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Comparative study of intramuscular and intraskeletal osteogenesis by recombinant human bone morphogenetic protein-2
Comparative study of intramuscular and intraskeletal osteogenesis by recombinant human bone morphogenetic protein-2 Yasunori Okubo, DDS,a Kazuhisa Bessho, DDS, DMSc,b Kazuma Fujimura, DDS, DMSc,b Kenji Kusumoto, MD, DMSc,c Yutaka Ogawa, MD, DMSc,d Yoshiaki Tani, DDS, DMSc,e and Tadahiko Iizuka, DDS, DMSc,f Kyoto, Japan KYOTO UNIVERSITY AND KANSAI MEDICAL UNIVERSITY
Objective. The purpose of this study was to compare the osteoinducing activity of recombinant human bone morphogenetic protein-2 (rhBMP-2) at intramuscular and intraskeletal sites in rats.
Study design. Five µg of rhBMP-2 was implanted into the right calf muscle of each of 20 rats and into a hole (4 mm in diameter, 1.5 mm in depth) that was made in the mandibular body of each of 20 other rats, with atelopeptide type I collagen as a carrier. The alkaline phosphatase activity and calcium content were quantitatively analyzed 1, 3, 7, and 21 days after the implantation of rhBMP-2 into either mandibular bone (in the intraskeletal group) or calf muscle (in the intramuscular group). The new bone formation was evaluated histologically 21 days after implantation. Results. On days 1 and 3, the alkaline phosphatase activity and calcium content in the intraskeletal group showed no significant differences from those in the intramuscular group. On the 7th and 21st days after implantation, however, the alkaline phosphatase activity and calcium content in the intraskeletal group were significantly higher than those in the intramuscular group. Histometry of the microscopic views showed that the mean trabecular area was 0.87 mm2 in the intramuscular group and 2.66 mm2 in the intraskeletal group. Conclusions. These results suggest that the new bone formation stimulated by rhBMP-2 in the intraskeletal group was greater than in the intramuscular group.
(Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;87:34-8)
Ever since Urist1 demonstrated in 1965 that demineralized bone matrix, including bone morphogenetic protein (BMP), induces osteogenesis, many studies of BMP have been performed. Excellent methods of isolating and purifying of BMP have been reported.2,3 Atelopeptide type I collagen (CL) was reported to be an effective carrier of BMP.4 We have been investigating intramuscular osteoinduction by recombinant human BMP-2 (rhBMP-2) for the reconstruction of large bony defects. Currently, This study was supported in part by a Grant-in-Aid for General Scientific Research C (no. 09672044) from the Japanese Ministry of Education, Science, Sports and Culture. aPostgraduate Student, Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Kyoto University. bAssistant Professor, Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Kyoto University. cAssociate Professor, Department of Plastic and Reconstructive Surgery, Kansai Medical University. dProfessor and Director, Department of Plastic and Reconstructive Surgery, Kansai Medical University. eProfessor and Head, Research Center for Biomedical Engineering, Kyoto University. fProfessor and Chairman, Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Kyoto University. Received for publication Oct. 27, 1997; returned for revision Dec. 17, 1997; accepted for publication Mar. 19, 1998. Copyright © 1999 by Mosby, Inc. 1079-2104/99/$8.00 + 0 7/12/90495
34
however, the frequency of the application of rhBMP2 is greater for small bony defects than for large bony defects, on the basis of preclinical studies. If rhBMP2 is to be applied in small bony defects, preclinical studies of rhBMP-2 should be carried out on the basis of the premise that osteoconduction and osteoinduction participate in osteogenesis. There are some studies of the osteogenesis by rhBMP-2 at intraskeletal sites 5,6 ; however, there have been no comparative studies of the osteogenesis by rhBMP-2 at various sites. In this study, we compared the osteogenesis by rhBMP-2 in relatively small bony defects to clarify whether osteoconduction can participate in the osteogenesis only by the osteoinduction of rhBMP-2 at intramuscular sites.
MATERIAL AND METHODS Animals We used an animal model that has been described previously.4,7-11 Each of 40 Wistar rats (all male, 10 weeks of age, and weighing 230 to 260 g) was randomly assigned to one of two groups of 20 rats each; one group was designated the intramuscular group (IMG), and the other was designated the intraskeletal group (ISG). The rats were fed rodent chow (Certified Diet MF; Oriental Koubo, Inc., Tokyo, Japan) preoperatively and postoperatively.
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Okubo et al 35
Fig. 1. Five µg of rhBMP-2 mixed with 3 mg of CL was lyophilized, and diskal specimens were implanted into a hole made in rat mandibular bone.
Implant material A supply of rhBMP-2 (Genetic Institute, Cambridge, Mass.) was donated by Yamanouchi Pharmaceutical Co., Ltd. (Tokyo, Japan). The rhBMP-2 was suspended in a buffer (pH 4.5) of 5 mmol/L glutamic acid, 2.5% glycine, 0.5% sucrose, and 0.01% Tween 80 and kept at a temperature of –80° C until needed, at which time it was thawed at room temperature. CL (pH 3.0; Cellmatrix LA; Nitta Gelatin, Inc., Osaka, Japan) was used as a carrier. Five µg of rhBMP-2 mixed with 3 mg of CL was lyophilized (EYELA FDU-830; Tokyo Rikakikai, Inc., Tokyo, Japan). The material was compressed in the injection syringe to diskal form (4 mm in diameter, 1.5 mm in thickness). Surgical procedure All rats were anesthetized with intraperitoneal administration of sodium pentobarbital (5.0 mg per 100 g of body weight). After desquamation of the periosteum of each rat in the ISG, a hole (4 mm in diameter, 1.5 mm in depth) was made with a round bur at the buccal side of the mandibular body at the molar region. After disinfection of the operative region, a lyophilized diskal specimen was implanted into a right calf muscle pouch of each rat in the IMG and into the hole in the mandibular bone (Fig. 1) of each rat in the ISG. The fascia and skin were sutured in the IMG, and the periosteum, fascia, and skin were sutured in the ISG. At 1, 3, 7, and 21 days after the implantation (n = 5 on each day in each group), rats were killed by an over-
dose of sodium pentobarbital. The implanted region was then excised, together with the surrounding tissue. Each excised specimen was removed and cut into halves, one for the histologic analysis and the other for the biochemical analysis.
Histologic analysis The specimens with peripheral tissues were fixed in 10% formalin neutral buffer solution (pH 7.4), demineralized in ethylenediamine tetraacetic acid, and embedded in paraffin. They were cut into 4 µm-thick sections and stained with hematoxylin and eosin. Analysis on micrographs The trabecular area and the percentage of trabeculum occupying the overall lump on histologic micrographs were measured on films by means of a computer system with Photoshop (version 3.0J; Adobe, Mountain View, Calif) and NIH-image (version 1.58). Biochemical examination The palpable nodule with a layer of peripheral tissues was excised. Samples for the quantitative analysis were weighed and then homogenized in 0.25 mol/L sucrose in a Polytron homogenizer (Bio-Mixer, type ABM; Nissei Inc., Osaka, Japan). The sediment was demineralized in 0.5 N HCl, and the calcium content of the soluble fraction was determined by the orthocresolphthalein complexone method.12 The alkaline phosphatase (ALP) activity and total protein in the resultant
36 Okubo et al
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY January 1999
Table I. Quantitative amount of new bone formation in IMG and ISG Mean (SD)
IMG (n = 5)
ISG (n = 5)
Trabecular area (mm2) Trabecular occupation in overall lump
0.87 (0.10) 19.3 (8.3)
2.66 (0.13) 49.4 (10.1)
bone, lining osteoblasts and a few osteoclasts were observed. The amounts of fatty marrow and anginoid tissues in the marrow tissue in the IMG were greater than those in the ISG. No cartilage or chondrocytes were seen in this figure. In the ISG, trabecular bone tissue and much bone matrix were observed (Fig. 2, B). There was less marrow tissue in the induced bone tissue than in the host bone tissue or the induced bone tissue in the IMG. A few osteoblasts and osteoclasts were observed, and there were fewer osteoblasts than in the IMG. The results of the computer-image analysis of the trabecular area and the percentage of the trabeculum occupying the overall lumps area are summarized in Table I.
Fig. 2. Histologic view of new bone formation in rat in IMG (A) and ISG (B) 21 days after implantation of rhBMP-2. M, Calf muscle of host; HB, host bone; BM, bone marrow; NB, bone matrix (decalcified by ethylenediamine tetraacetic acid; hematoxylin-eosin, original magnification ×100).
supernatant were determined by the 4-nitrophenylphosphate method.13 The calcium content (µg/mg of tissue) and the ALP activity (IU/mg of protein) were used as indices of bone formation.
Statistical analysis The Student t test was used to examine the statistical significance of differences in the values of ALP activity and calcium content between the IMG and ISG. The test was performed at the 95% confidence interval between the two groups at 1, 3, 7, and 21 days after implantation. RESULTS Histologic findings Twenty-one days after the implantation, light-microscopic examination disclosed new bone formation at the implanted sites in the muscle and in the bone. In the IMG, a considerable amount of trabecular bone tissue was observed (Fig. 2, A). Around the trabecular
Biochemical indices The ALP activity and calcium content after implantation in the muscle or bone are shown in Fig. 3, A and B, respectively. In the IMG, the ALP activity continuously increased until the 21st day, whereas in the ISG it was increased less markedly at the 7th day and was slightly decreased at the 21st day. The calcium content in both the IMG and ISG was markedly increased on day 21. No significant difference between the IMG and ISG was recognized for the calcium content on days 1 or 3. However, the ALP activity in the ISG was significantly higher than that in the IMG on days 3, 7, and 21 (day 3, p < 0.02; day 7, p < 0.0002; day 21, p < 0,002). The calcium content in the ISG was significantly higher than that in the IMG on days 7 and 21 (day 7, p < 0.0006; day 21, p < 0.0001). DISCUSSION Various preparations of BMP implanted in animals cause new bone formation at intraskeletal sites, skull defects,14-16 ulnar defects,17 and spinal fusions.18 In 1993, Gerhart et al.19 reported that femoral segmented defects in sheep were healed through use of rhBMP-2. In the oral and maxillofacial surgery fields, there are some reports of osteogenesis at intraskeletal sites.5,6 Intramuscular osteoinduction was reported.9 Since the recombinant DNA technique of synthesizing rhBMP-2 was established,20 rhBMP-2 has been applied in several clinical fields. Indeed, reconstruction of bony defects, fracture reduction, and augmentation are all
Okubo et al 37
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 87, Number 1
ulocytes, adipocytes, and osteoblasts.10,21 On the other hand, in intramuscular implantation of BMP-2, the differentiation of myoblasts is not to myocytes but rather to osteoblasts.22 It is thought that osteoconduction and osteoinduction are the essential processes in the healing of a skeletal defect with free bone grafts. Osteoinduction is characterized by graft-derived factors that actively stimulate osteogenic activity. Osteoconduction occurs when the graft acts passively as a scaffold on which new bone is deposited by the host.19 BMP promotes osteogenesis in intraskeletal sites as well as in ectopic sites. In this study, 5 µg of rhBMP-2, a relatively low dose, induced adequate new bone formation in the muscle. However, there were significant differences between the ISG and IMG with respect to ALP activity and calcium content on days 7 and 21, although there were no significant differences at day 3. Takagi and Urist23 observed new bone 3 mm from the edge of the bony defect 4 weeks after they had made skull defects (8 mm in diameter) in rats. In the same experiment, the defects were filled with new bone in the groups in which BMP was used. Inasmuch as our previous study9 revealed that the degree of bone formation is dependent on the dose of rhBMP-2 in the implants, we suggest that rhBMP-2 is potentially very useful in skeletal reconstruction as a biomaterial with osteoinductive activity. Further histologic studies on rhBMP-2 in the early stage of implantation are needed.
Fig. 3. Values of ALP activity/total protein (A) and calcium content/tissue weight (B) in IMG and ISG at days 1, 3, 7, and 21 after implantation of rhBMP-2. Significant difference between IMG and ISG is indicated (asterisk).
potential uses of rhBMP-2. However, for the clinical application of rhBMP-2, it is necessary to examine osteoinduction at the sites frequently indicated in clinical practice. It is reported that as little as 2 µg of rhBMP-2 induces bone formation in muscle.9 In this study, we found that the osteoinducing activity of rhBMP-2 at the intramuscular sites showed histologic and quantitative differences from that at the intraskeletal sites. The histologic findings suggested that the maturation of the induced bone in the ISG occurred earlier than that in the IMG. The ALP activity in the ISG reached its peak at day 7, but that in the IMG did not reach its peak before day 21. The peak calcium content in both groups was seen at day 21. In an intraskeletal site, BMP acts on immature mesenchymal cells, including osteoblasts, which then leads to osteogenesis. Immature mesenchymal cells are differentiated from committed progenitors into fibroblasts, retic-
We express our gratitude for the free use of NIH image software (version 1.58) for the computer-image analysis. The recombinant human BMP-2 was donated by Yamanouchi Pharmaceutical Co., Ltd. (Tokyo, Japan). REFERENCES 1. Urist MR. Bone formation by autoinduction. Science 1965;150:893-9. 2. Bessho K, Tagawa T, Murata M. Purification of bone morphogenetic protein derived from bovine bone matrix. Biochem Biophys Res Commun 1989;165:595-601. 3. Urist MR, Huo YK, Brownell AG, Hohl WM, Buyske J, Lietze A, et al. Purification of bovine bone morphogenetic protein by hydroxyapatite chromatography. Proc Natl Acad Sci U S A 1994;81:371-5. 4. Bessho K. Purification and characterization of bone morphogenetic protein. Mie Medical Journal 1990;40:61-71. 5. Boyne PJ. Animal studies of application of rhBMP-2 in maxillofacial reconstruction. Bone 1996;19:83-92. 6. Wozney JM. The potential role of bone morphogenetic proteins in periodontal reconstruction. J Periodontol 1995;66:506-10. 7. Bessho K, Tagawa T, Murata M. Comparison of bone matrixderived bone morphogenetic proteins from various animals. J Oral Maxillofac Surg 1992;50:496-501. 8. Bessho K, Iizuka T. Activity and solubility of bone morphogenetic protein derived from porcine bone matrix. Br J Oral Maxillofac Surg 1994;32:86-90. 9. Fujimura K, Bessho K, Kusumoto K, Ogawa Y, Iizuka T. Experimental studies on bone inducing activity of composites of atelopeptide type I collagen as a carrier for ectopic osteoinduction by rhBMP-2. Biochem Biophys-Res Commun 1995;208:316-22. 10. Kusumoto K, Bessho K, Fujimura K, Konishi Y, Ogawa Y,
38 Okubo et al
11.
12. 13. 14.
15. 16.
17.
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Iizuka T. Comparative study of bone marrow induced by purified BMP and recombinant human BMP-2. Biochem Biophys Res Commun 1995;215:205-11. Kusumoto K, Bessho K, Fujimura K, Akioka J, Ogawa Y, Iizuka T. Comparison of ectopic osteoinduction in vivo by recombinant human BMP-2 and recombinant xenopus BMP-4/7 heterodimer. Biochem Biophys Res Commun 1997;239:575-9. Connetry HV, Briggs AR. Determination of serum calcium by means of orthocresolphthalein complexone. Am J Clin Pathol 1966;45:290-6. The Executive Board in Analytical Section and the Committee on Enzymes in Analytical Section of the Japan Society of Clinical Chemistry (JSCC). Draft 1, Step 2. JSCC; 1988. p. 11-9. Ferguson D, Davis WL, Urist MR, Hurt WC, Allen EP. Bovine bone morphogenetic protein (bBMP) fraction-induced repair of craniotomy defects in the rhesus monkey (Macaca speciosa). Clin Orthop 1987;219:251-8. Lindholm TC, Lindholm TS, Alitalo I, Urist MR. Bovine bone morphogenetic protein (bBMP) induced repair of skull trephine defects in sheep. Clin 0rthop 1988;227:265-8. Urist MR, Nilsson O, Rasmussen J, Hirota W, Lovell T, Schmarlzreid T, et al. Bone regeneration under the influence of a bone morphogenetic protein (BMP) beta tricalcium phosphate (TCP) composite in skull trephine defects in dogs. Clin Orthop 1987;214:295-304. Johnson EE, Urist MR, Schmalzried TP, Chotivichit A, Huang HK, Finerman GA. Autogeneic cancellous bone grafts in extensive segmental ulnar defects in dogs: effects of xenogeneic bovine bone
18. 19. 20. 21. 22.
23.
morphogenetic protein without and with interposition of soft tissues and interruption of blood supply. Clin Orthop 1989;243:254-65. Lovell TP, Dawson EG, Nilsson OS, Urist MR. Augmentation of spinal fusion with bone morphogenetic protein in dogs. Clin Orthop 1989;243:266-74. Gerhart TN, Kirker Head CA, Kriz MJ, et al. Healing segmental femoral defects in sheep using recombinant human bone morphogenetic protein. Clin Orthop 1993;293:317-26. Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, et al. Novel regulators of bone formation: molecular clones and activities. Science 1988;242:1528-34. Whyte MP. In: Peck WA, editor. Bone and mineral research. 6th ed. New York: Elsevier; 1989. p. 175-218. Katagiri T, Yamaguchi A, Komaki M, et al. Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage [published erratum appears in J Cell Biol 1995 Feb;128:following 713]. J Cell Biol 1994;127:1755-66. Takagi K, Urist MR. The reaction of the dura to bone morphogenetic protein (BMP) in repair of skull defects. Ann Surg 1982;196:100-9.
Reprint requests: Yasunori Okubo, DDS Department of Oral and Maxillofacial Surgery, Faculty of Medicine Kyoto University 54 Kawahara-cho, Shogoin, Sakyo-ku Kyoto 606 Japan
AVAILABILITY OF JOURNAL BACK ISSUES As a service to our subscribers, copies of back issues of Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics for the preceding 5 years are maintained and are available for purchase from the publisher, Mosby, at a cost of $13.00 per issue until inventory is depleted. The following quantity discounts are available: 25% off on quantities of 12 to 23; one third off on quantities of 24 or more. Please write to Mosby, Inc., Subscription Services, 11830 Westline Industrial Drive, St. Louis, MO 63146-3318, or call (800) 453-4351 or (314) 453-4351 for information on availability of particular issues. If unavailable from the publisher, photocopies of complete issues are available from UMI, 300 N. Zeeb Rd., Ann Arbor, MI 48106; (313) 761-4700.
Objective. The purpose of this study was to compare the osteoinducing activity of recombinant human bone morphogenetic protein-2 (rhBMP-2) at intramuscular and intraskeletal sites in rats.
Study design. Five µg of rhBMP-2 was implanted into the right calf muscle of each of 20 rats and into a hole (4 mm in diameter, 1.5 mm in depth) that was made in the mandibular body of each of 20 other rats, with atelopeptide type I collagen as a carrier. The alkaline phosphatase activity and calcium content were quantitatively analyzed 1, 3, 7, and 21 days after the implantation of rhBMP-2 into either mandibular bone (in the intraskeletal group) or calf muscle (in the intramuscular group). The new bone formation was evaluated histologically 21 days after implantation. Results. On days 1 and 3, the alkaline phosphatase activity and calcium content in the intraskeletal group showed no significant differences from those in the intramuscular group. On the 7th and 21st days after implantation, however, the alkaline phosphatase activity and calcium content in the intraskeletal group were significantly higher than those in the intramuscular group. Histometry of the microscopic views showed that the mean trabecular area was 0.87 mm2 in the intramuscular group and 2.66 mm2 in the intraskeletal group. Conclusions. These results suggest that the new bone formation stimulated by rhBMP-2 in the intraskeletal group was greater than in the intramuscular group.
(Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;87:34-8)
Ever since Urist1 demonstrated in 1965 that demineralized bone matrix, including bone morphogenetic protein (BMP), induces osteogenesis, many studies of BMP have been performed. Excellent methods of isolating and purifying of BMP have been reported.2,3 Atelopeptide type I collagen (CL) was reported to be an effective carrier of BMP.4 We have been investigating intramuscular osteoinduction by recombinant human BMP-2 (rhBMP-2) for the reconstruction of large bony defects. Currently, This study was supported in part by a Grant-in-Aid for General Scientific Research C (no. 09672044) from the Japanese Ministry of Education, Science, Sports and Culture. aPostgraduate Student, Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Kyoto University. bAssistant Professor, Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Kyoto University. cAssociate Professor, Department of Plastic and Reconstructive Surgery, Kansai Medical University. dProfessor and Director, Department of Plastic and Reconstructive Surgery, Kansai Medical University. eProfessor and Head, Research Center for Biomedical Engineering, Kyoto University. fProfessor and Chairman, Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Kyoto University. Received for publication Oct. 27, 1997; returned for revision Dec. 17, 1997; accepted for publication Mar. 19, 1998. Copyright © 1999 by Mosby, Inc. 1079-2104/99/$8.00 + 0 7/12/90495
34
however, the frequency of the application of rhBMP2 is greater for small bony defects than for large bony defects, on the basis of preclinical studies. If rhBMP2 is to be applied in small bony defects, preclinical studies of rhBMP-2 should be carried out on the basis of the premise that osteoconduction and osteoinduction participate in osteogenesis. There are some studies of the osteogenesis by rhBMP-2 at intraskeletal sites 5,6 ; however, there have been no comparative studies of the osteogenesis by rhBMP-2 at various sites. In this study, we compared the osteogenesis by rhBMP-2 in relatively small bony defects to clarify whether osteoconduction can participate in the osteogenesis only by the osteoinduction of rhBMP-2 at intramuscular sites.
MATERIAL AND METHODS Animals We used an animal model that has been described previously.4,7-11 Each of 40 Wistar rats (all male, 10 weeks of age, and weighing 230 to 260 g) was randomly assigned to one of two groups of 20 rats each; one group was designated the intramuscular group (IMG), and the other was designated the intraskeletal group (ISG). The rats were fed rodent chow (Certified Diet MF; Oriental Koubo, Inc., Tokyo, Japan) preoperatively and postoperatively.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 87, Number 1
Okubo et al 35
Fig. 1. Five µg of rhBMP-2 mixed with 3 mg of CL was lyophilized, and diskal specimens were implanted into a hole made in rat mandibular bone.
Implant material A supply of rhBMP-2 (Genetic Institute, Cambridge, Mass.) was donated by Yamanouchi Pharmaceutical Co., Ltd. (Tokyo, Japan). The rhBMP-2 was suspended in a buffer (pH 4.5) of 5 mmol/L glutamic acid, 2.5% glycine, 0.5% sucrose, and 0.01% Tween 80 and kept at a temperature of –80° C until needed, at which time it was thawed at room temperature. CL (pH 3.0; Cellmatrix LA; Nitta Gelatin, Inc., Osaka, Japan) was used as a carrier. Five µg of rhBMP-2 mixed with 3 mg of CL was lyophilized (EYELA FDU-830; Tokyo Rikakikai, Inc., Tokyo, Japan). The material was compressed in the injection syringe to diskal form (4 mm in diameter, 1.5 mm in thickness). Surgical procedure All rats were anesthetized with intraperitoneal administration of sodium pentobarbital (5.0 mg per 100 g of body weight). After desquamation of the periosteum of each rat in the ISG, a hole (4 mm in diameter, 1.5 mm in depth) was made with a round bur at the buccal side of the mandibular body at the molar region. After disinfection of the operative region, a lyophilized diskal specimen was implanted into a right calf muscle pouch of each rat in the IMG and into the hole in the mandibular bone (Fig. 1) of each rat in the ISG. The fascia and skin were sutured in the IMG, and the periosteum, fascia, and skin were sutured in the ISG. At 1, 3, 7, and 21 days after the implantation (n = 5 on each day in each group), rats were killed by an over-
dose of sodium pentobarbital. The implanted region was then excised, together with the surrounding tissue. Each excised specimen was removed and cut into halves, one for the histologic analysis and the other for the biochemical analysis.
Histologic analysis The specimens with peripheral tissues were fixed in 10% formalin neutral buffer solution (pH 7.4), demineralized in ethylenediamine tetraacetic acid, and embedded in paraffin. They were cut into 4 µm-thick sections and stained with hematoxylin and eosin. Analysis on micrographs The trabecular area and the percentage of trabeculum occupying the overall lump on histologic micrographs were measured on films by means of a computer system with Photoshop (version 3.0J; Adobe, Mountain View, Calif) and NIH-image (version 1.58). Biochemical examination The palpable nodule with a layer of peripheral tissues was excised. Samples for the quantitative analysis were weighed and then homogenized in 0.25 mol/L sucrose in a Polytron homogenizer (Bio-Mixer, type ABM; Nissei Inc., Osaka, Japan). The sediment was demineralized in 0.5 N HCl, and the calcium content of the soluble fraction was determined by the orthocresolphthalein complexone method.12 The alkaline phosphatase (ALP) activity and total protein in the resultant
36 Okubo et al
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY January 1999
Table I. Quantitative amount of new bone formation in IMG and ISG Mean (SD)
IMG (n = 5)
ISG (n = 5)
Trabecular area (mm2) Trabecular occupation in overall lump
0.87 (0.10) 19.3 (8.3)
2.66 (0.13) 49.4 (10.1)
bone, lining osteoblasts and a few osteoclasts were observed. The amounts of fatty marrow and anginoid tissues in the marrow tissue in the IMG were greater than those in the ISG. No cartilage or chondrocytes were seen in this figure. In the ISG, trabecular bone tissue and much bone matrix were observed (Fig. 2, B). There was less marrow tissue in the induced bone tissue than in the host bone tissue or the induced bone tissue in the IMG. A few osteoblasts and osteoclasts were observed, and there were fewer osteoblasts than in the IMG. The results of the computer-image analysis of the trabecular area and the percentage of the trabeculum occupying the overall lumps area are summarized in Table I.
Fig. 2. Histologic view of new bone formation in rat in IMG (A) and ISG (B) 21 days after implantation of rhBMP-2. M, Calf muscle of host; HB, host bone; BM, bone marrow; NB, bone matrix (decalcified by ethylenediamine tetraacetic acid; hematoxylin-eosin, original magnification ×100).
supernatant were determined by the 4-nitrophenylphosphate method.13 The calcium content (µg/mg of tissue) and the ALP activity (IU/mg of protein) were used as indices of bone formation.
Statistical analysis The Student t test was used to examine the statistical significance of differences in the values of ALP activity and calcium content between the IMG and ISG. The test was performed at the 95% confidence interval between the two groups at 1, 3, 7, and 21 days after implantation. RESULTS Histologic findings Twenty-one days after the implantation, light-microscopic examination disclosed new bone formation at the implanted sites in the muscle and in the bone. In the IMG, a considerable amount of trabecular bone tissue was observed (Fig. 2, A). Around the trabecular
Biochemical indices The ALP activity and calcium content after implantation in the muscle or bone are shown in Fig. 3, A and B, respectively. In the IMG, the ALP activity continuously increased until the 21st day, whereas in the ISG it was increased less markedly at the 7th day and was slightly decreased at the 21st day. The calcium content in both the IMG and ISG was markedly increased on day 21. No significant difference between the IMG and ISG was recognized for the calcium content on days 1 or 3. However, the ALP activity in the ISG was significantly higher than that in the IMG on days 3, 7, and 21 (day 3, p < 0.02; day 7, p < 0.0002; day 21, p < 0,002). The calcium content in the ISG was significantly higher than that in the IMG on days 7 and 21 (day 7, p < 0.0006; day 21, p < 0.0001). DISCUSSION Various preparations of BMP implanted in animals cause new bone formation at intraskeletal sites, skull defects,14-16 ulnar defects,17 and spinal fusions.18 In 1993, Gerhart et al.19 reported that femoral segmented defects in sheep were healed through use of rhBMP-2. In the oral and maxillofacial surgery fields, there are some reports of osteogenesis at intraskeletal sites.5,6 Intramuscular osteoinduction was reported.9 Since the recombinant DNA technique of synthesizing rhBMP-2 was established,20 rhBMP-2 has been applied in several clinical fields. Indeed, reconstruction of bony defects, fracture reduction, and augmentation are all
Okubo et al 37
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 87, Number 1
ulocytes, adipocytes, and osteoblasts.10,21 On the other hand, in intramuscular implantation of BMP-2, the differentiation of myoblasts is not to myocytes but rather to osteoblasts.22 It is thought that osteoconduction and osteoinduction are the essential processes in the healing of a skeletal defect with free bone grafts. Osteoinduction is characterized by graft-derived factors that actively stimulate osteogenic activity. Osteoconduction occurs when the graft acts passively as a scaffold on which new bone is deposited by the host.19 BMP promotes osteogenesis in intraskeletal sites as well as in ectopic sites. In this study, 5 µg of rhBMP-2, a relatively low dose, induced adequate new bone formation in the muscle. However, there were significant differences between the ISG and IMG with respect to ALP activity and calcium content on days 7 and 21, although there were no significant differences at day 3. Takagi and Urist23 observed new bone 3 mm from the edge of the bony defect 4 weeks after they had made skull defects (8 mm in diameter) in rats. In the same experiment, the defects were filled with new bone in the groups in which BMP was used. Inasmuch as our previous study9 revealed that the degree of bone formation is dependent on the dose of rhBMP-2 in the implants, we suggest that rhBMP-2 is potentially very useful in skeletal reconstruction as a biomaterial with osteoinductive activity. Further histologic studies on rhBMP-2 in the early stage of implantation are needed.
Fig. 3. Values of ALP activity/total protein (A) and calcium content/tissue weight (B) in IMG and ISG at days 1, 3, 7, and 21 after implantation of rhBMP-2. Significant difference between IMG and ISG is indicated (asterisk).
potential uses of rhBMP-2. However, for the clinical application of rhBMP-2, it is necessary to examine osteoinduction at the sites frequently indicated in clinical practice. It is reported that as little as 2 µg of rhBMP-2 induces bone formation in muscle.9 In this study, we found that the osteoinducing activity of rhBMP-2 at the intramuscular sites showed histologic and quantitative differences from that at the intraskeletal sites. The histologic findings suggested that the maturation of the induced bone in the ISG occurred earlier than that in the IMG. The ALP activity in the ISG reached its peak at day 7, but that in the IMG did not reach its peak before day 21. The peak calcium content in both groups was seen at day 21. In an intraskeletal site, BMP acts on immature mesenchymal cells, including osteoblasts, which then leads to osteogenesis. Immature mesenchymal cells are differentiated from committed progenitors into fibroblasts, retic-
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Reprint requests: Yasunori Okubo, DDS Department of Oral and Maxillofacial Surgery, Faculty of Medicine Kyoto University 54 Kawahara-cho, Shogoin, Sakyo-ku Kyoto 606 Japan
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