ORIGINAL ARTICLE
Equine and Porcine Bone Substitutes in Maxillary Sinus Augmentation: A Histological and Immunohistochemical Analysis of VEGF Expression Stefano Tetè, MD, DDS,* Vincenzo Luca Zizzari, DDS,† Raffaele Vinci, MD, DDS,‡ Susi Zara, PhD,† Umberto Di Tore, DDS,* Marco Manica, DDS,§ Amelia Cataldi, MD,† Carmen Mortellaro, MD, DDS,||¶ Adriano Piattelli, MD, DDS,* and Enrico Gherlone, MD, DDS‡ Abstract: The aim of this work was to investigate the morphological structure and the expression of vascular endothelial growth factor (VEGF) after maxillary sinus augmentation through equine and porcine bone substitutes in humans. Ten patients showing edentulous posterior maxilla underwent maxillary sinus augmentation through particulate equine bone substitute and 10 patients through particulate porcine bone substitute. At the moment of implants insertion, 6 months after grafting, bone specimens were withdrawn and processed for morphological and immunohistochemical analyses. Notwithstanding the almost comparable clinical performances of both bone substitutes, histological results showed a better integration when an equine bone substitute was used compared to a porcine one. In particular, evident signs of particles resorption were observed in equine bone substitute group specimens compared to porcine ones. Immunohistochemical analysis showed a statistically significant increase of VEGF expression in equine compared to porcine bone substitute group specimens. These results showed both bone substitutes to achieve comparable clinical performance, indicating their successful use for bone regenerative procedures. However, in the same experimental time, equine group specimens showed evident resorption phenomena, whereas no or little signs of resorption were evident in the porcine group specimens. However, a more rapid and intense vascularization was achieved in equine bone substitute group, as demonstrated by immunohistochemical analysis for VEGF expression. Even if differences in vascularization significantly affect the clinical performance of a heterologous bone substitute, its ability to be resorbed is also very important in influencing long-term integration and long-term predictability of implant-prosthetic rehabilitation in regenerated sites. From the *Departments of Medical, Oral and Biotechnological Sciences and †Pharmacy, University “G. d’Annunzio,” Chieti-Pescara; ‡Department of Dentistry, University “Ateneo Vita-Salute S. Raffaele,” Milan; §Pistoia (private practice); ||Department of Medical Science, Faculty of Medicine, University of Eastern Piedmont, Novara; and ¶Oral Surgery Unit, Regina Margherita Pediatric Hospital, Turin, Italy. Received June 4, 2013. Accepted for publication January 7, 2014. Address correspondence and reprint requests to Stefano Tetè, MD, DDS, Department of Medical, Oral and Biotechnological Sciences, University “G. d’Annunzio,” Via dei Vestini, 31-66100 Chieti, Italy; E-mail: [email protected] The authors report no conflicts of interest. Copyright © 2014 by Mutaz B. Habal, MD ISSN: 1049-2275 DOI: 10.1097/SCS.0000000000000679
Key Words: Equine bone substitute, porcine bone substitute, maxillary sinus augmentation, immunohistochemical analysis, VEGF (J Craniofac Surg 2014;25: 835–839)
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o allow implant-supported rehabilitation of edentulous ridges affected by severe bone atrophy, bone regeneration procedures are often necessary. Especially in the posterior maxilla, after teeth extraction, alveolar bone undergoes remodeling phenomena together with maxillary sinus hyperpneumatization, resulting in an insufficient bone height to ensure stability to implant insertion.1 A classification of residual bone in the posterior maxilla was introduced with the purpose of considering, besides the width and the height of the residual alveolar bone, also the relationship between upper and lower jaw after the remodeling phenomena take place.2 To correct severe bone defects when the intermaxillary relationship is maintained and to have suitable prosthetic results, maxillary sinus augmentation procedure represents a valid treatment solution.3,4 In recent years, different materials have been proposed and used to ensure bone regeneration through grafting the maxillary sinus: intraoral or extraoral autologous bone, homologous grafts, heterologous grafts, alloplastic grafts, or a combination of these.5–8 Autologous bone graft is still considered the gold standard for bone regeneration. However, the morbidity involved with its use may induce many patients to refuse this treatment. Instead, different heterologous bone substitutes were utilized for sinus augmentation.9 As previously reported, heterologous biomaterials were found to be as clinically efficient as autologous bone for their osteoconductive potential, though not much is known about their capability to be fully resorbed or about the time they need to be entirely substituted by newly formed bone.7 As the biological response of the host tissue can be related to the biomaterial derivation and to the treatment it underwent before its clinical use, attention was addressed on the interactions occurring between bone substitutes and host tissue.9–11 Different heterologous biomaterials have been proposed for clinical use in oral surgery; bovine-derived bone substitutes represent one of the most popular and well-documented categories. They were described to have osteoconductive properties and to be well integrated in host bone tissue as shown in both in vitro histological and clinical studies.12 However, the possibility of prion disease transmission associated with the use of this source of heterologous bone led to other different sources for bone substitutes.13,14 In literature, few data are reported concerning the use of equine or porcine bone substitutes. Recent studies described equine-derived bone as being able to induce osteoblast differentiation, to be resorbed in vitro by osteoclasts, and to be successfully used in mandibular ridge augmentation.15,16 On the other hand, porcine-derived bone may be successfully used to obtain osteogenesis in guided bone regeneration techniques.17,18 Independently by their derivation, all bone substitutes from a nonhuman mammal species must undergo a series of deantigenation processes to make them suitable for clinical use by reducing antigenicity
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and any possible risks of cross-infections. One of the most utilized way to completely eliminate the organic components (sugars, lipids, proteins) of an animal-derived bone tissue is the thermal deantigenation at high temperatures (about 500–1200°C). Even if it allows the removal of the entire organic component, thus avoiding the onset of an immune response of the host, this treatment also modifies the mineral structure of bone hydroxyapatite, and thus the resulting biomaterial usually possesses a reduced resorption potential. On the contrary, equine-derived bone substitutes are deantigenated by a proteolytic process through digestive enzymes at about 37°C, selective for the organic component, which leaves unaltered the ability of the biomaterial to be reabsorbed in vivo.19 The host tissue response to a bone substitute may be evaluated by immunohistochemical analysis for the expression of molecules specifically involved in the bone healing process.11 In this process, vascular endothelial growth factor (VEGF) plays an important role. VEGF is produced by endothelial cells and osteoblasts, and is involved in initial bone remodeling phases because it regulates osteoblast evolution.20,21 VEGF is also capable of inducing the growth of new blood vessels and plays a significant role in the preservation and development of endothelial fenestrations,22,23 thus supporting new blood vessels formation in the site of grafting, which is a fundamental phase for graft integration. The aim of this work was to evaluate the morphological structure and the expression of VEGF after maxillary sinus augmentation through equine and porcine bone substitutes in humans.
PATIENTS AND METHODS Patients Twenty patients, 8 males and 12 females, aged between 51 and 63 years, with inadequate bone volume in the posterior maxilla, classified as class C according to Chiapasco’s classification of the posterior maxilla,2 who were scheduled for maxillary sinus augmentation procedures before implants placement, were included in this study. All patients gave written informed consent in accordance with the Local Ethics Committee, in compliance with Italian legislation and with the code of Ethical Principles for Medical Research involving Human Subjects of the World Medical Association (Declaration of Helsinki). The patients were randomly divided into 2 groups. Ten patients (3 males, 7 females; age range 51–59) underwent maxillary sinus augmentation procedure through a particulate bone substitute of equine origin (BioBone Osteoconductor Mix; BioSAF IN S.r.l., Ancona, Italy), and 10 patients (5 males, 5 females; age range 52–63) underwent maxillary sinus augmentation procedure through a particulate bone substitute of swine origin (Gen-Os; Tecnoss, Turin, Italy). BioBone Osteoconductor Mix is a resorbable, collagen-deprived, and deantigenated osteoconductive biomaterial, obtained after deantigenation at 37°C in a humid atmosphere, made up of a corticocancellous mixture of equine origin (particle width 0.5–1 mm). Gen-Os are commercially available collagenated swine-derived corticocancellous bone chips, with particle width between 250 and 1000 μm. Before surgical procedures, patients underwent complete medical anamnesis and radiographic examinations, including orthopantomography and cone beam computed tomography. All the selected patients had healthy systemic conditions, including the absence of any diseases that would contraindicate oral surgery. The exclusion criteria were uncontrolled periodontal disease, sinusitis, severe illness, unstable diabetes, drug abuse, a history of head and neck irradiation, and chemotherapy. Moreover, antibiotic therapy (amoxicillin 875 mg/ clavulanic acid 125 mg, Augmentin, 2 g an hour before) was administered to all patients preoperatively.
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Surgical Procedures The soft tissue was incised and flapped back to expose the underlying lateral wall of the maxillary sinus. The bone wall was then removed with a piezoelectric instrument (Easy Surgery; BioSAF IN S.r.l.) exposing the Schneiderian membrane. The membrane was then cut back from the inner face of the lower portion of the maxillary sinus cavity. The newly formed space within the bone cavity of the sinus inferior to the intact membrane was filled with the heterologous bone substitute and then covered by a collagen membrane (BioBone Collagen Membrane; BioSAF IN S.r.l.). Closure of the surgical access was obtained with 3-0 sutures (Silkam/Virgin Silk; B. Braun Melsungen AG, Germany). For all patients, the postoperative therapy protocol comprised administration of antibiotic (Augmentin) 2 g/day for 10 days, nonsteroidal analgesic drug (ketoprofen, OKI; Dompè, L’Aquila, Italy) at a dose of 200 mg twice daily for 3 days, and cortisone (Betametason, Bentelan; Defiante Farmaceutico, Madeira, Portugal) 4 mg/day for 2 days and 2 mg on day 3. Moreover, soft diet and oral hygiene, including rinsing 3 times daily with 0.2% chlorhexidine (Corsodyl; GlaxoSmithKline) mouthwash and the application at the surgical site of 1% chlorhexidine gel (Corsodyl), were prescribed. Sutures were removed 10 days after the intervention and postoperative check-ups scheduled weekly for the first month, and then monthly by clinical and radiographical examination with periapical x-rays in the grafted area. As postoperative healing was uneventful for all the patients, after about 6 months they all underwent a second surgery for implant placement. The implant features (diameter and length) were decided according to the anatomic situation individuated by clinical and radiographic examinations. However, all implants inserted had diameters between 3.3 mm and 4.5 mm, and lengths between 9 mm and 13 mm. During implant insertion, bone samples were retrieved by a 3-mm-diameter and 8-mm-height trephine bur under sterile saline solution irrigation at the sites of implant placement to obtain significant specimens of bone regenerated with both heterologous bone substitutes. Pharmacological protocol included antibiotic (Augmentin 2 g an hour before) and nonsteroidal analgesic drugs to be taken as required.
Light Microscopy and Immunohistochemical Analysis Bone specimens, fixed in phosphate-buffered formalin solution, were decalcified in 10% tetrahydrated EDTA solution according to data sheet (MIELODEC kit; Bio-Optica, Milan, Italy), dehydrated through ascending alcohol concentrations, and then paraffin-embedded, to be sectioned at 5 μm thick. Sections were then dewaxed (alcohols progressively lower concentrations) and processed for hematoxylineosin staining and for immunohistochemical analysis. Tissue sections with hematoxylin-eosin staining were observed to detect the presence of area of tissue remodeling. To detect VEGF, immunohistochemical analysis was performed on 5-μm-thick tissue sections for each experimental specimen, by means of Ultravision LP Detection System HRP Polymer and DAB Plus Chromogen (Lab Vision Thermo, Fremont, CA). Sections were incubated in the presence of rabbit polyclonal anti-VEGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and successively in the presence of specific HRP-conjugated secondary antibody. Peroxidase was developed using diaminobenzidine chromogen and nuclei were hematoxylin counterstained. Negative controls were performed by omitting the primary antibody. Samples were then observed by means of Leica DM 4000 light microscopy (Leica Cambridge Ltd, Cambridge, UK) equipped with a Leica DFC 320 camera (Leica Cambridge Ltd) for computerized images. © 2014 Mutaz B. Habal, MD
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
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VEGF in Equine and Porcine Grafts
Image Analysis and Statistical Evaluation After digitizing the images derived from hematoxylin-eosin stained sections, the presence of native bone tissue was recognized by the presence of osteocyte lacunae-containing cells, whereas the newly formed bone tissue was recognized by the absence of lacunae. Densitometric evaluation was obtained by measuring the percentage area (±SD) determined by direct visual evaluation of 10 fields (mean values) for each of 5 slides per specimen at 20 magnification. After digitizing the images derived from immunohistochemical stained sections, QWin Plus 3.5 software (Leica Cambridge Ltd) was used to evaluate VEGF expression. Image analysis of protein expression was performed through the quantification of threshold area for brown color, as average value per 10 fields, randomly chosen, for each sample at light microscope observation. Negative control images were randomly chosen. The statistical significance was evaluated by the Wilcoxon and Mann-Whitney tests, using R Software, version 2.12.1 for Mac and setting P = 0.05. After collecting results, the mean data were reported and shown in a histogram using Excel 2008 for Mac.
RESULTS Morphological analysis was performed by light microscope after hematoxylin-eosin staining (Fig. 1). A better integration was shown when an equine bone substitute is used compared to a porcine one, and more evident signs of particles resorption were observed in equine bone substitute group specimens compared to porcine ones. In particular, hematoxylin-eosin staining highlights new bone formation in equine bone substitute group along with areas of native bone tissue and connective fibers. Moreover, the grafted biomaterial particles are easily distinguishable because of lack of cellular structure, and they appear in part resorbed and well integrated with the newly formed bone tissue. Abundant areas of newly formed bone tissue are recognizable for the lack of bone lacunae. In samples from the porcine bone substitute group, the bone particles are still clearly distinguishable from native bone tissue for absence of viable cells. The size of the bone particles appears quite unchanged and no clear evidence of resorption is detected. Samples from equine bone substitute group showed a significant higher amount of newly formed bone tissue compared to the native tissue than in porcine ones (Fig. 2). VEGF expression modifications occurring after different bone graft insertion and the evaluation of clinical applicability and integration ability of these grafts were then carried out. Densitometric values obtained from immunohistochemical analysis reveal a statistically significant increase in VEGF expression in samples from equine compared to porcine bone substitute group (P < 0.05) (Fig. 3).
FIGURE 2. Graphic representation of densitometric evaluation of newly formed bone tissue and native bone in equine and porcine bone substitutes specimens. Data are expressed as area % (±SD) determined by direct visual evaluation of 10 fields (mean values) for each of 5 slides per specimen at 20 magnification. Group A, bone tissue specimens obtained from equine bone substitute grafted area; Group B, bone tissue specimens obtained from porcine bone substitute grafted area. *Group A newly formed bone tissue versus group B newly formed bone tissue, P < 0.05; group A native bone tissue versus group B native bone tissue, P < 0.05.
implants placed in regenerated sites.24,25 In our study, we evaluated the success of regenerative therapy directly at the time of implant placement, that is 6 months after grafting, by histological and immunohistochemical analyses. Treatment of edentulous sites in the posterior maxilla requires a correct approach to obtain a functional implant-supported restoration. This goal may be achieved through adequate reconstruction by regenerative techniques with the use of bone substitutes. Even if the best performance of autologous bone grafts was widely demonstrated,23,26 clinicians are constantly searching for a heterologous bone substitute that permits to combine the osteoregenerative features of autologous bone, eliminating at the same time the limits imposed by the need for a second surgical intervention for withdrawal.10 Moreover, most heterologous bone substitutes currently used in clinical practice do not show ideal characteristics for bone regeneration such
DISCUSSION Most previous studies, to assess the success of a maxillary sinus augmentation procedure, take into consideration the survival rate of
FIGURE 1. Hematoxylin and eosin staining of equine and porcine bone substitutes specimens. Magnification 20 (inset 40). Group A: bone tissue specimens obtained from equine bone substitute grafted area; group B: bone tissue specimens obtained from porcine bone substitute grafted area.
FIGURE 3. Immunohistochemical analysis of VEGF expression in equine and porcine bone substitutes specimens. Magnification 20. Group A, bone tissue specimens obtained from equine bone substitute grafted area; Group B, bone tissue specimens obtained from porcine bone substitute grafted area; C (−), negative control; D, graphic representation of VEGF positive area % (±SD) densitometric analysis determined by direct visual counting of 10 fields (mean values) for each of 5 slides per specimen at 20 magnification. *Group A versus group B, P < 0.05.
© 2014 Mutaz B. Habal, MD
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as biological safety; osteogenic, osteoinductive, and angiogenic potentials; long shelf-life; no size restrictions; and reasonable cost.27,28 Heterologous biomaterials of different origins are successfully used as filling material for maxillary sinus augmentation.29,30 As previously described, heterologous bone particles work only as osteoconductive biomaterial and they usually undergo slow resorption.31,32 A biomaterial exhibiting no or scarce resorption could cause problems for a correct bone regeneration because of lower osteogenesis capability in respect to native autologous bone during the remodeling phase.33 The biological behavior of a heterologous bone substitute is also affected by the treatment it undergoes before in vivo utilization. Most heterologous bone substitutes of animal origin, such as bovine and porcine, are treated by thermal deantigenation to make them suitable for clinical use by reducing antigenicity and possible risks of cross-infections. However, this treatment is known to alter the mineral structure of bone hydroxyapatite, thus reducing the osteoclastic remodeling rate of the bone substitute. Clinically, the performance of the biomaterial is not significantly affected by this kind of treatment, as the bone substitute acts principally as a bioinert scaffold. In fact, bovine and porcine bone substitutes have been successfully used for years in guided bone regeneration techniques, even if residual biomaterial particles could be detected after many years from grafting.20,33 On the contrary, equine-derived bone substitutes are deantigenated by a proteolytic, low-temperature process, selective for the organic component, that seems to increase the capabilities of a bone substitute subjected to this kind of process to be resorpted.34 In fact, recent scientific evidence shows that biomaterials treated in this way undergo resorption and remodeling in about 12 months.20 In literature, only a few studies have reported on the use of an equine bone substitute, which appeared to be able to induce osteoblasts differentiation, to be resorbed in vitro by osteoclasts, and to be safely used in mandibular bone regeneration.20,35 The aim of this work was to investigate the morphological aspect and the expression of VEGF after maxillary sinus augmentation through equine and porcine bone substitutes in humans. Light microscopic analysis strongly evidenced that sites treated with the equine bone substitute showed good integration between host tissue and graft. Moreover, after a mean healing period of 6 months, the grafted biomaterial seemed to have activated intense remodeling phenomena, as confirmed by the existence of large areas of newly formed bone tissue, while particles of the porcine-derived bone substitute showed only few signs of resorption. To support these morphological aspects, the occurrence of new angiogenic processes was evaluated by checking VEGF expression. Vascularization has a crucial role in the regulation of bone remodeling and repair. New blood vessel formation is necessary to allow circulating osteoblast precursors to reach the remodeling site.20 Moreover, VEGF is also involved in supporting osteoblast growth during the initial phase of bone graft integration.36 In the present study, VEGF expression resulted higher in sites treated with equine bone substitute than in those treated with the porcine one, indicating that neoangiogenic phenomena occurred both earlier and an intense way between the 2 groups at the same healing time. Even if long-term results are not yet available to evaluate host-tissue response after a longer healing period, the present results indicate that both equine- and porcine-derived bone substitutes could be successfully used for regenerative therapy of intraoral bone defects. Still aware that differences in vascularization significantly affect the clinical performance of a heterologous bone substitute, its ability to be resorbed is also very important in influencing long-term integration and long-term predictability of implant-prosthetic rehabilitation in regenerated sites. In fact, the higher resorption capabilities of the equine-derived bone substitute compared to porcine-derived one,
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which probably could be due to the deantigenation process this biomaterial undergoes, could represent an added value to this category of bone substitute.
ACKNOWLEDGMENTS The authors thank the FIRB-Accordi di Programma 2010, “Processi degenerativi dei tessuti mineralizzati del cavo orale, impiego di biomateriali e controllo delle interazioni con i microrganismi dell’ambiente,” Prof. A. Cataldi, for the fellowship attributed to V.L.Z.
REFERENCES 1. Tallgren A. The continuing reduction of the residual alveolar ridges in complete denture wearers: a mixed-longitudinal study covering 25 years. J Prosthet Dent 2003;89:427–435 2. Chiapasco M, Zaniboni M. Methods to treat the edentulous posterior maxilla: implants with sinus grafting. J Oral Maxillofac Surg 2009;67:867–871 3. Ariji Y, Kuroki T, Moriguchi S, et al. Age changes in the volume of the human maxillary sinus: a study using computed tomography. Dentomaxillofac Radiol 1994;23:163–168 4. Jensen J, Sindet-Pedersen S, Oliver AJ. Varying treatment strategies for reconstruction of maxillary atrophy with implants: results in 98 patients. J Oral Maxillofac Surg 1994;52:210–216 5. Tetè S, Vinci R, Zizzari VL, et al. Maxillary sinus augmentation procedures through equine-derived biomaterial or calvaria autologous bone: immunohistochemical evaluation of OPG/RANKL in humans. Eur J Histochem 2013;57:60–65 6. De Leonardis D, Pecora GE. Augmentation of the maxillary sinus with calcium sulfate: one-year clinical report from a prospective longitudinal study. Int J Oral Maxillofac Implants 1999;14:869–878 7. Mangano C, Scarano A, Iezzi G, et al. Maxillary sinus augmentation using an engineered porous hydroxyapatite: a clinical, histological, and transmission electron microscopy study in man. J Oral Implantol 2006;32:122–131 8. Aguirre-Zorzano LA, Rodriguez-Tojo MJ, Aguirre-Urizar JM. Maxillary sinus lift with intraoral autologous bone and B-tricalcium phosphate: histological and histomorphometric clinical study. Med Oral Patol Oral Cir Bucal 2007;2:532–536 9. Jensen OT, Shulman LB, Block MS, et al. Report of the sinus consensus conference of 1996. Int J Oral Maxillofac Implants 1998; 13(Suppl):11–45 10. Cho YR, Gosain AK. Biomaterials in craniofacial reconstruction. Clin Plast Surg 2004;31:377–385 11. Tetè S, Zara S, Vinci R, et al. Vascular endothelial growth factor and e-nitric oxide synthase-mediated regenerative response occurring upon autologous and heterologous bone grafts. Int J Immunopathol Pharmacol 2009;22:1105–1116 12. Tetè S, Mastrangelo F, Carone L, et al. Morphostructural analysis of human follicular stem cells on highly porous bone hydroxyapatite scaffold. Int J Immunopathol Pharmacol 2007;20:819–826 13. Sogal A, Tofe AJ. Risk assessment of bovine spongiform encephalopathy transmission through bone graft material derived from bovine bone used for dental applications. J Periodontol 1999;70:1053–1063 14. Kim Y, Nowzari H, Rich SK. Risk of prion disease transmission through bovine-derived bone substitutes: a systematic review. Clin Implant Dent Relat Res 2011. doi: 10.1111/j.1708-8208.2011.00407.x 15. Perrotti V, Nicholls BM, Piattelli A. Human osteoclast formation and activity on an equine spongy bone substitute. Clin Oral Implants Res 2009;20:17–23 16. Artese L, Piattelli A, Di Stefano DA, et al. Sinus lift with autologous bone alone or in addition to equine bone: an immunohistochemical study in man. Implant Dent 2011;20:383–388 17. Traini T, Degidi M, Sammons R, et al. Histologic and elemental microanalytical study of anorganic bovine bone substitution following sinus floor augmentation in humans. J Periodontol 2008;79:1232–1340
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Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
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18. Orsini G, Scarano A, Piattelli M, et al. Histologic and ultrastructural analysis of regenerated bone in maxillary sinus augmentation using a porcine bone-derived biomaterial. J Periodontol 2006;77:1984–1990 19. Pagnutti S, Maggi S, Di Stefano DA, et al. An enzymatic deantigenation process allows achieving physiological remodeling and even osteopromoting bone grafting materials. Biotechnol & Biotechnol 2007;21:4 20. Gerber HP, Vu TH, Ryan AM, et al. VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat Med 1999;5:623–628 21. Hiltunen MO, Ruuskanen M, Huuskonen J, et al. Adenovirus-mediated VEGF-A gene transfer induces bone formation in vivo. FASEB J 2003;17:1147–1149 22. Yokomori H, Oda M, Yoshimura K, et al. Vascular endothelial growth factor increases fenestral permeability in hepatic sinusoidal endothelial cells. Liver Int 2003;23:467–475 23. Darland DC, Massingham LJ, Smith SR, et al. Pericyte production of cell-associated VEGF is differentiation-dependent and is associated with endothelial survival. Dev Biol 2003;264:275–288 24. Del Fabbro M, Rosano G, Taschieri S. Implant survival rates after maxillary sinus augmentation. Eur J Oral Sci 2008;116:497–506 25. Pjetursson BE, Tan WC, Zwalhem M, et al. A systematic review of the success of sinus floor elevation and survival of implants inserted in combination with sinus floor elevation. J Clin Periodontol 2008;35:216–240 26. Crespi R, Vinci R, Capparè P, et al. Calvarial versus iliac crest for autologous bone graft material for a sinus lift procedure: a histomorphometric study. Int J Oral Maxillofac Implants 2007;22:527–532 27. Tetè S, Vinci R, Zara S, et al. Long-term evaluation of maxillary reconstruction by iliac bone graft. J Craniofac Surg 2011; 22:1702–1707
VEGF in Equine and Porcine Grafts
28. Tetè S, Vinci R, Zara S, et al. Atrophic jaw reconstruction by means of calvarial bone graft: long-term results. J Craniofac Surg 2010;21:1147–1152 29. Tetè S, Zara S, Zizzari VL, et al. Immunohistochemical analysis of matrix metalloproteinase-9, vascular endothelial growth factor, bone sialoprotein and i-nitric oxide synthase in calvaria vs. iliac crest bone grafts. Clin Oral Implants Res 2012;23:1254–1260 30. Finkemeier CG. Bone-grafting and bone-graft substitutes. J Bone Joint Surg Am 2002;84-A:454–464 31. John HD, Wenz B. Histomorphometric analysis of natural bone mineral for maxillary sinus augmentation. Int J Oral Maxillofac Implants 2004;19:199–207 32. Hallman M, Lundgren S, Sennerby L. Histological analysis of clinical biopsies taken 6 months and 3 years after maxillary sinus floor augmentation with 80% bovine hydroxyapatite and 20% autogenous bone mixed with fibrin glue. Clin Implant Dent Relat Res 2001;3:87–96 33. Cypher TJ, Grossman JP. Biological principles of bone graft healing. J Foot Ankle Surg 1996;35:413–417 34. Mordenfeld A, Hallman M, Johansson CB, et al. Histological and histomorphometrical analyses of biopsies harvested 11 years after maxillary sinus floor augmentation with deproteinized bovine and autogenous bone. Clinical Oral Implants Research 2010;21:961–970 35. Di Stefano DA, Artese L, Iezzi G, et al. Alveolar ridge regeneration with equine spongy bone: a clinical, histological, and immunohistochemical case series. Clin Implant Dent Relat Res 2009;11:90–100 36. Gerstenfeld LC, Cullinane DM, Barnes GL, et al. Fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation. J Cell Biochem 2003;88:873–884
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Equine and Porcine Bone Substitutes in Maxillary Sinus Augmentation: A Histological and Immunohistochemical Analysis of VEGF Expression Stefano Tetè, MD, DDS,* Vincenzo Luca Zizzari, DDS,† Raffaele Vinci, MD, DDS,‡ Susi Zara, PhD,† Umberto Di Tore, DDS,* Marco Manica, DDS,§ Amelia Cataldi, MD,† Carmen Mortellaro, MD, DDS,||¶ Adriano Piattelli, MD, DDS,* and Enrico Gherlone, MD, DDS‡ Abstract: The aim of this work was to investigate the morphological structure and the expression of vascular endothelial growth factor (VEGF) after maxillary sinus augmentation through equine and porcine bone substitutes in humans. Ten patients showing edentulous posterior maxilla underwent maxillary sinus augmentation through particulate equine bone substitute and 10 patients through particulate porcine bone substitute. At the moment of implants insertion, 6 months after grafting, bone specimens were withdrawn and processed for morphological and immunohistochemical analyses. Notwithstanding the almost comparable clinical performances of both bone substitutes, histological results showed a better integration when an equine bone substitute was used compared to a porcine one. In particular, evident signs of particles resorption were observed in equine bone substitute group specimens compared to porcine ones. Immunohistochemical analysis showed a statistically significant increase of VEGF expression in equine compared to porcine bone substitute group specimens. These results showed both bone substitutes to achieve comparable clinical performance, indicating their successful use for bone regenerative procedures. However, in the same experimental time, equine group specimens showed evident resorption phenomena, whereas no or little signs of resorption were evident in the porcine group specimens. However, a more rapid and intense vascularization was achieved in equine bone substitute group, as demonstrated by immunohistochemical analysis for VEGF expression. Even if differences in vascularization significantly affect the clinical performance of a heterologous bone substitute, its ability to be resorbed is also very important in influencing long-term integration and long-term predictability of implant-prosthetic rehabilitation in regenerated sites. From the *Departments of Medical, Oral and Biotechnological Sciences and †Pharmacy, University “G. d’Annunzio,” Chieti-Pescara; ‡Department of Dentistry, University “Ateneo Vita-Salute S. Raffaele,” Milan; §Pistoia (private practice); ||Department of Medical Science, Faculty of Medicine, University of Eastern Piedmont, Novara; and ¶Oral Surgery Unit, Regina Margherita Pediatric Hospital, Turin, Italy. Received June 4, 2013. Accepted for publication January 7, 2014. Address correspondence and reprint requests to Stefano Tetè, MD, DDS, Department of Medical, Oral and Biotechnological Sciences, University “G. d’Annunzio,” Via dei Vestini, 31-66100 Chieti, Italy; E-mail: [email protected] The authors report no conflicts of interest. Copyright © 2014 by Mutaz B. Habal, MD ISSN: 1049-2275 DOI: 10.1097/SCS.0000000000000679
Key Words: Equine bone substitute, porcine bone substitute, maxillary sinus augmentation, immunohistochemical analysis, VEGF (J Craniofac Surg 2014;25: 835–839)
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o allow implant-supported rehabilitation of edentulous ridges affected by severe bone atrophy, bone regeneration procedures are often necessary. Especially in the posterior maxilla, after teeth extraction, alveolar bone undergoes remodeling phenomena together with maxillary sinus hyperpneumatization, resulting in an insufficient bone height to ensure stability to implant insertion.1 A classification of residual bone in the posterior maxilla was introduced with the purpose of considering, besides the width and the height of the residual alveolar bone, also the relationship between upper and lower jaw after the remodeling phenomena take place.2 To correct severe bone defects when the intermaxillary relationship is maintained and to have suitable prosthetic results, maxillary sinus augmentation procedure represents a valid treatment solution.3,4 In recent years, different materials have been proposed and used to ensure bone regeneration through grafting the maxillary sinus: intraoral or extraoral autologous bone, homologous grafts, heterologous grafts, alloplastic grafts, or a combination of these.5–8 Autologous bone graft is still considered the gold standard for bone regeneration. However, the morbidity involved with its use may induce many patients to refuse this treatment. Instead, different heterologous bone substitutes were utilized for sinus augmentation.9 As previously reported, heterologous biomaterials were found to be as clinically efficient as autologous bone for their osteoconductive potential, though not much is known about their capability to be fully resorbed or about the time they need to be entirely substituted by newly formed bone.7 As the biological response of the host tissue can be related to the biomaterial derivation and to the treatment it underwent before its clinical use, attention was addressed on the interactions occurring between bone substitutes and host tissue.9–11 Different heterologous biomaterials have been proposed for clinical use in oral surgery; bovine-derived bone substitutes represent one of the most popular and well-documented categories. They were described to have osteoconductive properties and to be well integrated in host bone tissue as shown in both in vitro histological and clinical studies.12 However, the possibility of prion disease transmission associated with the use of this source of heterologous bone led to other different sources for bone substitutes.13,14 In literature, few data are reported concerning the use of equine or porcine bone substitutes. Recent studies described equine-derived bone as being able to induce osteoblast differentiation, to be resorbed in vitro by osteoclasts, and to be successfully used in mandibular ridge augmentation.15,16 On the other hand, porcine-derived bone may be successfully used to obtain osteogenesis in guided bone regeneration techniques.17,18 Independently by their derivation, all bone substitutes from a nonhuman mammal species must undergo a series of deantigenation processes to make them suitable for clinical use by reducing antigenicity
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Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
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and any possible risks of cross-infections. One of the most utilized way to completely eliminate the organic components (sugars, lipids, proteins) of an animal-derived bone tissue is the thermal deantigenation at high temperatures (about 500–1200°C). Even if it allows the removal of the entire organic component, thus avoiding the onset of an immune response of the host, this treatment also modifies the mineral structure of bone hydroxyapatite, and thus the resulting biomaterial usually possesses a reduced resorption potential. On the contrary, equine-derived bone substitutes are deantigenated by a proteolytic process through digestive enzymes at about 37°C, selective for the organic component, which leaves unaltered the ability of the biomaterial to be reabsorbed in vivo.19 The host tissue response to a bone substitute may be evaluated by immunohistochemical analysis for the expression of molecules specifically involved in the bone healing process.11 In this process, vascular endothelial growth factor (VEGF) plays an important role. VEGF is produced by endothelial cells and osteoblasts, and is involved in initial bone remodeling phases because it regulates osteoblast evolution.20,21 VEGF is also capable of inducing the growth of new blood vessels and plays a significant role in the preservation and development of endothelial fenestrations,22,23 thus supporting new blood vessels formation in the site of grafting, which is a fundamental phase for graft integration. The aim of this work was to evaluate the morphological structure and the expression of VEGF after maxillary sinus augmentation through equine and porcine bone substitutes in humans.
PATIENTS AND METHODS Patients Twenty patients, 8 males and 12 females, aged between 51 and 63 years, with inadequate bone volume in the posterior maxilla, classified as class C according to Chiapasco’s classification of the posterior maxilla,2 who were scheduled for maxillary sinus augmentation procedures before implants placement, were included in this study. All patients gave written informed consent in accordance with the Local Ethics Committee, in compliance with Italian legislation and with the code of Ethical Principles for Medical Research involving Human Subjects of the World Medical Association (Declaration of Helsinki). The patients were randomly divided into 2 groups. Ten patients (3 males, 7 females; age range 51–59) underwent maxillary sinus augmentation procedure through a particulate bone substitute of equine origin (BioBone Osteoconductor Mix; BioSAF IN S.r.l., Ancona, Italy), and 10 patients (5 males, 5 females; age range 52–63) underwent maxillary sinus augmentation procedure through a particulate bone substitute of swine origin (Gen-Os; Tecnoss, Turin, Italy). BioBone Osteoconductor Mix is a resorbable, collagen-deprived, and deantigenated osteoconductive biomaterial, obtained after deantigenation at 37°C in a humid atmosphere, made up of a corticocancellous mixture of equine origin (particle width 0.5–1 mm). Gen-Os are commercially available collagenated swine-derived corticocancellous bone chips, with particle width between 250 and 1000 μm. Before surgical procedures, patients underwent complete medical anamnesis and radiographic examinations, including orthopantomography and cone beam computed tomography. All the selected patients had healthy systemic conditions, including the absence of any diseases that would contraindicate oral surgery. The exclusion criteria were uncontrolled periodontal disease, sinusitis, severe illness, unstable diabetes, drug abuse, a history of head and neck irradiation, and chemotherapy. Moreover, antibiotic therapy (amoxicillin 875 mg/ clavulanic acid 125 mg, Augmentin, 2 g an hour before) was administered to all patients preoperatively.
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Surgical Procedures The soft tissue was incised and flapped back to expose the underlying lateral wall of the maxillary sinus. The bone wall was then removed with a piezoelectric instrument (Easy Surgery; BioSAF IN S.r.l.) exposing the Schneiderian membrane. The membrane was then cut back from the inner face of the lower portion of the maxillary sinus cavity. The newly formed space within the bone cavity of the sinus inferior to the intact membrane was filled with the heterologous bone substitute and then covered by a collagen membrane (BioBone Collagen Membrane; BioSAF IN S.r.l.). Closure of the surgical access was obtained with 3-0 sutures (Silkam/Virgin Silk; B. Braun Melsungen AG, Germany). For all patients, the postoperative therapy protocol comprised administration of antibiotic (Augmentin) 2 g/day for 10 days, nonsteroidal analgesic drug (ketoprofen, OKI; Dompè, L’Aquila, Italy) at a dose of 200 mg twice daily for 3 days, and cortisone (Betametason, Bentelan; Defiante Farmaceutico, Madeira, Portugal) 4 mg/day for 2 days and 2 mg on day 3. Moreover, soft diet and oral hygiene, including rinsing 3 times daily with 0.2% chlorhexidine (Corsodyl; GlaxoSmithKline) mouthwash and the application at the surgical site of 1% chlorhexidine gel (Corsodyl), were prescribed. Sutures were removed 10 days after the intervention and postoperative check-ups scheduled weekly for the first month, and then monthly by clinical and radiographical examination with periapical x-rays in the grafted area. As postoperative healing was uneventful for all the patients, after about 6 months they all underwent a second surgery for implant placement. The implant features (diameter and length) were decided according to the anatomic situation individuated by clinical and radiographic examinations. However, all implants inserted had diameters between 3.3 mm and 4.5 mm, and lengths between 9 mm and 13 mm. During implant insertion, bone samples were retrieved by a 3-mm-diameter and 8-mm-height trephine bur under sterile saline solution irrigation at the sites of implant placement to obtain significant specimens of bone regenerated with both heterologous bone substitutes. Pharmacological protocol included antibiotic (Augmentin 2 g an hour before) and nonsteroidal analgesic drugs to be taken as required.
Light Microscopy and Immunohistochemical Analysis Bone specimens, fixed in phosphate-buffered formalin solution, were decalcified in 10% tetrahydrated EDTA solution according to data sheet (MIELODEC kit; Bio-Optica, Milan, Italy), dehydrated through ascending alcohol concentrations, and then paraffin-embedded, to be sectioned at 5 μm thick. Sections were then dewaxed (alcohols progressively lower concentrations) and processed for hematoxylineosin staining and for immunohistochemical analysis. Tissue sections with hematoxylin-eosin staining were observed to detect the presence of area of tissue remodeling. To detect VEGF, immunohistochemical analysis was performed on 5-μm-thick tissue sections for each experimental specimen, by means of Ultravision LP Detection System HRP Polymer and DAB Plus Chromogen (Lab Vision Thermo, Fremont, CA). Sections were incubated in the presence of rabbit polyclonal anti-VEGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and successively in the presence of specific HRP-conjugated secondary antibody. Peroxidase was developed using diaminobenzidine chromogen and nuclei were hematoxylin counterstained. Negative controls were performed by omitting the primary antibody. Samples were then observed by means of Leica DM 4000 light microscopy (Leica Cambridge Ltd, Cambridge, UK) equipped with a Leica DFC 320 camera (Leica Cambridge Ltd) for computerized images. © 2014 Mutaz B. Habal, MD
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
The Journal of Craniofacial Surgery • Volume 25, Number 3, May 2014
VEGF in Equine and Porcine Grafts
Image Analysis and Statistical Evaluation After digitizing the images derived from hematoxylin-eosin stained sections, the presence of native bone tissue was recognized by the presence of osteocyte lacunae-containing cells, whereas the newly formed bone tissue was recognized by the absence of lacunae. Densitometric evaluation was obtained by measuring the percentage area (±SD) determined by direct visual evaluation of 10 fields (mean values) for each of 5 slides per specimen at 20 magnification. After digitizing the images derived from immunohistochemical stained sections, QWin Plus 3.5 software (Leica Cambridge Ltd) was used to evaluate VEGF expression. Image analysis of protein expression was performed through the quantification of threshold area for brown color, as average value per 10 fields, randomly chosen, for each sample at light microscope observation. Negative control images were randomly chosen. The statistical significance was evaluated by the Wilcoxon and Mann-Whitney tests, using R Software, version 2.12.1 for Mac and setting P = 0.05. After collecting results, the mean data were reported and shown in a histogram using Excel 2008 for Mac.
RESULTS Morphological analysis was performed by light microscope after hematoxylin-eosin staining (Fig. 1). A better integration was shown when an equine bone substitute is used compared to a porcine one, and more evident signs of particles resorption were observed in equine bone substitute group specimens compared to porcine ones. In particular, hematoxylin-eosin staining highlights new bone formation in equine bone substitute group along with areas of native bone tissue and connective fibers. Moreover, the grafted biomaterial particles are easily distinguishable because of lack of cellular structure, and they appear in part resorbed and well integrated with the newly formed bone tissue. Abundant areas of newly formed bone tissue are recognizable for the lack of bone lacunae. In samples from the porcine bone substitute group, the bone particles are still clearly distinguishable from native bone tissue for absence of viable cells. The size of the bone particles appears quite unchanged and no clear evidence of resorption is detected. Samples from equine bone substitute group showed a significant higher amount of newly formed bone tissue compared to the native tissue than in porcine ones (Fig. 2). VEGF expression modifications occurring after different bone graft insertion and the evaluation of clinical applicability and integration ability of these grafts were then carried out. Densitometric values obtained from immunohistochemical analysis reveal a statistically significant increase in VEGF expression in samples from equine compared to porcine bone substitute group (P < 0.05) (Fig. 3).
FIGURE 2. Graphic representation of densitometric evaluation of newly formed bone tissue and native bone in equine and porcine bone substitutes specimens. Data are expressed as area % (±SD) determined by direct visual evaluation of 10 fields (mean values) for each of 5 slides per specimen at 20 magnification. Group A, bone tissue specimens obtained from equine bone substitute grafted area; Group B, bone tissue specimens obtained from porcine bone substitute grafted area. *Group A newly formed bone tissue versus group B newly formed bone tissue, P < 0.05; group A native bone tissue versus group B native bone tissue, P < 0.05.
implants placed in regenerated sites.24,25 In our study, we evaluated the success of regenerative therapy directly at the time of implant placement, that is 6 months after grafting, by histological and immunohistochemical analyses. Treatment of edentulous sites in the posterior maxilla requires a correct approach to obtain a functional implant-supported restoration. This goal may be achieved through adequate reconstruction by regenerative techniques with the use of bone substitutes. Even if the best performance of autologous bone grafts was widely demonstrated,23,26 clinicians are constantly searching for a heterologous bone substitute that permits to combine the osteoregenerative features of autologous bone, eliminating at the same time the limits imposed by the need for a second surgical intervention for withdrawal.10 Moreover, most heterologous bone substitutes currently used in clinical practice do not show ideal characteristics for bone regeneration such
DISCUSSION Most previous studies, to assess the success of a maxillary sinus augmentation procedure, take into consideration the survival rate of
FIGURE 1. Hematoxylin and eosin staining of equine and porcine bone substitutes specimens. Magnification 20 (inset 40). Group A: bone tissue specimens obtained from equine bone substitute grafted area; group B: bone tissue specimens obtained from porcine bone substitute grafted area.
FIGURE 3. Immunohistochemical analysis of VEGF expression in equine and porcine bone substitutes specimens. Magnification 20. Group A, bone tissue specimens obtained from equine bone substitute grafted area; Group B, bone tissue specimens obtained from porcine bone substitute grafted area; C (−), negative control; D, graphic representation of VEGF positive area % (±SD) densitometric analysis determined by direct visual counting of 10 fields (mean values) for each of 5 slides per specimen at 20 magnification. *Group A versus group B, P < 0.05.
© 2014 Mutaz B. Habal, MD
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
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as biological safety; osteogenic, osteoinductive, and angiogenic potentials; long shelf-life; no size restrictions; and reasonable cost.27,28 Heterologous biomaterials of different origins are successfully used as filling material for maxillary sinus augmentation.29,30 As previously described, heterologous bone particles work only as osteoconductive biomaterial and they usually undergo slow resorption.31,32 A biomaterial exhibiting no or scarce resorption could cause problems for a correct bone regeneration because of lower osteogenesis capability in respect to native autologous bone during the remodeling phase.33 The biological behavior of a heterologous bone substitute is also affected by the treatment it undergoes before in vivo utilization. Most heterologous bone substitutes of animal origin, such as bovine and porcine, are treated by thermal deantigenation to make them suitable for clinical use by reducing antigenicity and possible risks of cross-infections. However, this treatment is known to alter the mineral structure of bone hydroxyapatite, thus reducing the osteoclastic remodeling rate of the bone substitute. Clinically, the performance of the biomaterial is not significantly affected by this kind of treatment, as the bone substitute acts principally as a bioinert scaffold. In fact, bovine and porcine bone substitutes have been successfully used for years in guided bone regeneration techniques, even if residual biomaterial particles could be detected after many years from grafting.20,33 On the contrary, equine-derived bone substitutes are deantigenated by a proteolytic, low-temperature process, selective for the organic component, that seems to increase the capabilities of a bone substitute subjected to this kind of process to be resorpted.34 In fact, recent scientific evidence shows that biomaterials treated in this way undergo resorption and remodeling in about 12 months.20 In literature, only a few studies have reported on the use of an equine bone substitute, which appeared to be able to induce osteoblasts differentiation, to be resorbed in vitro by osteoclasts, and to be safely used in mandibular bone regeneration.20,35 The aim of this work was to investigate the morphological aspect and the expression of VEGF after maxillary sinus augmentation through equine and porcine bone substitutes in humans. Light microscopic analysis strongly evidenced that sites treated with the equine bone substitute showed good integration between host tissue and graft. Moreover, after a mean healing period of 6 months, the grafted biomaterial seemed to have activated intense remodeling phenomena, as confirmed by the existence of large areas of newly formed bone tissue, while particles of the porcine-derived bone substitute showed only few signs of resorption. To support these morphological aspects, the occurrence of new angiogenic processes was evaluated by checking VEGF expression. Vascularization has a crucial role in the regulation of bone remodeling and repair. New blood vessel formation is necessary to allow circulating osteoblast precursors to reach the remodeling site.20 Moreover, VEGF is also involved in supporting osteoblast growth during the initial phase of bone graft integration.36 In the present study, VEGF expression resulted higher in sites treated with equine bone substitute than in those treated with the porcine one, indicating that neoangiogenic phenomena occurred both earlier and an intense way between the 2 groups at the same healing time. Even if long-term results are not yet available to evaluate host-tissue response after a longer healing period, the present results indicate that both equine- and porcine-derived bone substitutes could be successfully used for regenerative therapy of intraoral bone defects. Still aware that differences in vascularization significantly affect the clinical performance of a heterologous bone substitute, its ability to be resorbed is also very important in influencing long-term integration and long-term predictability of implant-prosthetic rehabilitation in regenerated sites. In fact, the higher resorption capabilities of the equine-derived bone substitute compared to porcine-derived one,
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which probably could be due to the deantigenation process this biomaterial undergoes, could represent an added value to this category of bone substitute.
ACKNOWLEDGMENTS The authors thank the FIRB-Accordi di Programma 2010, “Processi degenerativi dei tessuti mineralizzati del cavo orale, impiego di biomateriali e controllo delle interazioni con i microrganismi dell’ambiente,” Prof. A. Cataldi, for the fellowship attributed to V.L.Z.
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Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
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VEGF in Equine and Porcine Grafts
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Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
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