphonate, another patient showed signs of chronic sinusitis, while the last one excluded presented a very thin lateral bone wall that would make the harvest of the bone impossible.

As a consequence, 12 patients, 4 females and 8 males with an average age of 59.1 years, were recruited and randomly allocated to the two study groups: 6 patients in the test group (no membrane) and 6 patients to the control group (with membrane) (Table 1). Only a maxillary sinus was augmented. None of the patients left the study for the following 6 months of follow-up, which means, in this case, the period of time from the augmentation surgery to implant placement. For each patient the healing phase was uneventful, except for a patient who presented a mild hematoma which resolved spontaneously.

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Assessed for eligibility (n=lS)

Excluded (n=3) - not meeting inclusion criteria (n=2) - other reason (n=1, lateral wall extremely thin)

Patients randomized (n=12)

Analysed patients belonging to the test group (n=6)

Analysed patients belonging to control group (n=6)

tabLE 1 Flow diagram showing the recruitment and randomization of patients.

The mean dimensions of the lateral window were similar in both groups. In the non-membrane group (test group) the mean height (H) was 10.0±1.3 mm, the mean length (L) was 18.1±1.6 mm while the mean bone wall thickness was 0.7±0.2 mm; in the membrane group (control) the mean height was 9.8±1.2 mm, the mean length was 17.7±1.8 mm while 0.6±0.5 mm was the mean bone wall thickness (Table 2).

As far as the histological examination is concerned, newly formed bone, connective tissue and osteoblasts were detected around graft particles in both groups (Fig. 7, 8). In addition, in the analyzed specimens the presence of osteoclasts close to the grafted material was observed.

Moreover the histomorphometric measures showed that in the membrane group, the mean amount of newly formed bone was 30.4±15.8% (median 28.2%), the connective tissue counted for a mean value of 50.3±18.9% (median 46.7%), while 18.2±20.4% (median 8.8%) was the amount of residual graft particles; on the other hand, in the non-membrane group, 28.0±19.5% (median 17.3%) was the mean amount of newly formed bone, the mean quantity of connective tissue was 59.2±15.6% (median 50.8%) and about residual graft particles a mean value of 12.8±12.6% (median 17.1%) was registered (Table 3). This data show that no significant difference was detected in the histomorphometrical evaluation between the two groups (p=0,85).

Clinical parameters Osteotomy window length(L) Osteotomy window height (H) Lateral bone wall width

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Test group (no membrane) (mean ± SD) 18.1 ± 1.6 mm 10.0 ± 1.3 mm 0.7 ± 0.2 mm

fig. 7 Histologic section of a biopsy taken from the control group 6 months after augmentation. Corticocancellous porcine bone particles are surrounded by woven bone. The marrow spaces are rich in cells and blood vessels. Magnification: bar = 100 µm

fig. 8 Histologic section of a biopsy taken from the test group 6 months after augmentation. Residual porcine bone particles were covered with new bone, showing ongoing bone formation, i.e., osteoblastic seams. The soft tissues were without signs of inflammation and showing a high density of newly formed vessels. Magnification: bar = 100 µm.

DISCUSSION The aim of this study was to assess whether covering the lateral window with a reabsorbable collagenous barrier membrane during augmentation of the maxillary sinus

Control Group (membrane) (mean ± SD) 17.7 ± 1.8 mm 9.8 ± 1.2 mm 0.6 ± 0.5 mm

tabLE 2 Clinical parameters (mean ±standard deviation) in test group and in control group.

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Histomorphometric measures Newly formed bone Connective Tissue Residual graft particles

Test Group (no membrane) (mean + SD, %) (median, %) 28.0 ± 19.5% (17.3%) 59.2 ± 15.6% (50.8%) 12.8 ± 12.6% (17.1%)

floor with a mixture of porcine bone and autologous bone in 1:1 ratio is beneficial for bone regeneration.

Considerable controversy exists regarding the use of membrane in terms of benefits to implant survival and treatment success. Some researchers found no differences in implant survival rates between membrane covered and uncovered groups(17,24), while others reported higher implant survival rates when the lateral walls is covered by a membrane(16,19,25). However, as stated previously, a direct comparison among different studies is extremely hard due to the multiplicity of used protocols.

Moreover, some authors stated that an unfavorable healing is obtained if a sinus lift surgery is performed without covering the lateral wall with a membrane(26).

Indeed, the main adverse consequence of non using a membrane is the graft particle displacement and the encleftation (i.e. the proliferation of connective tissue into the sinus cavity), which would hamper the new bone formation thereby ensuring a minor bone to implant contact due to a minor amount of osseointegration.

Our data showed that the mean amount of newly formed bone was 30.4±15.8% for the patients whose lateral wall was covered with a membrane, whilst in the nonmembrane group the mean amount of newly formed bone was 28.0±19.5%. As far as the amount of connective tissue is concerned, we found that in the membrane group it counted for a mean value of 50.3±18.9%, while in the test group the mean amount was 59.2±15.6%. Finally, about residual graft particles, we registered in the nonmembrane group a value of 12.8±12.6% and on the other hand in the membrane group a value of 18.2±20.4%.

These data are not statistically significant because of the small number of patients recruited but, despite it, they underline a trend which shows a little influence of the membrane upon the formation of new bone. However, the lack of a membrane seems to lead to a higher soft tissues penetration in the lateral bone defect, thereby facilitating a major resorption of the residual graft particles.

In addition, the results of the histomorphometric evaluation could be influenced by the harvest technique of the bone biopsy (i.e from the lateral bone wall or from the bone crest) (27) and this could explain the great heterogeneity of observed data in the literature.

Regarding the membrane, some authors compared the use of a resorbable collagen membrane with the use of a resorbable PRF membrane, finding no substantial differences in terms of better healing and/or amount of vital bone formation (27). A recent study in dogs

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Control Group (membrane) (mean + SD, %) (median, %) 30.4 ± 15.8% (28.2%) 50.3 ± 18.9% (46.7%) 18.2 ± 20.4% (8.8%)

tabLE 3 Histomorphometric measurements showing the mean values and variations in the test and the control group.

compared the effectiveness of a membrane employed in two different ways. In one study group, the membrane was placed over the lateral bone window while in the other group (experimental) it was placed at the areas of the lateral osteotomy window, extending over the apex of implants to the posterior bone wall. The authors found that 24 weeks after implant placement, in the experimental group the amount of lamellar bone had increased and the biomaterial particles were significantly fewer, claiming that the pressure of the Schneider’s membrane could play a key role in bone resorption (28).

Many papers analyzed the resorption of graft material, showing that an important amount of graft particles could be found in patients’ augmented maxillary sinus even after 11 years (29, 30). However, porcine-derived bone has a slightly higher resorption rate as confirmed by our data and by other studies (31, 33). This study found no difference in vital bone formation between membrane covered and uncovered group, contrasting with Tarnow et al. (19), who found a higher amount of vital bone in sites covered with membrane with respect to sites left uncovered. In our opinion, data emerging from this study can be explained in part with a lack of revascularization in the area of the membrane. In fact, branches of maxillary artery, which are the main source of blood for the maxillary bone, are included in the elevated flap and, during its repositioning, blood supply may not be able to reabsorb the grafted material; this could explain the higher value of residual graft particles found in the membrane group.

On the other hand, membrane could prevent soft tissues from leaking into the grafted area, allowing a major bone formation to occur without soft tissues interference.

CONCLUSION Although further studies with strict and accepted protocols are needed in order to improve our knowledge on bone regeneration and sinus lift augmentation, data emerging from this study show that the use of a membrane does not substantially improve the healing of the surgical area, yet highlighting a higher amount of graft particles resorption and amount of connective tissue in uncovered areas as compared to covered ones.

In the end, our study has several strengths such as a very rigorous protocol ensuring no bias during the randomization procedure, the results analysis and during the harvest of bone biopsies. This work has also some

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limitations such as paucity of sample size who could have been the reason of a low significance of our results.

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