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Effect of recombinant human bone morphogenetic protein-2 on bone formation in alveolar ridge defects in dogs
Int. J. Oral Maxillofac. Surg. 2002; 31: 66–72 doi:10.1054/ijom.2001.0133, available online at http://www.idealibrary.com on
Research and emerging technologies: Osteobiology
Effect of recombinant human bone morphogenetic protein-2 on bone formation in alveolar ridge defects in dogs
H. Nagao1, N. Tachikawa1, T. Miki2, M. Oda2, M. Mori2, K. Takahashi3, S. Enomoto1,2 1
Clinic for Oral Implantology, University Hospital, Faculty of Dentistry, Tokyo Medical and Dental University, Tokyo, Japan; 2Second Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Tokyo Medical and Dental University, Tokyo, Japan; 3Yamanouchi Pharmaceutical Co. Ltd, Ibaraki, Japan
H. Nagao, N. Tachikawa, T. Miki, M. Oda, M. Mori, K. Takahashi, S. Enomoto: Effect of recombinant human bone morphogenetic protein-2 on bone formation in alveolar ridge defects in dogs. Int. J. Oral Maxillofac. Surg. 2002; 31: 66–72. 2002 International Association of Oral and Maxillofacial Surgeons Abstract. This study was designed to evaluate the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) combined with poly D, L lactic-co-glycolic acid (PLGA)/gelatin sponge complex (PGS) on the formation of bone in critically sized marginal defects of the mandible in dogs. Three months after extraction of the pre-molar teeth, rectangular bone defects (1087 mm) were made in both sides of the mandible. A PGS block soaked in rhBMP-2 (400 g/ml) was implanted into one defect (BMP (+) group). As control, an untreated PGS block was implanted into the contralateral defect (BMP () group). 2, 4, 8, and 12 weeks after implantation, the defects were examined. In the BMP (+) group, newly formed bone was found in all defects from 4 weeks onward and was marked at 12 weeks. In contrast, the BMP () group showed no appreciable new bone formation, even at 12 weeks. Moreover, density of newly formed bone in the BMP (+) group was similar to that of the surrounding cortical bone at 12 weeks. These findings suggest that rhBMP-2/PGS is an effective bone substitute for reconstructive surgery of the dog mandible.
Restoration of bone is often required after dental and oral surgical procedures. Treatment of cysts, tumours, and fractures of the jaw can result in bone defects. Such defects must be repaired with bone grafts or bone substitutes to ensure a good structural and functional outcome. Conventionally, fresh autogenous bone grafts have been used to repair oral and maxillofacial bone defects. However, the need for surgery of the donor site and the limited supply of bone available have led to the development of various alternative materials to autogenous and allogenic bone grafts. 0901-5027/02/010066+07 $35.00/0
These materials include type I collagen4, hydroxyapatite7, tricalcium phosphate12, and isolated bone marrow cells10. Recent reports suggest that synthetic bone grafting preparations with properties similar to those of autogenous bone may be available soon. In 1965, Urist described ectopic bone formation after i.m. implantation of demineralized bone matrix in rats. The factor responsible for this effect was later named bone morphogenetic protein (BMP)22. Many attempts have been made to purify BMP from demineralized bone of
Key words: bone morphogenetic proteins; dogs; mandible; bone substitutes. Accepted for publication 28 May 2001
various animals, however pure BMP has not yet been obtained. Recently, the molecular biologic techniques enabled the production of very pure specific proteins. In 1988, W successfully synthesized recombinant human BMP (rhBMP-2)24. RhBMP-2 is known to induce osteogenesis and many experimental studies have shown that rhBMP-2 promotes bone healing17. Clinically, rhBMP-2 is expected to be used to fill bone defects and promote healing of fractures. The availability of rhBMP-2 has led to many studies
2002 International Association of Oral and Maxillofacial Surgeons
Alveolar bone repair by rhBMP-2 evaluating the functional roles of BMPs and the development of substitutes for bone grafts. When used for bone repair, rhBMP-2 requires a suitable carrier to prevent rapid diffusion of the protein. However, an effective bone repair system for rhBMP-2 has yet to be established, precluding the use of this protein in humans. Moreover, detailed studies of the properties of newly formed bone induced by rhBMP-2 are lacking. Recently, PGS [poly D, L lactic-coglycolic acid (PLGA)/gelatin sponge complex] has been developed as a new carrier for rhBMP-2. This carrier has a sponge-like consistency and readily adapts to surrounding structures, with minimal risk of leakage after implantation. It is also very biocompatible and biodegradable8,9. We have therefore attempted to develop combinations of rhBMP-2 and PGS that could be used in selected situations as alternatives to either autogenous or allogenic bone. This study was designed to examine the effect of rhBMP-2 in PGS implants on bone formation in critically sized defects of the mandible in dogs and to examine the properties of the newly formed bone.
Materials and methods Animals
Twelve adult beagle dogs without general or oral health problems, weighing 10 to 12 kg, were used in this study. Implant materials
The rhBMP-2 used in this experiment was manufactured by recombinant expression in Chinese hamster ovary cells at the Genetics Institute (Cambridge, MA, USA) and was purified to >98% purity. It was supplied by the Genetics Institute through Yamanouchi Pharmaceutical Co. Ltd (Tokyo, Japan). PGS [poly D, L lactic-co-glycolic acid (PLGA; molar ratio 50:50; MW 30 000)/ gelatin sponge complex] was used as the carrier for rhBMP-2. The weight ratio of PLGA:gelatin was 4:1 and the porosity was approximately 90%. PGS and PLGA were provided by Yamanouchi Co. Ltd Before implantation, PGS was soaked in rhBMP-2 solution (400 g/ml, 0.5 M L-arginine, 10 mM L-histidine, pH 6.5) and kept at room temperature for more than 30 min to permit incorporation of rhBMP-2. The same solution
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Fig. 1. Photograph of surgical procedure. (1) PGS carrier. (2) Defects measuring 1087 mm were made in both sizes of the mandible. (3) PGS blocks with or without rhBMP-2 were implanted into the defects. (4) The wounds were closed with nylon suture.
without rhBMP-2 was used as the carrier control. Operative procedure
All surgical procedures were performed under systemic (xylazine hydrochloride, 2 mg/kg and ketamine hydrochloride, 5 mg/kg i.v.) and local (2% lidocaine hydrochloride with 1/80 000 epinephrine) anaesthesia. The pre-molar teeth had been bilaterally extracted previously, and the ridges were allowed to heal for 3 months. After this period, marginal resection of the mandible was performed. A mucoperiosteal flap was raised from the mucogingival junction to the lingual gingiva. Rectangular bone defects were made in both sides of the mandible by a microengine (Fig. 1). The defects measured 1087 mm. These defects are large enough to be ‘critically sized’5 and cannot heal naturally, without some form of treatment or intervention. The bone defects were irrigated with sterile saline and the carrier was shaped to fit the size and shape of each defect. Then, a PGS block with rhBMP-2 was implanted into the bone defect on one side of the mandible (BMP (+) group). As control, PGS alone was implanted into the contralateral side (BMP () group). The wounds were closed with nylon sutures. Soft food was provided for 1 week after surgery. The dogs were killed 2, 4, 8, and 12 weeks after operation (n=3, respectively) and the defects were examined by means of radiographs on soft X-ray film, three-
dimensional computed tomographic (3D-CT) scans, histological specimens, and peripheral quantitative computed tomography (pQCT). Soft X-ray analysis
The defects were radiographed with a SRO-M50 soft X-ray unit (Sofron, Tokyo, Japan) operated at 40 KVp and 4 mA, with an exposure time of 2 min. Radiolucency of the defects was examined. 3D-CT scan analysis
In this study, 3D-CT with helical CT scanning was performed with a Somatom Plus CT scanner (Siemens, Forchheim, Germany) to examine the structure of the defects. Histologic procedures
The defects and adjacent host bone were obtained en bloc and were fixed in 10% buffered formalin. They were placed in a series of graded ethanol (70–100%), and embedded in Rigolac (Nissin EM Co. Ltd, Tokyo, Japan). Non-decalcified ground sections 30 m thick were prepared and stained with toluidine blue. pQCT analysis
To examine the properties of new bone induced by rhBMP-2, pQCT was performed with an XCT-960A quantitative computed tomograph (Stratec,
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Birkenfeld, Germany), and the bone mineral density (BMD) was measured at the midpoint of the reconstructed segments. Results Complications
All dogs survived for the duration of the study and no infections developed. However, in some dogs localized mild swelling was more marked in BMP (+) group sites than in BMP () group sites. Swelling resolved gradually and disappeared in about 1 week. The mucosal suture wounds healed well in all dogs. Soft X-ray analysis
In the BMP (+) group, complete healing of defects was confirmed radiographically. Figure 2 shows soft X-ray radiographs obtained 2, 4, 8, and 12 weeks after operation. In the BMP (+) group, a radiopaque pattern was observed from 4 weeks onward and was marked at 12 weeks. In the BMP () group, only a radiolucent pattern was observed in the mandibular defects, even after 12 weeks. 3D-CT scan analysis
Figure 3 shows the 3D-CT images obtained in each group 8 weeks after operation. In the BMP (+) group, new bone formation was observed in the defect. The contour of the newly formed bone approximated that of the surrounding mandible. There was close fusion between the old and new bone, resulting in excellent contact between the edges of the defect and the implant material. Furthermore, the height of the new bone was similar to that of the original bone crest. In the BMP () group, however, there was virtually no new bone formation. Histologic analysis
Figure 4 shows frontal sections of the defects stained with toluidine blue. The BMP (+) group at 2, 4, 8, and 12 weeks showed extensive bone formation throughout the defect. At 2 weeks, small islands of newly formed bone were seen in the defect. These islands increased time-dependently, and were larger at 8 weeks than at 4 weeks. The surface of the new bone was lined by osteoid and plump osteoblasts, indicating active bone formation. At 12 weeks, there was evidence of more vigorous bone formation, and the colour of the stained
Fig. 2. Radiographic time course of defect healing induced by rhBMP-2. The radiograph showed no appreciable change 2 weeks postoperatively as compared with immediately after operation. At 4 weeks, callus-like bone density was seen at the defect in the BMP (+) group. At 8 weeks, the density at the defect in the BMP (+) group increased. At 12 weeks, the density at the defect in the BMP (+) group increased markedly. In the BMP () group, only a radiolucent pattern was observed, even at 12 weeks.
new bone approximated that of the surrounding cortical bone. Residual carrier material was seen in some defects at 4 weeks, but had been completely resorbed at 8 weeks. In contrast, even at 12 weeks, the BMP () group showed only fibrous tissue in the defects, with no bone formation.
BMP (+) group at 4, 8, and 12 weeks was 376 mg/cm3, 526 mg/cm3 and 716 mg/cm3, respectively (n=3 per time). The mean BMD of the surrounding cortical bone was about 800 mg/cm3. The BMD of the newly formed bone increased time-dependently, and at 12 weeks was nearly similar to that of the surrounding cortical bone.
pQCT analysis
Figure 5 shows the pQCT images in the BMP (+) group at 2, 4, 8, and 12 weeks after operation. Mean BMD in the
Discussion Animal models play a critical role in the development of alternatives to
Alveolar bone repair by rhBMP-2
Fig. 3. 3D-CT image of the defect at 8 postoperative weeks. Distinct evidence of new bone formation and close fusion between the old and new bone were observed in the BMP (+) group.
autogenous bone grafting. Many studies in animals have shown that bone formation is induced after surgical implantation of BMP plus a carrier in mandibular segmental defects2,21, cleft palate defects3,11, alveolar ridge defects1,19, maxillary sinus14, and periodontal defects9,18. M et al. evaluated co-polymer poly (lactic-co-glycolide) and autogenous blood as carriers for rhBMP-2 in maxillary alveolar clefts in dogs and reported that wound setting and improved carrier design are important determinants of bone induction11. T et al. reported a new technique for preparing vascularized bone grafts from the lattissimus dorsi muscle of miniature pigs, using rhOP-1 and xenogenic bone mineral as a carrier20. N et al. demonstrated the ability of rhBMP2/absorbable collagen sponge (ACS) implants to induce substantial new bone formation in the maxillary sinus of goats and concluded that the rhBMP-2/ACS composite implant may be an acceptable alternative to traditional bone grafts and bone substitutes in patients undergoing maxillary sinus floor augmentation14. Moreover, B demonstrated in a pilot study in adult male rhesus monkeys (Macaca fascicularis) that rhBMP-2 can promote osseous regeneration of critically sized hemimandibulectomy defects2. The properties of BMP have thus been examined in a variety of species. We used rhBMP-2 to repair defects caused by marginal excision of the mandible in dogs. We selected this model for the following reasons: 1. Dogs of similar ages and breeds can readily be acquired.
2. Surgical procedures on the dog mandible are relatively simple. 3. Histological and radiographic studies can be performed easily. 4. This type of defect frequently occurs in patients. 5. The defect (1087 mm) is large enough to be critically sized5 and cannot healed naturally without treatment or intervention (pilot study). 6. Alveolar ridge augmentation is commonly performed to permit prosthetic reconstruction, such as the placement of dental implants. Dogs are much more sensitive to BMP than are primates, which may lead to overestimation of the bone-induction properties of BMP in humans. However, we consider this model to approximate clinical conditions close enough to permit evaluation of the effects of rhBMP-2 and its carrier. The clinical application of BMP requires a suitable carrier and delivery system for local, defined, and controllable bone formation. PLGA/blood clots11, collagen6, hydroxyapatite1, demineralized bone matrix18,25, biphasic calcium phosphate (BCP)15, and other materials have been tested as carriers for BMP. PLGA alone does not readily adsorb rhBMP-2, requiring combination with autogenous blood. However, PLGA/blood clots are difficult to manage when implanted into some types of defects11. Collagen is well suited as a carrier, but when implanted into humans, certain animal-derived collagens have the risks of disease transmis-
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sion or unexpected inflammation owing to the residual immunogenicity of xenogenic collagen molecules23. Detailed investigations of the type, form, origin, and telopeptide treatment of collagen are necessary to establish an optimal collagen carrier. Hydroxyapatite also does not appear to be the best carrier for BMP. B et al. evaluated ACS/ hydroxyapatite as a carrier for ridge augmentation of the dog mandible and reported that hydroxyapatite remained 12 weeks after operation and appeared to partially obstruct bone formation1. Moreover, hydroxyapatite is easily fractured when exposed to heavy loads, and when used to reconstruct the jawbone osseointegration of hydroxyapatite; dental implantion also remains a problem. Recently, M et al. evaluated rhBMP-2/atelocollagen in the parietal bone of rats and showed it to be an effective, biocompatible material when used for biological onlay implants13. O et al. evaluated the cross-linked structure of fibrillar and denatured atelocollagen sponge (DCFD-AS)/ rhBMP-2 in the rat and showed that the size, shape, and location of newly formed bone could be accurately controlled by the carrier. They also reported that the carrier was safe16. We used PGS as a carrier/delivery system. This carrier is sponge-like and easily adapts to the shape of implantation sites, with little risk of leakage. PGS also can provide space for ridge augmentation because it is stiff and easy to use, i.e. rhBMP-2 solution is simply applied to the PGS carrier, which is then implanted into the defect. The toxicity of PGS has been tested by general toxicity studies, intracutaneous reaction tests, sensitization tests, and mutagenicity tests, and no adverse reactions have been noted. When PGS with rhBMP-2 was implanted subcutaneously in rats, new bone having a similar size and shape as the original carrier was induced, with no appreciable swelling. When PGS was implanted into segmental defects of the ulna in rabbits, radiopacity was seen 2 weeks postoperatively, and bone fusion was observed in 3 to 4 weeks. Evaluation of bone density by pQCT revealed that bone had newly formed towards the centre of the carrier 6 weeks after operation. New bone in the centre of the defects was resorbed, probably forming cortical bone and medullary canals. K et al. used PGS as a carrier to promote the regeneration of periodontal tissue after applying rhBMP-2 to horizontal circumferential defects in dogs
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Nagao et al. and reported that rhBMP-2/PGS can considerably enhance periodontal tissue regeneration9. I et al. evaluated rhBMP-2/PGS as a carrier for the repair of ulnar segmental defects in dogs and reported that this combination may be a useful bone graft substitute for orthopaedic reconstruction8. For clinical use, a carrier must be (1) non-cytotoxic, (2) non-carcinogenic, (3) non-immunogenic, (4) nonproinflammatoric, (5) biodegradable, (6) easy to process and mold, (7) able to be manufactured with high reproducibility, and (8) without contraindications. PGS gelatin sponges are used to control bleeding (Spongel, Yamanouchi Co. Ltd, Tokyo, Japan), and are recognized to be safe. PGS satisfies the conditions described earlier and may therefore be suited for clinical application. We examined the efficacy of rhBMP-2 with PGS and studied the properties of newly formed bone in mandibular defects in dogs. We combined 0.4 g/mm3 of rhBMP-2 with PGS. BMP-induced bone formation is affected by the concentration of BMP, species, age, and the type of the defect6,11,17. The dose of rhBMP-2 used in this study was based on the findings of previous studies8,9. Our results demonstrated that rhBMP-2/PGS promotes substantial new bone formation in mandibular defects, with no immune or other adverse reactions in the dog. Histologically, non-demineralized ground sections showed islands of immature trabecular bone throughout the defects 4 weeks postoperatively. PGS remained structurally stable for several weeks, acting as a scaffold for adhesion, proliferation, and differentiation of osteoprogenitor cells. The sponge-like form of PGS may have contributed to the vascularization of newly formed connective tissue. After
Fig. 4. Photomicrograph of histologic sections of the defects (toluidine blue, original magnification 1). At 2 postoperative weeks, slight bone formation was seen some parts of the defect in the BMP (+) group. At 4 weeks, small islands of new bone were seen throughout the defect in the BMP (+) group. At 8 weeks, new bone increased and matured at the defect in the BMP (+) group. At 12 weeks, the colour of the stained new bone approximated that of the surrounding cortical bone at the defect in the BMP (+) group. In contrast, BMP () group showed only fibrous tissue in the defect, even at 12 weeks. At 2 and 4 weeks, residual carrier material was seen in the defect, but had been completely resorbed at 8 weeks.
Alveolar bone repair by rhBMP-2
Fig. 5. pQCT image of defects in the BMP (+) group 2, 4, 8, and 12 weeks after operation. The white part is the densest area. Mean BMD at 4, 8, and 12 weeks was 376 mg/cm3, 526 mg/cm3, and 716 mg/cm3, respectively (all n=3). Newly formed bone developed towards the centre of the defect.
maturation of the new bone, within 8 to 12 weeks postoperatively, staining intensity was similar to that of the surrounding cortical bone. The presence of osteoid suggested active bone deposition by osteoblasts, and there was no evidence of inflammation. The rhBMP-2 implants probably stimulated the rapid influx and transformation of primitive mesenchymal cells into osteoblasts, resulting in the extensive formation of bone in the defects. Generally, BMP induces new bone formation mainly through the endochondral pathway, and BMP released from a carrier material initially induces cartilage formation
around the carrier. Therefore, endochondral ossification may have been the major route for osteogenesis. However, specimens at 2 and 4 weeks showed no distinct evidence of cartilage, suggesting that endochondral ossification was not involved. To clarify the mechanism of osteogenesis, examinations at earlier time points may be necessary. Residual PGS was found in some defects after 4 weeks; however, PGS was completely absorbed by 8 weeks and was replaced by new bone. The bone growth associated with the rhBMP-2/PGS implants in our study was rapid. This early replacement by host bone may be an advantage
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of rhBMP-2/PGS as compared with bone grafts. We performed 3D-CT 8 weeks after surgery to confirm the characteristics of newly formed bone. Conventional X-ray film is often used to evaluate bone defects. In our study, soft X-ray photographs suggested that bone formed in the defects in a time-dependent manner. To eliminate the effects of superimposed bone and soft tissue and to examine the complex structure of the defects, 3D-CT analysis was done. The newly formed bone had a smooth contour and showed evidence of mineralization. There was close fusion between the old and new bone, making it difficult to distinguish the rhBMP-2 implant from the surrounding bone. Bone density was examined by pQCT to evaluate the properties of new bone induced by rhBMP-2. pQCT is widely used in clinical and experimental studies. This technique can non-nvasively and selectively measure bone density in regions such as the distal radius or proximal tibia, which are rich in trabecular bone. To our knowledge, no study has used pQCT analysis to assess ridge augmentation of the dog mandible. In our study, pQCT showed that the density of bone newly formed in the defect in the BMP (+) group increased with time, reaching a level similar to that of the surrounding cortical bone at 12 weeks. Although not included in this report, examinations at 56 weeks showed no signs of bone resorption, and bone density at the defect was similar to that of the surrounding cortical bone. New bone formation seemed to proceed from the host bone and the periosteum in the BMP (+) group. In contrast, no bone was formed, and the defects were filled with fibrous tissue in the BMP () group and in unfilled defects (data not included). Our results suggest that the combination of rhBMP-2 and PGS is an effective bone substitute, providing good morphological and functional outcomes when used for reconstructive surgery of the mandible. However, newly formed bone induced by rhBMP-2 implants must be examined under actual clinical conditions before firm conclusions can be drawn. The final goal of reconstructive surgery of the mandible is the restoration of shape and masticatory function. An understanding of the properties of newly formed bone induced by rhBMP-2 is essential when establishing indications for rhBMP-2, such as the promotion of
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osteogenesis before the placement of dental implants. Our study showed that new bone induced by rhBMP-2 is morphologically similar to the surrounding cortical bone. However, long-term observations of the time course of newly formed bone are considered necessary. Acknowledgments. We thank K. Takahashi, S. Kokubo, K. Nozaki, and S. Fukushima for their technical advice and support, especially in managing the experimental animals. The authors also thank Yamanouchi Pharmaceutical Co. Ltd, Tokyo, Japan, for providing the rhBMP-2 and the carrier material. References 1. B EP, D ME, G L, S RG, R GE, W UM. Ridge augmentation following implantation of recombinant human bone morphogenetic protein-2 in the dog. J Periodontol 2000: 71: 488–496. 2. B PJ. Animal studies of application of rhBMP-2 in maxillofacial reconstruction. Bone 1996: 19(1 Suppl): 83S–92S. 3. B PJ, N R, N A. Human recombinant BMP-2 in osseous reconstruction of simulated cleft palate defects. Br J Oral Maxillofac Surg 1998: 36: 84–90. 4. H GA, S JM, S JP, J JA J, D CG, S NE, G GT, K DF, S TM. Utilization of type I collagen gel, demineralized bone matrix, and bone morphogenetic protein-2 to enhance autologous bone lumbar spinal fusion. J Neurosurg 1997: 86: 93–100. 5. H JO, K JC. The critical size defect as an experimental model to test bone repair materials. J Craniofac Surg 1990: 1: 60–68. 6. H JO, S JM, B DC, S R, J SP, Z HD, W J. Recombinant human bone morphogenetic protein-2 and collagen for bone regeneration. J Biomed Mater Res 1998: 43: 356–364. 7. H RE, R SM. Porous hydroxyapatite as a bone graft substitute in alveolar ridge augmentation: a histometric study. Int J Oral Maxillofac Surg 1987: 16: 718–728.
8. I T, M M, N R, M S, K T, K S, Y S, S N. Repair of ulnar segmental defect by recombinant human bone morphogenetic protein-2 in dogs. J Vet Med Sci 1998: 60: 451–458. 9. K A, O S, T K, Y S, I I. Periodontal regeneration by application of recombinant human bone morphogenetic protein-2 to horizontal circumferential defects created by experimental periodontitis in beagle dogs. J Periodontol 1997: 68: 103–109. 10. M RE, M RI, E WJ, H G, M TI. A comparison of particulate allogeneic and particulate autogenous bone grafts into maxillary alveolar clefts in dogs. J Oral Maxillofac Surg 1984: 42: 3–9. 11. M M, H J, R E, W J. Maxillary alveolar cleft repair in dogs using recombinant human bone morphogenetic protein-2 and a polymer carrier. Plast Reconstr Surg 1996: 98: 247–259. 12. MA MP, G PW, L TA. Tricalcium phosphate as a bone graft substitute in trauma: preliminary report. J Orthop Trauma 1988: 2: 333–339. 13. M M, H BZ, S T, I S, N N, A M. Bone augmentation by recombinant human BMP-2 and collagen on adult rat parietal bone. Int J Oral Maxillofac Surg 1999: 28: 232–237. 14. N M, K-H C, N M, W JA, P R, G D. Bone formation in the goat maxillary sinus induced by absorbable collagen sponge implants impregnated with recombinant human bone morphogenetic protein-2. Int J Periodont Rest Dent 1996: 16: 8–19. 15. O S, K A, H T, S T, I I. Ectopic bone formation by biphasic calcium phosphate (BCP) combined with recombinant human bone morphogenetic protein-2 (rhBMP-2). J Med Dent Sci 1997: 44: 53–62. 16. O S, M N, K R, O S, K S, F K. A carrier for clinical use of recombinant human BMP-2: dehydrothermally cross-linked composite of fibrillar and denatured atelocollagen sponge. Int J Oral Maxillofac Surg 1998: 27: 129–134. 17. S JM, H K, W SR, H JO. Bone morphogenetic
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proteins: an update on basic biology and clinical relevance. J Orthop Res 1999: 17: 269–278. S TJ, N L, T DN, F E, T TJ, J L, W JM, W UM. Periodontal repair in dogs: evaluation of rhBMP-2 carriers. Int J Periodont Rest Dent 1996: 16: 524–537. S TJ, F E, T DN, R MD, W UM. Bone morphogenetic protein-2 for peri-implant bone regeneration and osseointegration. Clin Oral Impl Res 1997: 8: 367–374. T H, J S, R DR. Mandibular reconstruction in miniature pigs with prefabricated vascularized bone grafts using recombinant human osteogenic protein- 1: a preliminary study. Int J Oral Maxillofac Surg 1999: 28: 461–463. T DM, O’G K, H DM, D D, T TJ, W J. Mandibular reconstruction using bone morphogenetic protein 2: long-term follow-up in a canine model. Laryngoscope 1999: 109: 1481–1489. U MR. Bone formation by autoinduction. Science 1965: 150: 893–899. V L MJ, W PM, O D LH, D PJ, F J, N P. Secondary cytotoxicity of cross-linked dermal sheep collagens during repeated exposure to human fibroblasts. Biomaterials 1992: 13: 1017–1024. W JM, R V, C AJ, M LM, W MJ, K RW, H RM, W EA. Novel regulators of bone formation: molecular clones and activities. Science 1988: 242: 1528–1534. Y AW, L JM, F EJ, R V, W JM, W EA. The healing of segmental bone defects, induced by recombinant human bone morphogenetic protein (rhBMP-2). A radiographic, histological, and biomechanical study in rats. J Bone Joint Surg 1992: 74: 659–670.
Address: Hiroshi Nagao, DDS, PhD Clinic for Oral Implant, University Hospital Faculty of Dentistry Tokyo Medical and Dental University 1-5-45, Yushima, Bunkyo-ku Tokyo 113-8549, Japan Tel: +81 3 5803 5773 Fax: +81 3 5803 5774 E-mail: [email protected]
Research and emerging technologies: Osteobiology
Effect of recombinant human bone morphogenetic protein-2 on bone formation in alveolar ridge defects in dogs
H. Nagao1, N. Tachikawa1, T. Miki2, M. Oda2, M. Mori2, K. Takahashi3, S. Enomoto1,2 1
Clinic for Oral Implantology, University Hospital, Faculty of Dentistry, Tokyo Medical and Dental University, Tokyo, Japan; 2Second Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Tokyo Medical and Dental University, Tokyo, Japan; 3Yamanouchi Pharmaceutical Co. Ltd, Ibaraki, Japan
H. Nagao, N. Tachikawa, T. Miki, M. Oda, M. Mori, K. Takahashi, S. Enomoto: Effect of recombinant human bone morphogenetic protein-2 on bone formation in alveolar ridge defects in dogs. Int. J. Oral Maxillofac. Surg. 2002; 31: 66–72. 2002 International Association of Oral and Maxillofacial Surgeons Abstract. This study was designed to evaluate the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) combined with poly D, L lactic-co-glycolic acid (PLGA)/gelatin sponge complex (PGS) on the formation of bone in critically sized marginal defects of the mandible in dogs. Three months after extraction of the pre-molar teeth, rectangular bone defects (1087 mm) were made in both sides of the mandible. A PGS block soaked in rhBMP-2 (400 g/ml) was implanted into one defect (BMP (+) group). As control, an untreated PGS block was implanted into the contralateral defect (BMP () group). 2, 4, 8, and 12 weeks after implantation, the defects were examined. In the BMP (+) group, newly formed bone was found in all defects from 4 weeks onward and was marked at 12 weeks. In contrast, the BMP () group showed no appreciable new bone formation, even at 12 weeks. Moreover, density of newly formed bone in the BMP (+) group was similar to that of the surrounding cortical bone at 12 weeks. These findings suggest that rhBMP-2/PGS is an effective bone substitute for reconstructive surgery of the dog mandible.
Restoration of bone is often required after dental and oral surgical procedures. Treatment of cysts, tumours, and fractures of the jaw can result in bone defects. Such defects must be repaired with bone grafts or bone substitutes to ensure a good structural and functional outcome. Conventionally, fresh autogenous bone grafts have been used to repair oral and maxillofacial bone defects. However, the need for surgery of the donor site and the limited supply of bone available have led to the development of various alternative materials to autogenous and allogenic bone grafts. 0901-5027/02/010066+07 $35.00/0
These materials include type I collagen4, hydroxyapatite7, tricalcium phosphate12, and isolated bone marrow cells10. Recent reports suggest that synthetic bone grafting preparations with properties similar to those of autogenous bone may be available soon. In 1965, Urist described ectopic bone formation after i.m. implantation of demineralized bone matrix in rats. The factor responsible for this effect was later named bone morphogenetic protein (BMP)22. Many attempts have been made to purify BMP from demineralized bone of
Key words: bone morphogenetic proteins; dogs; mandible; bone substitutes. Accepted for publication 28 May 2001
various animals, however pure BMP has not yet been obtained. Recently, the molecular biologic techniques enabled the production of very pure specific proteins. In 1988, W successfully synthesized recombinant human BMP (rhBMP-2)24. RhBMP-2 is known to induce osteogenesis and many experimental studies have shown that rhBMP-2 promotes bone healing17. Clinically, rhBMP-2 is expected to be used to fill bone defects and promote healing of fractures. The availability of rhBMP-2 has led to many studies
2002 International Association of Oral and Maxillofacial Surgeons
Alveolar bone repair by rhBMP-2 evaluating the functional roles of BMPs and the development of substitutes for bone grafts. When used for bone repair, rhBMP-2 requires a suitable carrier to prevent rapid diffusion of the protein. However, an effective bone repair system for rhBMP-2 has yet to be established, precluding the use of this protein in humans. Moreover, detailed studies of the properties of newly formed bone induced by rhBMP-2 are lacking. Recently, PGS [poly D, L lactic-coglycolic acid (PLGA)/gelatin sponge complex] has been developed as a new carrier for rhBMP-2. This carrier has a sponge-like consistency and readily adapts to surrounding structures, with minimal risk of leakage after implantation. It is also very biocompatible and biodegradable8,9. We have therefore attempted to develop combinations of rhBMP-2 and PGS that could be used in selected situations as alternatives to either autogenous or allogenic bone. This study was designed to examine the effect of rhBMP-2 in PGS implants on bone formation in critically sized defects of the mandible in dogs and to examine the properties of the newly formed bone.
Materials and methods Animals
Twelve adult beagle dogs without general or oral health problems, weighing 10 to 12 kg, were used in this study. Implant materials
The rhBMP-2 used in this experiment was manufactured by recombinant expression in Chinese hamster ovary cells at the Genetics Institute (Cambridge, MA, USA) and was purified to >98% purity. It was supplied by the Genetics Institute through Yamanouchi Pharmaceutical Co. Ltd (Tokyo, Japan). PGS [poly D, L lactic-co-glycolic acid (PLGA; molar ratio 50:50; MW 30 000)/ gelatin sponge complex] was used as the carrier for rhBMP-2. The weight ratio of PLGA:gelatin was 4:1 and the porosity was approximately 90%. PGS and PLGA were provided by Yamanouchi Co. Ltd Before implantation, PGS was soaked in rhBMP-2 solution (400 g/ml, 0.5 M L-arginine, 10 mM L-histidine, pH 6.5) and kept at room temperature for more than 30 min to permit incorporation of rhBMP-2. The same solution
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Fig. 1. Photograph of surgical procedure. (1) PGS carrier. (2) Defects measuring 1087 mm were made in both sizes of the mandible. (3) PGS blocks with or without rhBMP-2 were implanted into the defects. (4) The wounds were closed with nylon suture.
without rhBMP-2 was used as the carrier control. Operative procedure
All surgical procedures were performed under systemic (xylazine hydrochloride, 2 mg/kg and ketamine hydrochloride, 5 mg/kg i.v.) and local (2% lidocaine hydrochloride with 1/80 000 epinephrine) anaesthesia. The pre-molar teeth had been bilaterally extracted previously, and the ridges were allowed to heal for 3 months. After this period, marginal resection of the mandible was performed. A mucoperiosteal flap was raised from the mucogingival junction to the lingual gingiva. Rectangular bone defects were made in both sides of the mandible by a microengine (Fig. 1). The defects measured 1087 mm. These defects are large enough to be ‘critically sized’5 and cannot heal naturally, without some form of treatment or intervention. The bone defects were irrigated with sterile saline and the carrier was shaped to fit the size and shape of each defect. Then, a PGS block with rhBMP-2 was implanted into the bone defect on one side of the mandible (BMP (+) group). As control, PGS alone was implanted into the contralateral side (BMP () group). The wounds were closed with nylon sutures. Soft food was provided for 1 week after surgery. The dogs were killed 2, 4, 8, and 12 weeks after operation (n=3, respectively) and the defects were examined by means of radiographs on soft X-ray film, three-
dimensional computed tomographic (3D-CT) scans, histological specimens, and peripheral quantitative computed tomography (pQCT). Soft X-ray analysis
The defects were radiographed with a SRO-M50 soft X-ray unit (Sofron, Tokyo, Japan) operated at 40 KVp and 4 mA, with an exposure time of 2 min. Radiolucency of the defects was examined. 3D-CT scan analysis
In this study, 3D-CT with helical CT scanning was performed with a Somatom Plus CT scanner (Siemens, Forchheim, Germany) to examine the structure of the defects. Histologic procedures
The defects and adjacent host bone were obtained en bloc and were fixed in 10% buffered formalin. They were placed in a series of graded ethanol (70–100%), and embedded in Rigolac (Nissin EM Co. Ltd, Tokyo, Japan). Non-decalcified ground sections 30 m thick were prepared and stained with toluidine blue. pQCT analysis
To examine the properties of new bone induced by rhBMP-2, pQCT was performed with an XCT-960A quantitative computed tomograph (Stratec,
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Birkenfeld, Germany), and the bone mineral density (BMD) was measured at the midpoint of the reconstructed segments. Results Complications
All dogs survived for the duration of the study and no infections developed. However, in some dogs localized mild swelling was more marked in BMP (+) group sites than in BMP () group sites. Swelling resolved gradually and disappeared in about 1 week. The mucosal suture wounds healed well in all dogs. Soft X-ray analysis
In the BMP (+) group, complete healing of defects was confirmed radiographically. Figure 2 shows soft X-ray radiographs obtained 2, 4, 8, and 12 weeks after operation. In the BMP (+) group, a radiopaque pattern was observed from 4 weeks onward and was marked at 12 weeks. In the BMP () group, only a radiolucent pattern was observed in the mandibular defects, even after 12 weeks. 3D-CT scan analysis
Figure 3 shows the 3D-CT images obtained in each group 8 weeks after operation. In the BMP (+) group, new bone formation was observed in the defect. The contour of the newly formed bone approximated that of the surrounding mandible. There was close fusion between the old and new bone, resulting in excellent contact between the edges of the defect and the implant material. Furthermore, the height of the new bone was similar to that of the original bone crest. In the BMP () group, however, there was virtually no new bone formation. Histologic analysis
Figure 4 shows frontal sections of the defects stained with toluidine blue. The BMP (+) group at 2, 4, 8, and 12 weeks showed extensive bone formation throughout the defect. At 2 weeks, small islands of newly formed bone were seen in the defect. These islands increased time-dependently, and were larger at 8 weeks than at 4 weeks. The surface of the new bone was lined by osteoid and plump osteoblasts, indicating active bone formation. At 12 weeks, there was evidence of more vigorous bone formation, and the colour of the stained
Fig. 2. Radiographic time course of defect healing induced by rhBMP-2. The radiograph showed no appreciable change 2 weeks postoperatively as compared with immediately after operation. At 4 weeks, callus-like bone density was seen at the defect in the BMP (+) group. At 8 weeks, the density at the defect in the BMP (+) group increased. At 12 weeks, the density at the defect in the BMP (+) group increased markedly. In the BMP () group, only a radiolucent pattern was observed, even at 12 weeks.
new bone approximated that of the surrounding cortical bone. Residual carrier material was seen in some defects at 4 weeks, but had been completely resorbed at 8 weeks. In contrast, even at 12 weeks, the BMP () group showed only fibrous tissue in the defects, with no bone formation.
BMP (+) group at 4, 8, and 12 weeks was 376 mg/cm3, 526 mg/cm3 and 716 mg/cm3, respectively (n=3 per time). The mean BMD of the surrounding cortical bone was about 800 mg/cm3. The BMD of the newly formed bone increased time-dependently, and at 12 weeks was nearly similar to that of the surrounding cortical bone.
pQCT analysis
Figure 5 shows the pQCT images in the BMP (+) group at 2, 4, 8, and 12 weeks after operation. Mean BMD in the
Discussion Animal models play a critical role in the development of alternatives to
Alveolar bone repair by rhBMP-2
Fig. 3. 3D-CT image of the defect at 8 postoperative weeks. Distinct evidence of new bone formation and close fusion between the old and new bone were observed in the BMP (+) group.
autogenous bone grafting. Many studies in animals have shown that bone formation is induced after surgical implantation of BMP plus a carrier in mandibular segmental defects2,21, cleft palate defects3,11, alveolar ridge defects1,19, maxillary sinus14, and periodontal defects9,18. M et al. evaluated co-polymer poly (lactic-co-glycolide) and autogenous blood as carriers for rhBMP-2 in maxillary alveolar clefts in dogs and reported that wound setting and improved carrier design are important determinants of bone induction11. T et al. reported a new technique for preparing vascularized bone grafts from the lattissimus dorsi muscle of miniature pigs, using rhOP-1 and xenogenic bone mineral as a carrier20. N et al. demonstrated the ability of rhBMP2/absorbable collagen sponge (ACS) implants to induce substantial new bone formation in the maxillary sinus of goats and concluded that the rhBMP-2/ACS composite implant may be an acceptable alternative to traditional bone grafts and bone substitutes in patients undergoing maxillary sinus floor augmentation14. Moreover, B demonstrated in a pilot study in adult male rhesus monkeys (Macaca fascicularis) that rhBMP-2 can promote osseous regeneration of critically sized hemimandibulectomy defects2. The properties of BMP have thus been examined in a variety of species. We used rhBMP-2 to repair defects caused by marginal excision of the mandible in dogs. We selected this model for the following reasons: 1. Dogs of similar ages and breeds can readily be acquired.
2. Surgical procedures on the dog mandible are relatively simple. 3. Histological and radiographic studies can be performed easily. 4. This type of defect frequently occurs in patients. 5. The defect (1087 mm) is large enough to be critically sized5 and cannot healed naturally without treatment or intervention (pilot study). 6. Alveolar ridge augmentation is commonly performed to permit prosthetic reconstruction, such as the placement of dental implants. Dogs are much more sensitive to BMP than are primates, which may lead to overestimation of the bone-induction properties of BMP in humans. However, we consider this model to approximate clinical conditions close enough to permit evaluation of the effects of rhBMP-2 and its carrier. The clinical application of BMP requires a suitable carrier and delivery system for local, defined, and controllable bone formation. PLGA/blood clots11, collagen6, hydroxyapatite1, demineralized bone matrix18,25, biphasic calcium phosphate (BCP)15, and other materials have been tested as carriers for BMP. PLGA alone does not readily adsorb rhBMP-2, requiring combination with autogenous blood. However, PLGA/blood clots are difficult to manage when implanted into some types of defects11. Collagen is well suited as a carrier, but when implanted into humans, certain animal-derived collagens have the risks of disease transmis-
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sion or unexpected inflammation owing to the residual immunogenicity of xenogenic collagen molecules23. Detailed investigations of the type, form, origin, and telopeptide treatment of collagen are necessary to establish an optimal collagen carrier. Hydroxyapatite also does not appear to be the best carrier for BMP. B et al. evaluated ACS/ hydroxyapatite as a carrier for ridge augmentation of the dog mandible and reported that hydroxyapatite remained 12 weeks after operation and appeared to partially obstruct bone formation1. Moreover, hydroxyapatite is easily fractured when exposed to heavy loads, and when used to reconstruct the jawbone osseointegration of hydroxyapatite; dental implantion also remains a problem. Recently, M et al. evaluated rhBMP-2/atelocollagen in the parietal bone of rats and showed it to be an effective, biocompatible material when used for biological onlay implants13. O et al. evaluated the cross-linked structure of fibrillar and denatured atelocollagen sponge (DCFD-AS)/ rhBMP-2 in the rat and showed that the size, shape, and location of newly formed bone could be accurately controlled by the carrier. They also reported that the carrier was safe16. We used PGS as a carrier/delivery system. This carrier is sponge-like and easily adapts to the shape of implantation sites, with little risk of leakage. PGS also can provide space for ridge augmentation because it is stiff and easy to use, i.e. rhBMP-2 solution is simply applied to the PGS carrier, which is then implanted into the defect. The toxicity of PGS has been tested by general toxicity studies, intracutaneous reaction tests, sensitization tests, and mutagenicity tests, and no adverse reactions have been noted. When PGS with rhBMP-2 was implanted subcutaneously in rats, new bone having a similar size and shape as the original carrier was induced, with no appreciable swelling. When PGS was implanted into segmental defects of the ulna in rabbits, radiopacity was seen 2 weeks postoperatively, and bone fusion was observed in 3 to 4 weeks. Evaluation of bone density by pQCT revealed that bone had newly formed towards the centre of the carrier 6 weeks after operation. New bone in the centre of the defects was resorbed, probably forming cortical bone and medullary canals. K et al. used PGS as a carrier to promote the regeneration of periodontal tissue after applying rhBMP-2 to horizontal circumferential defects in dogs
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Nagao et al. and reported that rhBMP-2/PGS can considerably enhance periodontal tissue regeneration9. I et al. evaluated rhBMP-2/PGS as a carrier for the repair of ulnar segmental defects in dogs and reported that this combination may be a useful bone graft substitute for orthopaedic reconstruction8. For clinical use, a carrier must be (1) non-cytotoxic, (2) non-carcinogenic, (3) non-immunogenic, (4) nonproinflammatoric, (5) biodegradable, (6) easy to process and mold, (7) able to be manufactured with high reproducibility, and (8) without contraindications. PGS gelatin sponges are used to control bleeding (Spongel, Yamanouchi Co. Ltd, Tokyo, Japan), and are recognized to be safe. PGS satisfies the conditions described earlier and may therefore be suited for clinical application. We examined the efficacy of rhBMP-2 with PGS and studied the properties of newly formed bone in mandibular defects in dogs. We combined 0.4 g/mm3 of rhBMP-2 with PGS. BMP-induced bone formation is affected by the concentration of BMP, species, age, and the type of the defect6,11,17. The dose of rhBMP-2 used in this study was based on the findings of previous studies8,9. Our results demonstrated that rhBMP-2/PGS promotes substantial new bone formation in mandibular defects, with no immune or other adverse reactions in the dog. Histologically, non-demineralized ground sections showed islands of immature trabecular bone throughout the defects 4 weeks postoperatively. PGS remained structurally stable for several weeks, acting as a scaffold for adhesion, proliferation, and differentiation of osteoprogenitor cells. The sponge-like form of PGS may have contributed to the vascularization of newly formed connective tissue. After
Fig. 4. Photomicrograph of histologic sections of the defects (toluidine blue, original magnification 1). At 2 postoperative weeks, slight bone formation was seen some parts of the defect in the BMP (+) group. At 4 weeks, small islands of new bone were seen throughout the defect in the BMP (+) group. At 8 weeks, new bone increased and matured at the defect in the BMP (+) group. At 12 weeks, the colour of the stained new bone approximated that of the surrounding cortical bone at the defect in the BMP (+) group. In contrast, BMP () group showed only fibrous tissue in the defect, even at 12 weeks. At 2 and 4 weeks, residual carrier material was seen in the defect, but had been completely resorbed at 8 weeks.
Alveolar bone repair by rhBMP-2
Fig. 5. pQCT image of defects in the BMP (+) group 2, 4, 8, and 12 weeks after operation. The white part is the densest area. Mean BMD at 4, 8, and 12 weeks was 376 mg/cm3, 526 mg/cm3, and 716 mg/cm3, respectively (all n=3). Newly formed bone developed towards the centre of the defect.
maturation of the new bone, within 8 to 12 weeks postoperatively, staining intensity was similar to that of the surrounding cortical bone. The presence of osteoid suggested active bone deposition by osteoblasts, and there was no evidence of inflammation. The rhBMP-2 implants probably stimulated the rapid influx and transformation of primitive mesenchymal cells into osteoblasts, resulting in the extensive formation of bone in the defects. Generally, BMP induces new bone formation mainly through the endochondral pathway, and BMP released from a carrier material initially induces cartilage formation
around the carrier. Therefore, endochondral ossification may have been the major route for osteogenesis. However, specimens at 2 and 4 weeks showed no distinct evidence of cartilage, suggesting that endochondral ossification was not involved. To clarify the mechanism of osteogenesis, examinations at earlier time points may be necessary. Residual PGS was found in some defects after 4 weeks; however, PGS was completely absorbed by 8 weeks and was replaced by new bone. The bone growth associated with the rhBMP-2/PGS implants in our study was rapid. This early replacement by host bone may be an advantage
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of rhBMP-2/PGS as compared with bone grafts. We performed 3D-CT 8 weeks after surgery to confirm the characteristics of newly formed bone. Conventional X-ray film is often used to evaluate bone defects. In our study, soft X-ray photographs suggested that bone formed in the defects in a time-dependent manner. To eliminate the effects of superimposed bone and soft tissue and to examine the complex structure of the defects, 3D-CT analysis was done. The newly formed bone had a smooth contour and showed evidence of mineralization. There was close fusion between the old and new bone, making it difficult to distinguish the rhBMP-2 implant from the surrounding bone. Bone density was examined by pQCT to evaluate the properties of new bone induced by rhBMP-2. pQCT is widely used in clinical and experimental studies. This technique can non-nvasively and selectively measure bone density in regions such as the distal radius or proximal tibia, which are rich in trabecular bone. To our knowledge, no study has used pQCT analysis to assess ridge augmentation of the dog mandible. In our study, pQCT showed that the density of bone newly formed in the defect in the BMP (+) group increased with time, reaching a level similar to that of the surrounding cortical bone at 12 weeks. Although not included in this report, examinations at 56 weeks showed no signs of bone resorption, and bone density at the defect was similar to that of the surrounding cortical bone. New bone formation seemed to proceed from the host bone and the periosteum in the BMP (+) group. In contrast, no bone was formed, and the defects were filled with fibrous tissue in the BMP () group and in unfilled defects (data not included). Our results suggest that the combination of rhBMP-2 and PGS is an effective bone substitute, providing good morphological and functional outcomes when used for reconstructive surgery of the mandible. However, newly formed bone induced by rhBMP-2 implants must be examined under actual clinical conditions before firm conclusions can be drawn. The final goal of reconstructive surgery of the mandible is the restoration of shape and masticatory function. An understanding of the properties of newly formed bone induced by rhBMP-2 is essential when establishing indications for rhBMP-2, such as the promotion of
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osteogenesis before the placement of dental implants. Our study showed that new bone induced by rhBMP-2 is morphologically similar to the surrounding cortical bone. However, long-term observations of the time course of newly formed bone are considered necessary. Acknowledgments. We thank K. Takahashi, S. Kokubo, K. Nozaki, and S. Fukushima for their technical advice and support, especially in managing the experimental animals. The authors also thank Yamanouchi Pharmaceutical Co. Ltd, Tokyo, Japan, for providing the rhBMP-2 and the carrier material. References 1. B EP, D ME, G L, S RG, R GE, W UM. Ridge augmentation following implantation of recombinant human bone morphogenetic protein-2 in the dog. J Periodontol 2000: 71: 488–496. 2. B PJ. Animal studies of application of rhBMP-2 in maxillofacial reconstruction. Bone 1996: 19(1 Suppl): 83S–92S. 3. B PJ, N R, N A. Human recombinant BMP-2 in osseous reconstruction of simulated cleft palate defects. Br J Oral Maxillofac Surg 1998: 36: 84–90. 4. H GA, S JM, S JP, J JA J, D CG, S NE, G GT, K DF, S TM. Utilization of type I collagen gel, demineralized bone matrix, and bone morphogenetic protein-2 to enhance autologous bone lumbar spinal fusion. J Neurosurg 1997: 86: 93–100. 5. H JO, K JC. The critical size defect as an experimental model to test bone repair materials. J Craniofac Surg 1990: 1: 60–68. 6. H JO, S JM, B DC, S R, J SP, Z HD, W J. Recombinant human bone morphogenetic protein-2 and collagen for bone regeneration. J Biomed Mater Res 1998: 43: 356–364. 7. H RE, R SM. Porous hydroxyapatite as a bone graft substitute in alveolar ridge augmentation: a histometric study. Int J Oral Maxillofac Surg 1987: 16: 718–728.
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Address: Hiroshi Nagao, DDS, PhD Clinic for Oral Implant, University Hospital Faculty of Dentistry Tokyo Medical and Dental University 1-5-45, Yushima, Bunkyo-ku Tokyo 113-8549, Japan Tel: +81 3 5803 5773 Fax: +81 3 5803 5774 E-mail: [email protected]