- Email: [email protected]
Bone induction by Escherichia coli -derived recombinant human bone morphogenetic protein-2 compared with Chinese hamster ovary cell-derived recombinant human bone morphogenetic protein-2
British Journal of Oral and Maxillofacial Surgery (2000) 38, 645–649 © 2000 The British Association of Oral and Maxillofacial Surgeons doi: 10.1054/bjom.2000.0533
BRITISH JOURNAL OF ORAL
& M A X I L L O FAC I A L S U R G E RY
Bone induction by Escherichia coli-derived recombinant human bone morphogenetic protein-2 compared with Chinese hamster ovary cell-derived recombinant human bone morphogenetic protein-2 K. Bessho,* Y. Konishi,† S. Kaihara,‡ K. Fujimura,§ Y. Okubo,¶ T. Iizuka** *Assistant Professor; †Research Fellow; ‡Instructor; §Assistant Professor; ¶Graduate Student; **Professor and Chairman, Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan SUMMARY. Most recombinant human bone morphogenetic protein (rhBMP) is currently obtained from Chinese hamster ovary (CHO) cells. If rhBMP with more activity could be derived from Escherichia coli (E. coli), a large quantity of rhBMP could be produced at low cost. The bone-inducing ability of an E. coliderived rhBMP-2 (ErhBMP-2) variant with an N-terminal sequence was examined and compared with CHO cell-derived rhBMP-2 (CrhBMP-2). Two, 10, or 50 g of ErhBMP-2 or CrhBMP-2 was mixed with 3 mg of atelopeptide type I collagen as the carrier, and specimens were implanted into the calf muscle pouches of Wistar rats (n:5 in each group). Three weeks later, new bone had formed in all the ErhBMP-2-implanted and CrhBMP-2-implanted muscles. Radiographic and histological examinations showed that the bone induced by ErhBMP-2 had a large hollow bone matrix with more fatty marrow than the bone induced by CrhBMP-2. Quantitative analysis indicated that the activity of ErhBMP-2 was similar to that of CrhBMP-2, so ErhBMP-2 may be useful for inducing bone formation. © 2000 The British Association of Oral and Maxillofacial Surgeons
INTRODUCTION
Sephacryl S-200 in 6 M guanidinium chloride, 0.1 M Tris hydrochloric acid, 0.1 mmol EDTA, and 1 mmol dithiothreitol, pH 8. By extensive dialysis against water, the protein was refolded, concentrated, and enriched by CMSepharose chromatography.7 It was finally purified by FPLC (Fractogel EMD SO3-650, 50 mmol sodium acetate, pH 5, and 30% 2-propanol) eluted with a sodium chloride gradient from 0 to 1.5 M. After dialysis against water, the protein was lyophilized and stored at ⫺80°C. CrhBMP-2 (Genetics Institute Inc., Massachusetts, USA) was produced by a recombinant DNA technique.8 It was dissolved in a buffer (pH 4.5) containing 5 mmol glutamic acid, glycine 2.5%, sucrose 0.5% and Tween 80 0.01%, and stored at ⫺80°C.
Several bone morphogenetic proteins (BMPs) have become available by cloning and genetic engineering.1 However, there are still some problems with the supply and activity of recombinant human BMP (rhBMP).2 At present, most rhBMP is obtained from mammalian cells, such as Chinese hamster ovary (CHO) cells.3 If rhBMP with more activity could be derived from Escherichia coli (E. coli), a large quantity of rhBMP could be produced at low cost. In this study, we examined the bone-inducing ability of E. coli-derived rhBMP-2 (ErhBMP-2) and compared it with that of CHO cell-derived rhBMP-2 (CrhBMP-2) using bioassays.4
Bioassays MATERIAL AND METHODS
The study comprised two experimental groups, each tested with three different amounts of BMP. Samples of 2, 10, or 50 g of ErhBMP-2 or CrhBMP-2 were each mixed with 3 mg of porcine skin-derived atelopeptide type I collagen solution (3 mg/ml, pH 3.0, Nitta Gelatin Inc, Osaka, Japan) as carrier. Each mixed sample was then lyophilized into a disc (4 mm in diameter and 1.5 mm thick). These specimens were then implanted into the calf muscles of 10-week-old male Wistar rats (weight 230–260 g, n:5 in each group) that had been anaesthetized by an intraperitoneal injection of sodium pentobarbital. The fascia and skin were then closed. Three weeks later, soft radiographs (SRO-M50; Sofron Inc., Tokyo, Japan; 35 kV, 4 mA, 2 minutes) were taken of the implanted regions.
ErhBMP-2 and CrhBMP-2 The variant ErhBMP-2 was generated by exchanging the sequence coding the first 12 amino acids of rhBMP-25 (MAKHKQRKRLKS) for the sequence coding the first 13 amino acids in human interleukin 2. This created the new N-terminus MAPTSSSTKKTQL. The hybrid cDNA was integrated into the vector pRPR9fd.6 The synthesis of ErhBMP-2 was induced by a temperature shift to 42°C. The protein was purified from the inclusion-body fraction by extraction with 4 M guanidinium chloride (0.1 M Tris hydrochloric acid, pH 8, 0.1 mmol EDTA, 14 mmol 2-mercaptoethanol) and chromatography on 645
646
British Journal of Oral and Maxillofacial Surgery
The implants were then excised, and each was cut in half specimen with its surrounding muscle. One half was separated from the surrounding tissues, weighed, and homogenized in 0.25 M sucrose in a Polytron homogenizer (Bio-Mixer type ABM-1; Nissei Inc., Osaka, Japan). Alkaline phosphatase (ALP) activity and protein in the resultant supernatant were measured by the p-nitrophenyl phosphate method and pyrogallol red method, respectively. The sediments were demineralized in 0.5 M hydrochloric acid 10 ml, and the calcium content of the soluble fractions was measured by the o-cresolphthalein complex method. The ALP activity (IU/mg protein) and the calcium content (g/mg tissue) were used as indices of bone formation. The other half of the specimen with surrounding muscle was used for histological examination after being fixed in 10% formalin neutral buffer solution (pH 7.4), demineralized in EDTA, and embedded in paraffin. Sections 4 m thick were cut on a microtome, stained with haematoxylin and eosin, and examined under a light microscope. The entire implant areas and the bone matrix areas were measured on histological micrographs of slices near the centre of the implant using a computer system with Photoshop ver. 4.0J (Adobe Systems Inc., San Jose, CA, USA) and NIH Image ver. 1.61 (National Institutes of Health, Bethesda, MD, USA) software. The percentage of bone matrix in the entire area was calculated in each group. The control, type I collagen alone, was implanted and evaluated in a similar manner. The study was approved by the Animal Experiments Committee and followed the Guidelines for Animal Experiments of Kyoto University. RESULTS Soft radiographic findings Radio-opaque shadows were visible in the radiographs of all of the ErhBMP-2- and CrhBMP-2-implanted muscles (Fig. 1). The sizes of the shadows of the
Fig. 1 – Soft radiographs taken three weeks after implantation.
ErhBMP-2- and CrhBMP-2-implanted muscles increased with the amount of implanted ErhBMP-2 or CrhBMP-2, but were much larger in the ErhBMP-2-implanted muscles than in CrhBMP-2-implanted muscles. The opacity of the shadows in the ErhBMP-2 implanted muscles was strong on the outermost edge of the implant around the entire circumference, and weak at the core of the implant. The opaque shadows in the CrhBMP-2 implanted muscles were slightly more uniform than those in ErhBMP2-implanted muscles. These radio-opaque shadows were not visible in the controls. Biochemical analysis The volume of ErhBMP-2- and CrhBMP-2-induced bone increased with the amount of BMP implanted. The ALP activity and calcium content in the specimens are shown in Table 1. Student’s t-test was used to examine the significance of differences in the value of ALP activity and calcium content between ErhBMP-2 implanted groups and CrhBMP-2 implanted groups. The ALP activity of ErhBMP-2 was significantly higher than that of CrhBMP-2. The calcium content of ErhBMP-2 was similar to that of CrhBMP-2. Table 1 – Alkaline phosphatase (ALP) activity and calcium content in the specimens. Values are the mean (SD) of five specimens in each group ALP activity (IU/mg protein) Control ErhBMP-2 2 g 10 g 50 g CrhBMP-2 2 g 10 g 50 g
0.1
Calcium content (g/mg tissue) 1.0
1.6 (0.5)* 6.3 (0.9)* 19.9 (3.1)**
12.8 (1.9) 23.4 (3.5) 32.8 (5.2)
0.5 (0.3) 4.1 (0.7) 13.9 (2.0)
11.3 (1.6) 21.5 (3.4) 30.2 (4.8)
*P0.005, **P0.05 (Student’s t-test compared with CrhBMP-2 implanted group).
Bone induction by E. coli-derived rhBMP
647
Fig. 2 – Photomicrographs of control (A) and bone induced by (B) 2 g, (C) 10 g, (D) 50 g, of ErhBMP-2, or (E) 2 g, (F) 10 g, (G) 50 g, of CrhBMP-2 in the calf muscle pouch of Wistar rats 3 weeks after implantation (haematoxylin–eosin; original magnification, 100; bars:50 m). M, calf muscle of host; NB, new bone matrix; BM, bone marrow; F, fatty marrow; AT, angioid tissue; and C, collagen.
648
British Journal of Oral and Maxillofacial Surgery
Histological findings Light microscopy showed new bone surrounded by immature mesenchymal-type cells in all the ErhBMP-2and CrhBMP-2-implanted muscles, but there was no evidence of osteoinduction in the controls (Fig. 2A). However, the compositions of ErhBMP-2- and CrhBMP-2-induced bone differed. Implantation of 2 g of ErhBMP-2 resulted in bone matrix formation in only part of the area on the outermost edge of the intramuscular implant. Much collagen was present at the core of the implant (Fig. 2B). Implantation of ErhBMP-2 10 g resulted in bone matrix formation on the outermost edge of the implant around almost the entire circumference, and immature mesenchymal type cells surrounded the new bone matrix. There was a little collagen and much fatty marrow in the core of the implant (Fig. 2C). Implantation of 50 g of ErhBMP-2 resulted in the formation of bone matrix that was thicker than the bone matrix formed by 10 g of ErhBMP-2 on the outermost edge of the implant around almost the entire circumference, and immature mesenchymal type cells surrounded the new bone matrix. A little bone marrow containing angioid tissue and much fatty marrow was seen in the core of the implant (Fig. 2D). Implantation of 2 g of CrhBMP-2 resulted in slightly more bone matrix being formed than was formed by 2 g of ErhBMP-2 in only part of the area on the outermost edge of the intramuscular implant. There was much collagen present at the core of the implant (Fig. 2E). Implantation of 10 g of CrhBMP-2 resulted in bone matrix formation on the outermost edge of the implant around almost the entire circumference and in part of the area in the core of the implant, and immature mesenchymal type cells surrounded the new bone matrix. A little collagen and fatty marrow were present in the core of the implant (Fig. 2F). Implantation of 50 g of CrhBMP-2 resulted in bone matrix formation not only on the outermost edge but also deep within the implant. Immature mesenchymaltype cells were present both inside and outside the new bone, and there was also much angioid tissue containing bone marrow with only a little fatty marrow. No collagen was detected (Fig. 2G). Histomorphometric analysis The means of the entire implant area bone matrix area and percentage of the bone matrix occupying the entire implant area for each histological specimen are shown in Table 2.
DISCUSSION ErhBMP-2 induced ALP activity in the C3H10T1/2 fibroblast cell line and proteoglycan synthesis in embryonic chicken limb bud cells.5 However, there have been no previous reports of the bone-inducing activity of ErhBMP-2 in vivo. If ErhBMP-2 has sufficient boneinducing activity, a large quantity of rhBMP could be produced at low cost. This would have important applications for clinical use.
Table 2 – The means of the entire implant area (E), bone matrix area (B) and percentage of the bone matrix occupying the entire implant area (P) for each histological specimen. Values are the mean (SD) of five specimens in each group
Control ErhBMP-2 2 g 10 g 50 g CrhBMP-2 2 g 10 g 50 g
E (mm2)
B (mm2)
P (%)
6.9 (0.8)
0 (0)
0 (0)
5.6 (0.8) 13.6 (2.2) 44.9 (6.3)
0.6 (0.2) 2.6 (0.6) 8.8 (1.6)
11.0 (1.6) 19.2 (1.7) 19.5 (1.9)
1.7 (0.4) 10.2 (0.9) 12.9 (1.6)
0.2 (0.1) 2.3 (0.4) 4.2 (0.9)
11.8 (3.5) 22.8 (3.3) 32.1 (3.8)
The mature BMP-2 is a homodimer of two 114residue subunits representing the C-terminal sequence of a long precursor protein of 396 amino acids. One of the four N-glycosylation sites present in the proprotein is retained and used in the mature BMP-2 polypeptide chains.9 Recently, biological activity and physical binding constants were established using rhBMP-2 from CHO cells.3 In addition, a cDNA encoding mature human BMP-2 can be efficiently expressed in E. coli, and after renaturation a dimeric BMP-2 protein with a molecular weight of 26 000 was prepared with a purity of over than 98%. The ErhBMP-2 had been substituted by a dummy sequence of equal length for the N-terminal residues 1–12 of rhBMP-2. A heparin-binding site was identified in the N-terminal segments of dimetric rhBMP-2. The variant ErhBMP-2 showed biological activity, so the basic N-terminal domains of dimeric rhBMP-2 as heparin-binding sites are not obligatory for receptor activation.5 In the present study, soft radiographic shadows were much larger in the ErhBMP-2 than in CrhBMP-2 implanted groups. Histological examination showed that the bone tissue induced by ErhBMP-2 consisted of rich fatty marrow, much collagen, and comparatively poor bone matrix, while that induced by CrhBMP-2 had a little fatty marrow and collagen, and comparatively rich bone matrix. These findings suggest that ErhBMP-2 with collagen expands in vivo, because ErhBMP-2 does not have heparin-binding sites. Furthermore, ErhBMP-2 was delivered slowly from the expanded implant (ErhBMP-2 with collagen) because new bone matrix formed on the outermost edge of the implant. Much collagen was, therefore, seen at the core of the implant in the ErhBMP-2 implanted groups, and the ALP activity of these groups was higher than that of the CrhBMP-2 implanted groups at 3 weeks after implantation. Biochemical analysis showed that the bone-inducing activity of ErhBMP-2 was similar to that of CrhBMP-2. Histomorphometric analysis showed that the percentage of bone matrix occupying the entire implant area in the ErhBMP-2 implanted group was lower than that in the CrhBMP-2 implanted group. However, the bone matrix area in the ErhBMP-2 implanted group was larger than that in the CrhBMP-2 implanted group. These results indicate that ErhBMP-2 effectively induces bone formation under some conditions. Many patients with large bone defects resulting from tumours or injury have been treated by oral and
Bone induction by E. coli-derived rhBMP
maxillofacial surgeons. Even bioactive materials with osteoconductive ability, such as hydroxyapatite, are not sufficiently effective for them. Hence a biomaterial with bone-inducing ability is needed. BMP is the only cytokine with bone inducing ability, but the dose of BMP-2 required is much greater than that of other cytokines and growth factors.10 The fact that ErhBMP-2 is effective in inducing bone formation is therefore valuable. It should be possible to produce a large quantity of ErhBMP-2 at low cost for clinical use in the near future.
649
7. Cerletti N, McMaster GK, Cox D, Schmitz A, Meyhack B. Ciba-Geigy Ltd. Basel, Switzerland, European Patient Application 1991; 0433 225 A1. 8. Wozney JM, Rosen V, Celeste AJ et al. Novel regulators of bone formation: Molecular clones and activities. Science 1988; 242: 1528–1534. 9. Celeste AJ, Iannazzi JA, Taylor RC et al. Identification of transforming growth factor  family members present in bone-inductive protein purified from bovine bone. Proc Natl Acad Sci USA 1990; 87: 9843–9847. 10. Yamaguchi A, Katagiri T, Ikeda T et al. Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro. J Cell Biol 1991; 113: 681–687.
Acknowledgements This work was partly supported by a grant-in-aid for scientific research (B No. 12470437) of the Japanese Ministry of Education, Science, Sports and Culture. We thank Professor Walter Sebald, Würzburg University, Germany for supplying us with the ErhBMP-2, and the Yamanouchi Pharmaceutical Company, Tokyo, Japan for providing the CrhBMP-2.
References 1. Wang EA, Rosen V, Cordes P et al. Purification and characterization of other distinct bone-inducing factors. Proc Natl Acad Sci USA 1988; 85: 9484–9488. 2. Bessho K. Ectopic osteoinductive difference between purified bovine and recombinant human bone morphogenetic protein. In: Lindholm TS, ed. Bone Morphogenetic Proteins: Biology, Biochemistry and Reconstructive Surgery. Austin: RG Landes, 1996: 105–111. 3. Wang EA, Rosen V, D’Alessandro JS et al. Recombinant human bone morphogenetic protein induces bone formation. Proc Natl Acad Sci USA 1990; 87: 2220–2224. 4. 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. 5. Ruppert R, Hoffmann E, Sebald W. Human bone morphogenetic protein 2 contains a heparin-binding site which modifies its biological activity. Eur J Biochem 1996; 237: 295–302. 6. Wiegel U, Meyer M, Sebald W. Mutant proteins of human interleukin 2. Eur J Biochem 1989; 180: 295–300.
The Authors Kazuhisa Bessho DDS, DMSc Assistant Professor Yasuzo Konishi DDS Research Fellow Shinji Kaihara DDS Instructor Kazuma Fujimura DDS, DMSc Assistant Professor Yasunori Okubo DDS Graduate Student Tadahiko Iizuka DDS, DMSc Professor and Chairman Department of Oral and Maxillofacial Surgery Graduate School of Medicine Kyoto University Kyoto, Japan Correspondence and requests for offprints to: Kazuhisa Bessho DDS, DMSc, Assistant Professor, Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Tel: ;82 75 751 3405; Fax: ;81 75 761 9732; E-mail: [email protected] Paper received 14 January 2000 Accepted 24 July 2000
BRITISH JOURNAL OF ORAL
& M A X I L L O FAC I A L S U R G E RY
Bone induction by Escherichia coli-derived recombinant human bone morphogenetic protein-2 compared with Chinese hamster ovary cell-derived recombinant human bone morphogenetic protein-2 K. Bessho,* Y. Konishi,† S. Kaihara,‡ K. Fujimura,§ Y. Okubo,¶ T. Iizuka** *Assistant Professor; †Research Fellow; ‡Instructor; §Assistant Professor; ¶Graduate Student; **Professor and Chairman, Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan SUMMARY. Most recombinant human bone morphogenetic protein (rhBMP) is currently obtained from Chinese hamster ovary (CHO) cells. If rhBMP with more activity could be derived from Escherichia coli (E. coli), a large quantity of rhBMP could be produced at low cost. The bone-inducing ability of an E. coliderived rhBMP-2 (ErhBMP-2) variant with an N-terminal sequence was examined and compared with CHO cell-derived rhBMP-2 (CrhBMP-2). Two, 10, or 50 g of ErhBMP-2 or CrhBMP-2 was mixed with 3 mg of atelopeptide type I collagen as the carrier, and specimens were implanted into the calf muscle pouches of Wistar rats (n:5 in each group). Three weeks later, new bone had formed in all the ErhBMP-2-implanted and CrhBMP-2-implanted muscles. Radiographic and histological examinations showed that the bone induced by ErhBMP-2 had a large hollow bone matrix with more fatty marrow than the bone induced by CrhBMP-2. Quantitative analysis indicated that the activity of ErhBMP-2 was similar to that of CrhBMP-2, so ErhBMP-2 may be useful for inducing bone formation. © 2000 The British Association of Oral and Maxillofacial Surgeons
INTRODUCTION
Sephacryl S-200 in 6 M guanidinium chloride, 0.1 M Tris hydrochloric acid, 0.1 mmol EDTA, and 1 mmol dithiothreitol, pH 8. By extensive dialysis against water, the protein was refolded, concentrated, and enriched by CMSepharose chromatography.7 It was finally purified by FPLC (Fractogel EMD SO3-650, 50 mmol sodium acetate, pH 5, and 30% 2-propanol) eluted with a sodium chloride gradient from 0 to 1.5 M. After dialysis against water, the protein was lyophilized and stored at ⫺80°C. CrhBMP-2 (Genetics Institute Inc., Massachusetts, USA) was produced by a recombinant DNA technique.8 It was dissolved in a buffer (pH 4.5) containing 5 mmol glutamic acid, glycine 2.5%, sucrose 0.5% and Tween 80 0.01%, and stored at ⫺80°C.
Several bone morphogenetic proteins (BMPs) have become available by cloning and genetic engineering.1 However, there are still some problems with the supply and activity of recombinant human BMP (rhBMP).2 At present, most rhBMP is obtained from mammalian cells, such as Chinese hamster ovary (CHO) cells.3 If rhBMP with more activity could be derived from Escherichia coli (E. coli), a large quantity of rhBMP could be produced at low cost. In this study, we examined the bone-inducing ability of E. coli-derived rhBMP-2 (ErhBMP-2) and compared it with that of CHO cell-derived rhBMP-2 (CrhBMP-2) using bioassays.4
Bioassays MATERIAL AND METHODS
The study comprised two experimental groups, each tested with three different amounts of BMP. Samples of 2, 10, or 50 g of ErhBMP-2 or CrhBMP-2 were each mixed with 3 mg of porcine skin-derived atelopeptide type I collagen solution (3 mg/ml, pH 3.0, Nitta Gelatin Inc, Osaka, Japan) as carrier. Each mixed sample was then lyophilized into a disc (4 mm in diameter and 1.5 mm thick). These specimens were then implanted into the calf muscles of 10-week-old male Wistar rats (weight 230–260 g, n:5 in each group) that had been anaesthetized by an intraperitoneal injection of sodium pentobarbital. The fascia and skin were then closed. Three weeks later, soft radiographs (SRO-M50; Sofron Inc., Tokyo, Japan; 35 kV, 4 mA, 2 minutes) were taken of the implanted regions.
ErhBMP-2 and CrhBMP-2 The variant ErhBMP-2 was generated by exchanging the sequence coding the first 12 amino acids of rhBMP-25 (MAKHKQRKRLKS) for the sequence coding the first 13 amino acids in human interleukin 2. This created the new N-terminus MAPTSSSTKKTQL. The hybrid cDNA was integrated into the vector pRPR9fd.6 The synthesis of ErhBMP-2 was induced by a temperature shift to 42°C. The protein was purified from the inclusion-body fraction by extraction with 4 M guanidinium chloride (0.1 M Tris hydrochloric acid, pH 8, 0.1 mmol EDTA, 14 mmol 2-mercaptoethanol) and chromatography on 645
646
British Journal of Oral and Maxillofacial Surgery
The implants were then excised, and each was cut in half specimen with its surrounding muscle. One half was separated from the surrounding tissues, weighed, and homogenized in 0.25 M sucrose in a Polytron homogenizer (Bio-Mixer type ABM-1; Nissei Inc., Osaka, Japan). Alkaline phosphatase (ALP) activity and protein in the resultant supernatant were measured by the p-nitrophenyl phosphate method and pyrogallol red method, respectively. The sediments were demineralized in 0.5 M hydrochloric acid 10 ml, and the calcium content of the soluble fractions was measured by the o-cresolphthalein complex method. The ALP activity (IU/mg protein) and the calcium content (g/mg tissue) were used as indices of bone formation. The other half of the specimen with surrounding muscle was used for histological examination after being fixed in 10% formalin neutral buffer solution (pH 7.4), demineralized in EDTA, and embedded in paraffin. Sections 4 m thick were cut on a microtome, stained with haematoxylin and eosin, and examined under a light microscope. The entire implant areas and the bone matrix areas were measured on histological micrographs of slices near the centre of the implant using a computer system with Photoshop ver. 4.0J (Adobe Systems Inc., San Jose, CA, USA) and NIH Image ver. 1.61 (National Institutes of Health, Bethesda, MD, USA) software. The percentage of bone matrix in the entire area was calculated in each group. The control, type I collagen alone, was implanted and evaluated in a similar manner. The study was approved by the Animal Experiments Committee and followed the Guidelines for Animal Experiments of Kyoto University. RESULTS Soft radiographic findings Radio-opaque shadows were visible in the radiographs of all of the ErhBMP-2- and CrhBMP-2-implanted muscles (Fig. 1). The sizes of the shadows of the
Fig. 1 – Soft radiographs taken three weeks after implantation.
ErhBMP-2- and CrhBMP-2-implanted muscles increased with the amount of implanted ErhBMP-2 or CrhBMP-2, but were much larger in the ErhBMP-2-implanted muscles than in CrhBMP-2-implanted muscles. The opacity of the shadows in the ErhBMP-2 implanted muscles was strong on the outermost edge of the implant around the entire circumference, and weak at the core of the implant. The opaque shadows in the CrhBMP-2 implanted muscles were slightly more uniform than those in ErhBMP2-implanted muscles. These radio-opaque shadows were not visible in the controls. Biochemical analysis The volume of ErhBMP-2- and CrhBMP-2-induced bone increased with the amount of BMP implanted. The ALP activity and calcium content in the specimens are shown in Table 1. Student’s t-test was used to examine the significance of differences in the value of ALP activity and calcium content between ErhBMP-2 implanted groups and CrhBMP-2 implanted groups. The ALP activity of ErhBMP-2 was significantly higher than that of CrhBMP-2. The calcium content of ErhBMP-2 was similar to that of CrhBMP-2. Table 1 – Alkaline phosphatase (ALP) activity and calcium content in the specimens. Values are the mean (SD) of five specimens in each group ALP activity (IU/mg protein) Control ErhBMP-2 2 g 10 g 50 g CrhBMP-2 2 g 10 g 50 g
0.1
Calcium content (g/mg tissue) 1.0
1.6 (0.5)* 6.3 (0.9)* 19.9 (3.1)**
12.8 (1.9) 23.4 (3.5) 32.8 (5.2)
0.5 (0.3) 4.1 (0.7) 13.9 (2.0)
11.3 (1.6) 21.5 (3.4) 30.2 (4.8)
*P0.005, **P0.05 (Student’s t-test compared with CrhBMP-2 implanted group).
Bone induction by E. coli-derived rhBMP
647
Fig. 2 – Photomicrographs of control (A) and bone induced by (B) 2 g, (C) 10 g, (D) 50 g, of ErhBMP-2, or (E) 2 g, (F) 10 g, (G) 50 g, of CrhBMP-2 in the calf muscle pouch of Wistar rats 3 weeks after implantation (haematoxylin–eosin; original magnification, 100; bars:50 m). M, calf muscle of host; NB, new bone matrix; BM, bone marrow; F, fatty marrow; AT, angioid tissue; and C, collagen.
648
British Journal of Oral and Maxillofacial Surgery
Histological findings Light microscopy showed new bone surrounded by immature mesenchymal-type cells in all the ErhBMP-2and CrhBMP-2-implanted muscles, but there was no evidence of osteoinduction in the controls (Fig. 2A). However, the compositions of ErhBMP-2- and CrhBMP-2-induced bone differed. Implantation of 2 g of ErhBMP-2 resulted in bone matrix formation in only part of the area on the outermost edge of the intramuscular implant. Much collagen was present at the core of the implant (Fig. 2B). Implantation of ErhBMP-2 10 g resulted in bone matrix formation on the outermost edge of the implant around almost the entire circumference, and immature mesenchymal type cells surrounded the new bone matrix. There was a little collagen and much fatty marrow in the core of the implant (Fig. 2C). Implantation of 50 g of ErhBMP-2 resulted in the formation of bone matrix that was thicker than the bone matrix formed by 10 g of ErhBMP-2 on the outermost edge of the implant around almost the entire circumference, and immature mesenchymal type cells surrounded the new bone matrix. A little bone marrow containing angioid tissue and much fatty marrow was seen in the core of the implant (Fig. 2D). Implantation of 2 g of CrhBMP-2 resulted in slightly more bone matrix being formed than was formed by 2 g of ErhBMP-2 in only part of the area on the outermost edge of the intramuscular implant. There was much collagen present at the core of the implant (Fig. 2E). Implantation of 10 g of CrhBMP-2 resulted in bone matrix formation on the outermost edge of the implant around almost the entire circumference and in part of the area in the core of the implant, and immature mesenchymal type cells surrounded the new bone matrix. A little collagen and fatty marrow were present in the core of the implant (Fig. 2F). Implantation of 50 g of CrhBMP-2 resulted in bone matrix formation not only on the outermost edge but also deep within the implant. Immature mesenchymaltype cells were present both inside and outside the new bone, and there was also much angioid tissue containing bone marrow with only a little fatty marrow. No collagen was detected (Fig. 2G). Histomorphometric analysis The means of the entire implant area bone matrix area and percentage of the bone matrix occupying the entire implant area for each histological specimen are shown in Table 2.
DISCUSSION ErhBMP-2 induced ALP activity in the C3H10T1/2 fibroblast cell line and proteoglycan synthesis in embryonic chicken limb bud cells.5 However, there have been no previous reports of the bone-inducing activity of ErhBMP-2 in vivo. If ErhBMP-2 has sufficient boneinducing activity, a large quantity of rhBMP could be produced at low cost. This would have important applications for clinical use.
Table 2 – The means of the entire implant area (E), bone matrix area (B) and percentage of the bone matrix occupying the entire implant area (P) for each histological specimen. Values are the mean (SD) of five specimens in each group
Control ErhBMP-2 2 g 10 g 50 g CrhBMP-2 2 g 10 g 50 g
E (mm2)
B (mm2)
P (%)
6.9 (0.8)
0 (0)
0 (0)
5.6 (0.8) 13.6 (2.2) 44.9 (6.3)
0.6 (0.2) 2.6 (0.6) 8.8 (1.6)
11.0 (1.6) 19.2 (1.7) 19.5 (1.9)
1.7 (0.4) 10.2 (0.9) 12.9 (1.6)
0.2 (0.1) 2.3 (0.4) 4.2 (0.9)
11.8 (3.5) 22.8 (3.3) 32.1 (3.8)
The mature BMP-2 is a homodimer of two 114residue subunits representing the C-terminal sequence of a long precursor protein of 396 amino acids. One of the four N-glycosylation sites present in the proprotein is retained and used in the mature BMP-2 polypeptide chains.9 Recently, biological activity and physical binding constants were established using rhBMP-2 from CHO cells.3 In addition, a cDNA encoding mature human BMP-2 can be efficiently expressed in E. coli, and after renaturation a dimeric BMP-2 protein with a molecular weight of 26 000 was prepared with a purity of over than 98%. The ErhBMP-2 had been substituted by a dummy sequence of equal length for the N-terminal residues 1–12 of rhBMP-2. A heparin-binding site was identified in the N-terminal segments of dimetric rhBMP-2. The variant ErhBMP-2 showed biological activity, so the basic N-terminal domains of dimeric rhBMP-2 as heparin-binding sites are not obligatory for receptor activation.5 In the present study, soft radiographic shadows were much larger in the ErhBMP-2 than in CrhBMP-2 implanted groups. Histological examination showed that the bone tissue induced by ErhBMP-2 consisted of rich fatty marrow, much collagen, and comparatively poor bone matrix, while that induced by CrhBMP-2 had a little fatty marrow and collagen, and comparatively rich bone matrix. These findings suggest that ErhBMP-2 with collagen expands in vivo, because ErhBMP-2 does not have heparin-binding sites. Furthermore, ErhBMP-2 was delivered slowly from the expanded implant (ErhBMP-2 with collagen) because new bone matrix formed on the outermost edge of the implant. Much collagen was, therefore, seen at the core of the implant in the ErhBMP-2 implanted groups, and the ALP activity of these groups was higher than that of the CrhBMP-2 implanted groups at 3 weeks after implantation. Biochemical analysis showed that the bone-inducing activity of ErhBMP-2 was similar to that of CrhBMP-2. Histomorphometric analysis showed that the percentage of bone matrix occupying the entire implant area in the ErhBMP-2 implanted group was lower than that in the CrhBMP-2 implanted group. However, the bone matrix area in the ErhBMP-2 implanted group was larger than that in the CrhBMP-2 implanted group. These results indicate that ErhBMP-2 effectively induces bone formation under some conditions. Many patients with large bone defects resulting from tumours or injury have been treated by oral and
Bone induction by E. coli-derived rhBMP
maxillofacial surgeons. Even bioactive materials with osteoconductive ability, such as hydroxyapatite, are not sufficiently effective for them. Hence a biomaterial with bone-inducing ability is needed. BMP is the only cytokine with bone inducing ability, but the dose of BMP-2 required is much greater than that of other cytokines and growth factors.10 The fact that ErhBMP-2 is effective in inducing bone formation is therefore valuable. It should be possible to produce a large quantity of ErhBMP-2 at low cost for clinical use in the near future.
649
7. Cerletti N, McMaster GK, Cox D, Schmitz A, Meyhack B. Ciba-Geigy Ltd. Basel, Switzerland, European Patient Application 1991; 0433 225 A1. 8. Wozney JM, Rosen V, Celeste AJ et al. Novel regulators of bone formation: Molecular clones and activities. Science 1988; 242: 1528–1534. 9. Celeste AJ, Iannazzi JA, Taylor RC et al. Identification of transforming growth factor  family members present in bone-inductive protein purified from bovine bone. Proc Natl Acad Sci USA 1990; 87: 9843–9847. 10. Yamaguchi A, Katagiri T, Ikeda T et al. Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro. J Cell Biol 1991; 113: 681–687.
Acknowledgements This work was partly supported by a grant-in-aid for scientific research (B No. 12470437) of the Japanese Ministry of Education, Science, Sports and Culture. We thank Professor Walter Sebald, Würzburg University, Germany for supplying us with the ErhBMP-2, and the Yamanouchi Pharmaceutical Company, Tokyo, Japan for providing the CrhBMP-2.
References 1. Wang EA, Rosen V, Cordes P et al. Purification and characterization of other distinct bone-inducing factors. Proc Natl Acad Sci USA 1988; 85: 9484–9488. 2. Bessho K. Ectopic osteoinductive difference between purified bovine and recombinant human bone morphogenetic protein. In: Lindholm TS, ed. Bone Morphogenetic Proteins: Biology, Biochemistry and Reconstructive Surgery. Austin: RG Landes, 1996: 105–111. 3. Wang EA, Rosen V, D’Alessandro JS et al. Recombinant human bone morphogenetic protein induces bone formation. Proc Natl Acad Sci USA 1990; 87: 2220–2224. 4. 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. 5. Ruppert R, Hoffmann E, Sebald W. Human bone morphogenetic protein 2 contains a heparin-binding site which modifies its biological activity. Eur J Biochem 1996; 237: 295–302. 6. Wiegel U, Meyer M, Sebald W. Mutant proteins of human interleukin 2. Eur J Biochem 1989; 180: 295–300.
The Authors Kazuhisa Bessho DDS, DMSc Assistant Professor Yasuzo Konishi DDS Research Fellow Shinji Kaihara DDS Instructor Kazuma Fujimura DDS, DMSc Assistant Professor Yasunori Okubo DDS Graduate Student Tadahiko Iizuka DDS, DMSc Professor and Chairman Department of Oral and Maxillofacial Surgery Graduate School of Medicine Kyoto University Kyoto, Japan Correspondence and requests for offprints to: Kazuhisa Bessho DDS, DMSc, Assistant Professor, Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Tel: ;82 75 751 3405; Fax: ;81 75 761 9732; E-mail: [email protected] Paper received 14 January 2000 Accepted 24 July 2000