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Effect of hyperbaric oxygenation on bone induced by recombinant human bone morphogenetic protein-2
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British Journal of Oral and Maxillofacial Surgery (2001) 39, 91–95 © 2001 The British Association of Oral and Maxillofacial Surgeons doi: 10.1054/bjom.2000.0550, available online at http://www.idealibrary.com on
BRITISH
Journal of Oral and Maxillofacial Surgery
Effect of hyperbaric oxygenation on bone induced by recombinant human bone morphogenetic protein-2 Y. Okubo,* K. Bessho,† K. Fujimura,† K. Kusumoto,‡ Y. Ogawa,§ T. Iizuka¶ *Graduate Student; †Assistant Professors; ¶Professor and Chairman, Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Kyoto University, Kyoto; ‡Associate Professor; §Professor and Head, Department of Plastic and Reconstructive Surgery, Kansai Medical University, Osaka, Japan SUMMARY. We examined the effect of hyperbaric oxygen (HBO) therapy on the osteoinductive activity of recombinant human bone morphogenetic protein-2 (rhBMP-2), 5 g of which was implanted into the calf muscle of rats using atelopeptide type I collagen as a carrier. Thirty Wistar rats were divided equally to be given HBO or act as controls. New bone formation was measured radiographically, biochemically, and histologically 3, 7, and 21 days after implantation. In both groups, new bone formation was found on day 21. However, there was significantly more new bone in the HBO group. In the HBO group, cartilage was present at the outer edge of the implanted material on day 7. On days 7 and 21, the local tissue alkaline phosphatase activity and calcium content in the HBO group were significantly greater than in the control group. These results suggest that HBO accelerated the activity and rate of osteoinduction by rhBMP-2. © 2001 The British Association of Oral and Maxillofacial Surgeons
Implant material
INTRODUCTION
Recombinant human bone morphogenetic protein-2 (batch number 2DIIK026/28, Genetics Institute Inc., Massachusetts, USA) was provided by Yamanouchi Pharmaceutical Co., Ltd (Tokyo, Japan). The rhBMP-2 was suspended in a buffer (pH 4.5) of 5 mmol glutamic acid, 2.5% glycine, 0.5% sucrose and 0.01% Tween 80 and kept at a temperature of 980°C until it had thawed at room temperature. Atelopeptide type I collagen (Cellmatrix LA; Nitta Gelatin Inc., Osaka, Japan) was used as a carrier. rhBMP-2 5 g of mixed with 3 mg of collagen was lyophilized (EYELA FDU-830; Tokyo Rikakikai Inc., Tokyo, Japan), and compressed in an injection syringe to form discs (4 mm in diameter and 1.5 mm thick).
Hyperbaric oxygen (HBO) treatment is used to improve anoxia by increasing the amount of oxygen dissolved. It increases collagen synthesis, capillary ingrowth,1 neovascularization, and osteogenesis.2 Recently, the use of HBO to improve the rate of bone healing in conjunction with operations for dental implants, osteomyelitis, and osteonecrosis has increased. Bone morphogenetic protein (BMP) seems to be one of the most promising biomaterials for clinical use in reconstructive surgery for bony defects and augmentation. Many studies on osteoinduction by rhBMP-2 have been reported.3–5 However, for clinical application of rhBMP-2 in tissues with low blood supply, such as, scarred or irradiated tissue, it is necessary to evaluate the factors that increase osteoinduction by rhBMP-2. In this study, we compared osteoinduction by rhBMP-2 with and without HBO.
Operation All rats were anaesthetized with an intraperitoneal injection of sodium pentobarbital (5.0 mg/100 g body weight). After disinfection of the operative region, the lyophilized discs were implanted into a right calf muscle pouch. The fascia and skin were sutured.
MATERIAL AND METHODS Animals Thirty Wistar rats (male; 10 weeks old; weight, 230– 260 g) were divided into 2 groups of 15 animals. They were fed rodent chow (certified diet MF; Oriental Koubo Inc., Tokyo, Japan) before and after operation. The treatment of each animal was conducted according to the 1988 guidelines for animal experiments at Kyoto University.
HBO treatment The rats in the HBO group were placed in a pressure chamber (KHO-100; Kawasaki Engineering Inc., Hyogo, Japan) and exposed to a pressure of 2.0 atmospheres 91
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absolute pressure (ATA) 100% inspired flow oxygen for 60 minutes every day for 3, 7, or 21 days. During the first 15 minutes of HBO therapy, the pressure was increased to 2.0 ATA, and decompression proceeded for 15 minutes after the treatment. Radiographic evaluation Three, 7, and 21 days after the implantation, the rats were killed with an overdose of sodium pentobarbital. The implanted region then was excised together with the surrounding tissue and soft radiographs were taken (SOFRON; SRO-M50, Sofron Inc., Tokyo, Japan). Each specimen was then removed and cut into two halves, one for histological examination and the other for biochemical assays. Histological examination The specimens with surrounding tissues were fixed in 10% formalin neutral buffer solution (pH 7.4), demineralized in ethylenediamine tetra-acetic acid, and embedded in paraffin. They were cut into sections 4 m thick and stained with haematoxylin and eosin. For histomorphometric analysis, the trabecular area and the percentage of trabeculum occupying the overall area on histological micrographs on days 21 were measured on films using a computer system with Photoshop (version 5.0J; Adobe, Mountain View, CA, USA) and NIH image software (version 1.58). Biochemical assays The samples for quantitative analysis were weighed and then homogenized in 0.25 M sucrose in a Polytron
homogenizer (Bio-Mixer; type ABM, Nissei Inc., Osaka, Japan). The sediment was demineralized in 0.5 M hydrochloric acid, and the calcium content of the soluble fraction was estimated by the orthocresolphthalein complexone method.6 The alkaline phosphatase (ALP) activity and total protein in the resultant supernatant were estimated by the 4-nitrophenylphosphate method.7 The calcium content (g/mg of tissue) and the ALP activity (IU/mg of protein) were used as indices of bone formation.
Statistical analysis The results were presented as mean
RESULTS Radiographic findings On day 21, soft radiographs showed opaque shadows morphologically identical to the implanted specimens in both groups (Fig. 1). The oval shadows in the HBO group were larger and slightly more radio-opaque than those in the control group. On days 3 and 7 no radioopaque images were seen in either group.
Fig. 1 Soft radiographs in the HBO group and the control group were taken 3, 7, and 21 days after implantation.
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Histological findings On day 3 after the implantation there was no evidence of osteoinduction, and there was a large amount of the collagen pellet, which remained in a non-absorbable form, in both groups (Fig. 2A&B). In the HBO group, immature mesenchymal cells at the inner site proliferated more than in the control group. On day 7 after the implantation, in both groups, fibroblast-like spindle cells had appeared at the inner site of the implanted material. In the HBO group, cartilage tissue was seen at the outer edge of the implanted material. In the control group, no cartilage or chondrocytes were detected. The area of the collagen pellet in the HBO group, which remained in non-absorbable form, was smaller than in the control group (Fig. 3A&B), and the area in the HBO group into which immature mesenchymal cells had infiltrated was larger than in the control group. On day 21 after the implantation there was new bone formation in both groups. Lining osteoblasts and a few osteoclasts were seen around the trabecular bone in both groups. In the
control group there was trabecular bone tissue at the outer edge of the implanted material. In the HBO group, the trabecular area was larger than that in the control group. The bone marrow area in the HBO group, including fatty marrow in part, was wider than in the control group. A small amount of collagen remained in both groups (Fig. 4A&B). The area of trabecular bone in the HBO group was wider than that in the control group. The results of the micrograph analysis of the trabecular area and the percentage of the trabeculum occupying the overall area are shown in Table 1.
Biochemical values The ALP activity and calcium content of the HBO group and the control group are shown in Table 2. There were no significant differences between the two groups on day 3. However, the values of ALP activity and calcium content in the HBO group were significantly higher than in the control group on days 7 and 21.
Fig. 2 Histological findings in the (A) HBO and (B) control groups 3 days after implantation (M:muscle of the host, CL:atelopeptide type I collagen, haematoxylin and eosin; original magnification25).
Fig. 3 Histological findings in the (A) HBO and (B) control groups 7 days after implantation (M:muscle of the host, CL:atelopeptide type I collagen, C:cartilage, haematoxylin and eosin; original magnification 25).
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Fig. 4 Histological finding in the (A) HBO and (B) control groups 21 days after implantation (M:muscle of the host, CL:atelopeptide type I collagen, OB:osteoblast, BM:bone marrow, NB:newly formed bone, haematoxylin and eosin; original magnification 25).
Table 1 Histomorphometric findings in the two groups HBO group (n:15) Control group (n:15) Trabecular area (mm2) Trabecular occupation in overall area (%)
4.1 (0.4)*
2.2 (0.2)
30.1 (2.2)*
16.9 (1.2)
All values are expressed as means (SD) * P0.001 significantly different between the two groups.
DISCUSSION There have been some reports on the influence of HBO on bone healing.2,8,9 HBO accelerates fracture healing and osteogenesis, increases the partial pressure of oxygen in arterial blood, and increases the diffusion of oxygen from blood to tissues. Mainous8 found that an increase in the partial pressure of oxygen resulted in increased collagen formation and fibroblastic proliferation, capillary budding, osteoblastic and osteoclastic activity, callus formation, and mineralization. After the recombinant DNA technique of synthesizing rhBMP-2 had been established,10 we reported that rhBMP-2 had osteoinductive activity in vivo.3–5,11,12 In the present study, rhBMP-2 5 g induced new bone formation in both groups by day 21. However, the osteoinductive activity of rhBMP-2 in the two groups was radiographically, histologically, and quantitatively
different. Histologically, cartilage was induced at the outer edge of the implanted material in the HBO group, but not in the control group on day 7 after the implantation. On day 21 after the implantation, the area of induced bone in the HBO group was significantly wider than in the control group. In all evaluations, the osteoinductive activity in the HBO group was greater than in the control group and HBO treatment accelerated osteoinductive activity. Bone formation is influenced by many factors, of which blood supply may be the most important because the partial pressure of oxygen in the tissue is directly influenced by the blood supply at the site. The quantitative difference in the indices of bone formation between the HBO group and the control group might therefore be the results of the partial pressure of oxygen – the blood supply to the tissue around the implanted material. Shaw and Bassett13,14 showed that increasing oxygen tension caused cellular differentiation to osseous tissue. In clinical practice, BMP may be implanted in sites with low blood supply, including scarred or irradiated tissue. In our previous study, we found the amount of bone formation in ischaemic muscle tissue, prepared by ligating and cutting the feeding artery, was less than in normal muscle (unpublished observations). The present results suggest that HBO may accelerate osteoinduction by BMP in ischaemic tissues.
Table 2 Biochemical findings in the two groups HBO group (n:15) Days ALP activity (IU/mg protein) Ca content (g/mg tissue)
3
7
21
0.4 (0.2) 5.1 (0.7)** 7.5 (0.8)*
Control group (n:15) 3
7
21
0.3 (0.1)
3.0 (0.3) 3.8 (1.2)
0.5 (0.2) 1.4 (0.7)* 41.0 (3.6)** 0.2 (0.1)
0.3 (0.2) 24.0 (2.9)
All values are expressed as means (SD) * P0.05; ** P0.01 significantly different between the two groups.
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In conclusion, the present results suggest that HBO treatment accelerates the activity and rate of osteoinduction by rhBMP-2, and that hyperbaric oxygenation increases the effect of rhBMP-2 on the differentiation from immature mesenchymal cells to osteoblasts. These results may be useful when BMP is applied clinically in future. ACKNOWLEDGEMENTS We thank National Institute of Health for allowing us 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. Hunt TK, Pai MP. The effect of varying ambient oxygen tensions on the wound metabolism and collagen synthesis. Surg Gynecol Obstet 1972; 135: 561–563. 2. Nilsson P, Albrektson T, Granström G, Röckert HOE. The effect of hyperbaric oxygen treatment on bone generation: an experimental study using the bone harvest chamber in the rabbit. Int J Oral Maxillofac Implants 1988; 3: 43–48. 3. 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: 316–322. 4. Kusumoto K, Bessho K, Fujimura K, Konishi Y, Ogawa Y, Iizuka T. Comparative study of bone marrow induced by purified BMP and recombinant human BMP-2. Biochem Biophys Res Commun 1995; 215: 205–211. 5. 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: 575–579. 6. Connetry HV, Briggs AR. Determination of serum calcium by means of orthocresolphthalein complexone. Am J Clin Pathol 1966; 45: 290–296. 7. The executive board in analytical section, the committee of enzymes in analytical section of Japan Society of Clinical Chemistry. Provisional recommendation for measurement of catalytic concentration of alkaline phosphatase is serum. Japan Society of Clinical Chemistry 1988; 1: 11–19. 8. Mainous EG. Hyperbaric oxygen in maxillofacial osteomyelitis, osteoradionecrosis, and osteogenesis enhancement. In: Dais JC,
Hunt TK, eds. Hyperbaric oxygen therapy. Bethesda: Undersea Medical Society, 1977: 217–227. 09. Dahlin C, Linde A, Röckert H. Stimulation of early bone formation by the combination of an osteopromotive membrane technique and hyperbaric oxygen. Scand J Plast Reconstr Surg Hand Surg 1993; 27: 103–108. 10. Wozney JM, Rosen V, Celeste AJ et al. Novel regulators of bone formation: molecular clones and activities. Science 1988; 242: 1528–1534. 11. Okubo Y, Bessho K, Fujimura K et al. Comparative study of intramuscular and intraskeletal osteogenesis by recombinant human bone morphogenetic protein-2. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999; 87: 34–38. 12. Yoshida K, Bessho K, Fujimura K et al. Osteoinduction capability of recombinant human bone morphogenetic protein-2. J Craniomaxillofac Surg 1998; 26: 112–115. 13. Shaw JK, Bassett CAL. The effect of varying oxygen concentrations on osteogenesis and embryonic cartilage in vitro. J Bone Joint Surg 1967; 49A: 73–80. 14. Basset CAL. Current Concepts of Bone Formation. J Bone Joint Surg 1962; 44A: 1217–1244.
The Authors Yasunori Okubo DDS Graduate Student Kazuhisa Bessho DDS, DMSc Kazuma Fujimura DDS, DMSc Assistant Professors Tadahiko Iizuka DDS, DMsc Professor and Chairman Department of Oral & Maxillofacial Surgery Graduate School of Medicine Kyoto University Kyoto, Japan Kenji Kusumoto MD, DMSc Associate Professor Yutaka Ogawa MD, DMSc Professor and Head Department of Plastic and Reconstructive Surgery Kansai Medical University Osaka, Japan Correspondence and requests for offprints to: Yasunori Okubo DDS, Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Tel:;81 75 751 3410; Fax:;81 75 761 9732 Paper received 2 October 1999 Accepted 11 September 2000
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British Journal of Oral and Maxillofacial Surgery (2001) 39, 91–95 © 2001 The British Association of Oral and Maxillofacial Surgeons doi: 10.1054/bjom.2000.0550, available online at http://www.idealibrary.com on
BRITISH
Journal of Oral and Maxillofacial Surgery
Effect of hyperbaric oxygenation on bone induced by recombinant human bone morphogenetic protein-2 Y. Okubo,* K. Bessho,† K. Fujimura,† K. Kusumoto,‡ Y. Ogawa,§ T. Iizuka¶ *Graduate Student; †Assistant Professors; ¶Professor and Chairman, Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Kyoto University, Kyoto; ‡Associate Professor; §Professor and Head, Department of Plastic and Reconstructive Surgery, Kansai Medical University, Osaka, Japan SUMMARY. We examined the effect of hyperbaric oxygen (HBO) therapy on the osteoinductive activity of recombinant human bone morphogenetic protein-2 (rhBMP-2), 5 g of which was implanted into the calf muscle of rats using atelopeptide type I collagen as a carrier. Thirty Wistar rats were divided equally to be given HBO or act as controls. New bone formation was measured radiographically, biochemically, and histologically 3, 7, and 21 days after implantation. In both groups, new bone formation was found on day 21. However, there was significantly more new bone in the HBO group. In the HBO group, cartilage was present at the outer edge of the implanted material on day 7. On days 7 and 21, the local tissue alkaline phosphatase activity and calcium content in the HBO group were significantly greater than in the control group. These results suggest that HBO accelerated the activity and rate of osteoinduction by rhBMP-2. © 2001 The British Association of Oral and Maxillofacial Surgeons
Implant material
INTRODUCTION
Recombinant human bone morphogenetic protein-2 (batch number 2DIIK026/28, Genetics Institute Inc., Massachusetts, USA) was provided by Yamanouchi Pharmaceutical Co., Ltd (Tokyo, Japan). The rhBMP-2 was suspended in a buffer (pH 4.5) of 5 mmol glutamic acid, 2.5% glycine, 0.5% sucrose and 0.01% Tween 80 and kept at a temperature of 980°C until it had thawed at room temperature. Atelopeptide type I collagen (Cellmatrix LA; Nitta Gelatin Inc., Osaka, Japan) was used as a carrier. rhBMP-2 5 g of mixed with 3 mg of collagen was lyophilized (EYELA FDU-830; Tokyo Rikakikai Inc., Tokyo, Japan), and compressed in an injection syringe to form discs (4 mm in diameter and 1.5 mm thick).
Hyperbaric oxygen (HBO) treatment is used to improve anoxia by increasing the amount of oxygen dissolved. It increases collagen synthesis, capillary ingrowth,1 neovascularization, and osteogenesis.2 Recently, the use of HBO to improve the rate of bone healing in conjunction with operations for dental implants, osteomyelitis, and osteonecrosis has increased. Bone morphogenetic protein (BMP) seems to be one of the most promising biomaterials for clinical use in reconstructive surgery for bony defects and augmentation. Many studies on osteoinduction by rhBMP-2 have been reported.3–5 However, for clinical application of rhBMP-2 in tissues with low blood supply, such as, scarred or irradiated tissue, it is necessary to evaluate the factors that increase osteoinduction by rhBMP-2. In this study, we compared osteoinduction by rhBMP-2 with and without HBO.
Operation All rats were anaesthetized with an intraperitoneal injection of sodium pentobarbital (5.0 mg/100 g body weight). After disinfection of the operative region, the lyophilized discs were implanted into a right calf muscle pouch. The fascia and skin were sutured.
MATERIAL AND METHODS Animals Thirty Wistar rats (male; 10 weeks old; weight, 230– 260 g) were divided into 2 groups of 15 animals. They were fed rodent chow (certified diet MF; Oriental Koubo Inc., Tokyo, Japan) before and after operation. The treatment of each animal was conducted according to the 1988 guidelines for animal experiments at Kyoto University.
HBO treatment The rats in the HBO group were placed in a pressure chamber (KHO-100; Kawasaki Engineering Inc., Hyogo, Japan) and exposed to a pressure of 2.0 atmospheres 91
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absolute pressure (ATA) 100% inspired flow oxygen for 60 minutes every day for 3, 7, or 21 days. During the first 15 minutes of HBO therapy, the pressure was increased to 2.0 ATA, and decompression proceeded for 15 minutes after the treatment. Radiographic evaluation Three, 7, and 21 days after the implantation, the rats were killed with an overdose of sodium pentobarbital. The implanted region then was excised together with the surrounding tissue and soft radiographs were taken (SOFRON; SRO-M50, Sofron Inc., Tokyo, Japan). Each specimen was then removed and cut into two halves, one for histological examination and the other for biochemical assays. Histological examination The specimens with surrounding tissues were fixed in 10% formalin neutral buffer solution (pH 7.4), demineralized in ethylenediamine tetra-acetic acid, and embedded in paraffin. They were cut into sections 4 m thick and stained with haematoxylin and eosin. For histomorphometric analysis, the trabecular area and the percentage of trabeculum occupying the overall area on histological micrographs on days 21 were measured on films using a computer system with Photoshop (version 5.0J; Adobe, Mountain View, CA, USA) and NIH image software (version 1.58). Biochemical assays The samples for quantitative analysis were weighed and then homogenized in 0.25 M sucrose in a Polytron
homogenizer (Bio-Mixer; type ABM, Nissei Inc., Osaka, Japan). The sediment was demineralized in 0.5 M hydrochloric acid, and the calcium content of the soluble fraction was estimated by the orthocresolphthalein complexone method.6 The alkaline phosphatase (ALP) activity and total protein in the resultant supernatant were estimated by the 4-nitrophenylphosphate method.7 The calcium content (g/mg of tissue) and the ALP activity (IU/mg of protein) were used as indices of bone formation.
Statistical analysis The results were presented as mean
RESULTS Radiographic findings On day 21, soft radiographs showed opaque shadows morphologically identical to the implanted specimens in both groups (Fig. 1). The oval shadows in the HBO group were larger and slightly more radio-opaque than those in the control group. On days 3 and 7 no radioopaque images were seen in either group.
Fig. 1 Soft radiographs in the HBO group and the control group were taken 3, 7, and 21 days after implantation.
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Histological findings On day 3 after the implantation there was no evidence of osteoinduction, and there was a large amount of the collagen pellet, which remained in a non-absorbable form, in both groups (Fig. 2A&B). In the HBO group, immature mesenchymal cells at the inner site proliferated more than in the control group. On day 7 after the implantation, in both groups, fibroblast-like spindle cells had appeared at the inner site of the implanted material. In the HBO group, cartilage tissue was seen at the outer edge of the implanted material. In the control group, no cartilage or chondrocytes were detected. The area of the collagen pellet in the HBO group, which remained in non-absorbable form, was smaller than in the control group (Fig. 3A&B), and the area in the HBO group into which immature mesenchymal cells had infiltrated was larger than in the control group. On day 21 after the implantation there was new bone formation in both groups. Lining osteoblasts and a few osteoclasts were seen around the trabecular bone in both groups. In the
control group there was trabecular bone tissue at the outer edge of the implanted material. In the HBO group, the trabecular area was larger than that in the control group. The bone marrow area in the HBO group, including fatty marrow in part, was wider than in the control group. A small amount of collagen remained in both groups (Fig. 4A&B). The area of trabecular bone in the HBO group was wider than that in the control group. The results of the micrograph analysis of the trabecular area and the percentage of the trabeculum occupying the overall area are shown in Table 1.
Biochemical values The ALP activity and calcium content of the HBO group and the control group are shown in Table 2. There were no significant differences between the two groups on day 3. However, the values of ALP activity and calcium content in the HBO group were significantly higher than in the control group on days 7 and 21.
Fig. 2 Histological findings in the (A) HBO and (B) control groups 3 days after implantation (M:muscle of the host, CL:atelopeptide type I collagen, haematoxylin and eosin; original magnification25).
Fig. 3 Histological findings in the (A) HBO and (B) control groups 7 days after implantation (M:muscle of the host, CL:atelopeptide type I collagen, C:cartilage, haematoxylin and eosin; original magnification 25).
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Fig. 4 Histological finding in the (A) HBO and (B) control groups 21 days after implantation (M:muscle of the host, CL:atelopeptide type I collagen, OB:osteoblast, BM:bone marrow, NB:newly formed bone, haematoxylin and eosin; original magnification 25).
Table 1 Histomorphometric findings in the two groups HBO group (n:15) Control group (n:15) Trabecular area (mm2) Trabecular occupation in overall area (%)
4.1 (0.4)*
2.2 (0.2)
30.1 (2.2)*
16.9 (1.2)
All values are expressed as means (SD) * P0.001 significantly different between the two groups.
DISCUSSION There have been some reports on the influence of HBO on bone healing.2,8,9 HBO accelerates fracture healing and osteogenesis, increases the partial pressure of oxygen in arterial blood, and increases the diffusion of oxygen from blood to tissues. Mainous8 found that an increase in the partial pressure of oxygen resulted in increased collagen formation and fibroblastic proliferation, capillary budding, osteoblastic and osteoclastic activity, callus formation, and mineralization. After the recombinant DNA technique of synthesizing rhBMP-2 had been established,10 we reported that rhBMP-2 had osteoinductive activity in vivo.3–5,11,12 In the present study, rhBMP-2 5 g induced new bone formation in both groups by day 21. However, the osteoinductive activity of rhBMP-2 in the two groups was radiographically, histologically, and quantitatively
different. Histologically, cartilage was induced at the outer edge of the implanted material in the HBO group, but not in the control group on day 7 after the implantation. On day 21 after the implantation, the area of induced bone in the HBO group was significantly wider than in the control group. In all evaluations, the osteoinductive activity in the HBO group was greater than in the control group and HBO treatment accelerated osteoinductive activity. Bone formation is influenced by many factors, of which blood supply may be the most important because the partial pressure of oxygen in the tissue is directly influenced by the blood supply at the site. The quantitative difference in the indices of bone formation between the HBO group and the control group might therefore be the results of the partial pressure of oxygen – the blood supply to the tissue around the implanted material. Shaw and Bassett13,14 showed that increasing oxygen tension caused cellular differentiation to osseous tissue. In clinical practice, BMP may be implanted in sites with low blood supply, including scarred or irradiated tissue. In our previous study, we found the amount of bone formation in ischaemic muscle tissue, prepared by ligating and cutting the feeding artery, was less than in normal muscle (unpublished observations). The present results suggest that HBO may accelerate osteoinduction by BMP in ischaemic tissues.
Table 2 Biochemical findings in the two groups HBO group (n:15) Days ALP activity (IU/mg protein) Ca content (g/mg tissue)
3
7
21
0.4 (0.2) 5.1 (0.7)** 7.5 (0.8)*
Control group (n:15) 3
7
21
0.3 (0.1)
3.0 (0.3) 3.8 (1.2)
0.5 (0.2) 1.4 (0.7)* 41.0 (3.6)** 0.2 (0.1)
0.3 (0.2) 24.0 (2.9)
All values are expressed as means (SD) * P0.05; ** P0.01 significantly different between the two groups.
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In conclusion, the present results suggest that HBO treatment accelerates the activity and rate of osteoinduction by rhBMP-2, and that hyperbaric oxygenation increases the effect of rhBMP-2 on the differentiation from immature mesenchymal cells to osteoblasts. These results may be useful when BMP is applied clinically in future. ACKNOWLEDGEMENTS We thank National Institute of Health for allowing us 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. Hunt TK, Pai MP. The effect of varying ambient oxygen tensions on the wound metabolism and collagen synthesis. Surg Gynecol Obstet 1972; 135: 561–563. 2. Nilsson P, Albrektson T, Granström G, Röckert HOE. The effect of hyperbaric oxygen treatment on bone generation: an experimental study using the bone harvest chamber in the rabbit. Int J Oral Maxillofac Implants 1988; 3: 43–48. 3. 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: 316–322. 4. Kusumoto K, Bessho K, Fujimura K, Konishi Y, Ogawa Y, Iizuka T. Comparative study of bone marrow induced by purified BMP and recombinant human BMP-2. Biochem Biophys Res Commun 1995; 215: 205–211. 5. 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: 575–579. 6. Connetry HV, Briggs AR. Determination of serum calcium by means of orthocresolphthalein complexone. Am J Clin Pathol 1966; 45: 290–296. 7. The executive board in analytical section, the committee of enzymes in analytical section of Japan Society of Clinical Chemistry. Provisional recommendation for measurement of catalytic concentration of alkaline phosphatase is serum. Japan Society of Clinical Chemistry 1988; 1: 11–19. 8. Mainous EG. Hyperbaric oxygen in maxillofacial osteomyelitis, osteoradionecrosis, and osteogenesis enhancement. In: Dais JC,
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The Authors Yasunori Okubo DDS Graduate Student Kazuhisa Bessho DDS, DMSc Kazuma Fujimura DDS, DMSc Assistant Professors Tadahiko Iizuka DDS, DMsc Professor and Chairman Department of Oral & Maxillofacial Surgery Graduate School of Medicine Kyoto University Kyoto, Japan Kenji Kusumoto MD, DMSc Associate Professor Yutaka Ogawa MD, DMSc Professor and Head Department of Plastic and Reconstructive Surgery Kansai Medical University Osaka, Japan Correspondence and requests for offprints to: Yasunori Okubo DDS, Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Tel:;81 75 751 3410; Fax:;81 75 761 9732 Paper received 2 October 1999 Accepted 11 September 2000