JOB vol 1 N3 2010 by Paolo Trisi

ISSN: 2036-6795

Journal Journal of of Osteology Osteology and Biomaterials Biomaterials and The official Journal of Biomaterial Clinical and histological Research Association

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Journal of Osteology and Biomaterials The official journal of BioCRA - Biomaterial Clinical and histological Research Association President Giampiero Massei Deputy-president Alberto Rebaudi Editor in-chief Paolo Trisi, DDS PhD Scientfic director BioCRA, Pescara, Italy Associate Editor Gilberto Sammartino, MD DDS University of Naples Federico II, Naples, Italy Francesco Carinci, MD DMD University of Ferrara, Ferrara, Italy Assistant Editor Teocrito Carlesi, DDS Secretary BioCRA, Chieti, Italy Managing Editor Renato C. Barbacane, MD University G. d’Annunzio, Chieti, Italy

www.osteobiom.com

Scientific Director Paolo Trisi Secretary Teocrito Carlesi

Editorial Board

Roberto Abundo, Turin, Italy Mario Aimetti, Turin, Italy Moshe Ayalon, Hadera, Israel Luigi Ambrosio, Naples, Italy Massimo Balsamo, Thiene, Italy Francesco Benazzo, Pavia, Italy Ermanno Bonucci, Roma, Italy Mauro Bovi, Rome, Italy Maria Luisa Brandi, Firenze, Italy Paul W. Brown, Pennsylvania, USA Ranieri Cancedda, Genova, Italy Saverio Capodiferro, Bari, Italy Sergio Caputi, Chieti, Italy Chih-Hwa Chen, Keelung, Taiwan Joseph Choukroun, Nice, France Gabriela Ciapetti, Bologna, Italy Giuseppe Corrente, Turin, Italy Massimo Del Fabbro, Milan, Italy Marco Esposito, Manchester, UK Antonello Falco, Pescara, Italy Gianfranco Favia, Bari, Italy Paolo Filipponi, Umbertide, Italy Pier Maria Fornasari, Bologna, Italy Bruno Frediani, Siena, Italy Sergio Gandolfo, Turin, Italy David Garber, Atlanta, USA Zhimon Jacobson, Boston, USA Jack T Krauser, Boca Raton, USA Richard J. Lazzara, West Palm Beach, USA Lorenzo Lo Muzio, Foggia, Italy Gastone Marotti, Modena, Italy Christian T. Makary, Beirut, Lebanon

Gideon Mann, Jerusalem, Israel Ivan Martin, Basel, Switzerland Milena Mastrogiacomo, Genoa, Italy Anthony McGrath, Santmore, UK Alvaro Ordonez, Coral Gables, USA Zeev Ormianer, Tel-Aviv, Israel Carla Palumbo, Modena, Italy Sandro Palla, Zurich, Switzerland Ady Palti, Kraichtal, Germany Michele Paolantonio, Chieti, Italy Giorgio Perfetti, Chieti, Italy Adriano Piattelli, Chieti, Italy Domenique P. Pioletti, Lausanne, Switzerland Sergio Rosini, Pisa, Italy Ugo Ripamonti, Johannesburg, South Africa Henry Salama, Atlanta, USA Maurice Salama, Atlanta, USA Lucia Savarino, Bologna, Italy Arnaud Scherberich, Basel, Switzerland Nicola Marco Sforza, Bologna, Italy Christian FJ Stappert, New York, USA Marius Steigman, Neckargemünd, Germany Hiroshi Takayanagi, Tokyo, Japan Dennis Tarnow, San Francisco, USA Tiziano Testori, Milan, Italy Anna Teti, L’Aquila, Italy Oriana Trubiani, Chieti, Italy Alexander Veis, Thessaloniki, Greece Raffaele Volpi, Rome, Italy Giovanni Vozzi, Pisa, Italy Hom-Lay Wang, Michigan, USA Xuejun Wen, South Carolina, USA

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Journal of Osteology and Biomaterials

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Journal of Osteology and Biomaterials The official journal of BioCRA - Biomaterial Clinical and histological Research Association

contents

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Review articles

Static osteogenesis and dynamic osteogenesis: their relevance in dental bone implants and biomaterial osseointegration Gastone Marotti, Davide Zaffe, Marzia Ferretti, Carla Palumbo

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Original articles

Implant Stability Quotient (ISQ) vs direct in vitro measurement of primary stability (micromotion): effect of bone density and insertion torque Paolo Trisi, Teocrito Carlesi, Marco Colagiovanni, Giorgio Perfetti

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Novel injectable hydrogel scaffold for cartilage repair based on natural polymers

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Bone density evaluation after 5 years of implant rehabilitation in fibula free flap used for maxilla reconstruction

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Evaluation of bone resorption around implants inserted in calvaria autogenous bone grafts used for jaw reconstruction

Giorgio Mattei, Francesca Montemurro, Monica Mattioli-Belmonte, Giovanni Vozzi

Francesco Grecchi, Francesco Gallo, Giuseppe Rubino, Alessandro Motroni, Raffaella Bianco, Ilaria Zollino, Francesca Farinella, Ambra Girardi, Francesco Carinci

Roberto Cenzi, Laura Arduin, Ilaria Zollino, Claudia Casadio, Ambra Girardi, Francesca Farinella, Francesco Carinci

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Effect of implant-tooth distance on crestal bone resorption Matteo Danza, Ilaria Zollino, Anna Avantaggiato, Francesco Carinci

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Static osteogenesis and dynamic osteogenesis: their relevance in dental bone implants and biomaterial osseointegration Gastone Marotti, Davide Zaffe, Marzia Ferretti, Carla Palumbo*

The present report summarizes the results of a series of investigations carried out in our laboratory on intramembranous ossification occurring under normal conditions during skeletal organogenesis and osseointegrations of dental implants and biomaterials. No morphological differences were observed between normal and pathological conditions, since the same sequence of events were found. Inside the embryonic mesenchyme, or the connective tissue formed after bleeding due to surgery, cords of plum cells, displaying the typical osteoblastic structure, differentiate between the blood capillaries. These osteoblasts appear to be stationary since they do not move, but transform into osteocytes in the same site where they differentiated, thus giving origin to a trabecular woven-bone framework laid down by static osteogenesis (SO). Soon after, typical movable osteoblastic laminae differentiate along the surface of this SO-trabeculae and them thicken with lamellar bone formed by dynamic osteogenesis (DO). SO seems to depend on inductive stimuli and appears to be mechanically independent, whereas DO mainly depend on mechanical strains. Additionally, SO-bone is a bad quality bone because of its woven texture and high microporosity, due to the many osteocyte lacunae it contains, whereas DO-bone generally is a lamellar bone and thus mechanically much more resistant. The clinical implication of these findings with regards to the time of load application after prostheses/biomaterials implantation is discussed. (J Osteol Biomat 2010; 1:133-139)

Keywords: intramembranous ossification, static osteogenesis, dynamic osteogenesis, osseointegration, dental implants, biomaterials Department of Biomedical Sciences - Human Anatomy Section, University of Modena and Reggio Emilia Corresponding author: * Prof. Carla Palumbo Department of Biomedical Sciences - Human Anatomy Section University of Modena and Reggio Emilia Via del Pozzo 71- Area Policlinico 41100 MODENA Phone: +39.059.4224850Â ; Fax nÂ°: +39.059.4224861 e-mailÂ : carla.palumbo@unimore.it

INTRODUCTION In recent years remarkable progress has been made in bone dental implants with regards to surgical techniques and osteoinductive properties of different biomaterials. Notwithstanding the large numbers of investigations published in the literature and that of our knowledge has greatly improved in this field, the process of bone regeneration is not yet clearly defined, particularly as far as stages and timetable of formation of the different types of bone tissues (woven and lamellar bone) are concerned. According to our findings1 on normal bone histogenesis, intramembranous ossification takes place according to two subsequent stages of bone deposition that we respectively named static (SO) and dynamic osteogenesis (DO), because of the different behaviour of osteoblast activity. DO is the well known classical mechanism of bone formation; it depends on the secretory activity of monostratified osteoblastic laminae that move away from the osteogenic surface as osteoide secretion proceeds, and the osteocytes that remain entrapped within the preosseous matrix differentiate from the widening of the secretory territory of their parental osteoblasts2-6. On the contrary, SO, that we first described7,

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adjacent ones in the same cord, appears to be polarized in a different direction (Fig. 4A), contrary to what happens in the typical monostratified laminae where all osteoblasts are polarized in the same direction, i.e., towards the growing osteogenic surface. The most prominent feature of these osteoblasts is that, unlike those arranged in laminae, they do not move away from the osteogenic surface as bone matrix secretion proceeds, they are instead immobile elements. Thus, compared with the movable osteoblasts in the typical laminae (Fig. 5), the osteogenic cells in the cords are stationary osteoblasts since they directly transform into osteocytes at the same site where they differentiated (Fig. 4B): they secrete a preosseous matrix all around their cellular body that soon undergoes mineralization. The osteocytes to which they give origin are irregularly and tightly grouped inside confluent lacunae;

INTRAMEMBRANOUS OSSIFICATION Physiological conditions During normal body growth, intramembranous ossification occurs in the bones of the cranial vault, partly in clavicle and mandible and in perichondral centers of ossification surrounding the diaphysis of the cartilaginous models of long bones. In intramembranous ossification centers, the osteogenesis takes place in the highly cellular and

vascularized mesenchyme. The onset of ossification is morphologically characterized by the appearance of variously shaped (cuboidal, polygonal, globous) plump cells that differentiate at about midway between adjacent blood capillaries. The mean distance between the capillaries and the core of the bony trabeculae, where such plump cells differentiate, was found to be fairly constant (26-30mm) (Figs. 1-3). These cells soon take on the structure and ultrastructure of osteoblasts: they display a characteristic highly developed rough endoplasmic reticulum and a large Golgi apparatus. It is interesting to note that these osteoblasts were never found to form the typical monostratified laminae, usually observed along the osteogenic surface; they, instead, are irregularly arranged in cords of 2-3 layers of cells, connected to each other by gap junctions. Additionally, each osteoblast, with respect to the

Figure 1. Cross section at the mid-shaft level of the cartilaginous bud of a chick embryo tibia. The trabecular woven bone, surrounding the vessels, was laid down by SO; note that DO is not yet started (Light Microscopy-LM under ordinary light).

Figure 2. Cross section of the perichondral center of ossification in new born rabbit tibia. The arrows point two cords of stationary osteoblasts. The arrow-head indicates a lamina of movable osteoblast on the surface of an SO trabecula (LM under ordinary light).

is performed by unexpectedly stationary osteoblasts that transform into osteocytes in the same site where they differentiated, hence the term â&#x20AC;&#x153;staticâ&#x20AC;? osteogenesis. In the present review, the two above mentioned osteogenic stages of intramembranous ossification will be described under physiological conditions, as well as during bone regeneration around dental implants and different types of biomaterials.

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Figure 3: (A) Cords of stationary osteoblasts (arrows) differentiating on the tip of newly forming SO-trabeculae. (B) Laminae of movable osteoblasts (arrows) lining SO-trabeculae enclosing vascular capillaries. Note the high osteocyte cellularity inside the SO-trabecula below (LM under ordinary light).

Figure 4: Schematic drawings depicting the two stages sequentially occurring during intramembranous ossification: static osteogenesis (SO) and dynamic osteogenesis (DO). (A) Cords of stationary osteoblasts differentiated around 3 vascular capillaries; the different polarization of the osteoblasts is pointed by arrows. (B) Osteocytes, differentiated from stationary osteoblasts in (A), form a bony SO-trabecula. (C) Laminae of movable osteoblasts laying down by DO bone matrix on the surfaces of the SO trabecula. (D) As a result, the trabecular core, made up of mechanically low resistant woven bone, is reinforced by lamellar bone added on its surfaces (see text for further explanation).

they display a globous cell body and their short cytoplasmic processes (or dendrites), they symmetrically radiate all around their body, are functionally connected by simple contacts and gap junctions8. Briefly, the bone laid down during SO has the typical structure of woven bone. The further growth by SO of the intramembranous ossification centers proceeds following the same sequence of events: 1) differentiation of stationary osteoblasts irregularly arranged

in cords around the blood vessels, 2) secretion of preosseous matrix and in situ transformation of stationary osteoblasts into osteocytes, 3) mineralization of preosseous matrix and formation of very thin (10-15 mm thick) bony trabeculae usually containing no more than 2-3 layers of osteocytes. As these processes of SO are in progress at the periphery of the ossification centers, the compaction of the spaces surrounded by SO-trabeculae (the socalled primary Haversian spaces) takes

place by DO. Typical osteogenic laminae, made up of movable osteoblasts (whence the name of dynamic osteogenesis), all polarized in the same direction, differentiate along the surface of the trabeculae previously formed by stationary osteoblasts (Figs. 2, 3B, 4C). These movable osteoblastic laminae fill the primary Haversian spaces with primary Haversian systems (or primary osteons) by laying down concentric layers of lamellar bone. Briefly, the two osteogenic stages,

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we first showed to occur in sequence during intramembranous ossification, seem to have different functional meaning. SO, performed by stationary osteoblasts, allows the formation of a framework of trabecular woven bone, having the supporting function for the subsequent lamellar bone apposition by typical movable osteoblasts (Fig. 4D). Therefore, SO appears to be mainly devoted to the expansion of the ossification center and consequently to increasing bone size, whereas DO is mainly involved in bone compaction, i.e., in thickening the SO-trabeculae and consequently increasing their mechanical strength. Under the mechanical point of view, it is crucial to underline the differences in size, shape, distribution and density between the osteocytes in woven and those in lamellar bone9; in other words, between the osteocytes in SOtrabeculae and those in lamellar bone laid down by DO. Woven bone, compared with lamellar bone, consists of irregularly-arranged coarse bundles of collagen fibers and contains a higher number of globous or lentil shaped osteocytes, enclosed within larger lacunae, irregularly distributed in clusters (Fig. 6A). Under SEM the lacunae appear surrounded by a layer of loosely arranged collagen fibers. As a consequence, the areas where osteocytes are clustered have a loose collagen texture, whose microarchitecture looks like that of loose lamellae, whereas the matrix in between osteocytic areas contains thick bundles of densely-packed fibers arranged as in dense lamellae (see below). As shown by Marotti and coworkers,9-11 lamellar bone is made up of an orderly

Journal of Osteology and Biomaterials

Figure 5. Typical laminae of movable osteoblasts: (A) light microscopy (LM) under ordinary light; (B) transmission electron microscopy (TEM).

Figure 6. Scanning electron micrographs (SEM) of woven (A) and lamellar (B) human compact bone. The osteocytes are larger, more numerous and irregularly distributed in woven bone with respect to lamellar bone, where they are only located inside loose lamellae. Note that dense lamellae, alternating with the loose ones, are thinner and do not contain osteocyte lacunae.

alternation of collagen-rich (dense lamellae) and collagen-poor layers (loose lamellae) all having an highly interwoven collagen texture. Osteocytes have an almond-like shape and their lacunae are smaller and less numerous than those in woven bone. Since osteocytes were found to be enclosed in loose lamellae only, it was suggested that they should be recruited in successive groups, and that those pertaining to each group are distributed in a single plane, namely that corresponding to a loose lamella (Fig. 6B). On the basis of these findings, it was maintained that the difference in texture between woven and lamellar bone depends on the

distribution of osteocytes throughout the bone matrix, that is to say, in the manner of recruitment of the osteocyte-differentiating osteoblasts from the osteogenic laminae (for a more exhaustive account, see Marotti, 19969). To summarize, from the what has been said it clearly appears that the greater osteocytic microporosity, together with the coarse collagen texture and the high degree of mineralization, all account for the lower mechanical resistance and greater fragility of woven bone with respect to lamellar bone. In other words, during the 1st stage of intramembranous ossification, the woven bone laid down by static osteogen-

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Response of bone to dental implants and biomaterials Bone healing of injuries induced by dental implants or biomaterial application occurs by intramembranous ossification only; no cartilage and endochondral ossification are formed as appears in the literature and our studies. As is well known, two bone-to-implant contacts (BIC) are distinguished: a) primary, where the implant directly contacts the bone and later undergoes remodelling12,13; b) secondary, where the newly formed bone comes into contact with the prosthesis/biomaterial, lead-

ing to its osseointegration.13-15 Only the latter was analyzed in our studies. After the bleeding and inflammatory phases induced by surgery, the spaces between the bone and the implant, or biomaterials, where secondary osseointegration takes place, are filled with a highly cellular and vascularized fibrous connective tissue, similar to the embryonic mesenchyme. Afterwards, a typical static osteogenesis starts from the surrounding bone only (Figs. 7-10). Contrary to calcium-phosphate ceramic biomaterials, signs of osteogenesis were never observed to begin from metal implants, whatever their material and/or surface (smooth, rough, coated or uncoated).12-15 Cords of plum cells, displaying the typical osteoblastic structure, differentiate

Figure 7. Microradiograph of a titanium implant in a sheep mandible. Note in the squared area numerous SO-trabeculae growing from the preexistent bone.

Figure 8. Micrograph under LM ordinary light of the squared area in Fig. 7, showing SO-trabeculae of woven bone.

esis is a bad quality bone under a mechanical viewpoint and its resistance is improved during the 2nd stage by dynamic osteogenesis lamellar bone.

in between the blood capillaries and growth towards the implant/biomaterial.15-18 These osteoblasts appear to be stationary since they do not move, but transform into osteocytes in the same site where they differentiated, thus giving origin to a framework of trabecular woven-bone laid down by SO. Soon after, typical movable osteoblastic laminae differentiate along the surface of this SO-trabeculae and thicken them with lamellar bone laid down by DO. Briefly, it appears from our investigations that, at least under the morphological viewpoint, the process of bone healing recapitulates the same sequence of events occurring during bone histogenesis.

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CONCLUSIONS Intramembranous ossification independent of whether it occurs during normal bone histogenesis or bone repair is characherized by two subsequent stages of bone formation that we named static osteogenesis (SO) and dynamic osteogenesis (DO). We believe that different factors and signals are sequentially involved in the two types of osteogenesis. The first phase (SO) appears to be mechanically-independent, since no osteocytes (i.e. primary bone mechanosensors, as is now generally accepted) are present at its inception. SO seems rather to depend on inductive stimuli (cytokines, such as endothelin 1 or growth factors such as PDGF, VEGF, etc.) set free from the adjacent vessels and the bleeding. On the contrary the second phase (DO), appears to be driven by mechanical strains sensed by the osteocytes contained in SO-trabeculae. In relations to this it has been suggested that woven bone can form without pre-existing osteocytes, whereas lamellar bone can only be laid down on the surface of bone containing osteocyes.9 As reported here in the second section, the bone laid down during SO is a bad quality bone, under a mechanical point of view, because of its woven texture and high lacuno-canalicular microporosity. Such high density osteocytic lacunae in SO-bone depends on the fact that the osteocytes they contain differentiate from stationary osteoblasts tightly arranged in cords. The fact that inside the cords osteoblast movement is trivial, implies the formation of very narrow septa of disorderly interwoven collagen fibers in between the osteocytes. On the contrary, the bone laid

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graft graft

graft

Figure 9. Micrograph under LM ordinary light showing the initial stage of osseointegration around graft . The arrows point to SO-bone growing inside the connective tissue surrounding the biomaterial.

graft

Figure 10. Micrograph under LM ordinary light showing graft osseointegration. The area outlined by the dotted line corresponds to the core of the trabecula laid down by SO, made up of woven-bone with many globous osteocytes. The arrow points to the lamellar-bone subsequently laid down by DO.

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down by dynamic osteogenesis generally is of lamellar type and thus mechanically much more resistant. In conclusion, the first stages of bone healing is characterized by the deposition of a bad quality bone, due to inductive stimuli; thus loading appears to be useless or sometimes even dangerous during SO; this fact is empirically shown by clinical practice. On the contrary mechanical loading, which is known to greatly enhance osteoblast activity, becomes very important after DO starts. Therefore it becomes crucial in clinical practice to know how long SO goes on before DO starts, namely to establish when a bad quality bone is reinforced with a bone actually capable of resisting mechanical loading.

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of the mechanical properties of the bone surrounding dental implants. Biomaterials 2002; 23:9-17. 13. Zaffe D, Rodriguez y Baena R, Rizzo S, Brusotti C, Soncini M, Pietrabissa R, Cavani F, Quaglini V. Behavior of the bone-titanium interface after push-in testing: A morphological study. J Biomed Mater Res 2003; 64A:365-371. 14. Zaffe D, Bertoldi C, Consolo U. Accumulation of aluminum in lamellar bone after implantation of titanium plates, Ti-6Al-4 screws, hydroxyapatite granules. Biomaterials 2004; 25:3837-3844. 15. Zaffe D. Some considerations on biomaterials and bone Micron 2005; 36:583-592.

4. Palumbo C. A three-dimensional ultrastructural study of osteoid-osteocytes in the tibia of chick embryos. Cell Tissue Res 1986; 246:125-131.

16. Zaffe D, Leghissa GC, Pradelli J, Botticelli AR. Histological study on sinus lift grafting by Fisiograft and Bio-Oss J Mater Sci Mater Med 2005; 16:789-793.

5. Palumbo C, Palazzini S, Zaffe D, Marotti G. Osteocyte differentiation in the tibia of newborn rabbit: an ultrastructural study of the formation of cytoplasmic processes. Acta Anat 1990; 137:350-358.

17. Consolo U, Zaffe D, Bertoldi C, Ceccherelli G. PRP activity on maxillary sinus floor augmentation by autologous bone. Clin Oral Implant Res 2007; 18:252-262.

6. Palumbo C, Palazzini S, Marotti G. Morphological study of intercellular junctions durino osteocyte differentiation. Bone 1990; 11:401-406.

18. Bertoldi C., Zaffe D., Consolo U. Polylactide/polyglycolide copolymer in bone defect healing in humans. Biomaterials 2008; 29:1817-1823.

7. Marotti G, Ferretti M, Palumbo C, Benincasa M. Static and dynamic bone formation and the mechanism of collagen fiber orientation. Bone 1999; 25:156. 8. Palumbo C, Ferretti M, Marotti G. Osteocyte dendrogenesis in static and dynamic bone formation: an ultrastructural study. Anat Rec 2004; 278A:474-480. 9. Marotti G. The structure of bone tissues and the cellular control of their deposition. It J Anat Embryol 1996, 101(4):25-79. 10. Marotti G. A new theory of bone lamellation. Calcif Tissue Int 1993; 53(1):S47-S56. 11. Marotti G, Muglia MA, Palumbo C. Structure and function of lamellar bone. Clin Rheumatol 1994; 13(1):63-68. 12. Soncini M, Rodriguez-y-Baena R, Pietrabissa R, Quaglini V, Rizzo S, Zaffe D. Experimental procedure for the evaluation

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Implant Stability Quotient (ISQ) vs direct in vitro measurement of primary stability (micromotion): effect of bone density and insertion torque Paolo Trisi PhD1,2,Teocrito Carlesi DDS1, Marco ColagiovanniDDS3 , Giorgio Perfetti MD, DDS3

Objectives: Measuring peak insertion torque in relation to different bone densities, the present study seeks to determine whether micromotion at the boneimplant interface is related to the ISQ values. Materials and methods: A total of 30 Tixos Implants (Leader SRL, Cinisello B., Milan, Italy) were used. Implants were placed in fresh bovine bone samples representing three density categories: hard, normal and soft (H-N-S). Customized electronic equipment connected to a PC was used to register the peak and insertion torque data. A loading device, consisting of a digital force gauge and a digital micrometer was used to measure the micromovements of the implant during the application of 25 N lateral forces. Resonance Frequency Analysis was calculated using the “Osstell ISQ”, the latest version of the Osstell instruments, and the values were recorded in ISQ units. The data were analyzed for statistical significance by Spearman’s rank correlation coefficient tests. Results: Correlation coefficient showed a high dependency between the observed micromovement and ISQ values (ρ=-0.72 r2=0.52 P=< 0.0001). This correlation was found in all the types of bone, in the Hard bone ρ=-0.89 r2=0.80 P=0.0002, in the Normal bone ρ=-0.91 r2=0.82 P=0.0007 and in the Soft bone ρ=-0.73 r2=0.53 P=0.016 where the correlation was less powerful. The statistical analysis showed significant correlation between ISQ and torque-in (ρ=0.38 r2=0.14 P=0.0394) and between Torque-in values and micromotion (ρ= -0.49 r2=0.24 P=< 0.0059). Conclusions: Results showed a high dependence between the observed micromotion and the ISQ values, indicating that micromotion decreased with increasing ISQ values. Contrarily, increasing the peak insertion torque increased the ISQ values. (J Osteol Biomat 2010; 1:141-151)

Key Words: bone density, dental implant, immediate loading, insertion torque, micromotion, primary stability, ISQ. Bio.C.R.A.,Scientific Director of the Biomaterial Clinical and histological Research Association, Pescara, Italy; 2 Laboratory of Biomaterials and Biomechanics, Galeazzi Orthopaedic Institute, University of Milano, Italy; 3 Department of Oral Surgery, University of Chieti-Pescara, Italy; 1

Corresponding author: *Teocrito Carlesi DDS; Biomaterial Clinical and histological Research Association, Via Silvio Pellico 68, 65132 Pescara, Italy. Tel:+39 085 28432; Fax: +39 085 28427; e-mail: t.carlesi@email.it

INTRODUCTION Over the past few decades, implant rehabilitation has attracted increasing attention in dentistry as a result of improved success rates reported in the literature. More recently, the possibility of immediate functional loading of implants has been explored with particular success for the anterior mandible and with lesser success for the upper jaw and posterior mandible1. The cause of failure in these cases has not been attributed to immediate loading itself, but rather to the micromotion at the interface induced by the immediate loading, which, in turn, could ultimately be responsible for the failure of osseointegration of immediately loaded implants2. These same mechanisms are thought to be responsible for the failures of fracture healing according to the strain theory3. Implant stability depends on the direct mechanical connection between its surface and the surrounding bone and can be divided into primary and secondary stability. Classically, the clinical parameter relative to micromotion is ‘primary stability,’ which has been defined as “a sufficiently strong initial bone–implant fixation”4. Primary stability is achieved when the implant is positioned into the host bone site such that it is well seated. The success of this adaptation,

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Figure 1. Implant with a mount-transfer used for the micromotion analysis.

Figure 2. Digital torque wrench.

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however, depends on several factors, including the density and dimension of the host bone, the implant geometry and the surgical technique used. Secondary stability is attained when new bone forms at the implant interface. Given the importance of implant stability, it appears obvious that every implantologistâ&#x20AC;&#x2122;s normal instrumentation should include a tool to measure the stability of the implant. Recently, new methods were developed to non-destructively measure the implant stability, there are literature reports on percussion tests, radiographs (BMD), cutting resistance, torque-in, impact hammer method (Periotest) and even resonance frequency analysis (RFA).5 Among these, the RFA is the most used in experimental studies, as well as in clinical practice, but many aspects still need to be clarified. Resonance Frequency Analysis developed by Meredith et al. 6 uses specified resonance characteristics of acoustically excited implants and utilises a small L-shaped transducer which is screwed onto an implant fixture or abutment. The transducer comprises 2 piezoceramic elements, one of which vibrates by a sinusoidal signal (5 to 15 kHz). The other serves as a receptor for the signal. Resonance peaks from the received signal indicate the first flexural (bending) resonance frequency of the measured object. The specific value that indicates the implant stability of a given situation is called the resonance frequency.6,7 In vitro and in vivo studies have suggested that this resonance peak may be used to assess implant stability in a quantitative manner.8,9 In the first European Osseointegration

Association Consensus Conference held in 2006 10, some authors sustained that a single measure using RFA does not define the characteristics of the bone-implant interface and does not offer any reliable quantitative evaluation of the degree of osseointegration. Not only that, but the RFA would not have any prognostic validity on the development of the instability. These authors assert that the validity and reliability of RFA, from a clinical point of view, still remain to be demonstrated, for every implant system, such as the ISQ values which indicate the stability or the risk of loss of stability of the precise implant system. Research evidences suggest that elevated values of ISQ in a specific implant indicates that the implant is stable, and if ISQ values remain high the stability is maintained; while low values of ISQ, or a lowering of the values with time would indicate risk of instability of the implant.11 However, there are still many aspects that need clarification. Although these instruments are widely used and appear in the scientific literature, the ISQ values were never directly compared to implant micromotion.12 In the present study, the micromovement of implants inserted in freshly slaughtered bovine bone samples of different densities was measured using a previously published experimental model13 and compared to the ISQ values to evaluate its statistical relationship.

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Figure 3. Osstell ISQ Instrument.

Figure. 4. Schematic drawing of the micromotion-testing tool. The bone specimen is located in the middle with the implant in place. On the right side, the digital force gauge is powered on the implant long abutment, and on the left side, the micrometer reveals the movement of the implant.

MATERIALS AND METHODS The test was performed on 2cm X 2cm samples of fresh humid bovine bone representative of the following quality categories: Hard, Normal and Soft (H-N-S). The bone qualities were selected according to (1) drilling resistance14 and (2) a preliminary histological analysis of the bone structure. Hard bone is dense with a completely compact structure. Normal bone is average hard bone with a 2–3mm cortical layer and a normal cancellous structure inside. Soft bone has low drilling resistance and a 1mm cortical layer with a low-density cancellous structure. Tixos implants (Leader SRL, Cinisello B.mo, Milan, Italy) 3.3mm in diameter and 11.5 mm in length were specifically utilized for this study. Each implant was fitted with a mount-transfer of 11mm in length to allow for the application of the lateral load. (Fig.1) The implants were placed according to the manufacturer’s instructions using the appropriate burs. A customized digitally

controlled hand wrench was used to measure the peak insertion torque. In addition, electronic equipment consisting of a digital handoperated torque wrench (Fig.2), equipped with a calibrated strain gauge and connected to a PC reading the peak insertion torque value every 0.5 ms, was customized for this study. To obtain the peak insertion torque, the signal was subsequently evaluated by the MECODAREC software (ATech s.r.l., Bergamo, Italy). After implant placement, the smart peg type 32 was screwed onto the implant (Integration Diagnostics AB, Göteborg, Sweden) and ISQ was measured using the new “Osstell ISQ” device. (Fig. 3) After ISQ was measured, the bone blocks were fixed on a customized loading device for evaluation of micromovement (Fig. 4). This device consists of a digital force gauge [Akku Force Cadet (range of 0–90N and accuracy of 0.5%), Ametek, Largo, FL, USA] and, on the opposite side, a digital micrometer (Mitutoyo Digimatic Micrometer,

Kawasaki, Japan) that detects the micromovements of the implant under lateral load application, as previously published 13. Horizontal forces of 25 N/ mm were tested on each implant, and the lateral movement of the mountingdevice was measured by the digital micrometer 10 mm above the crest. On each implant, the load application was repeated five times for 2 s, simulating the occlusal load in a patient’s mouth. The average value of these five measurements was calculated for each implant. A total of 30 implants were tested in groups of 10 implants, in each bone quality including hard, normal and soft as defined above. The linear Pearson coefficient of correlation (ρ) was applied to test the relationship between the micromotion vs. ISQ values and between the torque-in vs. micromotion. To the normality test which evaluates the deviation from the Gaussian distribution, was applied the D’Agostino and Pearson test. The one-way non-para-

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N° Sample Torque In (N Cm) ISQ (V-D) Microm (µm) Bone type H H 1 150 68 130 H2 120 75 56 H3 113 77,5 44 H4 58 77,5 42 H5 46 77,5 67 H6 110 78 39 H7 137 72 70 H8 100 73,5 58 H9 50 70 88 H10 89 76,5 47 H11 128 75 55 Mean ± SD 100.09 ± 35.53 74.59 ± 3.37 63.27 ± 26.36 Table 1. The values of Torque-in (N cm), values of RFA (ISQ) and micromotion (µm) of implants loaded with 25N lateral force for each implant at the time of the placement in relation to the Hard bone.

N° Sample Bone type N M1 M2 M3 M4 M5 M6 M7 M8 M9 Mean ± SD

Torque In (N Cm) ISQ (V-D) 50 72 64 73 10 67,5 20 69 140 76 113 76,5 76 73,5 125 76 68 75,5 74.00 ± 45.00 73.22 ± 3.23

Microm (µm ) 120 100 384 250 77 88 100 83 92 143.77 ± 104.48

Table 2. The values of Torque-in (N cm), values of RFA (ISQ) and micromotion (µm) of implants loaded with 25N lateral force for each implant at the time of the placement in relation to the Normal bone.

N° Sample Bone type S S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Mean ± SD

Torque In (N Cm) ISQ (V-D) 36 68,5 62 74 47 73,5 46 73 27 71 32 71,5 40 73 46 74 38 74,5 46 75 42.00 ± 9.74 72.8 ± 1.96

Microm (µm ) 130 49 60 80 148 146 95 83 96 80 96.70 ± 34.22

Table 3. The values of Torque-in (N cm), values of RFA (ISQ) and micromotion (µm) of implants loaded with 25N lateral force for each implant at the time of the placement in relation to the soft bone.

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metric Kruskal-Wallis test was used to verify if there were difference in the mean of the different bone densities groups, in term of ISQ values, torquein values and micromotion and the Dunn’s Multiple Comparison Test was used to verify the different between each group. RESULTS 11 implants were placed in the group of Hard bone, 9 implants in Normal bone and 10 implants in Soft bone. All data are reported in Tables 1-2-3. The D’Agostino and Pearson normality test showed the data were normally distributed. The linear Pearson Coefficient of correlation between all micromotion data and the relative ISQ values was ρ=-0.72 and r2=0.52, with a P=< 0.0001, statistically highly significant. (Fig. 5). When the correlation was plotted for each different type of bone, again these variables were significantly correlated. The correlation in Hard bone between micromotion and ISQ was highly significant (ρ=-0.89, r2=0.80, P=0.0002) (Fig. 6). In Normal bone the correlation was highly significant too (ρ=-0.91, r2=0.82, P=0.0007) (Fig. 7), as well as in Soft bone (ρ=-0.73, r2=0.53, P=0.016) (Fig.8). Looking at the data, it was possible to note that the distribution in the Soft bone group was more scattered with more outliers, while in the Medium and Hard bone the data distribution was more linear with few outliers. When correlating the insertion torque to the ISQ values, the linear Pearson Coefficient of correlation showed a less strong correlation (ρ=0.38, r2=0.14,

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ISQ vs. Micromotion

Figure 5. Regression analysis and Spearman’s rank correlation coefficient shows a correlation between the micromotion values and the ISQ values indicating that micromotion decreased with increasing ISQ values The linear Pearson Coefficient of correlation (ρ) and (r2) values for the all values of Torque-In and ISQ was ρ=-0.72 r2=0.52 P=< 0.0001

Figure 6. In the Hard bone, regression analysis and Spearman’s rank correlation coefficient shows a high dependence between all values of the micromotion and the ISQ values, indicating that micromotion decreased with increasing ISQ values ρ=-0.89 r2=0.80 P=0.0002; the variables are correlated and test is statistically significant.

Figure 7. In the Hard bone, regression analysis and Spearman’s rank correlation coefficient shows a high dependence between all values of the micromotion and the ISQ values, indicating that micromotion decreased with increasing ISQ values ρ=-0.91 r2=0.82 P=0.0007; the variables are correlated and test is statistically significant.

Figure 8. In the soft bone, regression analysis and Spearman’s rank correlation coefficient shows a high dependence between all values of the micromotion and the ISQ values, indicating that micromotion decreased with increasing ISQ values ρ=-0.73 r2=0.53 P=0.016; the variables are correlated and test is statistically significant.

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Figure 9. Regression analysis and Spearman’s rank correlation coefficient showed a correlation between the torque-in values and the ISQ values indicating that torque-in increases with increasing ISQ values. The linear Pearson Coefficient of correlation (ρ) and (r2) values for all the values of Torque-In and ISQ was ρ=0.38 r2=0.14 P=0.0394.

P=0.0394), even if it was statistically significant (Fig.9). When correlating the insertion torque to the micromotion, the linear Pearson Coefficient of correlation showed also a good correlation (ρ= -0.49, r2=0.24, P=< 0.0059) (Fig.10). The Anova test demonstrated that the ISQ data from the different bone density groups were not statistically significant (fig.11), but the micromotion and torque-in values between the different groups were statistically different (p<0.001). DISCUSSION It was suggested that the success of immediate loading on implants is not related to either immediate or delayed loading15,16, but a critical micromotion threshold could be responsible for the peri-implant bone loss. For this reason, recently, primary stability has gained

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Figure 10. Regression analysis and Spearman’s rank correlation coefficient showed a high dependence between the torque-in values and the micromotion values indicating that torque-in increases with decreasing micromotion values. The linear Pearson Coefficient of correlation (ρ) and (r2) values for all the values of Torque-In and micromotion (Test-3) was ρ= -0.49 r2=0.24 P=< 0.0059.

more and more interest in the scientific and clinical world of dental implantology. Primary stability is nothing more than the absence of micromotion immediately after implant placement. Unfortunately, nowadays there is no instrument which can directly measure the amount of micromotion in the mouth of the patients. The RFA (resonance frequency analysis) is not a direct measure of the primary stability, but it measures the stiffness of the structure connected to the instrument. Despite the fact that RFA is today the most used method to measure primary stability in experimental studies, as well as in clinical practice, it has never been tested in direct relation to micromotion. To our knowledge the present study is the first that compares the ISQ value to the implant micromotion, which is the only direct measurement of the primary stability.

The results of the present study indicate that the ISQ value is related to the amount of implant micromotion with a statistically significant correlation. When combining the samples from all the bone density groups together, the r2 measured 0.52, meaning that 52% of the variance in the ISQ values could be explained by variations in the micromotion. When the same analysis was plotted for the Hard and Normal Bone groups separately, this r2 value raised up to 80% (H group r2=0.80 and in N group r2= 0.82), while in the Soft bone group this chance was much lower (53%, r2=0.53). This means that the “Osstell ISQ” is more suitable for analyzing the primary stability in Hard and Medium bone than in Soft bone. In addition, when comparing ISQ from the different groups of bone density, the average values were not statistically different,

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while the mean values of micromotion and insertion torque were statistically different between these groups. Looking at figure 5 it is possible to observe how, in many cases, the same ISQ values correspond to different amount of micromotion (Fig. 5). One possible explanation of these results, is the fact that when loading an implant which is not integrated, as in the present case, it is possible that in our model, there is not only a shift between the implant and the bone itself, but also an elastic movement of the bone itself. This elasticity is different between different bone density and this could explain the different sensitivity of the ISQ between the different bone densities. Another option could be the hypothesis that the RFA is more sensitive to the amount of cortical bone anchorage than of the trabecular bone, as suggested by other studies.17-20 In a study by Trisi et al. a correlation was found between the values of ISQ and the number of threads in contact with the compact bone, both on the crest and along the whole bone-implant interface; in some samples compact bone was found on the apex or along the lateral surface.17 Similar results were found in a experimental study in cadaver jaws18 in which the histomorphometric analysis was performed at the time of implant placement. The authors found that the BMD values, TBPf (trabecular bone pattern factor), BV/TV (density of trabecular bone) were not related to ISQ; while the BIC measured on the lingual aspect of the implants was positively correlated with ISQ values, and the correlation increased only when implants were in contact with

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Figure 11. The Anova test demonstrated that the ISQ data from the different bone density groups were not statistically significant (fig.11), but the micromotion and torque-in values between the different groups were statistically different (p<0.001)

the cortical bone. A similar positive correlation (the height of the cortical passage implants vs ISQ) was found by Miyamoto et al.19, who measured digitally (Computed Tomography) the thickness of the cortical bone at the implant sites (mesial and distal). In another human study a micro-CT was used for measurements of the BVD (bone volume density) and BCT (bone trabecular connectivity) of the implant site before the insertion of the implant, no significant correlation was found with values of ISQ.20 The results of the present study support the hypotheses that RFA is more sensitive to the rigidity of the boneimplant complex within the compact bone than in cancellous bone. Measuring the primary stability of an implant being subject to immediate load is of utmost important since it has been shown that micromotion in the

healing phase may be detrimental to the interfacial bone. In the past, it was generally agreed that implant interface failure was a consequence of bone resorption due to excessive load.3 In contrast to this hypothesis, a series of experiments have been conducted where both the displacement and the load were controlled, and it was clear that the resorption was induced by instability, even when only small loads were applied.3,21,22 These experiments showed that, in cortical bone, a displacement of only a few micrometers at the bone interface can induce resorption of the bone surface. This resorption process increases the distance between the mobile surfaces, thus placing deformation or ‘strain’ on regenerating tissues.3,22 The basic working hypothesis of this ‘strain’ theory3 is that, when bone segments are tightly compressed, leaving only a small gap

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between them, then almost no movement should be allowed at their interface. Otherwise, even movements in the micrometer range could induce a stretch, or a strain, that could destroy the new cells and vessels forming in the gap. In such a case, osteoclasts enter the gap and begin to reabsorb bone in order to increase the space over the critical threshold of strain of the regenerated tissue. A similar mechanism can be hypothesized to be involved in the failure of immediately loaded implants. Previous animal studies reported a micromotion above the range fo 50-100 micrometers could induce bone resorption at the interface, thus producing fibrosis and ultimately failures of immediately loaded endosseous implants. 16,23,24 Human studies assert a correlation between the values of RFA (ISQ) measured at the time of implant placement and the values of insertion torque25-27. In other studies, this correlation was confirmed for Hounsfield values of the implant site calculated using TC28-29. Currently, 2 RFA machines are in clinical use: the Osstelltm device (Integration Diagnostics AB, Göteborg, Sweden) and Implomates (Bio Tech One,Taipei,Taiwan). Osstell combines the transducer, computerized analysis and the excitation source into one machine closely resembling the model used by Meredith. In the early studies, the Hertz signal was used as a measurement unit. 6-9,30 Later, Osstell created the implant stability quotient (ISQ) as a measurement unit in place of Hertz. Resonance frequency values ranging from 3500 to 8500 Hz are translated into an ISQ of 0 to 100, where a high

Journal of Osteology and Biomaterials

value indicates greater stability, whereas a low value implies instability. The manufacturer’s guidelines suggest that a successful implant typically has an ISQ greater than 65. An ISQ < 50 may indicate potential failure or increased risk of failure.31 Only very wide ranges are hypothesized since there are many variables that come into play. Literature data help identify the numerous factors that can influence such measurements, as for example, the characteristics of the bone tissue (density and quality), mono and bicortical anchoring of the implant,32 the inclination of the transducer,33 the effective length of the implant above the bone crest, the diameter of the implant, the micro and macro geometry of the implant 34. The results of the present study showed that the ISQ values is statistically related to the micromotion and to the insertion torque values, indicating that ISQ increases with decreasing micromotion values and with increasing torque-in values. There are, however, literature reports that demonstrate a lack of correlation between the Torque-In test and ISQ values measured at implant insertion on cadavers18,35 in humans27,36,37 and in dogs38. In the first study27 the cutting-torque at the crest (first third of implant insertion) was related to the ISQ, while the overall insertion torque values was not related to the ISQ. The peak insertion torque measures the maximum torque of insertion obtained during implant placement until it is totally lodged in its site. Such a procedure may be influenced by the preparation of host bone sites, the bone density and the type of implant

(self-tapping or not, cone-shaped or cylindrical, surface roughness). Trisi et al. found that high insertion torque values correspond to a high degree of primary stability of an implant, and increasing the peak insertion torque reduces the level of implant micromotion.13 In addition, micromotion in soft bone was found to be consistently high and in the soft bone it was not possible to achieve more than 35 N/cm of peak insertion torque, when placing standard cylindrical screw type implants with a blasted surface. As well, in the present study the correlation between torque-in values and the implant micromobility values was also demonstrated, but as opposed to the Trisi et al. study13 it was possible to attain peak insertion torque values in soft bone, up to 62 Ncm. This could be explained by the fact that the implant surface used in the present study is a laser-microfused titanium powder one with a much higher surface roughness able to increase the grip during implant insertion. It was also shown that insertion torque values are correlated to bone mineral density (BMD) of the receiving bone site, obtained by measuring TC or micro TC39-41, or by the sensitivity of the operator during the preparation of the surgical site14. Such measurements have therefore been considered valid instruments for the determination of the quality of the implant site, and can foresee good primary stability of the implant. It must be pointed out that in the present study the ISQ was measured using the latest version of the machine, i.e. the new “Ostell ISQ” instrument, which is claimed to be less sensitive to

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electromagnetic noise. This fact should be considered when evaluating the results of the present study in comparison to previously published papers. In conclusion, the present study shows an inverse correlation between the ISQ and micromotion, demonstrating that it is able to understand if an implant is more stable than another. Nevertheless similar values of ISQ were found for quite different micromotions and for this reason the ISQ value cannot be taken as an absolute substitute of the micromobility of an implant. It must be underlined that the present is an in vitro study and the results cannot be directly transferred to clinical applications. Specific clinical follow-up studies are necessary to confirm the hypotheses that a certain amount of micromotion could be responsible for implant failures under immediate loading conditions.

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ACKNOWLEDGEMENTS: The authors wish to thank Novaxa Leader Italia for providing the implants used in this study, and Osstell AB for providing the Ostell ISQ. The study was funded by the Biomaterial Clinical Research Association (Bio.C.R.A.).

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healing in the rabbit tibia. Clin Oral Implant Res 1997;8:234–243. 10. Aparicio C, Lang NP, Rangert B. Validity and clinical significance of biomechanical testing of implant/bone interface. Clin Oral Impl Res 2006;17 (supp.):2–7.

2. Szmukler-Moncler S, Salama H, Reingewirtz Y, Dubruille JH. Timing of loading and effect of micromotion on bonedental implant interface: a review of experimental literature. J Biomed Mater Res 1998;43:192–203.

11. Glauser R, Sennerby L, Meredith N, Rée A, Lundgren AK, Gottlow J, Hämmerle CH. Resonance frequency analysis of implants subjected to immediate or early functional occlusal loading. Successful vs. failing implants. Clin Oral Impl Res 2004;15:428–434.

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4. Roberts WE. Bone dynamics of osseointegration, ankylosis, and tooth movement. J Indiana Dent Assoc 1999;78:24–32. 5. Atsumi M, Park SH, Wang HL. Methods used to assess implant stability: current status. Int J Oral Maxillofac Implants 2007;22:743-754. 6. Meredith N, Alleyne D, Cawley P. Quantitative determination of the stability of the implant-tissue interface using resonance frequency analysis. Clin Oral Implants Res 1996;7:261–267. 7. Glauser R, Meredith N. Diagnostic possibility for the evaluation of the implant stability (in German). Implantologie 2001;9:147–159. 8. Meredith N, Book K, Friberg B, Jemt T, Sennerby L. Resonance frequency measurements of implant stability in vivo. A cross-sectional and longitudinal study of resonance frequency measurements on implants in the edentulous and partially dentate maxilla. Clin Oral Implant Res 1997;8:226-233. 9. Meredith N, Shagaldi F, Alleyne D, Sennerby L, Cawley P. The application of resonance frequency measurements to study the stability of titanium implants during

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13. Trisi P, Perfetti G, Baldoni E, Berardi D, Colagiovanni M, Scogna G. Implant micromotion is related to peak insertion torque and bone density. Clin Oral Impl Res. 20, 2009; 467–471. 14. Trisi P, Rao W. Bone classification: clinical-histomorphometric comparison. Clin Oral Implants Res 1999;10: 1–7. 15. Brunski JB. In vivo bone response to biomechanical loading at the bone/dental– implant interface. Adv Dent Res 1999;13: 99– 119. 16. Szmukler-Moncler S, Piattelli A, Favero GA, Dubruille JH. Considerations preliminary to the application of early and immediate loading protocols in dental mplantology. Clin Oral Implants Res 2000; 11: 12–25. 17. Trisi P, Carlesi T, Rocci M, Rocci A. ISQ (RFA) vs BIC and torque: a histomorphometric and biomechanical analysis in humans. J Osteol Biomat 2010; 1:81-91.

of surgery-clinical, prospective, biomechanical, and imaging study. Bone 2005;37:776– 780. 20. Huwiler MA, Pjetursson BE, Bosshardt DD, et al. Resonance frequency analysis in relation to jawbone characteristics and during early healing of implant installation. Clin Oral Impl Res 2007;18(3):275-280. 21. Ganz R, Perren SM, Ruter A. Mechanical induction of bone resorption. Fortschr Kiefer Gesichtschir 1975:19: 45–48. 22. Hente R, Lechner J, Fuechtmeier B, Schlegel U, Perren SM. Der Einfluss einer zeitlich limitierten kontrollierten Bewegung auf die Fraktureilung. Hefte Unfallchirurg 2001: 283: 23–24. 23. Soballe K, Brockstedt-Rasmussen H, Hansen ES, Bunger C. Hydroxyapatite coating modifies implant membrane formation. Controlled micromotion studied in dogs. Acta Orthop Scand 1992: 63: 128–140. 24. Soballe K, Hansen ES, Brockstedt-Rasmussen H, Bunger C. Hydroxyapatite coating converts fibrous tissue to bone around loaded implants. J Bone Joint Surg Br. 1993: 75: 270–278. 25. Turkyilmaz I. A comparison between insertion torque and resonance frequency in the assessment of torque capacity and primary stability of Branemark system implants. J Oral Rehabil 2006;33:754-759. 26. Alsaadi G, Quirynen M, Michiels K, Jacobs R, van Steenberghe D. A biomechanical assessment of the relation between the oral implant stability at insertion and subjective bone quality assessment. J Clin Periodontol 2007;34:359–366.

18. Nkenke E, Hahn M, Weinzierl K, et al. Implant stability and histomorphometry: a correlation study in human cadavers using stepped cylinder implants. Clin Oral Implants Res 2003 ;14:601–609.

27. Friberg B, Sennerby L, Meredith N, Lekholm U. A comparison between cutting torque and resonance frequency measurements of maxillary implants. A 20- months clinical study. Int J Oral Maxillofac Surg 1999;28:297–303.

19. Miyamoto I, Tsuboi Y, Wada E, et al. Influence of cortical bone thickness and implant length on implant stability at the time

28. Turkyilmaz I, Tözüm TF, Tumer C, Ozbek EN. Assessment of correlation between computerized tomography values of the

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bone, and maximum torque and resonance frequency values at dental implant placement. J Oral Rehabil 2006;33:881-888. 29. Turkyilmaz I, Tumer C, Ozbek EN, Tozum TF. Relations between the bone density values from computerized tomography, and implant stability parameters: a clinical study of 230 regular platform implants. J Clin Periodontol 2007;34:716–722. 30. Meredith N. Assesment of implant stability as a prognostic determinant. Int J Prosthodont 1998;11:491-501. 31. Gahleitner A, Monov G. Assessment of bone quality: techniques, procedures and limitations. In: Watzek G, editors. Implants in Qualitatively Compromised Bone, Chicago: Quintessence; 2004:55-66. 32. Pattijn V, Van Lierde C, Van der Perre G, Naert L, Vander Sloten J. The resonance frequency and mode shapes of dental implants: rigid body behaviour versus bending behaviour. A numerical approach. J Biomech 2006;39:939–947. 33. Veltri M, Balleri P, Ferrari M. Influence of transducer orientation on Osstell stability measurements of osseointegrated implants. Clin Implant Dent Relat Res 2007;9:60-4.

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final torque capacity of standard and TiUnite single-tooth implants under immediate loading. Int J Oral Maxillofac Implants 2004;19:578-85 38. Schliephake H, Sewing A, Aref A. Resonance frequency measurements of implant stability in the dog mandible: experimental comparison with histomorphometric data. Int J Oral Maxillofac Surg 2006;35:941-946. 39. Beer A, Gahleitner A, Holm A, Tschabitscher M, Homolka P. Correlation of insertion torques with bone mineral density from dental quantitative CT in the mandible. Clin Oral Implants Res 2003;14:616-620. 40. Homolka P, Beer A, Birkfellner W, Nowotny R, Gahleitner A, Tschabitscher. M, Bergmann H. Bone mineral density measurement with dental quantitative CT prior to dental implant placement in cadaver mandibles: pilot study. Radiology 2002;224:247-252. 41. Rebaudi A, Koller B, Laib A, Trisi P. Microcomputed tomographic analysis of the peri-implant bone. Int J Periodontics Restorative Dent 2004;24:316-325.

34. Atsumi M, Park SH, Wang HL. Methods used to assess implant stability: current status. Int J Oral Maxillofac Implants 2007;22:743-754. 35. Akkocaoglu M, Uysal S, Tekdemir I, Akca K, Cehreli MC. Implant design and intraosseous stability of immediately placed implants: a human cadaver study. Clin Oral Implants Res 2005;16:202-209. 36. Rabel A, Köhler SG, Schmidt-Westhausen AM. Clinical study on the primary stability of two dental implant systems with resonance frequency analysis. Clin Oral Invest 2007;11:257-265. 37. Cunha HA, Francischone CE, Filho HN, de Oliveira RC. A comparison between cutting torque and resonance frequency in the assessment of primary stability and

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BioCRA

153 Original article

Novel injectable hydrogel scaffold for cartilage repair based on natural polymers Giorgio Mattei1, Francesca Montemurro1,2, Monica Mattioli-Belmonte3, Giovanni Vozzi1,2*

Among the principal problems of cartilage regeneration emerges its low mitotic activity and the problem that the regenerated tissue normally does not present the same mechanical properties of hyaline cartilage. Several synthetic and natural polymers have been used for cartilage repair. The research proposed in this paper is based on the use of a natural injectable hydrogel, composed of a polymeric matrix, which gives mechanical consistency and embeds a protein able to attract condrocytes in order to colonize the engineered structure. Different hydrogel scaffolds were realized and analyzed by SEM microscopy. They were mechanically characterized in terms of stress-strain, swelling and creep behavior and their respective visco-elastic mechanical models were derived. Moreover extrudability test has been performed and collagen release in aqueous environment has been evaluated by UV spectrophotometry. (J Osteol Biomat 2010; 1:153-161)

Key words: hydrogel, genepin, mechanical characterisation, cartilage regeneration, scaffold

Interdepartmental Research Center “E.Piaggio”, Faculty of Engineering - University of Pisa, Via Diotisalvi 2, 56126 Pisa, Italy. 2 Department of Chemical Engineering, Industrial Chemistry and Materials Science, University of Pisa, Via Diotisalvi 2, 56126 Pisa, Italy. 3 Department of Molecular Pathology and Innovative Therapies - Histology, Marche Polytechnic University, Via Tronto 10/A, 60020 Ancona, Italy. 1

Corresponding author: *PhD Eng. Giovanni Vozzi Interdepartmental Research Center “E.Piaggio” Faculty of Engineering - University of Pisa Department of Chemical Engineering Industrial Chemistry and Materials Science- University of Pisa, Via Diotisalvi 2, 56126 Pisa, Italy E-mail: g.vozzi@centropiaggio.unipi.it

INTRODUCTION In recent years, Tissue Engineering has been the topic of many research papers. The main goal is to engineer natural tissue by combining autologous cells and three-dimensional biopolymers (scaffolds), which present chemical, physical and topological properties similar to that of natural tissue1. This approach can be applied to treatment of injuries, lesions and degenerative diseases of cartilage. Among the principal problems of cartilage regeneration emerges its low mitotic activity and the problem that the regenerated tissue normally does not present the same mechanical properties of hyaline cartilage2. Several types of cartilage scaffolds have been realized: nonwoven networks3 and foams4 made of α-hydroxy polyesters, polyglactins5, of hyaluronan alkyl esters6, of photopolymerizable hydrogels7 and of collagen and glycosaminoglycanes8. Scaffolds with cells entrapped within fibrin or alginate gels have also been proposed9. Some products, such as Hyaff®-11 (FIDIA Advanced Biopolymers, Italy) are already commercialized. Collagen represents one of the most used biomaterials in this area. It is known that chondrocytes embedded in collagen gel preserve their phenotype and produce GAG for six weeks. More-

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Figure 1. Gelling time of gelatin as a function of genipin content as fraction of total weight of protein solution.

b Figure 2. (a) Stress-strain curves of agar matrix samples; (b) Stress-strain curves of gelatin matrix samples

Figure 3. Youngâ&#x20AC;&#x2122;s moduli of experimental samples

Journal of Osteology and Biomaterials

over, cell enzymes can recognize collagen and degrade it allowing new tissue growth10. The principal problem with the use of this material is its production in large amounts without any pathogens. For this reason, synthetic biopolymers have been principally investigated. They are industrially manufactured and their chemical and mechanical properties can be easily modulated. In addition, they can be biodegradable and they can include growth factors or drugs, able to improve cell activities such as differentiation and growth. However, only few polymers have been accepted for clinical applications by the FDA11. Actually, several studies have focused on interactions between chondrocytes and FDA approved polymers, such as polyglycolic acid (PGA), polylactic acid (PLLA) and copolymer, poly(lactic-co-glycolic acid) (PLGA). Fibrous non-woven PGA scaffolds have been extensively used for articular cartilage tissue engineering because they have a high degradation rate and high porosity, they induce high rates of initial cell growth, they maintain chondrocyte phenotype and they induce ECM secretion similar to that presented by health hyaline cartilage12. PLLA has also been investigated for cartilage repair, but cell growth and ECM synthesis were lower than in PGA13. Other polyesters such as PCL (Polycaprolactone) and PPF (polypropylene fumarate) have been used as alternatives in order to tailor the mechanical and degradable properties to that of natural tissue. Recently, PPF or poly(ethylene fumarate) (OPF) has also been evaluated for use as thermoreversible hydrogel scaffolds for articular cartilage engineering14. This paper is focused on the development and

mechanical characterization of a novel injectable natural hydrogel for cartilage tissue repair. Injectable materials do not require invasive surgery, so they reduce most of the complications normally present in routine surgery. Injectable alginate scaffolds cross-linked with calcium and incorporating chondrocytes, have been studied. These systems induce cartilage repair, but they are unstable and unable to reproduce a functional tissue repair15. This response perhaps is due to immunogenic effect of alginate that induces the increase of lymphocytes and anti-alginate antibodies. Synthetic injectable polymers have also been tested as matrixes for chondrocyte grafts, such as polyethylenoxide (PEO) and copolymers with propylene PEOco-PO, that have shown a more physiological cartilage regeneration respect to PGA or alginate scaffolds16. The research proposed in this paper is based on the use of a biocompatible hydrogel composed of a polymeric matrix, such as agar or gelatin, which embeds a chemo-attractor factor to promote cell colonization, such as collagen cross-linked with a natural cross-linker, genipin, and not cross-linked. The main problem with the use of hydrogel scaffolds is their rapid dissolution in an aqueous environment, for this reason it is important to evaluate their swelling. To avoid collagen dissolution in a biological environment, gluteraldehyde (GTA) can be used as a cross-linking agent, but its cytotoxic properties are well known17. For this reason we decided to use genipin, a natural cross-linker extracted by Gardenia Giasminoide Ellis. The formed structures were mechani-

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f Figure 4. Creep curve of (a) cross-linked collagen; (b) agar; (c) agar and not cross-linked collagen; (d) agar and cross-linked collagen; (e) gelatin; (f) gelatin and not cross-linked collagen; (g) gelatin and pre cross-linked collagen

g cally characterized with stress-strain, swelling and creep tests and their respective viscoelastic mechanical models were derived. Also, the mechanical compression behavior was investigated with the â&#x20AC;&#x153;Gaboâ&#x20AC;? method. This analysis is particularly important for these constructs because normally cartilage is subjected to compression forces in the human body. Moreover, extrudabil-

ity tests were performed and collagen release in aqueous environment was evaluated by UV spectrophotometry. MATERIALS AND METHODS Materials Collagen was extracted from Wistar rat tails18. The concentration of the collagen solution, obtained by UV spectrophotometric analysis (BMG Labtec, Ita-

ly), is 2.37 mg/ml, its pH is 2.9 and it has a density of 1.070 g/cm3. 1% w/v agar (Sigma-Aldrich, Italy) solution in milliQ water and 5% w/v gelatin from porcine skin type A (Sigma-Aldrich, Italy) in milliQ water were prepared. Genipin (GP) (Challenge Bio Products Co., Ltd, Taiwan) was used as a cross-linking agent for collagen and gelatin solutions. GP was added in different concentrations,

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Figure 5. Dynamic loading curve of (a) Cross-linked collagen; (b) Agar; (c) Agar and not cross-linked collagen; (d) Agar and cross-linked collagen

between 0.5% and 3.5% (wt/wt) of the gelatin solution weight. The best result in terms of gelling time was obtained with 2.5% (wt/wt) concentration. An alternative collagen cross-linking method was also used. It is based on the use of DMEM (Dulbecco’s Modified Eagle’s Medium) culture medium and on 0.002M N-(dimethylaminopropyl)N’-ethylcarbodiimide hydrochloride (EDC) and 0.05M N-hydroxysuccinimide (NHS) solutions in milliQ water (Sigma-Aldrich, Italy). Constructs Preparation - Collagen cross-linking Two methods for collagen cross-linking have been studied: one based on the use of DMEM culture medium and EDC: NHS solution, while the second is based on the use of genipin. In the first method, DMEM was added to the collagen solution in a 1:9 weight ratio and rested for one hour in order to complete the cross-linking reaction. After that, the supernatant is removed.

Journal of Osteology and Biomaterials

DMEM induces a weak collagen crosslinking, unstable in physiological solution19. In fact, the cross-linking reversibility can be activated increasing the solution pH from 2.9 to basic values. To avoid this problem, the gel obtained after initial DMEM cross-linking is dipped in 0.05M EDC : 0.0002 NHS solution for 4 hours. At the end, EDC:NHS solution is removed and an excess of a 0.1M Na2HPO4 (Sigma-Aldrich, Italy) solution in water is added for 1 hour to obtain a uniform gel. The resulting collagen gel is rinsed for 30 minutes in milliQ water to remove all impurities. This method was adopted to realize cross-linked collagen (CC) constructs. In the second method, 2.5% (wt/wt) Genipin, calculated respect to the weight of collagen solution, was added. The mixture was heated at 50°C under continuous stirring until the cross-linking process started. The cross-linking reaction starts when the solution becomes brownish and finishes when the color becomes blue. This solution was

cast on a Petri dish and rested at room temperature for 48 hours. This method was used to cross-link both gelatin and collagen. - Agar matrix constructs 1% w/v agar solution in water was used pure or mixed with collagen (crosslinked and not) in 1:1 weight ratio. The three solutions were casted in 29 mm diameter Petri dishes, so three samples were realized: pure agar (A), agar and not cross-linked collagen (ANCC) and agar and cross-linked collagen (ACC) with DMEM and EDC-NHS. Because agar starts to gel at 45 °C, the collagen was added to it at around 36 °C, thus avoiding the collagen denaturation. Samples were hermetically closed to prevent dehydration and stored in fridge at 4 °C. - Gelatin matrix constructs 5% w/v gelatin solution in MilliQ water was cross-linked by adding 2.5% (wt/ wt) GP, respect to the gelatin solution weight. Three series of samples were realized by casting Gelatin and GP solu-

Mattei G. et al. 157

Figure 6. Time decrease of temperature of agar solution

Figure 7. Polymer mass flow out (Q) of syringe without the needle as function of stepper motor angular speed (ω) at several temperature

Figure 8. Comparison between the mass flow out of a syringe with and without the needle

Figure 9. Comparison between the mass flow out of ANCC and water

Figure 10. GNCC mass-flow out as function of angular speed, ω

tion (G), Gelatin and GP solution mixed dish and mounted on the transducer, with not cross-linked collagen (GNCC) so its deformations were measured. and Gelatin and GP solution mixed with The sampling rate was fixed at 1 Hz. pre cross-linked collagen (GPCC). The After one minute of acquisition with and GNCC solution was obtained by mixing no load, the offset (t ) evaluated l realwas l 0real (t ) was filled with deionized collagen and gelatin solutions in a 1:1 the Petriplate l real weight ratio (1g of collagen solution water until the sample0 was completely So,realthe measured time-dewas added to 1g of gelatin solution) covered. real ( )  l t l0 was equal to: at about 36 °C and adding 2.5% GP,re(t ) pendentrealdeformation l0 spect to total weight of solution. GPCC real l (t )  l 0real ( )   t solution was obtained by mixing 1g of l 0real gelatin solution to 1g of DMEM EDCreal (t ) is obtained by adding NHS pre cross-linked collagen and then where l 2.5% (wt/wt) GP was added respect to the offset to the measured length l (t ) whole weight of the mixed solution. Af- so it represents the elongation of the ter 48 hours of casting all samples were sample at time t. uniform and mechanically stable. The test was performed until a stable plateau was reached, so the swelling Collagen release time constants were derived for each Collagen released by ANCC and GNCC sample through the analysis of the obsamples into surrounding aqueous en- tained data. vironment was analyzed with UV spec- Stress-strain test allows the assessing trophotometry (BMG Labtec, Italy). A of Young modulus of each sample. Off2.9 cm diameter and 4.5 mm height set determination was obtained on the cylindrical sample was completely basis of previous results. The sampling dipped in a Petri dish containing 60 ml time was the same of the previous test. of deionized water. At different times The weights were applied every 3 min(1 m, 2m, 5 m, 10m, 15 m, 30 m, 1h, 2h, utes, until a maximum weight of 500g. 4h, 8h, 24h ) 0.5 ml of water bath was Acquired data were averaged on 180 sampled and analyzed at 225 nm with samples (3 minutes per each load, at an UV-Vis spectrophotometer. 1Hz sampling) to determine the elongation, so elastic moduli were evaluSwelling tensile stress-strain, ated as stress-strain behavior. and creep tests Creep test was performed in aqueous These tests were performed with the bath after the swelling test on each Ugo Basile 7006 isotonic transducer sample, so as to have stabilized its orig(Ugo Basile, Italy) connected to the inal length. In this way the initial length real l 0real (1   ) ,creep  l 0 MP35 acquisition platform (BIOPAC of the sample was equalendswellin to: g Systems Inc, Italy). The Swelling test real l 0real (1   endswelling ) ,creep  l 0 allows the evaluation of swelling of a sample dipped in water. Once the initial l real real length l 0 of the sample was meas- where 0 ,is the initial length of samreal real 1   endswelling )was the deformation ured, the latter was placed in la0 ,Petri creep  l 0ple (and

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reached in the plateau area after swelling. The sampling frequency was fixed at 2 Hz. The weight necessary to cause 1% linear deformation of a sample was derived from its stress-strain graph. Dynamic loading test Dynamic loading tests were performed E*  E 'iE ' ' with GABO Eplexor® 150N (GABO QUALIMETER Testanlagen GmbH, Germany). The elastic modulus in compression (not the Young modulus obtained from stress-strain tests) can be written as:

E*  E 'iE ' ' where E ' is the elastic component (“storage modulus”) and E ' ' the dissipative one (“loss modulus”). The transition from the elastic behavior to the beginning of viscous phenomena can be determined by:

tan  

E'' E'

Following parameters were adopted in the setup of experiment: - Contact force: 0.10 N E ' ' causes a 2% - Static load: one that tan   static strain on theEsample ' - Static and dynamic load max forces: 1.50 N - Static and dynamic load tolerance: 0.50% - Temperature: 25°C - Temperature tolerance: 2.00°C - Sweep type: log - Frequency sweep: 0.1 to 100 Hz (20.67 Hz/dec) - Number of sampling: 63 Each sample presents a cylindrical shape with 12.10 mm diameter and 5 mm height.

Journal of Osteology and Biomaterials

Extrudability test This test was performed to verify the extrudability of the realized polymeric hydrogels with a commercial syringe. A 10 ml syringe was filled with the polymer solution and then mounted on a motorized micro-positioner where a stepper motor drive the motion of the syringe piston. The mass outflow out of the syringe was determined by controlling the speed of the stepper motor. This experiment was performed for some temperatures of the polymer solution. RESULTS AND DISCUSSION Gelation results Initially we analyzed gelation time as function of GP concentration. From these tests we observed that after 2.5% GP concentration a plateau was reached (Fig. 1). This result suggested that this value is the maximum quantity of GP to add to cross-link all protein free sites. Collagen release Spectrophotometric analysis showed that no collagen was released from the scaffold matrix into the surrounding aqueous environment. This confirmed the result on gelation time previously reported. After 2.5% GP concentration all protein present in the construct were completely cross-linked. According to this evidence, no graph are shown in this paper. Stress-strain test and Swelling test From stress-strain graphs it is possible to note that all samples present an elastic behavior (Fig. 2a and 2b). In particular, elastic moduli of constructs

obtained by adding cross-linked or not cross-linked collagen inside gelatin or agar matrix are lower than those of pure matrices (Fig. 3). It could be due to the overall steric dimensions of CC and NCC included in the polymeric matrix. Moreover, from the swelling test analysis (Table 1) it is possible to note that samples that have gelatin as a matrix did not present swelling. This was due to a complete cross-linking of gelatin that created a structure that was not permeable to aqueous solutions. Instead, samples with agar matrix presented a low swelling relaxation. The swelling was due to the porous structure of the agar matrix and to the presence of CC and NCC which increased the dimension of these pores, and allowed water perfusion within the structure. This hypothesis was confirmed by reduced relaxation times in samples which embed CC. Creep test The tested materials showed a mechanical behavior describable with the Kelvin model, expressible as:      spring   Voigt  0  0 (1  e t /  ) k1 k 2 This model fits very well with the obtained experimental data (Fig. 4a-g). From Table 2, it is possible to note that the elastic constants (k1, k2) are similar to the experimental elastic moduli. In addition, samples with agar matrix show a short creep time due to the large water content within them. The presence of cross-linked and not crosslinked collagen produces a considerable increase in viscosity. This fact could be explained by the theory of “recruitment of the fibers”20. Collagen fibers

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Figure 11. SEM micrographs of (a) Gelatin (1000x magnification); (b) Genipin and not cross-linked collagen (35x magnification); (c) Genipin and not crosslinked collagen (250x magnification); (d) Genipin and not cross-linked collagen (500x magnification); (e) Genipin and not cross-linked collagen (1000x magnification)

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are arranged in a complex structure, thus their alignment along the direction of traction is a slow and gradual process which results in an increase in viscous behavior. This aspect explains why samples with gelatin matrix have the highest constant of viscosity (and longest characteristic creep time) when collagen was embedded in them respect to pure cross-linked gelatin. Dynamic loading test All samples demonstrated a marked increase in the compression modulus with increasing deformation rate. At high-speed deformation samples have an elastic behavior, while for low-speed deformation their behavior is similar to a viscous liquid. This trend in compression modulus is justified because polymeric chains could not orient along the loading direction because of the increase in the frequency of stimulation. Viscous behavior involves inner flows of polymeric chains which is associated with the conversion of mechanical energy into heat, so the temperature rises. For all samples there is a nearly perfect overlap between the “storage” and the overall modulus. For this reason the samples principally present an elastic behavior and, therefore, losses due to viscosity are very limited. The “loss” modulus is significantly lower than the “storage” one in the considered frequency range, and, consequently, tan(δ), the ratio between the “loss” and “storage” modulus, is close to zero. Tan (δ), “loss” and “storage” modulus, are shown for cross-linked collagen and for agar-matrix samples in the 0-10 Hz frequency range (Fig. 5a-d). Tan (δ) is stable at about 0.1 already at a frequency of 1 Hz, that is the typical

Journal of Osteology and Biomaterials

Sample

Final swelling strain (%)

Relaxation time (s)

4 2.4 1.5 3 0 0 0

2000 8000 1500 1000 ∞ ∞ ∞

CC A ANCC ACC G GNCC GPCC Table 1. Swelling test result

CC ANCC G GPCC

- Cross-linked collagen - Agar + not cross-linked collagen - Gelatin + GP - Gelatin + pre cross-linked collagen + GP

k1 (KPa) 5.05 32.26 k2 (KPa) 2.71 14.13 η (KPa×s) 83.95 158.39 τ (s) 30.98 11.21 Table 2. Creep test result

CC ANCC G GPCC

A - Agar ACC - Agar + cross-linked collagen GNCC - Gelatin + not cross-linked collagen + GP

ANCC

ACC

GNCC

GPCC

9.68 6.42 74.21 11.56

7.23 5.39 144.07 26.73

101.22 157.50 2148.30 13.64

86.16 75.50 17667.00 234.00

90.09 54.22 13934.54 257.00

- Cross-linked collagen - Agar + not cross-linked collagen - Gelatin + GP - Gelatin + pre cross-linked collagen + GP

A - Agar ACC - Agar + cross-linked collagen GNCC - Gelatin + not cross-linked collagen + GP

frequency value corresponding to normal walking. This result suggests that all samples present an elastic mechanical behavior.

the polymeric mixture was not affected by temperature (Fig. 7) and the mass outflow (Q) increases by increasing the stepper motor angular speed (ω). Comparing the polymeric mixture extrusion with and without the syringe needle (Fig. 8), there is no significant load loss due to shear stress. This behavior is typical of Newtonian fluids and it is confirmed in figure 9 through a comparison in the extrusion of ANCC and water, which is the Newtonian fluid for excellence. This suggests that the polymeric matrix does not affect the extrusion rheology. Mass outflow of GNCC is shown in figure 10 as function of angular speed (ω).

Extrudability test Particular attention was devoted to agar-matrix-based constructs, because their cross-linking is temperature-dependent and may affect the rheological properties, so the analysis of the extrudability of these hydrogel was performed at several temperatures. The time-temperature curve is shown in figure 6. The analysis of the extrudability starts at 36 °C, which is the temperature at which the two components are mixed. The decreasing in time of the temperature of the hydrogel within a commercial syringe follows an exponential behavior, with a characteristic time τ, equal to 667 s. The extrusion of

Mattei G. et al. 161

SEM micrographies - Gelatin + GP At 1000x magnification, SEM Micrograph (figure 11a) shows gelatin fibers cross-linked by genipin (GP). At minor magnification the matrix appears compact. The morphology of GPCC construct is compact and non regular due to protein aggregates (Fig. 11b-c). This confirms the mechanical and the swelling results. At high magnification it is possible to note a morphology close to that observed for cross-linked gelatin (Fig. 11d-e). CONCLUSIONS The aim of this work is the development of an innovative injectable natural polymeric system for cartilage repair applications. In this paper we realized two different hydrogels composed of a matrix (Agar or Gelatin) with cross-linked or not cross-linked collagen inside it. Collagen works as a chemo-attractant for cell colonization and cell function activation. Because common crosslinking agents, such as GTA, are cytotoxic, we used a natural cross-linking agent, GP. These constructs have been mechanically tested in order to evaluate their possible application for cartilage tissue engineering. Because these constructs are hydrogel, their swelling and their extrudability were analyzed. All experimental results suggested that Gelatin + Cross-linked Collagen constructs could be used as an innovative injectable hydrogel for cartilage repair. The elastic modulus of the constructs does not mimic that of natural tissue, but their high biocompatibility and the presence of chemo-attractors within them should help to produce fast cell colonization.

REFERENCES 1. Langer R, Vacanti JP. Tissue engineering. Science 2003;260:920-926. 2. Oâ&#x20AC;&#x2122; Driscoll SW. The healing and regeneration of articular cartilage. J Bone Jt Surg 1998;80-A:1795-1812. 3. Freed LE, Marquis JC, Nohria A, Emmanual J, Mikos AG,Langer R. Neocartilage formation in vitro and in vivo using cells cultured onsyn thetic biodegradable polymers. J Biomed Mater 1993;27:11-23. 4. Shastri VP, Martin I, Langer R. Macroporous polymer foams by hydrocarbontemplating. Proc Natl Acad Sci USA 2000;97:1970-1975. 5. Marijnissen WJ, van Osch GJ, Aigner J, et al. Alginate as a chondrocyte-delivery substance in combination with a nonwoven scaffold for cartilage tissue engineering. Biomaterials 2002;23:1511-1517. 6. Campoccia D, Doherty P, Radice M, Brun P, Abatangelo G,Williams DF. Semisynthetic resorbable materials from hyaluronan esterification. Biomaterials 1998;19:2101-2127. 7. Bryant SJ, Anseth KS. Hydrogel properties influence ECM production by chondrocytes photoencapsulated in poly(ethylene glycol) hydrogels. J Biomed Mater Res 2002;59: 6372. 8. Lee CR, Breinan HA, Nehrer S, Spector M. Articular cartilage chondrocytes in type I and type II collagenâ&#x20AC;&#x201C;GAG matrices exhibit contractile behavior in vitro. Tissue Engineering 2000;6:555-565. 9. Ameer GA, Mahmood TA, Langer R. A biodegradable composite scaffold for cell transplantation. J Orthop Res 2002;20:16-19. 10. Kimura T, Yasui N, Ohsawa S, Ono K. Chondrocytes embedded in collagen gels maintain cartilage phenotype during long-term cultures. Clin Orthop Rel Res 1984;186:231-239.

12. Grande DA, Halberstadt C, Naughton G, Schwartz R, Manji R. Evaluation of matrix scaffolds for tissue engineering of articular cartilage grafts. J Biomed Mater Res 1997;34:211-220. 13. Sittinger M, Reitzel D, Dauner M, Hierlemann H, Hammer C, Kastenbauer E, Planck H, Burmester GR, Bujia J. Resorbable polyesters in cartilage engineering: affinity and biocompatibility of polymer fiber structures to chondrocytes. J Biomed Mater Res 1996;33:57-63. 14. Soo-Hong Lee, Heungsoo Shin. Matrices and scaffolds for delivery of bioactive molecules in bone and cartilage tissue engineering. Advanced Drug Delivery Reviews 2007;59:339-359. 15. Paige KT, Cima LG, Yaremchuk MJ, Schloo BL, Vacanti JP, Vacanti CA. De novo cartilage generation using calcium alginatechondrocyte constructs. Plast Reconstr Surg 1996;97:168-180. 16. Sims CD, Butler PEM, Casanova R, Lee BT, Randolph MA, Lee WPA, Vacanti CA, Yaremchuk MJ. Injectable cartilage using polyethylene oxide polymer substrates. Plast Reconstr Surg 1996;98:843-850. 17. Sung H, et al., Feasibility study of a natural crosslinking reagent for biological tissue fixation. J Biomed Mater Res, 1998;42:560567. 18. Elsdale T, Bard J. Collagen substrata for studies on cell behavior. J Cell Biol 1972;54:626-637. 19. Marzec E, Pietrucha K. The effect of different methods of cross-linking of collagen on its dielectric properties. Biophys Chem 2008;132:89-96. 20. Schwartz MH, et al. A microstructural model for the elastic response of articular cartilage. Journal of Biomechanics 1994;27:865-873.

11. Thomson RC, Wake MC, Yaszemski MJ, Mikos AG. Biodegradable polymer scaffolds to regenerate organs. Adv Polym Sci 1995;122:245-274.

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Bone density evaluation after 5 years of implant rehabilitation in fibula free flap used for maxilla reconstruction Francesco Grecchi MD1, Francesco Gallo MD1, Giuseppe Rubino MD1, Alessandro Motroni MS2, Raffaella Bianco MD1, Ilaria Zollino MD3, Francesca Farinella3, Ambra Girardi4, Francesco Carinci MD3* Introduction: Segmental resections of jaws secondary to malignant or benign causes, can lead to extensive composite defects, which result in a dramatic loss in quality of life with a significant morbidity for patients. Since there are non specific studies that evaluated bone density (BD) over time in patients who underwent maxillary reconstruction by means of fibula free flap (FFF), we therefore decided to perform a retrospective study. Materials and Methods: Two patients underwent upper jaw reconstruction by means of FFF and 12 implants (6 per patient) were inserted after 6 months. After an additional 6 months fixtures were loaded and CT evaluation was performed at 5 years follow-up. Pearsonâ&#x20AC;&#x2122;s chi-square test was used to investigate differences in bone density (i.e. BD) between native and grafted bone and between peri-implant and bone far from fixtures. Results: BD of FFF is higher then BD of native bone. Peri-implant BD is equal to BD far from fixtures. Conclusions: CT scan is a valuable and accurate pre-operative and follow-up method to obtain information on bone quality and quantity (i.e. volume of available bone). (J Osteol Biomat 2010; 1:163-169)

Key words: Jaw, reconstruction, fibula, homograft, resorption, bone, density.

Dept of Maxillofacial Surgery, Galeazzi Hospital, Milano, Italy; AMIRG (Applied Medical Imaging Research Group, Milan - Italy) 3 Maxillofacial Surgery, University of Ferrara, Ferrara, Italy; 4 Department of Histology, Embryology and Applied Biology, University of Bologna, Bologna, Italy 1 2

Corresponding authors: * Prof. Francesco Carinci MD Chair of Maxillofacial Surgery Arcispedale S. Anna, Corso Giovecca 203, 44100 Ferrara, ITALY E-mail: crc@unife.it; Web: www.carinci.org Phone/Fax: +39.0532.455582

INTRODUCTION Segmental resections of jaws secondary to malignant or benign causes, can lead to extensive composite defects, which result in a dramatic loss in quality of life with a significant morbidity for the patients.1 In the reconstruction of segmental defects, the vascularized flap proves extremely valuable because the large, poorly perfused regions can be treated successfully.2,3 These flaps have become a valuable means for the rehabilitation of such patients because they allow the immediate reconstruction of defects despite unfavourable local conditions such as large defects and irradiation.3 Vascularized flaps can either be free (i.e. needing re-anastomosing at the recipient site) or pedicled, staying attached at the supplying blood vessels that are mobilized allowing transpositioning. The biologic advantage of providing its own blood supply makes it more resistant to infection and promotes wound healing.2 Different donor sites such as the iliac crest, the fibula, the scapula, and the radius have been recently suggested for use. For jaws reconstruction, vascularized fibula free flap (FFF) seems to be the best choice owing to its length, its height, and the possibility to reshape and restore the mandibular/maxillary

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Figure 1. The virtual probe extracts the bone density around implants and in unloaded bone.

arch.4 FFF has many advantages for jaw reconstruction compared with any other vascularized bone grafts. The flap can be easily shaped with osteotomies, according to the defect size. The pedicle of the FFF is always long and large enough for microanastomoses.5,6 FFF, with its high cortical content, is very stable due to the high mechanical rigidity of the cortical bone. Additionally, the cortical bone has a high content of bone morphogenetic proteins, which act osteoinductively, promoting the bone healing process.6-8 Furthermore, this type of bone reconstruction should widely allow an implant-retained prosthesis.9 Full dental rehabilitation in this reconstructed environment can be challenging as most of these patients have oncological resections, frequent medi-

Journal of Osteology and Biomaterials

cal co-morbidity and often require radiotherapy. Insufficient bone height, altered soft tissue and loss of mucosal sensation can impair the success of a conventional tissue-borne prosthesis.10,11 However, operative safety and success have to be enhanced because risks such as inadequate osseous support or compromise and infringing upon important anatomic structures are to be avoided.12 By using the computer-guided implantology method, anatomic limitations and bone quantity and quality for implant insertion can be evaluated precisely.13 Thorough presurgical diagnostics with three dimensions (3D) radiographic techniques, such as computerized tomographic (CT) technology, provides all the information necessary without proneness to errors leading to potentially

serious complications.14 Cone beam computed tomography (CBCT) greatly reduces patientsâ&#x20AC;&#x2122; exposure to radiation, and reformats the raw data into DICOM data (Digital Imaging and Communications in Medicine). DICOM data are imported into simulation software that enables the manipulation of multiplanar reconstructed slices and threedimensional volume renderings.15,16 Moreover, an additional advantage by using the output DICOM file obtained from CT evaluation is that bone density in the region of the implant placement can also be calculated. Since there are no specific studies that evaluate bone density over time in patients who underwent maxillary reconstruction by means of FFF, we decided to perform a retrospective study to evaluate the clinical outcome by using

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Figure 2. Upper jaw with fibular free flap reconstruction and inserted implants.

DICOM files derived from CT evaluations and elaborated them with a specific computer program.17 MATERIALS AND METHODS Patients In the period between January and March 2005, two patients (one female and one male) with a median age of 60 years were operated at the Galeazzi Hospital, Milan, Italy, and FFF maxillary reconstruction was performed. After 6 months, 12 implants were inserted in FFF (6 per patient) and after an additional 6 months, a fixed prosthetic rehabilitation was performed. Informed written consent, approved by the local Ethics Committee, was obtained from patients to use their data for research purposes. CT check-up was performed after 5 years of loading. Subjects were screened according to

the following inclusion criteria: controlled oral hygiene and the absence of any lesions in the oral cavity; in addition, the patients had to agree to participate in a post-operative check-up program. Data collection Radiographic examinations were done with the use of CT scans. The DICOM data were processed with a medical imaging software (3Diagnosys 3.0 - 3DIEMME, Italy) which gives the possibility to use a virtual probe to extract the bone density values in the desired regions and export them in Excel tables for statistical analysis (Fig. 1 and 2). The virtual probes were set in the following regions in the final follow-up CT scans: 1. Native bone (pre-maxilla) 2. Bone graft (far from implants) 3. Around implants to analyze both the graft and native bone around implants

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4. Airways (calibration probe) An area of 1 mm thickness was extracted with this probe and exported to Excel for further analysis. In order to place the probes in the same position in subsequent CT scans, the bone volumes extracted from these exams were exported in STL file format and superimposed according to a â&#x20AC;&#x153;best-fitâ&#x20AC;? algorithm (Geomagic Studio 11 - Geomagic Corp., USA) based on the bone regions not affected by the surgery. After the repositioning of every DICOM stack in the same reference system, the data collected by the probes were extracted and analyzed for statistical processing. The data extracted is expressed in Hounsfield Units, being the processed CT scans calibrated according to waterbased phantoms. Implants A total of 12 implants were inserted in 2 patients, all in the maxilla. Implants were inserted to replace 3 incisors, 3 cuspids, 7 premolars and 1 molar. Surgical and prosthetic technique The patients underwent the same surgical protocol. An antimicrobial prophylaxis was administered with 2000 mg Amoxicillin before surgery and 1000 mg twice daily for 7 days starting 1 hour before surgery. The implants were inserted after 6 months from FFF operation. The implant platform was positioned at the natural alveolar crest level. Sutures were removed 10 days after surgery. Then 16 weeks after implant insertion, the provisional prosthesis was provided and the final restoration was usually delivered within an additional 8 weeks. All patients were included in a strict hygiene regimen.

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COMPUTER TOMOGRAPHY HOUNSFIELD (HU) PATIENTS

PROBE 5 YEARS FOLLOW-UP POST LOADING Bone grafted far from implant

801,52 ± 477,51

Bone grafted around implant

553,57 ± 621,66

Native bone

538,26 ± 236,51

Calibration

-958,21 ± 34,00

Bone grafted far from implant

988,41 ± 347,08

Bone grafted around implant

839,69 ± 666,74

Native bone

927,34 ± 258,37

Calibration

-992,15 ± 29,57

Table 1. Reports the median bone density (BD): in columns are reported the BD whereas in rows are reported the type of bone (native, grafted around implants and grafted far from implants).

Statistical Analysis Pearson’s chi-square test was used to investigate difference in bone density between native and FFF, as well as in the peri-implants and far from the fixture areas. RESULTS Table 1 reports the median bone density (BD): in columns are reported the BD whereas in rows are reported the type of bone (native, grafted around implants and grafted far from implants). BD of FFF has the higher value, about 25% more dense if compared to native BD. Peri-implant FFF BD is higher compare to BD of grafted bone located far from fixtures.

Journal of Osteology and Biomaterials

DISCUSSION Rehabilitation of patients with orofacial defects after large jaw resection poses a common and challenging problem in maxillofacial surgery.18,19 The introduction of the osseointegration concept and its application for dental rehabilitation has greatly improved the rehabilitative potential for such patients.20-22 The development of endosseous implants in vascularized fibula free flap (FFF) has eliminated many problems related to the retention and stability of conventional prosthesis23,24 and reduced prosthetic rehabilitation problems in irradiated patients with reduced salivary flow.25 The advantages of the adjunctive use of implants after jaw reconstruction with

a FFF became apparent quite soon, with reports from Huryn et al.26 and Sclaroff et al.27 highlighting this procedure. Reports about the long-term results of implants placed in FFF are rare, often being limited to only a few years, as reported by Rohner et al.28 in a 3-year follow-up study or by Guerleck et al.29 reporting on a 47-month followup (average).The FFF presents many advantages, but a limitation may be insufficient bone height (rarely more than 13 mm) for the reconstruction of both the skeletal base and the alveolar ridge. It presents some problems from a prosthetic point of view, particularly in cases of partial mandibular resection with residual dentition on the healthy side.5,30 The distance between the im-

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plant shoulder and the occlusal plane is large, leading to an unfavourable crown-to-implant ratio. This situation also creates a significant difference in the level of the alveolar crest between the residual mandible and the reconstructed part, therefore causing functional and aesthetic problems.5,31 Computer-assisted surgery is known to enhance safety in dental implantology.32 A potentially important application is the exact placement of dental implants in partially or completely edentulous patients with insufficient bone height because the long-term prognosis of implant supported oral restorations, concerning functionality and implant stability, depends to a large extent on implant anchorage in the jawbone.33 By using three dimensions (3D) images it is possible virtually to place implants in the bone in precise relation to their position in the final prosthesis.34 Moreover, to obtain information about available bone and bone structure/density, both conventional radiographic procedures and computed topographic methods can be used.35-39 Our results demonstrated that loaded regions have a bone density comparable to native bone, whereas those located far from the implants are higher. Since BD changes over time, CT scan represents a valuable method to obtain information about bone quality in addition to bone quantity (i.e. volume). It potentially allows not only to detect the most favourable time for implantation after bone reconstruction, to compare grafted bone quality to natural bone, but also to verity bone â&#x20AC;&#x153;maturationâ&#x20AC;? over time.

The inception of dental implants and the increasing improvement of this procedure have contributed greatly to the successful treatment of FFF rehabilitation. Nonetheless, it must be kept in mind that impeccable oral hygiene and regular recall visits are prerequisites for the lasting success of this treatment.

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ACKNOWLEDGMENT This work was supported by FAR from the University of Ferrara (FC), Ferrara, Italy, and from PRIN 2008 (F.C.).

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REFERENCES 1. Hundepool AC, Dumans AG, Hofer SO, Fokkens NJ, Rayat SS, van der Meij EH, et al. Rehabilitation after mandibular reconstruction with fibula free-flap: clinical outcome and quality of life assessment. Int J Oral Maxillofac Surg 2008;37:1009-1013. 2. Disa JJ, Hidalgo DA, Cordeiro PG, Winters RM, Thaler H. Evaluation of bone height in osseous free flap mandible reconstruction: an indirect measure of bone mass. Plast Reconstr Surg 1999;103:1371-1377. 3. Pogrel MA, Podlesh S, Anthony JP, Alexander J. A comparison of vascularized and nonvascularized bone grafts for reconstruction of mandibular continuity defects. J Oral Maxillofac Surg 1997;55:1200-1206. 4. Ferri J, Piot B, Ruhin B, Mercier J. Advantages and limitations of the fibula free flap in mandibular reconstruction. J Oral Maxillofac Surg 1997;55:440-448. 5. Wu YQ, Huang W, Zhang ZY, Zhang ZY, Zhang CP, Sun J. Clinical outcome of dental implants placed in fibula-free flaps for orofacial reconstruction. Chin Med J (Engl) 2008;121:1861-1865. 6. Gbara A, Darwich K, Li L, Schmelzle R, Blake F. Long-term results of jaw reconstruction with microsurgical fibula grafts and dental implants. J Oral Maxillofac Surg 2007;65:1005-1009. 7. Urist MR, Mc LF. Osteogenetic potency and new-bone formation by induction in transplants to the anterior chamber of the eye. J Bone Joint Surg Am 1952;34-A:443476. 8. Garg AK. Biology, Harvesting, Grafting For Dental Implants. Rationale and Clinical Applications. Chicago: Quintessence Publishing Co; 2004. 620 p. 9. Bodard AG, Bemer J, Gourmet R, Lucas R, Coroller J, Salino S, et al. [Dental implants and microvascular free fibula flap: 23 patients]. Rev Stomatol Chir Maxillofac 2008;109:363-366. 10. Chiapasco M, Biglioli F, Autelitano L, Ro-

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meo E, Brusati R. Clinical outcome of dental implants placed in fibula-free flaps used for the reconstruction of maxillo-mandibular defects following ablation for tumors or osteoradionecrosis. Clin Oral Implants Res 2006;17:220-228. 11. Iizuka T, Hafliger J, Seto I, Rahal A, Mericske-Stern R, Smolka K. Oral rehabilitation after mandibular reconstruction using an osteocutaneous fibula free flap with endosseous implants. Factors affecting the functional outcome in patients with oral cancer. Clin Oral Implants Res 2005;16:69-79. 12. Holst S, Blatz MB, Eitner S. Precision for computer-guided implant placement: using 3D planning software and fixed intraoral reference points. J Oral Maxillofac Surg 2007;65:393-399. 13. Widmann G, Bale RJ. Accuracy in computer-aided implant surgery--a review. Int J Oral Maxillofac Implants 2006;21:305-313. 14. Azari A, Nikzad S, Kabiri A. Using computer-guided implantology in flapless implant surgery of a maxilla: a clinical report. J Oral Rehabil 2008;35:690-694. 15. Kramer FJ, Baethge C, Swennen G, Rosahl S. Navigated vs. conventional implant insertion for maxillary single tooth replacement. Clin Oral Implants Res 2005;16:6068. 16. Degidi M, Piattelli A, Carinci F. Immediate loaded dental implants: comparison between fixtures inserted in postextractive and healed bone sites. J Craniofac Surg 2007;18:965-971. 17. Tarnow DP, Wallace SS, Testori T, Froum SJ, Motroni A, Prasad HS. Maxillary sinus augmentation using recombinant bone morphogenetic protein-2/acellular collagen sponge in combination with a mineralized bone replacement graft: a report of three cases. Int J Periodontics Restorative Dent;30:139-149. 18. Hayter JP, Cawood JI. Oral rehabilitation with endosteal implants and free flaps. Int J Oral Maxillofac Surg 1996;25:3-12.

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19. Chang YM, Santamaria E, Wei FC, Chen HC, Chan CP, Shen YF, et al. Primary insertion of osseointegrated dental implants into fibula osteoseptocutaneous free flap for mandible reconstruction. Plast Reconstr Surg 1998;102:680-688. 20. Aldegheri A, Beloni D, Blanc JL, Kaplanski P, Legre R, Zanaret M. [Dental rehabilitation using osseointegrated implants: treatment of oro-maxillo-facial cancer. A preliminary study of 7 cases]. Rev Stomatol Chir Maxillofac 1996;97:108-116. 21. Holzle F, Kesting MR, Holzle G, Watola A, Loeffelbein DJ, Ervens J, et al. Clinical outcome and patient satisfaction after mandibular reconstruction with free fibula flaps. Int J Oral Maxillofac Surg 2007;36:802-806. 22. Disa JJ, Winters RM, Hidalgo DA. Longterm evaluation of bone mass in free fibula flap mandible reconstruction. Am J Surg 1997;174:503-506. 23. Garrett N, Roumanas ED, Blackwell KE, Freymiller E, Abemayor E, Wong WK, et al. Efficacy of conventional and implant-supported mandibular resection prostheses: study overview and treatment outcomes. J Prosthet Dent 2006;96:13-24.

28. Rohner D, Jaquiery C, Kunz C, Bucher P, Maas H, Hammer B. Maxillofacial reconstruction with prefabricated osseous free flaps: a 3-year experience with 24 patients. Plast Reconstr Surg 2003;112:748-757. 29. Gurlek A, Miller MJ, Jacob RF, Lively JA, Schusterman MA. Functional results of dental restoration with osseointegrated implants after mandible reconstruction. Plast Reconstr Surg 1998;101:650-655; discussion 656-659. 30. Kurkcu M, Benlidayi ME, Kurtoglu C, Kesiktas E. Placement of implants in the mandible reconstructed with free vascularized fibula flap: comparison of 2 cases. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;105:36-40.

38. Lindh C, Nilsson M, Klinge B, Petersson A. Quantitative computed tomography of trabecular bone in the mandible. Dentomaxillofac Radiol 1996;25:146-150. 39. Muller R, Hahn M, Vogel M, Delling G, Ruegsegger P. Morphometric analysis of noninvasively assessed bone biopsies: comparison of high-resolution computed tomography and histologic sections. Bone 1996;18:215-220.

32. Ng FC, Ho KH, Wexler A. Computer-assisted navigational surgery enhances safety in dental implantology. Ann Acad Med Singapore 2005;34:383-388. 33. Birkfellner W, Solar P, Gahleitner A, Huber K, Kainberger F, Kettenbach J, et al. In-vitro assessment of a registration protocol for image guided implant dentistry. Clin Oral Implants Res 2001;12:69-78.

25. Raoul G, Ruhin B, Briki S, Lauwers L, Haurou Patou G, Capet JP, et al. Microsurgical reconstruction of the jaw with fibular grafts and implants. J Craniofac Surg 2009;20:2105-2117.

34. Spector L. Computer-aided dental implant planning. Dent Clin North Am 2008;52:761-775.

27. Sclaroff A, Haughey B, Gay WD, Paniello R. Immediate mandibular reconstruction and placement of dental implants. At the time of ablative surgery. Oral Surg Oral Med Oral Pathol 1994;78:711-717.

37. Dula K, Buser D, Porcellini B, Berthold H, Schwarz M. [Computed tomography/ oral implantology (I). Dental CT: a program for the computed tomographic imaging of the jaws: the principles and exposure technic]. Schweiz Monatsschr Zahnmed 1994;104:450-459.

31. Chiapasco M, Brusati R, Galioto S. Distraction osteogenesis of a fibular revascularized flap for improvement of oral implant positioning in a tumor patient: a case report. J Oral Maxillofac Surg 2000;58:14341440.

24. Adell R, Svensson B, Bagenholm T. Dental rehabilitation in 101 primarily reconstructed jaws after segmental resections--possibilities and problems. An 18-year study. J Craniomaxillofac Surg 2008;36:395-402.

26. Huryn JM, Zlotolow IM, Piro JD, Lenchewski E. Osseointegrated implants in microvascular fibula free flap reconstructed mandibles. J Prosthet Dent 1993;70:443446.

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35. Schultze-Mosgau S, Keweloh M, Wiltfang J, Kessler P, Neukam FW. Histomorphometric and densitometric changes in bone volume and structure after avascular bone grafting in the extremely atrophic maxilla. Br J Oral Maxillofac Surg 2001;39:439-447. 36. Clark DE, Danforth RA, Barnes RW, Burtch ML. Radiation absorbed from dental implant radiography: a comparison of linear tomography, CT scan, and panoramic and intra-oral techniques. J Oral Implantol 1990;16:156-164.

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Original article

Evaluation of bone resorption around implants inserted in calvaria autogenous bone grafts used for jaw reconstruction Roberto Cenzi,1 Laura Arduin,1 Ilaria Zollino,2 Claudia Casadio,2 Ambra Girardi,3 Francesca Farinella,2 Francesco Carinci2

Introduction: Insertion of endosseous implants is often difficult because of lack of supporting bone. In the case of severe jaw atrophy, a large volume of bone can be necessary to restore the alveolar ridge and calvarial bone grafts can be used to restore it. Calvarial grafts undergo partial resorption with time but few reports elucidated the rate of resorption. Materials and Methods: Twenty-nine grafts were inserted in 17 patients (6 males and 11 females with a median age of 45 years) at the Civil Hospital of Rovigo (Italy) between May 1992 and July 2009. Eleven (37.9%) grafts were inserted in the right maxillae, 8 (27.6%) in the left maxillae, 5 (17.2%) in the left mandible and 5 (17.2%) in the right mandible. Five (17.2%), 10 (34.5%) and 1 (3.4%) were inlay, onlay and veneer grafts, respectively. Three (10.3%), eight (27.6%), 2 (6.9%) were inlay plus onlay, inlay plus veneer and onlay plus veneer respectively. Results: Measurements were taken on pre-operative, post-operative and followup radiographs. In the maxilla the mean final alveolar bone height was about 20 mm with an average resorption of about 5 mm (20%), whereas in the mandible the mean final alveolar bone height was about 16 mm with an average resorption of about 6 mm (27%) after 43 months. A total of 175 implants were then inserted: 54 into the mandible and 121 into the maxilla. Nine different implant types were used. Implants were inserted to replace 29 incisors, 23 cuspids, 54 premolars and 69 molars. The mean postloading follow-up was 33 months. No implant was lost (i.e. survival rate SVR = 100%). Crestal bone resorption around implant neck was used as predictor of clinical outcome (i.e. success rate SCR). Higher bone resorption was detected in mandible and in molar region. Conclusion: Calvarial bone graft is a reliable material for alveolar bone restoration with a predicable average of resorption. However special care must be taken in planning the operation. (J Osteol Biomat 2010; 1:171-177)

Key words: Calvaria, graft, pre-prosthetic surgery, bone resorption. Department of Maxillofacial Surgery, Civil Hospital Rovigo, Rovigo, Italy; Department of Maxillofacial Surgery, University of Ferrara, Ferrara, Italy. 3 Department of Histology, Embriology and Applied Biology, University of Bologna, Bologna. Italy. 1 2

Corresponding author: * Prof.Francesco Carinci, MD - Chair of Maxillofacial Surgery University of Ferrara - Arcispedale S. Anna - Corso Giovecca, 203 44100 Ferrara ITALY - Phone/Fax: +39.0532.455582 E-mail: crc@unife.it; Web: www.carinci.org

Introduction Endosseous implants are the treatment of choice for restoring function and reconstructing most edentulous areas of the maxilla and mandible.1 Bone availability is the key for successful placement of endosseous implants2 and the anatomic limitations of the residual alveolar bone can cause problems.3 Less than ideal sites can result in an aesthetic and functional compromise3 because implant placement requires an adequate quantity and quality of bone.4 When the thickness of the bone between the sinus and alveolar crest is less than 10 mm, increasing the thickness of the alveolus sinus floor through grafting is necessary to support the required implants and prosthetic restoration. The grafting material chosen must provide adequate viable bone to stabilize the implant and encourage osseointegration.2 In many cases, however, this anatomic problem can be solved with autogenous bone grafts, which are the most predictable and successful material available.3 It is important to know which donor sites can consistently provide the most quantity of bone for a graft. The ideal recipient site should provide good blood supply, adequate bony contact, and the graft should be secured to minimize mobility.4

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position).12-15 The large cortical component of the calvarial bone determines an excellent mechanical strength and a slight tendency to resorption. Usually the calvarial donor site produces less discomfort to the patient than do the rib or iliac crest grafts, and the scar can be hidden in a better way. Since there are few reports available regarding the clinical outcome of calvarial autogenous grafts used for alveolar ridge reconstruction we therefore planned a retrospective study to evaluate the factors influencing the outcome of grafting and the degree of bone resorption. In addition, a series of 175 implants inserted were evaluated. Figure 1. The calvarial harvesting

MaterialS and Methods In the case of severe atrophy, surgeons can use iliac crest and calvaria bone grafts to harvest a significant amount of autogenous bone. However, embryology, histology, and mechanical proprieties of these two bone grafts are different and may affect the short- and long-term alveolar ridge augmentation. The final result in terms of stability of the implants and facial morphology depends on bone resorption.5,6 One of the main problems with the use of iliac bone graft is its high resorption rate.7,8 Some researchers have suggested that membranous bone grafts (calvaria) undergo less resorption than do endochondral grafts (ileum).9,10 A slower revascularization of the calvaria has been proposed as the explanation for the lesser and slower resorption.11 It has been hypothesized that the pattern of onlay bone graft resorption is mainly related to the graft microarchitecture (cortical and cancellous com-

Journal of Osteology and Biomaterials

Patients Between May 1992 and July 2009, 100 consecutive patients with severe atrophic mandible or maxilla (classes V and VI, according to Cawood and Howell16) were treated at the Civil Hospital, Rovigo, Italy. Among them 27 and 11 had iliac and mandibular grafts, respectively. Of the remaining 62, only 17 had complete data sets and were considered in this study for a mean follow-up of 43 months. There were 6 men (35.3%) and 11 women (64.7%) with a mean age of 45 years at the time of presentation. A total of 29 grafts were inserted with a median age for graft of 47 years. Gender for graft was 16 (55.2%) and 13 (44.8%) in females and males, respectively. Eleven (37.9%) grafts were inserted in the right maxillae, 8 (27.6%) in the left maxillae, 5 (17.2%) in the left mandible and 5 (17.2%) in the right

mandible. Five (17.2%), 10 (34.5%) and 1 (3.4%) were inlay, onlay and veneer grafts, respectively. Three (10.3%), eight (27.6%), 2 (6.9%) were inlay plus onlay, inlay plus veneer and onlay plus veneer respectively. Graft surgery All patients underwent the same surgical protocol. An antimicrobial prophylaxis was administered with 2000 mg Amoxicillin before surgery and 1000 mg three daily for 7 days starting 1 hour before surgery. Local anaesthesia was induced by infiltration with mepicain 2% with adrenalin 1:100.00 and post-surgical analgesic treatment was performed with 30 mg Ketorolac tromentamina (Toradol, Recordati Spa Italy) or 10 mg Paracetamol (Perfalgan, Bristol-Myers Squibb Srl Italy). Oral hygiene instructions were provided. The calvarial harvesting was performed by splitting the parietal diploe. Six to eight bone sticks with dimensions of approximately 30/40 mm x 10 mm were delimited by using a drill under abundant washing to cool the saw. The delimitation of splints was done by using an oscillating saw that cuts the calvaria at partial thickness, and then the detachment of grafts was performed by using a saw with a small blade at an angle of approximately 30 degrees and chisels (Fig. 1). The occasional discovery of small areas of dura mater did not create any complications and there was never any leaking of liquor; however, bone dust with fibrin glue was always placed to protect areas of exposed dura. The calvarial sampling area was then protected with titanium mesh fixed with micro screws. Inlay, onlay, and veneer grafts were

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Figures 2-3. Veneer grafts

Figure 4-5. Pre and post-surgical

performed in 5, 10, and 1 cases, respectively. Inlay plus onlay, inlay plus veneer and onlay plus veneer were performed in 3, 8 and 2 cases respectively. After making a crestal incision a mucoperiostal flap was elevated. Grafts were inserted according to the procedures recommended (Figs. 2-5). Sutures were removed after 10 days. Measurement of bone deficit The alveolar bone height (ABH) was measured according to Cawood and Howell16 recommendations. Measurements were made on pre-operative, post-operative, and follow-up orthopantomograms. In the upper jaw, the floor of the maxillary sinus was taken as the measurement of the upper limit, whereas the lower limit was the margin of the alveolar ridge. Three points were determined on the maxilla to measure the ABH at the floor of the maxillary sinus: point A, corresponding to the lower part of the mesial wall of the maxillary sinus; point P, corresponding to the distal wall; and point I, the median point between points A and P. For the premaxillary re-

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gion, the measurement from the floor of the nasal fossae to the margin of the alveolar ridge was taken (point N at the lower part of nasal floor). In the mandible, the line passing through the 2 mental foramina was taken as the inferior alveolar limit, whereas the free border of the alveolar ridge was taken as the superior limit. Point S was the mid point of the line joining the 2 mental foramina. To take measurements in the region of the body of the mandible, 3 points were determined: point M, corresponding to the mental foramen; point L, corresponding to the mandibular spine; and point K, corresponding to the mid point between point M and point L projected onto the mandibular canal. The bony region underlying the imaginary line joining these points was defined as the basal region; the region above the line was defined as the alveolar region (Fig.6). Eight measurements were taken in the maxilla (i.e., right and left A, I, P, and N) and 7 were taken in the mandible (i.e., right and left L, K, M, and S in midline). A single mean value was recorded at different phases for each patient: pre-

Ortopanthomographies

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surgery, post-surgery, and after an appropriate follow-up (at least 6 months). The recorded measurements were defined as the residual alveolar bone height (RABH), post-surgical alveolar bone height (PSABH), and final alveolar bone height (FABH), respectively. Implant surgery All patients underwent the same surgical protocol. An antimicrobial prophylaxis was administrated with 500 mg Amoxycillin twice a day for 5 days, starting 1 hour before surgery. Local anesthesia was induced by infiltration with Articaine/Epinephrine and postsurgical analgesic treatment was performed with 100 mg Nimesulid twice daily for 3 days. Oral hygiene instructions were provided. Implants were inserted in autogenous bone grafts after a 6 months healing period and were loaded after an additional 6 months. After a crestal incision a mucoperiosteal flap was elevated. Implants were inserted according to the procedures recommended. The implant platform was positioned at the alveolar crest level. Sutures were removed 14 days after surgery. After 24 weeks from the implant insertion, the provisional prosthesis was provided and the final restoration was usually delivered within an additional 8 weeks. All patients were included in a strict hygiene recall. Variables Several variables are investigated: demographic (age and gender), anatomic (upper/lower jaws and tooth site), implant (length and diameter and type). Primary and secondary predictors of clinical outcome were used. The prima-

Journal of Osteology and Biomaterials

Figure 6. Orthopantomogram showing reference points used for measures

ry predictor is the presence/absence of the implant at the end of the observation period. It is defined as the survival rate (i.e. SVR), which is the total number of implants still in place at the end of the follow-up period. The second predictor of outcome is the periimplant bone resorption. It is defined as the implant success rate (SCR) and is evaluated according to the absence of persisting peri-implant bone resorption greater than 1,5 mm during the first year of loading and 0,2 mm/years during the following years.17 Data collection methods and summary of operative methods Before surgery, radiographic examinations were done with the use of orthopantomographs. In each patient periimplant crestal bone levels were evaluated by the calibrated examination of orthopantomograph x-rays. Measure-

ments were recorded after surgery and at the end of the follow-up period. The measurements were carried out mesially and distally to each implant, calculating the distance between the implant’s platform and the most coronal point of contact between the bone and the implant. The bone level recorded just after the surgical insertion of the implant was the reference point for the following measurements. The measurement was rounded off to the nearest 0.1 mm. The radiographs were performed with a computer system (Gendex, KaVo ITALIA srl, Genova, Italia) and saved in uncompressed TIFF format for classification. Each file was processed with the Windows XP Professional operating system using Photoshop 7.0 (Adobe, San Jose, CA), and shown on a 17” SXGA TFT LCD display with a NVIDIA GÈ Force FX GO 5600, 64 MB video card (Acer Aspire 1703

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Table 1. Univariate analysis output. Variable Upper/lower jaw Implant site Length Diameter Implant type

SM-2.6). Having understood the known dimensions of the implant, it was possible to establish the distance from the mesial and distal edges of the implant platform to the point of bone-implant contact (expressed in tenths of a millimeter). The difference between the implant-abutment junction and the bone crestal level was defined as the Implant Abutment Junction (IAJ) and calculated at the time of operation and during the follow-up. The delta IAJ is the difference between the IAJ at the last check-up and the IAJ recorded just after the operation. Peri-implant probing was not performed because controversy still exists regarding the correlation between probing depth and implant success rates.18 Data analysis Disease-specific survival curves were calculated according to the productlimit method (Kaplan-Meier algorithm).{Dawson-Saunders, 1994 #26} Time zero was defined as the date of the implantâ&#x20AC;&#x2122;s insertion. Implants which are still in place (or have a crestal bone resorption value lower than the cutoff value) were included in the total number at risk of loss only up to the time of their last follow-up. Therefore the survival rate only changed when implant loss (or cut-off overcome) occurred. The calculated survival rate was

Log Rank 9.34 20.15 0.61 4.64 58.81

Degree of freedom 1 3 2 2 8

the maximum estimate of the true survival curve. Log rank testing was used to compare survival/success curves, generated by stratifications for a variable of interest. Cox regression analysis was then applied to determine the single contribution of covariates on the survival/ success rate. Cox regression analysis compares survival/success data while taking the statistical value of independent variables into account, such as age and sex, on the whether or not, that an event (i.e. implant loss, crestal bone resorption value overcome) is likely to occur. If the associated probability was less than 5% (p<.05), the difference was considered statistically significant. In the process of doing the regression analysis, odds ratio and 95% confidence bounds were calculated. Confidence bounds did not have to include the value ÂŤ1Âť.19 Stepwise Cox analysis allowed us to detect the variables most associated to implant survival and/or clinical success. Results At the time of presentation, the average RABH was 13.1 mm. Post-operatively, the average PSABH was 23.7 mm. At the last follow-up, the average FABH was 18.6 mm. In the maxilla the mean FABH was about 20 mm with an average resorption of about 5 mm

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Significance 0.0022 0.0002 0.7357 0.0982 0.0001

(20%) whereas in the mandible the mean FABH was about 16 mm with an average resorption of about 6 mm (27%) after 43 months. A total of 175 implants were inserted: 54 into the mandible and 121 into the maxilla. The mean post loading follow up was 33 months. There were 41 BiImplant (3i implants, Biomet Inc, US), 40 P1H (3i implants, Biomet Inc, US), 27 Biomax (3i implants, Biomet Inc, US), 10 Branemark (Nobel Biocare, Zurich, Switzerland), 12 Hexa (Biotec srl, Vicenza, Italy), 13 Geass (Geass srl, Udine, Italy), 16 Neoss (Neoss srl, Milan, Italy), 8 Camlog (Camlog Biotechnologies AG, Basel Switzerland), 8 Screwvent (Sweden & Martina srl, Padua, Italy). There were 74 standard length fixtures (i.e. 13 mm), 83 short and 18 long implants. There were 113 standard diameter fixtures (i.e. 3.75 mm), 31 narrow and 31 wide implants. Implants were inserted to replace 29 incisors, 23 cuspids, 54 premolars and 69 molars.The overall mean bone resorption around implants was 1.4 mm in a mean followup period of 33 months. No implant was lost in the post-operative period. Kaplan Meier algorithm demonstrates that site of graft (i.e. maxilla or mandible), implant site (i.e. incisors, cuspid, premolar and molar), and type of implant were statistically different (Table1).

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Table 2. Multivariate analysis output. Variable Age Gender Upper/lower jaw Implant site Implant type

Table 2 confirmed that the graft site (i.e. mandible) and implant site (i.e. molars) correlated with a statistically significant higher delta IAJ (i.e. deeper crestal bone loss) and thus a worst clinical outcome. Discussion In the present study, no graft over 29 failed with a success rate of 100%. No complications were reported in our study although calvarial bone harvest has to be done carefully. In fact, donorsite morbidity after bone harvesting in autologous grafts, still remains a crucial problem in alveolar ridge augmentation. Complications associated with calvarial bone harvesting are: the risk of epidural haematoma from damage to the sagittal or other dural sinuses, dural tears, limited shaping because of bone rigidity, and limited amount of cancellous bone. The risks of intracranial complications and donor site deformity can be minimized by using split calvarial instead of full thickness bone grafts.20 The skull as a donor site has further advantages in that skull chips or shavings as well as bone dust can be harvested to fill certain defects that do not require a formal graft, or can be used to augment a bone graft. The donor site defect can often be hidden by the hair, has a useful gradual curvature, and minimal resorption occurs.21

Journal of Osteology and Biomaterials

Significance p value 0.9990 0.1342 0.0001 0.0004 0.2628

One of the main problems with the use of iliac bone graft is its high resorption rate. Studies are available on autografts, from the same site (i.e. iliac crest) and from calvaria.7-9,22-24 Binger and Hell23 reported an average vertical loss of about 3 mm in the first year with the use of microsurgically vascularized bone grafts for mandible augmentation, whereas Verhoeven et al.24 reported a 36% mean resorption rate of the graft mainly during the first year. Some researchers have suggested that membranous bone grafts (calvaria) undergo less resorption than do endochondral grafts (i.e. iliac crest).9,10 greater volume maintenance has been reported for calvarial grafts than for iliac bone grafts (72% vs. 32%).5 Donovan et al.9 reported 85% retention of calvarial grafts compared with a 34% retention of grafted iliac bone, with calvarial onlay grafts showing more than a 2-fold greater radiographic density when compared with iliac grafts. Here we report an average resorption of 21.5%. This results is better then 33% at 40 months that was reported previously obtained on 47 calvarial grafts.22 In addition, 175 implants with a mean post loading follow-up of 33 months were evaluated. Survival rate was of 100% since no implant was lost. Crestal bone resorption around implant’s neck was considered as a predictor of

clinical outcome (i.e. success rate). The mean SCR was of 1.4 mm at the end of the observation period. Implants inserted in mandible and in molar region have an higher bone resorption around fixture’ neck and thus a worse clinical outcome. Conclusions In conclusion, calvarial bone is a valuable material for alveolar ridge augmentation, however special care must be taken in planning the operation. In addition, Implants inserted in calvarial grafts have an high survival and success rate.

Acknowledgments This work was supported by FAR form University of Ferrara (F.C.), Ferrara, Italy, from PRIN 2008 (F.C.) and from Regione Emilia-Romagna, Programma di Ricerca Regione-Università, 20072009, Area 1B: Patologia osteo-articolare: ricerca pre-clinica e applicazioni cliniche della medicina rigenerativa, Unità Operativa n. 14.

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References 1. Elo JA, Herford AS, Boyne PJ. Implant success in distracted bone versus autogenous bonegrafted sites. J Oral Implantol 2009;35:181-4. 2. Block MS, Kent JN. Sinus augmentation for dental implants: the use of autogenous bone. J Oral Maxillofac Surg 1997;55:1281-6. 3. Triplett RG, Schow SR. Autologous bone grafts and endosseous implants: complementary techniques. J Oral Maxillofac Surg 1996;54:48694. 4. Tolman DE. Reconstructive procedures with endosseous implants in grafted bone: a review of the literature. Int J Oral Maxillofac Implants 1995;10:275-94. 5. Chen NT, Glowacki J, Bucky LP et al. The roles of revascularization and resorption on endurance of craniofacial onlay bone grafts in the rabbit. Plast Reconstr Surg 1994;93:714-22; discussion 23-4.

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12. Hardesty RA, Marsh JL. Craniofacial onlay bone grafting: a prospective evaluation of graft morphology, orientation, and embryonic origin. Plast Reconstr Surg 1990;85:5-14; discussion 15. 13. Sullivan WG, Szwajkun P. Membranous versus endochondral bone. Plast Reconstr Surg 1991;87:1145. 14. Ozaki W, Buchman SR, Goldstein SA et al. A comparative analysis of the microarchitecture of cortical membranous and cortical endochondral onlay bone grafts in the craniofacial skeleton. Plast Reconstr Surg 1999;104:139-47. 15. Motoki DS, Mulliken JB. The healing of bone and cartilage. Clin Plast Surg 1990;17:527-44. 16. Cawood JI, Howell RA. A classification of the edentulous jaws. Int J Oral Maxillofac Surg 1988;17:232-6. 17. Albrektsson T, Zarb GA. Determinants of correct clinical reporting. Int J Prosthodont 1998;11:517-21.

6. Reinert S, Konig S, Bremerich A et al. Stability of bone grafting and placement of implants in the severely atrophic maxilla. Br J Oral Maxillofac Surg 2003;41:249-55.

18. Quirynen M, Naert I, van Steenberghe D et al. Periodontal aspects of osseointegrated fixtures supporting an overdenture. A 4-year retrospective study. J Clin Periodontol 1991;18:719-28.

7. Astrand P, Nord PG, Branemark PI. Titanium implants and onlay bone graft to the atrophic edentulous maxilla: a 3-year longitudinal study. Int J Oral Maxillofac Surg 1996;25:25-9.

19. Cox DR, Oakes D. Analysis of survival data. New York: Chapman & Hall 1984.

8. Vermeeren JI, Wismeijer D, van Waas MA. One-step reconstruction of the severely resorbed mandible with onlay bone grafts and endosteal implants. A 5-year follow-up. Int J Oral Maxillofac Surg 1996;25:112-5. 9. Donovan MG, Ondra SL, Illig JJ et al. Combined transmandibular-zygomatic approach and infratemporal craniotomy for intracranial skull base tumors. J Oral Maxillofac Surg 1993;51:754-8. 10. Pinholt EM, Solheim E, Talsnes O et al. Revascularization of calvarial, mandibular, tibial, and iliac bone grafts in rats. Ann Plast Surg 1994;33:193-7. 11. Johansson B, Grepe A, Wannfors K et al. A clinical study of changes in the volume of bone grafts in the atrophic maxilla. Dentomaxillofac Radiol 2001;30:157-61.

20. Zins JE, Whitaker LA. Membranous versus endochondral bone: implications for craniofacial reconstruction. Plast Reconstr Surg 1983;72:77885. 21. Smith JD, Abramson M. Membranous vs endochondrial bone autografts. Arch Otolaryngol 1974;99:203-5. 22. Carinci F, Farina A, Zanetti U et al. Alveolar ridge augmentation: a comparative longitudinal study between calvaria and iliac crest bone grafrs. J Oral Implantol 2005;31:39-45. 23. Binger T, Hell B. Resorption of microsurgically vascularized bone grafts after augmentation of the mandible. J Craniomaxillofac Surg 1999;27:82-5. 24. Verhoeven JW, Cune MS, Terlou M et al. The combined use of endosteal implants and iliac crest onlay grafts in the severely atrophic mandible: a longitudinal study. Int J Oral Maxillofac Surg 1997;26:351-7.

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BioCRA

Original article

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Effect of implant-tooth distance on crestal bone resorption Matteo Danza MD1, Ilaria Zollino MD2, Anna Avantaggiato MD2, Francesco Carinci MD2*

Introduction: Around dental implants exists a “biologic width” of about 3 mm that should be preserved in order not to have adverse effect on soft and hard tissues around the implant and tooth. Spiral family implants (SFIs) are a new type of fixtures and some of them have a reverse conical head that allows for an increased volume of crestal bone around the implant neck. Since there are no reports available in the literature we planned a retrospective study on a series of SFIs to evaluate their clinical outcome focusing on implant-tooth distance. Materials and Methods: One hundred sixtysix (166) implants were investigated. The mean follow-up was 14 months. Several host-, surgery-, and implantrelated factors were investigated. Independent samples T-test, Kaplan Meier algorithm and Cox regression were used to detect those variables associated with the clinical outcome. Results: Implants inserted in post-extractive sockets were the only variable that had a statistical significant impact on crestal bone resorption. Conclusion: Two millimeters between SFI implants is a safe distance in order not to cause crestal bone resorption. Post-extractive implant insertion is the major determinant in term of peri-implant bone resorption in a short follow-up period. (J Osteol Biomat 2010; 1:179-185)

Key words: Implant, biological width, bone resorption, biology.

1 2

Senior Lecturer, Dental School, University of Chieti, Chieti, Italy; Maxillofacial Surgery, University of Ferrara, Ferrara, Italy;

Corresponding author: * Prof. Francesco Carinci MD Chair of Maxillofacial Surgery Arcispedale S. Anna, Corso Giovecca 203, 44100 Ferrara, ITALY E-mail: crc@unife.it; Web: www.carinci.org Phone/Fax: +39.0532.455582

INTRODUCTION As osseointegration is considered essential for the anchoring of implants, the establishment of a supracrestal soft tissue seal for protection of the osseointegration is considered to be important for the success of the treatment.1 Therefore, different surgical and prosthetic management techniques of the hard and peri-implant soft tissue have been developed to achieve stable and predictable aesthetic and functional results.2 Mechanical loading, mainly the use of immediate restoration, has shown to play an important role in bone remodeling and formation3,4 and it has been advocated to preserve the dimensions of the alveolar ridge.5 Despite the high success rates reported in osseointegrated implants, achieving optimal peri-implant mucosa dimension is a challenging procedure and maintaining it over time can be an equally demanding task.6 Hermann et al.7 have emphasized that gingival aesthetics strongly depends on a stable and constant vertical dimension of healthy periodontal soft tissues, commonly referred to as “biologic width”. The concept of a defined biological width of the supracrestal soft tissue has been supported by clinical data from studies evaluating soft tis-

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sue dimensions at implant-supported single-tooth replacement,8,9 showing a height of proximal soft tissue (papilla height) of about 4 mm. The absence of the inter-proximal papilla can lead to cosmetic deformities, phonetic difficulty and food impaction.10 Besides, the predictable regeneration of the inter-proximal papilla adjacent to dental implants remains a complex challenge and the peri-implant mucosal response is not clearly understood.11 Thus, preservation of interdental papillae is essential for an aesthetic single tooth restoration and characterization of the components that affect the presence or the absence of the papilla is of great importance. Choquet et al.9 evaluated that when the distance from the base of the contact point to the alveolar crest was < 5 mm, the papilla was present 100% of the time. When the distance is 6 mm or greater, the papilla is present 50 percent of the time or less. According to Choquet, similar results were obtained by Gastaldo et al.12 that observed that when the distance between the tooth and the implant was 3, 3.5 or 4 mm the papilla was present most of the time. A spiral implant is a conical internal helix implant with a variable thread design which confers the characteristic of self drilling, self tapping and self bone condensing. The spiral implant family (SIF) is composed of two types of implants, the Spiral Implant (SPI) and the Spiral Flare Bevel (SFB) (Fig. 1 and 2). The latter has a reverse conical head that allows for an increased volume of crestal bone around the implant neck. The reverse conical neck is also available in different implant types, such as the Konus implant (http://www.konus-

Journal of Osteology and Biomaterials

Figure 1. SPI implant

Figure 2. SFB implant

group.com/index.html, MICROTECH s.r.l., Arzene Pordenone, Italy) that accounts for some additional benefits such as a closer placement of adjacent implants without compromising the health of the tissues and the aesthetic outcome. Because SFIs have been on the market for the last ten years (they are available as Nobelactive implant, Nobel Biocarre, Z端rich-Flughafen Switzerland ) and some studies were performed on SFIs,13-19 there are still no reports, to the best of our knowledge, on the effect of implant-tooth distance (i.e. implant-tooth biological width) on crestal bone resorption, a retrospective study on a series of SFIs was performed in order to analyze their clinical outcome.

age of 53 years were operated and 234 spiral family implants (SFIs, 3D Alpha Bio, Pescara, Italy) were inserted. The last check-up was performed in October 2008, with a mean follow-up of 13 months. Subjects were screened according to standard inclusion criteria:20-22 i.e. controlled oral hygiene and the absence of any lesions in the oral cavity; in addition, the patients had to agree to participate in a post-operative check-up program. Exclusion criteria were as follows: bruxism, smoking more than 20 cigarettes/day, localized radiation therapy of the oral cavity, antitumor chemotherapy, liver, blood and kidney diseases, immunosupressed patients, patients taking corticosteroids, pregnant women, inflammatory and autoimmune diseases of the oral cavity, and poor oral hygiene.

MATERIALS AND METHODS Patients In the period between May 2004 and November 2007, 86 patients (55 females and 31 males) with a median

Data collection Before surgery and in the follow-up period, radiographic examinations were

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done. In each patient, peri-implant crestal bone levels were evaluated by the calibrated examination of orthopantomograph x-rays (Ortoralix SD, Gendex, Milano, Italia). A periapical radiograph was impressed by means of a customized Rinn holder device. This device was necessary to maintain the X-ray cone perpendicular to a film pieced parallel to the long axis of the implant. The endoral x-rays were taken using a long x-ray tube at 70 Kw of power, and developed in acid in a dark room according to standard procedures; they were scanned, transferred to a computer and saved in an uncompressed TIFF format for classification. Each file was processed with the Window XP Professional operating system using the Photoshop 7.0 (Adobe, San Jose, CA), and shown on a 17” SXGA TFT LCD display with a NVIDIA GÈ Force FX GO 5600, 64 MB video card (Acer Aspire 1703 SM-2.6). Each image was modified using the fit-on-screen function (maximized screen) and the necessary adjustments in contrast, brightness and magnification were made. The measurements were taken at the highest level of resolution possible through the “grid and ruler” program options using various metric scales. Knowing the known dimensions of the implant and having located various points of reference on the profiles of the x-rayed fixtures (edge of the platform, bone crestal level, total length of the implant), it was possible to take linear measurements on the computer and thus execute a proportional metric calculation comparing the known dimensions of the implant’s geometric design with those of the examined x-ray images. This made it possible to

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Figure 3. Occlusal view of a SFB implant

establish the distance from the mesial and distal edges of the implant platform to the point of bone-implant contact plus the visible crown (expressed in tenths of a millimeter) as an expression of marginal bone resorption. The proportional calculation of the measurements also made it possible to establish, where present, any distortion in the x-ray images for further screening, thereby reducing the margin of error of the analysis to a minimum. Measurements were recorded before surgery, after surgery and at the end of the follow-up period. The measurements were carried out mesially and distally to each implant, calculating the distance between the implant abutment junction and the bone crestal level. The x-rays was calibrated by using an internal standard that was the implant’ length. The bone level recorded just after the surgical insertion of the implant was the reference point for the following measurements. The measurement was rounded off to the nearest 0.1 mm. In addition, the following parameters

were considered: absence of persisting pain or dysesthesia, absence of periimplant infection with suppuration, absence of mobility, and absence of persisting peri-implant bone resorption greater than 1,5 mm during the first year of loading and 0.2 mm/years during the following years.23 Implants A total of 234 SFIs were inserted: among them 166 were adjacent to teeth, 102 (61.4%) inserted in female and 64 in males (38.6%), and therefore were considered in this retrospective study. Patients’ median age was 52 ± 14 years (min-max 16-89 years) and average crestal bone resorption was 1.7 ± 0.3 mm (min-max 0-8 mm). The mean follow-up was 14 months. Twenty-two SPI and 144 SFB were inserted, 64 (38.6 %) in the mandible and 122 (61.4 %) in the maxilla. Implant diameter was 3.75, 4.2, 5 and 6 in 11 (6.6 %), 80 (48.2 %), 45 (27.1 %) and 30 (18.1 %) SFIs, respectively. Implant length was less than 13 mm, 13 mm and 16 mm in 66 (39.8 %), 52 (31.3

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%) and 48 (28.9 %) SFIs, respectively. Implants were inserted to replace 35 incisors (21.1%), 18 cuspids (10.8 %), 57 premolars (34.3 %) and 56 molars (33.7 %). Sixty-seven fixtures were inserted in post-extractive sockets and the remaining 99 in healed bone; 91 (54.8%) were immediately loaded. One hundred and fifty five (93.4%) bore fixed restorations whereas 11 (6.6%) had removable dentures. Surgical and prosthetic technique All patients underwent the same surgical protocol. An antimicrobial prophylaxis was administered with 500 mg Amoxycillin twice daily for 5 days starting 1 hour before surgery. Local anesthesia was induced by infiltration with articaine/epinephrine and post-surgical analgesic treatment was performed with 100 mg Nimesulid twice daily for 3 days. Oral hygiene instructions were provided. After making a crestal incision a mucoperiosteal flap was elevated. In several cases a mucotomy was performed. Implants were inserted according to the procedures recommended. The im-

Figure 5. Final prosthetic rehabilitation

Journal of Osteology and Biomaterials

Figure 4. Lateral view of the abutment showing the health status of the papilla

plant platform was positioned at the alveolar crest level. Sutures, if used, were removed 14 days after surgery. In case of delayed loading the provisional prosthesis was provided after 24 weeks from implant insertion and in all cases the final restoration was usually delivered within an additional 8 weeks (Figs.3-8). The number of prosthetic units (i.e. N.P.U. = implant/crown ratio) was about 0.84. The antagonists were natural teeth and prosthetic crowns in 82

(49.4%) and 84 (50.6%) cases, respectively. All patients were included in a strict hygiene regimen. Statistical Analysis Independent samples t-test was used to detect any statistical differences existing between two groups: fixtures with good clinical outcome and “failed” implants (i.e. those with peri-implant bone resorption greater than 1,5 mm during the first year of loading and 0,2 mm/years during the following years). In addition, univariate (i.e. Log rank test)24 and multivariate analyses (i.e. Cox algorithm)25 were used to detect those variables which have an impact on crestal bone resorption. RESULTS Ninety six implants had good clinical outcomes (peri-implant crestal bone resorption 1.75 ± 0.30 mm), whereas 70 failed (2.1 ± 2.6 mm). Independent samples t-Test did not detect any statistical difference (p = 0.088). Additional investigated variables were: implant length (lower, equal or higher than

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Figure 6. Rx performed to check the implant-abutment connection I

13 mm), diameter (3.75, 4.2, 5 and 6) and subtype (SFB and SPI); age and gender of patients; upper/lower jaw, site (incisors, canine, premolars and molars) and post-extractive/healed bone; type of prosthesis (removable vs. fixed), N.P.U. (divided as N.P.U.=1, 0.5â&#x2030;¤N.P.U.<1, N.P.U.<0.5), and type of antagonist element (prosthetic vs. natural tooth). In univariate analysis, post-extractive implants and N.P.U. were statistically significant (see Table 1, Kaplan Meier algorithm, Log rank=7.36 df=1 p=.0067 and Log rank=8.05 df=3 p=.0367). In multivariate analysis, only post-extractive implants have a significant adverse effect on crestal bone resorption (Table 2, Cox regression). DISCUSSION The object of modern implantology is to supply an excellent aesthetic, stable and healthy soft peri-implant tissue with minimum or non-existing resorp-

Figure 7. Rx performed to check the prosthetic rehabilitation K

tion of the bone crest. Hence, because the bone crest constitutes the base for the soft tissue seal, alterations in the peri-implant bone level will affect the position of the soft tissue margin, which in turn, will have a significant impact on the aesthetic outcome of the implant therapy 1. An aesthetically acceptable implant-supported restoration requires thorough surgical and prosthetic treatment planning.2 In two stage implantology the starting point of biological peri-implant width is represented by micro-gap of connection abutment/fixture. The link of the recovery screw or of the abutment generates apically to the interface a bone loss within 1.5 and 2 mm.26 The location and size of the inter-proximal contact area influence the vertical dimension of the inter-dental space between an implant and the adjacent tooth.2 Tarnow10 showed that inter-implant distance plays a very important role in influencing bone resorption.

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Figure 8. The 14 months Rx control L

The critical measure is identified in 3 mm, behind which you have a bone lose > 1.5 mm that can cause the absence of inter-implant papilla. Other more recent studies1,12 have confirmed the incidence that inter-implant distance and its bone resorption can lead to the formation of the papilla. Choquet et al.9 established that the papilla level around single-tooth implant restorations is mostly related to the bone level adjacent to the teeth and more specifically to the bone crest. The probability of achieving adequate papillae decreases when the distance of the crestal bone level of the adjacent teeth to the proposed contact of the restoration increases.27,28 Traini and coll.29 have evaluated and compared the microstructure of alveolar bone between implants inserted at different intervals. The results of the research have showed the importance of bone microstructure both for maintaining of osseointegration and for localiza-

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Table 1. Univariate analysis: post-extractive implants and N.P.U. have a significant p value. Variable Log Rank Degree of freedom Implant length 0.19 2 Implant diameter 0.34 3 Implant type 1.7 1 Maxilla/mandible 0.01 1 Implant site 7.59 3 Post-extractive 7.36 1 N.P.U. 8.5 3 Antagonist 3.74 1

Table 2. multivariate analysis. Variable Age Gender Post-extractive N.P.U.

tion of soft gingival tissues. Lazzara30 analyzed the horizontal dimension of bone resorption through medialization of the connection abutment/implant, verifying that the separation of the micro-gap from bone tissues induces a less vertical resorption. Our data do not detect significant statistical differences in crestal bone resorption over time between fixtures and teeth inserted at a lower or higher distance then 1.9 mm (mean value). This fact could be due to the implant type: in fact, the SFB has a reverse conical head that allows for an increased volume of crestal bone around the implant neck that accounts for some additional benefits such as a closer placement of adjacent implants, without compromising healthy tissues and the aesthetic outcome. Among the other studied variables (i.e. implant length, diameter and subtype;

Journal of Osteology and Biomaterials

Significance .9108 .9519 .1918 .9236 .0553 .0067 .0367 .0532

Significance p value .0144 .9509 .0107 .1038

age and gender of patients; upper/ lower jaw, site and post-extractive/ healed bone; type of prosthesis, N.P.U. and type of antagonist element), postextractive implants and N.P.U. were statistically significant in univariate analysis (Table 1), whereas in multivariate analysis, only post-extractive implants had a significant adverse effect on crestal bone resorption (Table 2). This datum can be due to the short follow-up (i.e. about 1 year) where post surgical crestal bone remodeling effect is higher. In conclusion, 2 mm between SFI implants is a safe distance in order not to cause crestal bone resorption. Post-extractive implant insertion is the major determinant in term of peri-implant bone resorption in a short followup period.

ACKNOWLEDGMENTS This work was supported by FAR (F.C.) from the University of Ferrara, and PRIN 2008 (F.C.). The authors declare that they have no conflict of interest.

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REFERENCE 1. Cardaropoli G, Wennstrom JL, Lekholm U. Peri-implant bone alterations in relation to inter-unit distances. A 3-year retrospective study. Clin Oral Implants Res 2003;14:430-6. 2. Lops D, Chiapasco M, Rossi A et al. Incidence of inter-proximal papilla between a tooth and an adjacent immediate implant placed into a fresh extraction socket: 1-year prospective study. Clin Oral Implants Res 2008;19:1135-40. 3. Degidi M, Petrone G, Lezzi G et al. Histologic evaluation of 2 human immediately loaded and 1 titanium implants inserted in the posterior mandible and submerged retrieved after 6 months. J Oral Implantol 2003;29:223-9. 4. Degidi M, Scarano A, Piattelli M et al. Bone remodeling in immediately loaded and unloaded titanium dental implants: a histologic and histomorphometric study in humans. J Oral Implantol 2005;31:18-24. 5. Rosenquist B, Grenthe B. Immediate placement of implants into extraction sockets: implant survival. Int J Oral Maxillofac Implants 1996;11:205-9. 6. Kan JY, Rungcharassaeng K, Umezu K et al. Dimensions of peri-implant mucosa: an evaluation of maxillary anterior single implants in humans. J Periodontol 2003;74:557-62. 7. Hermann JS, Buser D, Schenk RK et al. Biologic width around titanium implants. A physiologically formed and stable dimension over time. Clin Oral Implants Res 2000;11:1-11.

10. Tarnow DP, Cho SC, Wallace SS. The effect of inter-implant distance on the height of inter-implant bone crest. J Periodontol 2000;71:546-9. 11. Azzi R, Etienne D, Carranza F. Surgical reconstruction of the interdental papilla. Int J Periodontics Restorative Dent 1998;18:466-73. 12. Gastaldo JF, Cury PR, Sendyk WR. Effect of the vertical and horizontal distances between adjacent implants and between a tooth and an implant on the incidence of interproximal papilla. J Periodontol 2004;75:1242-6. 13. Danza M, Guidi R, Carinci F. Spiral family implants inserted in postextraction bone sites. Implant Dent 2009;18:270-8. 14. Danza M, Zollino I, Carinci F. Comparison between implants inserted with and without computer planning and custom model coordination. J Craniofac Surg 2009;20:1086-92. 15. Danza M, Guidi R, Carinci F. Comparison between implants inserted into piezo split and unsplit alveolar crests. J Oral Maxillofac Surg 2009;67:2460-5. 16. Danza M, Fromovich O, Guidi R et al. The clinical outcomes of 234 spiral family implants. J Contemp Dent Pract 2009;10:49-56. 17. Carinci F, Brunelli G, Danza M. Platform switching and bone platform switching. J Oral Implantol 2009;35:245-50. 18. Danza M, Riccardo G, Carinci F. Bone platform switching: a retrospective study on the slope of reverse conical neck. Quintessence Int 2010; 41:35-40.

8. Grunder U. Stability of the mucosal topography around single-tooth implants and adjacent teeth: 1-year results. Int J Periodontics Restorative Dent 2000;20:11-7.

19. Danza M, Grecchi F, Zollino I et al. Spiral implants bearing full-arch rehabilitation: analysis of clinical outcome. J Oral Implantol 2010; 16: in press

9. Choquet V, Hermans M, Adriaenssens P et al. Clinical and radiographic evaluation of the papilla level adjacent to single-tooth dental implants. A retrospective study in the maxillary anterior region. J Periodontol 2001;72:1364-71.

20. Degidi M, Piattelli A, Gehrke P et al. Clinical outcome of 802 immediately loaded 2-stage submerged implants with a new grit-blasted and acid-etched surface: 12-month follow-up. Int J Oral Maxillofac Implants 2006;21:763-8.

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21. Degidi M, Piattelli A, Carinci F. Immediate loaded dental implants: comparison between fixtures inserted in postextractive and healed bone sites. J Craniofac Surg 2007;18:965-71. 22. Degidi M, Piattelli A, Carinci F. Clinical outcome of narrow diameter implants: a retrospective study of 510 implants. J Periodontol 2008;79:49-54. 23. Albrektsson T, Zarb GA. Determinants of correct clinical reporting. Int J Prosthodont 1998;11:517-21. 24. Dawson-Saunders B, Trapp RG. Basic & Clinical Biostatistic. Norwalk: Appleton & Lange; 1994. 25. Cox DR, Oakes D. Analysis of survival data. New York: Chapman & Hall; 1984. 26. Novaes AB, Jr., de Oliveira RR, Muglia VA et al. The effects of interimplant distances on papilla formation and crestal resorption in implants with a morse cone connection and a platform switch: a histomorphometric study in dogs. J Periodontol 2006;77:1839-49. 27. Beagle JR. Surgical reconstruction of the interdental papilla: case report. Int J Periodontics Restorative Dent 1992;12:145-51. 28. Grunder U. The inlay-graft technique to create papillae between implants. J Esthet Dent 1997;9:165-8. 29. Traini T, Novaes AB, Jr., Papalexiou V et al. Influence of interimplant distance on bone microstructure: a histomorphometric study in dogs. Clin Implant Dent Relat Res 2008;10:1-10. 30. Lazzara RJ, Porter SS. Platform switching: a new concept in implant dentistry for controlling postrestorative crestal bone levels. Int J Periodontics Restorative Dent 2006;26:9-17.

Volume 1 - Number 3 - 2010

186 Errata corrige: Del Fabbro M, et al. J Osteol Biomat 2010;2:69-79 Implant survival in maxillary sinus augmentation. An updated systematic review. Table 1. Features of included studies Article

Year

Lozada et al.12 Keller et al.13 Zinner & Small14 Blomqvist et al.15 Froum et al.16 Peleg et al.17 Van den Bergh et al.18 Buchmann et al.19 De Leonardis & Pecora20 Olson et al.21 Tarnow et al.22 Van den Bergh et al.23 Wannfors et al.65 Bahat & Fontanessi24 Kahnberg et al.25 Tawil & Mawla26 Hallman et al.27 Engelke et al.28 McCarthy et al.29 Philippart et al.30 Rodriguez et al.31 Stricker et al.32 Hallman et al.33 Hatano et al.34 Iturriaga et al.36 John & Wenz35 Schwartz-Arad et al.37 Shlomi et al.38 Testori et al.39 Boyne et al.40 Simunek et al.41 Ewers42 Butz & Huys43 Wallace at al.44 Scarano et al.45 Peleg et al.46 Karabuda et al.47 Mardinger et al.48 Mangano et al.49 Marchetti et al.50 Galindo-Moreno et al.51 Aguirre-Zorzano et al.52 Minichetti et al.53 Lee et al.55 Kahnberg & Vannas-Lofqvist56 Lee et al.54 Yamamichi et al.57 Meyer et al.58 Ferreira et al.59 Torres et al.60 Triplett et al.61 Chaushu et al.62 Bettega et al.63 Cannizzaro et al.64

1993 1994 1996 1998 1998 1998 1998 1999 1999 2000 2000 2000 2000 2001 2001 2001 2002 2003 2003 2003 2003 2003 2004 2004 2004 2004 2004 2004 2004 2005 2005 2005 2005 2005 2006 2006 2006 2007 2007 2007 2007 2007 2008 2008 2008 2008 2008 2009 2009 2009 2009 2009 2009 2009

Study type CT CS CS CT CT CS CS CT CT RCT RCT CS RCT CS CS CT CS CS CS CS CS CS CS CS CS CT CS CT CT RCT CS CS CS CS CS CT CT CS RCT CS CS CS CS CT CS RCT CS CS CS RCT RCT CS RCT RCT

Graft material ABG/BS ABG ABG+BS ABG ABG+BS/BS ABG+BS ABG ABG BS ABG/ABG+BS/BS ABG/ABG+BS/BS BS ABG ABG+BS/BS ABG BS ABG/ABG+BS/BS ABG+BS ABG ABG BS ABG ABG+BS ABG+BS ABG ABG/ABG+BS/BS ABG/ABG+BS ABG/ABG+BS ABG/ABG+BS ABG/BS BS BS ABG+BS ABG+BS/BS ABG/ABG+BS/BS ABG/ABG+BS/BS BS BS BS ABG+BS ABG+BS ABG+BS BS ABG+BS ABG+BS ABG+BS BS BS BS BS ABG/ABG+BS/BS BS ABG ABG+BS

N° patients 60 20 50 50 78 20 42 50 57 29 12 24 40 62 26 29 21 83 18 18 15 41 20 191 58 38 70 63 22 44 24 118 20 51 94 731 91 109 40 30 70 22 42 52 36 41 69 20 314 87 160 28 18 20

N° N° IS (%) sinuses implants 69 158 91.77 23 66 92.42 66 215 98.60 75 202 84.16 113 215 98.14 20 55 100.00 62 161 100.00 75 167 100.00 65 130 98.46 45 120 97.50 24 55 96.36 30 69 100.00 80 150 84.00 83 313 92.65 39 91 61.54 30 61 85.25 36 111 90.99 118 211 94.79 27 79 79.75 25 58 91.38 24 70 92.86 66 183 99.45 30 108 86.11 191 361 94.18 79 223 100.00 38 103 96.12 81 212 95.75 73 253 90.91 26 63 96.83 88 219 83.11 97.78 24 45 209 614 95.60 100.00 22 56 97.78 64 135 98.34 144 362 97.94 731 2132 95.75 >91 259 100.00 129 294 >40 100 96.00 95.00 48 140 99.24 98 263 100.00 22 36 97.79 56 136 98.46 58 130 47 153 100.00 52 97 100.00 69 159 92.45 33 123 97.56 406 1025 98.15 144 286 97.55 240 492 82.32 29 72 94.44 36 111 100.00 20 44 88.64

Imm / del

RRH (mm)

imm/del imm imm del imm/del imm del imm imm/del imm/del del del imm/del imm/del imm imm/del del imm/del imm/del del imm imm/del del imm del imm/del imm/del imm/del imm/del del imm/del del imm/del del imm imm imm/del imm/del imm imm/del imm/del imm del imm/del del del imm/del del imm/del imm/del del imm del imm

<3 / ≥3 NR NR NR NR 1-2 <4 - 8 <5 1-7 NR NR 4-8 2-7 NR 1 - 5.5 (2.5) 4-8 <5 0.5 - 7 / 2 - 9 <4 / >4 1-3 <5 <5 / >5 <5 4-6 <5 <4 / ≥4 NR 2 - 8.3 <5 / >5 <6 <3 / >3 1 - 5 (3.6) <4 / >4 NR 3-5 1-7 <5 / >5 1-8 3.5 - 5 <4.5 / ≥4.5 <5 ≥5 NR <6 5-6 ≤5 ≤4 / 5 - 8 0 - 4.5 (3.3) ≤7 <4 / 4 - 7 5.44 / 5.51 1 - 4 (2.7) 1.8 - 6.3 / 2 - 5.3 3 - 6 (4.4)

Implant surface M/R M R M M/R R R M/R R R M/R R M M M M M R M R NR R M M R R R R R NR R R R NR NR R R R R M/R R R R R R R M/R NR M/R R NR R NR R

CT = clinical trial, CS = case series, RCT = randomized controlled trial, ABG = autogenous bone graft, BS = bone substitute, IS = implant survival, imm = immediate implant placement, del = delayed implant placement, RRH = residual ridge height, M = machined, R = rough, FU = follow-up, mo = months, NR = not reported

Journal of Osteology and Biomaterials

FU, range Mean FU (mo) (mo) up to 60 12.0 12 - 72 12.0 7 - >60 12.0 9 - 48 34.1 0 - 48 12.0 15 - 39 26.4 12 - 72 38.2 >36 - >60 60.0 12 12.0 5 - 71 38.2 0 - 60 12.0 12 - 72 34.6 12 12.0 12 - 96 37.3 12 - >60 39.8 12 - 40 22.4 18 18.0 0 - 60 12.0 17 - 66 37.5 12 - >48 31.5 6 - 36 12.0 15 - 40 27.4 60 60.0 up to 108 27.3 >17 17.0 18 18.0 4 - 84.8 43.6 24 24.0 18 - 60 44.0 36 36.0 12 - 23 16.4 up to 156 12.0 up to 84 12.0 >12 12.0 24 - 87 48.0 up to 108 69.0 8 - 72 36.0 20 20.0 18 18.0 12 - 60 12.0 24 24.0 27 - 53 35.0 27 - 54 27.0 6 - 27 13.0 12 - 60 34.8 12 12.0 36 36.0 24 - 72 48.0 3 - 72 42.0 24 24.0 24 24.0 11 - 46 27.0 12 12.0 12 12.0

Carla Palumbo Dominique Pioletti Fausto Polastri Sergio Rosini Gilberto Sammartino Carine Tabarani

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