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Bone regenerative issues related to bone grafting biomaterials
Chapter 8
Bone regenerative issues related to bone grafting biomaterials Alain Hoornaert*, Pierre Layrolle† *
CHU Nantes, Department of oral Implantology, Faculty of Dental Surgery, Nantes, France, INSERM, UMR 1238, PHY-OS, Bone Sarcomas and Remodeling of Calcified Tissues, Faculty of Medicine, University of Nantes, Nantes, France †
Between four and six million dental implants are implanted each year in Europe and volumes are increasing with the aging of the population. It is because tens of millions of European citizens are partially edentulous. However, on average, the placement of three out of ten implants requires the use of a bone augmentation procedure [1, 2]. The principle of guided bone regeneration (GBR) is to create a space between the bone defect and the gingival soft tissue to promote bone regeneration. This chapter reviews the different solutions used by oral surgeons in GBR [3, 4]. Autologous bone taken from the ramus or chin of the patient is most often used because it contains osteoprogenitor cells, growth factors, and a matrix for supporting bone regeneration. Nevertheless, this approach of bone transplantation requires the creation of two surgical sites with a significant extension of the operating time. In addition, the bone stock of the patient is limited and the autologous bone graft often has a significant and unpredictable resorption after its transplantation [5–8]. Allogeneic bone in the form of blocks or granules from authorized tissue banks is a first alternative to autologous bone grafting in GBR. Allogeneic bone is most often derived from femoral heads taken from living donors during hip prosthesis placement. This bone graft is exempt of any cellular element by various techniques such as freezing, lyophilization, demineralization, or washing with different solvents before sterilization by gamma irradiation. The allogeneic bone graft contains collagen (most often mineralized) and growth factors that will promote the adhesion and differentiation of osteoprogenitor cells and revascularization of the bone augmentation site. However, allogeneic bone is difficult to shape to the anatomy of the patient. The risks of immunological rejection or transmission of pathogens cannot be totally excluded with allogeneic bone grafts. In addition, the great variability in graft quality Dental Implants and Bone Grafts Materials and Biological Issues. https://doi.org/10.1016/B978-0-08-102478-2.00009-X Copyright © 2020 Elsevier Ltd. All rights reserved. 207
208 Dental implants and bone grafts materials and biological issues
d epending on the origin and the treatments performed cannot guarantee a consistency in the bone regeneration process [8, 9]. Xenografts from bovine, equine, or coral porites are very popular materials in GBR because they are the subject of many clinical publications. These animal-derived materials are composed of apatite calcium phosphate (CaP) whose composition and crystallinity are similar to the bone mineral. These materials have bioactivity and osteoconductive properties. Upon implantation, calcium and phosphate ions are released increasing the saturation of body fluids and leading to the precipitation on their surface of a biological carbonate apatite with proteins. This newly formed apatite layer favors the adhesion and proliferation of osteoprogenitor cells. Consequently, bone tissue grows in intimate contact with their surface. Nevertheless, these materials derived from animal resources do not allow bone regeneration of large volumes and have risks of rejection or transmission of pathogens [10–12]. Synthetic bone substitutes based on CaP are also used in GBR because they have also osteoconductive properties. By modulating their chemical composition and sintering temperature, their bioactivity and degradation can be controlled to a certain extent. It is well known that CaP biomaterials differ in solubility. For instance, β-TCP is more soluble than hydroxyapatite (HA) and mixtures called biphasic calcium phosphates (BCPs) are used to control their bioactivity. After implantation, BCP bioceramics partly dissolve while serving as substrate for biological apatite precipitation serving as scaffolds for bone regeneration. The extent of dissolution depends on the ß-TCP/HA ratio; the higher the ratio, the higher the extent of dissolution. The main attractive feature of bioceramics is their ability to form a direct bond with the host bone resulting in a strong interface compared with bioinert materials that form a fibrous tissue interface. The formation of this bone interface is due to interaction with cells and the formation of bone mineral by dissolution/precipitation processes. Nevertheless these biomaterials have insufficient GBR properties to regenerate large bone defects and are very often encapsulated by a fibrous tissue in oral surgery [13, 14]. A GBR membrane is frequently associated with the bone augmentation material. This membrane is placed between the bone filling material and the inner surface of the gingival flap. The action of the membrane is threefold: (1) to prevent the proliferation of the epithelial cells into the grafted site, (2) to promote the migration and differentiation of the bone cells, and (3) to oppose the resorption of the graft [15]. The first function of the membrane is to prevent the invasion of epithelial cells from colonizing the blood clot while the endosseous cells will regenerate bone. This phenomenon is called a barrier effect. The effectiveness of a membrane is therefore directly related to its ability to provide cellular impermeability for a sufficient period while maintaining its physical integrity. ln patients, primary alveolar bone healing takes around 6 weeks [16]. Ideally the membrane should cover the entire defect and effectively isolate it from the soft tissues from 2 to 6 weeks because cell colonization is no longer an issue,
Bone regenerative issues related to bone grafting biomaterials Chapter | 8 209
with bone tissue having already regenerated. It is therefore desired that the physical integrity of the membrane should remain for at least 6 weeks. Since 1996 the vast majority of membranes are based on collagen of animal origin (e.g., pig skin, bovine pericardium) and are chemically cross-linked to slow their resorption and increase their barrier function over time. Nevertheless these collagen membranes have insufficient mechanical strength, in particular in the wet environment of the mouth, to preserve the bone regeneration space. These collagen membranes can collapse in bone cavities under the pressure of the gum. Furthermore collagen is resorbed in a few weeks or even days when exposed to proteases of the saliva, whereas the GBR process generally requires 3 to 6 months to allow the insertion of dental implants. Cross-linked collagen membranes generally resorb more slowly than without treatment [17]. However, crosslinking agents such as glutaraldehyde lead to a decrease in the biocompatibility of the membrane because of their cytotoxic effects [18]. In principle materials known to be immunogenic should be avoided and special attention should be paid to the use of products of animal origin that by nature may contain allergens and carry the risk of contamination by transmission of pathogens. Among the synthetic resorbable polymers commonly used for manufacturing of implantable medical devices (e.g., sutures, surgical mesh), polylaclides (PLA), polyglycolides (PGA), polyhydroxyalkanoates (PHA), and polycaprolactones (PCL) are proven safe and effective in many clinical applications. These biomaterials degrade by hydrolysis in a predictable kinetic. Lactides are molecules found naturally in the body and they therefore cause very limited immune reactions after implantation [19]. Based on these demands, we have developed a new bi-layer, synthetic, resorbable membrane for GBR (Tisseos, Biomedical Tissues). This membrane is composed of polylactic-polyglycolic acid (PLGA), a polymer widely used in medical devices (e.g., resorbable sutures). Preclinical studies have demonstrated that this membrane is completely biocompatible, biodegradable, and guides bone regeneration. The membrane has a bilayer structure with a dense film for an optimal barrier effect and a layer of entangled microfibers whose structure is favorable to cell proliferation and GBR. This bilayer structure also provides mechanical properties facilitating its surgical placement and fixation to bone tissue. Finally, its chemical composition guarantees an optimal barrier effect during the gingival healing phase and a total resorption between 4 and 6 months; compatible with the time required for the GBR. Fig. 8.1 shows a clinical case of GBR with a bone substitute and the synthetic membrane. During the six-month period, the membrane has degraded while the biomaterial is still clearly visible. The reconstructed bone volume allowed the placement of dental implants while histological analysis shows an incomplete bone neoformation [20, 21]. In the European project REBORNE, we have proposed a therapeutic alternative to autologous bone grafting by using autologous MSCs amplified in culture and associated extemporaneously with CaP biomaterial [22]. These adult
210 Dental implants and bone grafts materials and biological issues
FIG. 8.1 Clinical case of GBR with beta-TCP biomaterial and synthetic resorbable PLGA membrane before implant placement; (A) bone defect, (B) apposition of beta-TCP granules, (C) membrane coverage, and (D) insertion of implants at 6 months.
stem cells are multi-potent, able to differentiate into osteoblasts, chondrocytes, or adipocytes and thus regenerate different tissues. However, these cells are present in a very small proportion (0.01%–0.001%) in human tissues and the proportion decreases sharply with age. Bone marrow is a natural reservoir of MSCs. These cells can be easily isolated from bone marrow by their adherence to culture-treated plastic dishes and multiplied in culture [22, 23]. We have standardized the production and transportation of clinical-grade MSCs in cell therapy units in France, Germany, Italy, and Spain (EFS Creteil, Toulouse, DRK Ulm, Policlinico Milan, UAM Hospital La Paz in Madrid). Cell culture is carried out in clean rooms in a culture medium containing human platelet lysate [24]. From 30 mL of bone marrow, several hundred million MSCs are produced in 2 to 3 weeks of culture. As shown in Fig. 8.2, human MSCs associated with calcium phosphate granules allow the formation of bone tissue after 8 weeks at the subcutaneous site in mice. This extemporaneous preparation of cells attached to the biomaterial can also regenerate bone defects of critical size in large animals. After having demonstrated the pre-clinical safety and efficacy of this cell therapy product, we have proposed a clinical protocol to ethical committees and regulatory agencies. In 2014 a clinical trial on mandibular bone crest augmentation with autologous MSCs associated with BCP biomaterial was approved in Bergen, Norway. In this clinical study, we demonstrated the feasibility and
Bone regenerative issues related to bone grafting biomaterials Chapter | 8 211
FIG. 8.2 Bone tissue regeneration; (A) human MSCs in culture, (B) extemporaneous attachment of MSCs onto CaP granules (methylene blue staining), (C) formation of ectopic bone in subcutaneous site of nude mice (Masson trichrome staining).
safety of this new therapeutic approach for regenerating bone volume in atrophied mandibles of 11 patients [25]. Five to 6 months after grafting autologous MSCs and BCP, bone augmentation was validated clinically by computed tomography (CT) imaging and histology of core biopsies (Fig. 8.3). Autologous MSCs expanded in vitro and attached to biphasic calcium phosphate granules induced significant new bone formation in the atrophied mandibles. Healing was uneventful, without adverse events. The regenerated bone volume was adequate for dental implant insertion and their osseointegration demonstrated by radio frequency analysis [26, 27]. The patients were satisfied with the aesthetic and functional outcomes. This novel augmentation procedure constituted the basis for carrying out a wide European randomized controlled trial challenging the gold standard in bone regeneration, autologous bone graft. We have recently obtained the financial support of the European Commission through the project MAXIBONE to conduct a late-stage clinical study. This randomized controlled trial will aim at comparing the safety and efficacy of autologous bone grafting with culture-expanded autologous bone marrow MSCs associated to a synthetic bone substitute covered by a resorbable membrane in alveolar bone augmentation procedures on 150 patients.
212 Dental implants and bone grafts materials and biological issues
FIG. 8.3 Clinical trial of bone augmentation on atrophied mandible with autologous culture- expanded MSCs and biomaterial; (A) preoperative CT scan, (B) 6-month CT scan, (C) micro- scanner, and (D) histology of core biopsy at 6 months after GBR (Masson trichrome staining).
The recruitment will be performed in ten major hospital centers, while the production of MSCs will be done in the German and French blood transfusion institutes. Medical imaging, direct measurements, and histology of core biopsies before dental implants will ensure the evaluation of bone regeneration. Cost-effective monitoring with a secured Internet platform (electronic case report form) will produce a clinical database for evaluation of safety, efficacy, and health costs in both arms. This project also supports the development of personalized biomaterials manufactured by 3D printing of CaP cement pastes from CT images. As shown in Fig. 8.4, these 3D scaffolds are customized to fit the anatomy of the patient. It is expected that the use of anatomic 3D scaffolds will efficiently promote the bone regeneration process with more favorable aesthetic outcomes than the conventional CaP granules. Furthermore the pore size and interconnection can be adjusted to allow cell colonization, vascularization, and bone regeneration while ensuring a sufficient strength in load-bearing areas based on finite element modeling. In addition, these 3D scaffolds are made at ambient temperatures by hydrolysis and precipitation of calcium-deficient apatite with similar composition and crystallinity as the bone mineral. These biomimetic features may favor
Bone regenerative issues related to bone grafting biomaterials Chapter | 8 213
FIG. 8.4 Personalized maxillofacial bone regenerative materials; (A) 3D printing of CaP cement pastes, (B) 3D scaffold fitting the anatomy of the patient’s mandible, (C) human MSCs grown on the 3D scaffold for 7 days, (D) bone tissue formation (in green) after subcutis implantation in nude mice for 8 weeks (Masson trichrome staining).
a complete substitution of the 3D scaffold by bone tissue. We have also shown that MSCs attached and proliferated on these 3D scaffolds and formed abundant bone formation in subcutis of nude mice. Further research will be conducted in mandibles of miniature pigs to demonstrate bone regeneration with 3D scaffolds and autologous MSCs in a pre-clinical model. The use of allogeneic MSCs are also being studied for bone regeneration [28]. For the moment, only MSCs derived from the patient’s own bone marrow have demonstrated their efficacy in bone regeneration [29], but in the future we plan to develop allogeneic transplants by using MSCs from donors selected and stored in cell banks. One can imagine cultivating these cells in bioreactors in order to produce large quantities, which the surgeon would have at will for bone regeneration in patients. This approach would avoid the heavy logistics in the autologous scenario, with a roundtrip of cells between the hospital where they are taken and the approved cell therapy center where they are grown for 2 to 3 weeks. Fully characterized and potent allogeneic cells may also decrease the current high prices of cell therapy products for the benefits of the patients requiring a bone regeneration procedure before dental implants.
214 Dental implants and bone grafts materials and biological issues
Acknowledgments We acknowledge the European Commission for financial support through the projects REBORNE, ORTHOUNION, PARAGEN, and MAXIBONE.
References [1] Chiapasco M, Casentini P, Zaniboni M. Bone augmentation procedures in implant dentistry. Int J Oral Maxillofac Implants 2009;24(Suppl):237–59. [2] Lutz R, Neukam FW, Simion M, Schmitt CM. Long-term outcomes of bone augmentation on soft and hard-tissue stability: a systematic review. Clin Oral Implants Res 2015;26(Suppl 11):103–22. [3] Al-Nawas B, Schiegnitz E. Augmentation procedures using bone substitute materials or autogenous bone – a systematic review and meta-analysis. Eur J Oral Implantol 2014;7(Suppl 2(2)):S219–34. [4] Jensen SS, Terheyden H. Bone augmentation procedures in localized defects in the alveolar ridge: clinical results with different bone grafts and bone-substitute materials. Int J Oral Maxillofac Implants 2008;24(Suppl):218–36. [5] Bauer TW, Muschler GF. Bone graft materials: an overview of the basic science. Clin Orthop Relat Res 2000;371:10. [6] Rissolo AR, Bennett J. Bone grafting and its essential role in implant dentistry. Dent Clin N Am 1998;42(1):91–116. [7] Smolka W, Eggensperger N, Carollo V, Ozdoba C, Iizuka T. Changes in the volume and density of calvarial split bone grafts after alveolar ridge augmentation. Clin Oral Implants Res 2006;17(2):149–55. [8] Al-Abedalla K, Torres J, Cortes ARG, Wu X, Nader SA, Daniel N, et al. Bone augmented with allograft onlays for implant placement could be comparable with native bone. J Oral Maxillofac Surg 2015;73(11):2108–22. [9] Beitlitum I, Artzi Z, Nemcovsky CE. Clinical evaluation of particulate allogeneic with and without autogenous bone grafts and resorbable collagen membranes for bone augmentation of atrophic alveolar ridges. Clin Oral Implants Res 2010;21(11):1242–50. [10] Vance GS, Greenwell H, Miller RL, Hill M, Johnston H, Scheetz JP. Comparison of an allograft in an experimental putty carrier and a bovine-derived xenograft used in ridge preservation: a clinical and histologic study in humans. Int J Oral Maxillofac Implants 2004;19(4):491–7. [11] Sculean A, Nikolidakis D, Nikou G, Ivanovic A, Chapple ILC, Stavropoulos A. Biomaterials for promoting periodontal regeneration in human intrabony defects: a systematic review. Periodontol 2000 2015;68(1):182–216. [12] Sanz M, Vignoletti F. Key aspects on the use of bone substitutes for bone regeneration of edentulous ridges. Dent Mater 2015;31(6):640–7. [13] Rodella LF, Favero G, Labanca M. Biomaterials in maxillofacial surgery: membranes and grafts. Int J Biomed Sci 2011;7(2):81–8. [14] Sanz M, Vignoletti F. Key aspects on the use of bone substitutes for bone regeneration of edentulous ridges. Dent Mater 2015;31(6):640–7. [15] Linde A, Alberius P, Dahlin C, Bjurstam K, Sundin Y. Osteopromotion: a soft-tissue exclusion principle using a membrane for bone healing and bone neogenesis. J Periodontol 1993;64(11 Suppl):1116–28. [16] Retzepi M, Donos N. Guided bone regeneration: biological principle and therapeutic applications. Clin Oral Implants Res 2010;21(6):567–76.
Bone regenerative issues related to bone grafting biomaterials Chapter | 8 215 [17] Rothamel D, Schwarz F, Sager M, Herten M, Sculean A, Becker J. Biodegradation of differently cross-linked collagen membranes: an experimental study in the rat. Clin Oral Implants Res 2005;16(3):369–78. [18] Lee SW, Kim SG. Membranes for the guided bone regeneration. Maxillofac Plast Reconstr Surg 2014;36(6):239–46. [19] Gentile P, Chiono V, Tonda-Turo C, Ferreira AM, Ciardelli G. Polymeric membranes for guided bone regeneration. Biotechnol J 2011;6(10):1187–97. [20] Hoornaert A, D’Arros C, Heymann M-F, Layrolle P. Biocompatibility, resorption and biofunctionality of a new synthetic biodegradable membrane for guided bone regeneration. Biomed Mater 2016;11(4):045012. [21] Martin-Thomé H, Bourdin D, Strube N, Saffarzadeh A, Morlock J-F, Campard G, et al. Clinical safety of a new synthetic resorbable dental membrane: a case series study. J Oral Implantol 2018;44(2):138–45. [22] Brennan MÁ, Renaud A, Amiaud J, Rojewski MT, Schrezenmeier H, Heymann D, et al. Preclinical studies of bone regeneration with human bone marrow stromal cells and biphasic calcium phosphate. Stem Cell Res Ther 2013;5(5):114. [23] Brennan MÁ, Renaud A, Gamblin A-L, D’Arros C, Nedellec S, Trichet V, et al. 3D cell culture and osteogenic differentiation of human bone marrow stromal cells plated onto jet-sprayed or electrospun micro-fiber scaffolds. Biomed Mater 2015;10(4):045019. [24] Chevallier N, Anagnostou F, Zilber S, Bodivit G, Maurin S, Barrault A, et al. Osteoblastic differentiation of human mesenchymal stem cells with platelet lysate. Biomaterials 2009;31(2):270–8. [25] Gjerde C, Mustafa K, Hellem S, Rojewski M, Gjengedal H, Yassin MA, et al. Cell therapy induced regeneration of severely atrophied mandibular bone in a clinical trial. Stem Cell Res Ther 2018;9(1):213. [26] Rozé J, Babu S, Saffarzadeh A, Gayet-Delacroix M, Hoornaert A, Layrolle P. Correlating implant stability to bone structure. Clin Oral Implants Res 2009;20(10):1140–5. [27] Rozé J, Hoornaert A, Layrolle P. Correlation between primary stability and bone healing of surface treated titanium implants in the femoral epiphyses of rabbits. J Mater Sci Mater Med 2014;25(8):1941–51. [28] Rosset P, Deschaseaux F, Layrolle P. Cell therapy for bone repair. Orthop Traumatol Surg Res 2014;100(1 Suppl):S107–12. [29] Gamblin A-L, Brennan MÁ, Renaud A, Yagita H, Lézot F, Heymann D, et al. Bone tissue formation with human mesenchymal stem cells and biphasic calcium phosphate ceramics: the local implication of osteoclasts and macrophages. Biomaterials 2014;35(36):9660–7.
Bone regenerative issues related to bone grafting biomaterials Alain Hoornaert*, Pierre Layrolle† *
CHU Nantes, Department of oral Implantology, Faculty of Dental Surgery, Nantes, France, INSERM, UMR 1238, PHY-OS, Bone Sarcomas and Remodeling of Calcified Tissues, Faculty of Medicine, University of Nantes, Nantes, France †
Between four and six million dental implants are implanted each year in Europe and volumes are increasing with the aging of the population. It is because tens of millions of European citizens are partially edentulous. However, on average, the placement of three out of ten implants requires the use of a bone augmentation procedure [1, 2]. The principle of guided bone regeneration (GBR) is to create a space between the bone defect and the gingival soft tissue to promote bone regeneration. This chapter reviews the different solutions used by oral surgeons in GBR [3, 4]. Autologous bone taken from the ramus or chin of the patient is most often used because it contains osteoprogenitor cells, growth factors, and a matrix for supporting bone regeneration. Nevertheless, this approach of bone transplantation requires the creation of two surgical sites with a significant extension of the operating time. In addition, the bone stock of the patient is limited and the autologous bone graft often has a significant and unpredictable resorption after its transplantation [5–8]. Allogeneic bone in the form of blocks or granules from authorized tissue banks is a first alternative to autologous bone grafting in GBR. Allogeneic bone is most often derived from femoral heads taken from living donors during hip prosthesis placement. This bone graft is exempt of any cellular element by various techniques such as freezing, lyophilization, demineralization, or washing with different solvents before sterilization by gamma irradiation. The allogeneic bone graft contains collagen (most often mineralized) and growth factors that will promote the adhesion and differentiation of osteoprogenitor cells and revascularization of the bone augmentation site. However, allogeneic bone is difficult to shape to the anatomy of the patient. The risks of immunological rejection or transmission of pathogens cannot be totally excluded with allogeneic bone grafts. In addition, the great variability in graft quality Dental Implants and Bone Grafts Materials and Biological Issues. https://doi.org/10.1016/B978-0-08-102478-2.00009-X Copyright © 2020 Elsevier Ltd. All rights reserved. 207
208 Dental implants and bone grafts materials and biological issues
d epending on the origin and the treatments performed cannot guarantee a consistency in the bone regeneration process [8, 9]. Xenografts from bovine, equine, or coral porites are very popular materials in GBR because they are the subject of many clinical publications. These animal-derived materials are composed of apatite calcium phosphate (CaP) whose composition and crystallinity are similar to the bone mineral. These materials have bioactivity and osteoconductive properties. Upon implantation, calcium and phosphate ions are released increasing the saturation of body fluids and leading to the precipitation on their surface of a biological carbonate apatite with proteins. This newly formed apatite layer favors the adhesion and proliferation of osteoprogenitor cells. Consequently, bone tissue grows in intimate contact with their surface. Nevertheless, these materials derived from animal resources do not allow bone regeneration of large volumes and have risks of rejection or transmission of pathogens [10–12]. Synthetic bone substitutes based on CaP are also used in GBR because they have also osteoconductive properties. By modulating their chemical composition and sintering temperature, their bioactivity and degradation can be controlled to a certain extent. It is well known that CaP biomaterials differ in solubility. For instance, β-TCP is more soluble than hydroxyapatite (HA) and mixtures called biphasic calcium phosphates (BCPs) are used to control their bioactivity. After implantation, BCP bioceramics partly dissolve while serving as substrate for biological apatite precipitation serving as scaffolds for bone regeneration. The extent of dissolution depends on the ß-TCP/HA ratio; the higher the ratio, the higher the extent of dissolution. The main attractive feature of bioceramics is their ability to form a direct bond with the host bone resulting in a strong interface compared with bioinert materials that form a fibrous tissue interface. The formation of this bone interface is due to interaction with cells and the formation of bone mineral by dissolution/precipitation processes. Nevertheless these biomaterials have insufficient GBR properties to regenerate large bone defects and are very often encapsulated by a fibrous tissue in oral surgery [13, 14]. A GBR membrane is frequently associated with the bone augmentation material. This membrane is placed between the bone filling material and the inner surface of the gingival flap. The action of the membrane is threefold: (1) to prevent the proliferation of the epithelial cells into the grafted site, (2) to promote the migration and differentiation of the bone cells, and (3) to oppose the resorption of the graft [15]. The first function of the membrane is to prevent the invasion of epithelial cells from colonizing the blood clot while the endosseous cells will regenerate bone. This phenomenon is called a barrier effect. The effectiveness of a membrane is therefore directly related to its ability to provide cellular impermeability for a sufficient period while maintaining its physical integrity. ln patients, primary alveolar bone healing takes around 6 weeks [16]. Ideally the membrane should cover the entire defect and effectively isolate it from the soft tissues from 2 to 6 weeks because cell colonization is no longer an issue,
Bone regenerative issues related to bone grafting biomaterials Chapter | 8 209
with bone tissue having already regenerated. It is therefore desired that the physical integrity of the membrane should remain for at least 6 weeks. Since 1996 the vast majority of membranes are based on collagen of animal origin (e.g., pig skin, bovine pericardium) and are chemically cross-linked to slow their resorption and increase their barrier function over time. Nevertheless these collagen membranes have insufficient mechanical strength, in particular in the wet environment of the mouth, to preserve the bone regeneration space. These collagen membranes can collapse in bone cavities under the pressure of the gum. Furthermore collagen is resorbed in a few weeks or even days when exposed to proteases of the saliva, whereas the GBR process generally requires 3 to 6 months to allow the insertion of dental implants. Cross-linked collagen membranes generally resorb more slowly than without treatment [17]. However, crosslinking agents such as glutaraldehyde lead to a decrease in the biocompatibility of the membrane because of their cytotoxic effects [18]. In principle materials known to be immunogenic should be avoided and special attention should be paid to the use of products of animal origin that by nature may contain allergens and carry the risk of contamination by transmission of pathogens. Among the synthetic resorbable polymers commonly used for manufacturing of implantable medical devices (e.g., sutures, surgical mesh), polylaclides (PLA), polyglycolides (PGA), polyhydroxyalkanoates (PHA), and polycaprolactones (PCL) are proven safe and effective in many clinical applications. These biomaterials degrade by hydrolysis in a predictable kinetic. Lactides are molecules found naturally in the body and they therefore cause very limited immune reactions after implantation [19]. Based on these demands, we have developed a new bi-layer, synthetic, resorbable membrane for GBR (Tisseos, Biomedical Tissues). This membrane is composed of polylactic-polyglycolic acid (PLGA), a polymer widely used in medical devices (e.g., resorbable sutures). Preclinical studies have demonstrated that this membrane is completely biocompatible, biodegradable, and guides bone regeneration. The membrane has a bilayer structure with a dense film for an optimal barrier effect and a layer of entangled microfibers whose structure is favorable to cell proliferation and GBR. This bilayer structure also provides mechanical properties facilitating its surgical placement and fixation to bone tissue. Finally, its chemical composition guarantees an optimal barrier effect during the gingival healing phase and a total resorption between 4 and 6 months; compatible with the time required for the GBR. Fig. 8.1 shows a clinical case of GBR with a bone substitute and the synthetic membrane. During the six-month period, the membrane has degraded while the biomaterial is still clearly visible. The reconstructed bone volume allowed the placement of dental implants while histological analysis shows an incomplete bone neoformation [20, 21]. In the European project REBORNE, we have proposed a therapeutic alternative to autologous bone grafting by using autologous MSCs amplified in culture and associated extemporaneously with CaP biomaterial [22]. These adult
210 Dental implants and bone grafts materials and biological issues
FIG. 8.1 Clinical case of GBR with beta-TCP biomaterial and synthetic resorbable PLGA membrane before implant placement; (A) bone defect, (B) apposition of beta-TCP granules, (C) membrane coverage, and (D) insertion of implants at 6 months.
stem cells are multi-potent, able to differentiate into osteoblasts, chondrocytes, or adipocytes and thus regenerate different tissues. However, these cells are present in a very small proportion (0.01%–0.001%) in human tissues and the proportion decreases sharply with age. Bone marrow is a natural reservoir of MSCs. These cells can be easily isolated from bone marrow by their adherence to culture-treated plastic dishes and multiplied in culture [22, 23]. We have standardized the production and transportation of clinical-grade MSCs in cell therapy units in France, Germany, Italy, and Spain (EFS Creteil, Toulouse, DRK Ulm, Policlinico Milan, UAM Hospital La Paz in Madrid). Cell culture is carried out in clean rooms in a culture medium containing human platelet lysate [24]. From 30 mL of bone marrow, several hundred million MSCs are produced in 2 to 3 weeks of culture. As shown in Fig. 8.2, human MSCs associated with calcium phosphate granules allow the formation of bone tissue after 8 weeks at the subcutaneous site in mice. This extemporaneous preparation of cells attached to the biomaterial can also regenerate bone defects of critical size in large animals. After having demonstrated the pre-clinical safety and efficacy of this cell therapy product, we have proposed a clinical protocol to ethical committees and regulatory agencies. In 2014 a clinical trial on mandibular bone crest augmentation with autologous MSCs associated with BCP biomaterial was approved in Bergen, Norway. In this clinical study, we demonstrated the feasibility and
Bone regenerative issues related to bone grafting biomaterials Chapter | 8 211
FIG. 8.2 Bone tissue regeneration; (A) human MSCs in culture, (B) extemporaneous attachment of MSCs onto CaP granules (methylene blue staining), (C) formation of ectopic bone in subcutaneous site of nude mice (Masson trichrome staining).
safety of this new therapeutic approach for regenerating bone volume in atrophied mandibles of 11 patients [25]. Five to 6 months after grafting autologous MSCs and BCP, bone augmentation was validated clinically by computed tomography (CT) imaging and histology of core biopsies (Fig. 8.3). Autologous MSCs expanded in vitro and attached to biphasic calcium phosphate granules induced significant new bone formation in the atrophied mandibles. Healing was uneventful, without adverse events. The regenerated bone volume was adequate for dental implant insertion and their osseointegration demonstrated by radio frequency analysis [26, 27]. The patients were satisfied with the aesthetic and functional outcomes. This novel augmentation procedure constituted the basis for carrying out a wide European randomized controlled trial challenging the gold standard in bone regeneration, autologous bone graft. We have recently obtained the financial support of the European Commission through the project MAXIBONE to conduct a late-stage clinical study. This randomized controlled trial will aim at comparing the safety and efficacy of autologous bone grafting with culture-expanded autologous bone marrow MSCs associated to a synthetic bone substitute covered by a resorbable membrane in alveolar bone augmentation procedures on 150 patients.
212 Dental implants and bone grafts materials and biological issues
FIG. 8.3 Clinical trial of bone augmentation on atrophied mandible with autologous culture- expanded MSCs and biomaterial; (A) preoperative CT scan, (B) 6-month CT scan, (C) micro- scanner, and (D) histology of core biopsy at 6 months after GBR (Masson trichrome staining).
The recruitment will be performed in ten major hospital centers, while the production of MSCs will be done in the German and French blood transfusion institutes. Medical imaging, direct measurements, and histology of core biopsies before dental implants will ensure the evaluation of bone regeneration. Cost-effective monitoring with a secured Internet platform (electronic case report form) will produce a clinical database for evaluation of safety, efficacy, and health costs in both arms. This project also supports the development of personalized biomaterials manufactured by 3D printing of CaP cement pastes from CT images. As shown in Fig. 8.4, these 3D scaffolds are customized to fit the anatomy of the patient. It is expected that the use of anatomic 3D scaffolds will efficiently promote the bone regeneration process with more favorable aesthetic outcomes than the conventional CaP granules. Furthermore the pore size and interconnection can be adjusted to allow cell colonization, vascularization, and bone regeneration while ensuring a sufficient strength in load-bearing areas based on finite element modeling. In addition, these 3D scaffolds are made at ambient temperatures by hydrolysis and precipitation of calcium-deficient apatite with similar composition and crystallinity as the bone mineral. These biomimetic features may favor
Bone regenerative issues related to bone grafting biomaterials Chapter | 8 213
FIG. 8.4 Personalized maxillofacial bone regenerative materials; (A) 3D printing of CaP cement pastes, (B) 3D scaffold fitting the anatomy of the patient’s mandible, (C) human MSCs grown on the 3D scaffold for 7 days, (D) bone tissue formation (in green) after subcutis implantation in nude mice for 8 weeks (Masson trichrome staining).
a complete substitution of the 3D scaffold by bone tissue. We have also shown that MSCs attached and proliferated on these 3D scaffolds and formed abundant bone formation in subcutis of nude mice. Further research will be conducted in mandibles of miniature pigs to demonstrate bone regeneration with 3D scaffolds and autologous MSCs in a pre-clinical model. The use of allogeneic MSCs are also being studied for bone regeneration [28]. For the moment, only MSCs derived from the patient’s own bone marrow have demonstrated their efficacy in bone regeneration [29], but in the future we plan to develop allogeneic transplants by using MSCs from donors selected and stored in cell banks. One can imagine cultivating these cells in bioreactors in order to produce large quantities, which the surgeon would have at will for bone regeneration in patients. This approach would avoid the heavy logistics in the autologous scenario, with a roundtrip of cells between the hospital where they are taken and the approved cell therapy center where they are grown for 2 to 3 weeks. Fully characterized and potent allogeneic cells may also decrease the current high prices of cell therapy products for the benefits of the patients requiring a bone regeneration procedure before dental implants.
214 Dental implants and bone grafts materials and biological issues
Acknowledgments We acknowledge the European Commission for financial support through the projects REBORNE, ORTHOUNION, PARAGEN, and MAXIBONE.
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