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Effect of Elcatonin on Osteoinduction by Recombinant Human Bone Morphogenetic Protein-2
Biochemical and Biophysical Research Communications 269, 317–321 (2000) doi:10.1006/bbrc.2000.2294, available online at http://www.idealibrary.com on
Effect of Elcatonin on Osteoinduction by Recombinant Human Bone Morphogenetic Protein-2 Yasunori Okubo,* ,1 Kazuhisa Bessho,* Kazuma Fujimura,* Kenji Kusumoto,† Yutaka Ogawa,† and Tadahiko Iizuka* *Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507 Japan; and †Department of Plastic and Reconstructive Surgery, Kansai Medical University, Moriguchi, Osaka 570-8507 Japan
Received January 31, 2000
To evaluate the effect of elcatonin on osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2), 5 g of rhBMP-2 was implanted into intramuscular sites of rats. For 14 days after the implantation, elcatonin was administered intraperitoneally with total dosage of 80 U, 8 U, and 0.8 U, respectively. For the control group, only physiological saline was administered. At 21 days after implantation, the area of the oval shadow in the radiologic findings depended on the elcatonin dose and the amount of trabecular bone and the number of osteoblasts observed in the histologic findings depended on the dosage of elcatonin. The values of ALP activity and Ca content also showed an elcatonin dose dependency. These results suggested that elcatonin is effective in enhancing osteoinduction by rhBMP-2 within the dose range of this study, and that elcatonin has an anabolic effect on osteoblasts in addition to an antiresorptive effect. © 2000 Academic Press
fibroblast cell line, by proteoglycan synthesis in embryonic chicken limb bud cells (8) and by osteogenesis in vivo (7). Calcitonin is a peptide hormone secreted by the parafollicular C-cells of the thyroid in response to a hypocalmic signal (9, 10). Calcitonin inhibits bone resorption by inactivation of osteoclasts, which then display morphological modifications and loss of mobility (11). Elcatonin is a derivative of eel calcitonin synthesized by substituting an ethylene bond for the disulfide bond (12–14). It has also been reported from in vivo and in vitro studies that elcatonin suppresses bone resorption (14, 15). In the clinical field, elcatonin is used currently for the treatment of Paget’s disease, and osteoporosis. However, the role of this hormone in producing an anabolic effect on osteoblasts is not yet fully understood. In this study, we evaluated the effect of elcatonin on osteoinduction by rhBMP-2, especially the anabolic effect on osteoblasts. MATERIALS AND METHODS
Bone morphogenetic protein (BMP) plays important roles in the migration of osteoblast progenitor cells, proliferation of mesenchymal cells, and differentiation to chondrogenic and osteogenic cells (1, 2). Since the synthesis of rhBMP-2 (3), many studies on osteoinduction by rhBMP-2 have been performed (4 –7). Recently, Escherichia coli (E. coli)-derived rhBMP-2 has been prepared at greater than 98% purity and in a largely homogeneous form (8). E. coli-derived rhBMP-2 was functionally active as demonstrated by the induction of alkaline phosphatase (ALP) activity in the C3H10T1/2 Abbreviations used: BMP, bone morphogenetic protein; ALP, alkaline phosphatase; Ca, calcium; E. coli, Escherichia coli; CL, atelopeptide type I collagen. 1 To whom correspondence should be addressed. Fax: ⫹81-75-7619732. E-mail: [email protected].
Animals. Twenty Wistar rats (male; 10 weeks old; weight 240 –260 g) were used. Four groups, consisting of a high elcatonin group (HEG), medium elcatonin group (MEG), low elcatonin group (LEG) and control group (CG), were established with 5 rats in each group. They were fed rodent chow (certified diet MF; Oriental Koubo Inc., Tokyo, Japan). The treatment of each animal was conducted according to the 1988 guidelines for animal experiments at Kyoto University. Implant material. rhBMP-2 derived from E. coli was obtained from W. Sebald (Wu¨rzburg University, Germany) (8). Atelopeptide type-I collagen (CL) (pH 3.0) (Cellmatrix LA; Nitta Gelatin Inc., Osaka, Japan) was used as a carrier. 5 g 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 discal form (4 mm in diameter, 1.5 mm in thickness). As the pharmacological agent, 14-day doses of elcatonin (Elcitonin; Asahi Chemical Industry Co., Ltd., Tokyo, Japan) were prepared, 80 U for HEG, 8 U for MEG, and 0.8 U for LEG. The total volume of the elcatonin agent in physiological saline solution was 0.2 ml for each rhBMP-2 implanted groups. The elcatonin solution was placed into a
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0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Vol. 269, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
mini-osmotic pump (Alzet Model 2002; ALZA Co., CA) that would pump out the solution continuously at a constant rate of 0.5 l/h for 14 days. For CG, only 0.2 ml of physiological saline was placed into the mini-osmotic pump. Surgical procedure. All rats were anesthetized with intraperitoneal administration of sodium pentobarbital (5.0 mg per 100 g of body weight). The lyophilized discal specimens were implanted into a right calf muscle. After the implantation, the fascia and skin were sutured. A 1-cm-long incision was made in the paramedian abdominal wall, including the skin, muscle, and the peritoneum and the mini-osmotic pump, which had been previously prepared for each group, was inserted into the peritoneal space. The abdominal wall was then closed by suturing layer by layer. Radiographic evaluation. Twenty-one days after the implantation, all rats were sacrificed with an overdose of sodium pentobarbital. The implanted region was excised with the surrounding tissue and a radiograph was taken (SOFRON; SRO-M50, Sofron Inc. Tokyo, Japan). Each excised specimen was removed and then cut into 2 halves, one for histological analysis and the other for biochemical analysis. Histological analysis. The specimens with peripheral tissues were fixed in 10% formalin neutral buffer solution (pH 7.4), demineralized in EDTA, and embedded in paraffin. They were cut into 4-m-thick sections and stained with hematoxylin and eosin. Biochemical examination. The samples for the biochemical examination were weighed, 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 N HCl, and the Ca content of the soluble fraction was determined by the orthocresolphthalein complexone method (16). The alkaline phosphatase (ALP) activity and total protein in the resultant supernatant were determined by the 4-nitrophenylphosphate method (17). The Ca content (g/mg tissue) and the ALP activity (IU/mg protein) were used as indices of bone formation. Statistical analysis. The results are presented as mean ⫾ standard deviation (SD). The statistical analysis of differences in the value of ALP activity and Ca content was performed by analysis of variance (ANOVA), followed by Fisher’s comparison test.
RESULTS Radiographic Findings The soft tissue radiographs revealed opaque shadows morphologically identical to the implanted specimens (Fig. 1). These opaque shadows were observed in each of the specimens of all groups. The area of the oval shadow on the X-ray film was larger in HEG, MEG, LEG, and CG. Histological Findings In HEG (Fig. 2A), there was a relatively vigorous trabecular bone on the outermost edge of the implanted material. Lining osteoblasts were observed around the trabecular bone. Bone marrow, including angioid tissue, was rich at the central side of the trabecular bone. Fatty marrow occupied a major part of the marrow tissue. In MEG (Fig. 2B), there was trabecular bone on the outermost edge of the implanted material. The trabecular bone was thinner than that in HEG. The number of osteoblasts was lower than that in HEG. Bone mar-
FIG. 1. Soft X-ray view of osteoinduction 21 days after the implantation.
row included fatty tissue. Collagen fibers remained at the center of the implanted material. In LEG (Fig. 2C), less trabecular bone and marrow were observed compared to the respective amounts in MEG and HEG. At the central side of the newly formed trabeculae, a small area of bone marrow was observed. The number of osteoblasts was lower than that in HEG and MEG. The amount of remnant collagen fibers was greater than that in MEG or HEG. In CG (Fig. 2D), especially, the amount of trabecular bone was clearly less than in the other groups and few osteoblasts were observed. Biochemical Indices The ALP activity and Ca content after implantation in each group are shown in Fig. 3. The values of ALP activity were 5.87 ⫾ 0.43 (mean ⫾ SD IU/mg protein) in CG, 6.41 ⫾ 0.37 in LEG, 7.10 ⫾ 0.37 in MEG, and 7.37 ⫾ 0.50 in HEG. The value was highest in HEG and lowest in CG. The values of Ca content were 25.0 ⫾ 1.61 (mean ⫾ SD g/mg tissue) in CG, 26.6 ⫾ 0.96 in LEG, 29.0 ⫾ 0.60 in MEG, and 31.3 ⫾ 1.56 in HEG. The ALP activity and Ca content in HEG were highest and lowest in CG. In HEG and MEG, the values of ALP activity and Ca content were significantly lower than in CG and LEG. DISCUSSION In this study, we evaluated the effect of elcatonin on osteoinduction by rhBMP-2 radiologically, histologically, and biochemically. The concentration of elcatonin administered in MEG is a common clinical dose. Elcatonin is a synthetic analogue of eel calcitonin that differs from the natural peptide hormone by replacement of the disulphide bridge with an ethylene
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FIG. 2. Histological views of four groups 21 days after the implantation. (A) HEG, (B) MEG, (C) LEG, (D) CG. (HE stain, original magnification ⫻50.) (M, muscle of the host; BM, bone marrow; NB, newly formed bone; OB, osteoblast; CL, atelopeptide type I collagen.)
bridge and deletion of the N-terminal amino group (12). In most studies, potent inhibition by calcitonin of bone resorption induced by the experimental models was observed, resulting in an increase in bone mass. Histomorphometric and biochemical studies indicate maintenance of skeletal integrity, and it can, therefore, be concluded that the reduction in resorptive activity and increase in bone mass act together to reduce bone fragility and subsequent fracture risk (18). However, calcitonin accelerated the proliferation of osteoblasts and enhanced their function (19). Hori et al. (20) also reported that when beagle dogs were administered a low dose (1 U/kg) or a high dose (5 U/kg) of elcatonin for 13 consecutive weeks, both bone resorption and bone formation were suppressed in the high dose group, but bone resorption was reduced and bone formation was increased in the low dose group. On the other hand, it was reported that short-term treatment with calcitonin did not stimulate osteoblast activity, on the contrary, it exerted a negative effect on osteoblastic bone formation
and mineralization (21). In the radiologic findings, the area of the oval shadow depended on the dosage of elcatonin. In the histological findings, the amount of trabecular bone and the number of osteoblasts depended on the dose of elcatonin. There appeared to be little difference in these findings between CG and LEG, and between MEG and HEG. However, it was suspected that there is a marked difference between LEG and MEG. The values of ALP activity and Ca content, which can be thought of as indices of osteogenesis, showed a dose-dependent fashion of elcatonin. In particular, ALP activity in MEG and HEG, which can be thought of as an index of osteoblast activity, was significantly higher than that in CG. The biochemical results almost paralleled the histological and radiological findings. BMP-2 promotes the differentiation of immature mesenchymal cells into osteoblastic precursor cells (22). BMP-2 has high osteoinducive activity and maintains the vertebrate skeleton. The results of this study
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FIG. 3. Values of ALP activity/total protein (A) and Ca content/tissue weight (B) 21 days after the implantation. Data are means ⫾ SD. *, p ⬍ 0.05; **, p ⬍ 0.01.
suggested that immature mesenchymal cells at the intramuscular site are differentiated to osteoblasts by rhBMP-2, that the proliferation of the osteoblasts and their function are enhanced by elcatonin and that the effect of elcatonin shows a dose-dependent fashion within the range of the dosages used in this study. Primarily, osteoinducive activity at higher concentrations should be evaluated. However, the kinetics of calcitonin fibrillation showed a linear dependence of the logarithm of fibrillation time versus that of concentration (23). Aggregation and fibrillation were minimized by avoiding the use of highly concentrated calcitonin solution (24). In our data, aggregation occurred at 500 U/ml of elcatonin. These results suggested that elcatonin is effective in enhancing osteoinduction by rhBMP-2, and that elcatonin has an anabolic effect on osteoblasts in addition to an anti-resorptive effect. ACKNOWLEDGMENT The authors thank Asahi Chemical Industry Co., Ltd. (Tokyo, Japan) for providing Elcatonin (Elcitonin).
REFERENCES 1. Wozney, J. M., Rosen, V., Celeste, A. J., Mitsock, L. M., Whitters, M. J., Kriz, R. W., Hewick, R. M., and Wang, E. A. (1988) Science 242, 1528 –1534. 2. Reddi, A. H. (1997) Cytokine Growth Factor. Rev. 8, 11–20.
3. Reddi, A. H., and Cunningham, N. S. (1993) J. Bone Miner. Res. 8, 499 –502. 4. Fujimura, K., Bessho, K., Kusumoto, K., Ogawa, Y., and Iizuka, T. (1995) Biochem. Biophys. Res. Commun. 208, 316 –322. 5. Kusumoto, K., Bessho, K., Fujimura, K., Konishi, Y., Ogawa, Y., and Iizuka, T. (1995) Biochem. Biophys. Res. Commun. 215, 205–211. 6. Okubo, Y., Bessho, K., Fujimura, K., Kusumoto, K., Ogawa, Y., Tani, Y., and Iizuka, T. (1999) Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 87, 34 –38. 7. Okubo, Y., Bessho, K., Fujimura, K., Konishi, Y., Kusumoto, K., Ogawa, Y., and Iizuka, T. (1999) Int. J. Oral. Maxillofac. Surg., in press. 8. Ruppert, R., Hoffmann, E., and Sebald, W. (1996) Eur. J. Biochem. 237, 295–302. 9. Copp, D. H., Cameron, E. C., Cheney, B. A., Davidson, A. F. G., and Henze, K. G. (1962) Endocrinology 70, 683– 649. 10. Foster, G. V., Baghdianz, A., Kumar, M. A., Slake, E., Soliman, H. A., and MacIntyre, I. (1964) Nature 202, 1303–1305. 11. Chambers, T. J., Chambers, J. C., Symonds, J., and Darby, J. A. (1986) J. Clin. Endocrinol. Metab. 63, 1080 –1085. 12. Morikawa, T., Munakata, E., Sakakibara, S., Noda, T., and Otani, M. (1976) Experientia 32, 1104 –1106. 13. Otani, M., Kitazawa, S., Yamaguchi, H., Noda, T., and Watanabe, S. (1978) Clin Med. 20, 2217–2222. 14. Orimo, H. (1990) in Osteoporosis (Christiansen, C., and Overgard, K., Eds.), pp. 2091–2095, Handelstrykkeriet, Aalborg. 15. Yamamoto, I., Morita, R., Fukunaga, M., Dokoh, S., Shigeno, C., Torizuka, K., and Noda, T. (1981) Endocrinology 108, 698 –702. 16. Connetry, H. V., and Briggs, A. R. (1966) Am. J. Clin. Pathol. 45, 290 –296. 17. The executive board in analytical section, the committee of
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enzymes in analytical section of Japan Society of Clinical Chemistry (JSCC). (1988) J.S.C.C. 1, 11–19. 18. Wallach, S., Rousseau, G., Martin, L., and Azria, M. (1999) Bone 25, 509 –513. 19. Farley, J. R., Tarbaux, N. M., Hall, S. L., Linkhart, T. A., and Baylink, D. J. (1988) Endocrinology 123, 159 –167. 20. Hori, M, Takahashi, H., Konno, T., Inoue, J., Haba, T., Noda, T., and Fujimoto, K. (1984) Oyo Yakuri (Pharmacometrics) 28, 609 – 614.
21. Monier-Faugere, M. C., Geng, Z., Qi, Q., Arnala, I., and Malluche, H. H. (1996) J. Bone Miner. Res. 11, 446 – 455. 22. Yamaguchi, A., Katagiri, T., Ikeda, T., Wozney, J. M., Rosen, V., Wang, E. A., Kahn, A. J., Suda, T., and Yoshiki, S. (1991) J. Cell Biol. 113, 681– 687. 23. Arvinte, T., Cudd, A., and Drake, A. F. (1993) J. Biol. Chem. 268, 6415– 6422. 24. Cudd, A., Arvinte, T., Das, R. E. G., Chinni, C., and Macintyre, I. (1995) J. Pharm. Sci. 84, 717–719.
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Effect of Elcatonin on Osteoinduction by Recombinant Human Bone Morphogenetic Protein-2 Yasunori Okubo,* ,1 Kazuhisa Bessho,* Kazuma Fujimura,* Kenji Kusumoto,† Yutaka Ogawa,† and Tadahiko Iizuka* *Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507 Japan; and †Department of Plastic and Reconstructive Surgery, Kansai Medical University, Moriguchi, Osaka 570-8507 Japan
Received January 31, 2000
To evaluate the effect of elcatonin on osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2), 5 g of rhBMP-2 was implanted into intramuscular sites of rats. For 14 days after the implantation, elcatonin was administered intraperitoneally with total dosage of 80 U, 8 U, and 0.8 U, respectively. For the control group, only physiological saline was administered. At 21 days after implantation, the area of the oval shadow in the radiologic findings depended on the elcatonin dose and the amount of trabecular bone and the number of osteoblasts observed in the histologic findings depended on the dosage of elcatonin. The values of ALP activity and Ca content also showed an elcatonin dose dependency. These results suggested that elcatonin is effective in enhancing osteoinduction by rhBMP-2 within the dose range of this study, and that elcatonin has an anabolic effect on osteoblasts in addition to an antiresorptive effect. © 2000 Academic Press
fibroblast cell line, by proteoglycan synthesis in embryonic chicken limb bud cells (8) and by osteogenesis in vivo (7). Calcitonin is a peptide hormone secreted by the parafollicular C-cells of the thyroid in response to a hypocalmic signal (9, 10). Calcitonin inhibits bone resorption by inactivation of osteoclasts, which then display morphological modifications and loss of mobility (11). Elcatonin is a derivative of eel calcitonin synthesized by substituting an ethylene bond for the disulfide bond (12–14). It has also been reported from in vivo and in vitro studies that elcatonin suppresses bone resorption (14, 15). In the clinical field, elcatonin is used currently for the treatment of Paget’s disease, and osteoporosis. However, the role of this hormone in producing an anabolic effect on osteoblasts is not yet fully understood. In this study, we evaluated the effect of elcatonin on osteoinduction by rhBMP-2, especially the anabolic effect on osteoblasts. MATERIALS AND METHODS
Bone morphogenetic protein (BMP) plays important roles in the migration of osteoblast progenitor cells, proliferation of mesenchymal cells, and differentiation to chondrogenic and osteogenic cells (1, 2). Since the synthesis of rhBMP-2 (3), many studies on osteoinduction by rhBMP-2 have been performed (4 –7). Recently, Escherichia coli (E. coli)-derived rhBMP-2 has been prepared at greater than 98% purity and in a largely homogeneous form (8). E. coli-derived rhBMP-2 was functionally active as demonstrated by the induction of alkaline phosphatase (ALP) activity in the C3H10T1/2 Abbreviations used: BMP, bone morphogenetic protein; ALP, alkaline phosphatase; Ca, calcium; E. coli, Escherichia coli; CL, atelopeptide type I collagen. 1 To whom correspondence should be addressed. Fax: ⫹81-75-7619732. E-mail: [email protected].
Animals. Twenty Wistar rats (male; 10 weeks old; weight 240 –260 g) were used. Four groups, consisting of a high elcatonin group (HEG), medium elcatonin group (MEG), low elcatonin group (LEG) and control group (CG), were established with 5 rats in each group. They were fed rodent chow (certified diet MF; Oriental Koubo Inc., Tokyo, Japan). The treatment of each animal was conducted according to the 1988 guidelines for animal experiments at Kyoto University. Implant material. rhBMP-2 derived from E. coli was obtained from W. Sebald (Wu¨rzburg University, Germany) (8). Atelopeptide type-I collagen (CL) (pH 3.0) (Cellmatrix LA; Nitta Gelatin Inc., Osaka, Japan) was used as a carrier. 5 g 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 discal form (4 mm in diameter, 1.5 mm in thickness). As the pharmacological agent, 14-day doses of elcatonin (Elcitonin; Asahi Chemical Industry Co., Ltd., Tokyo, Japan) were prepared, 80 U for HEG, 8 U for MEG, and 0.8 U for LEG. The total volume of the elcatonin agent in physiological saline solution was 0.2 ml for each rhBMP-2 implanted groups. The elcatonin solution was placed into a
317
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Vol. 269, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
mini-osmotic pump (Alzet Model 2002; ALZA Co., CA) that would pump out the solution continuously at a constant rate of 0.5 l/h for 14 days. For CG, only 0.2 ml of physiological saline was placed into the mini-osmotic pump. Surgical procedure. All rats were anesthetized with intraperitoneal administration of sodium pentobarbital (5.0 mg per 100 g of body weight). The lyophilized discal specimens were implanted into a right calf muscle. After the implantation, the fascia and skin were sutured. A 1-cm-long incision was made in the paramedian abdominal wall, including the skin, muscle, and the peritoneum and the mini-osmotic pump, which had been previously prepared for each group, was inserted into the peritoneal space. The abdominal wall was then closed by suturing layer by layer. Radiographic evaluation. Twenty-one days after the implantation, all rats were sacrificed with an overdose of sodium pentobarbital. The implanted region was excised with the surrounding tissue and a radiograph was taken (SOFRON; SRO-M50, Sofron Inc. Tokyo, Japan). Each excised specimen was removed and then cut into 2 halves, one for histological analysis and the other for biochemical analysis. Histological analysis. The specimens with peripheral tissues were fixed in 10% formalin neutral buffer solution (pH 7.4), demineralized in EDTA, and embedded in paraffin. They were cut into 4-m-thick sections and stained with hematoxylin and eosin. Biochemical examination. The samples for the biochemical examination were weighed, 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 N HCl, and the Ca content of the soluble fraction was determined by the orthocresolphthalein complexone method (16). The alkaline phosphatase (ALP) activity and total protein in the resultant supernatant were determined by the 4-nitrophenylphosphate method (17). The Ca content (g/mg tissue) and the ALP activity (IU/mg protein) were used as indices of bone formation. Statistical analysis. The results are presented as mean ⫾ standard deviation (SD). The statistical analysis of differences in the value of ALP activity and Ca content was performed by analysis of variance (ANOVA), followed by Fisher’s comparison test.
RESULTS Radiographic Findings The soft tissue radiographs revealed opaque shadows morphologically identical to the implanted specimens (Fig. 1). These opaque shadows were observed in each of the specimens of all groups. The area of the oval shadow on the X-ray film was larger in HEG, MEG, LEG, and CG. Histological Findings In HEG (Fig. 2A), there was a relatively vigorous trabecular bone on the outermost edge of the implanted material. Lining osteoblasts were observed around the trabecular bone. Bone marrow, including angioid tissue, was rich at the central side of the trabecular bone. Fatty marrow occupied a major part of the marrow tissue. In MEG (Fig. 2B), there was trabecular bone on the outermost edge of the implanted material. The trabecular bone was thinner than that in HEG. The number of osteoblasts was lower than that in HEG. Bone mar-
FIG. 1. Soft X-ray view of osteoinduction 21 days after the implantation.
row included fatty tissue. Collagen fibers remained at the center of the implanted material. In LEG (Fig. 2C), less trabecular bone and marrow were observed compared to the respective amounts in MEG and HEG. At the central side of the newly formed trabeculae, a small area of bone marrow was observed. The number of osteoblasts was lower than that in HEG and MEG. The amount of remnant collagen fibers was greater than that in MEG or HEG. In CG (Fig. 2D), especially, the amount of trabecular bone was clearly less than in the other groups and few osteoblasts were observed. Biochemical Indices The ALP activity and Ca content after implantation in each group are shown in Fig. 3. The values of ALP activity were 5.87 ⫾ 0.43 (mean ⫾ SD IU/mg protein) in CG, 6.41 ⫾ 0.37 in LEG, 7.10 ⫾ 0.37 in MEG, and 7.37 ⫾ 0.50 in HEG. The value was highest in HEG and lowest in CG. The values of Ca content were 25.0 ⫾ 1.61 (mean ⫾ SD g/mg tissue) in CG, 26.6 ⫾ 0.96 in LEG, 29.0 ⫾ 0.60 in MEG, and 31.3 ⫾ 1.56 in HEG. The ALP activity and Ca content in HEG were highest and lowest in CG. In HEG and MEG, the values of ALP activity and Ca content were significantly lower than in CG and LEG. DISCUSSION In this study, we evaluated the effect of elcatonin on osteoinduction by rhBMP-2 radiologically, histologically, and biochemically. The concentration of elcatonin administered in MEG is a common clinical dose. Elcatonin is a synthetic analogue of eel calcitonin that differs from the natural peptide hormone by replacement of the disulphide bridge with an ethylene
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FIG. 2. Histological views of four groups 21 days after the implantation. (A) HEG, (B) MEG, (C) LEG, (D) CG. (HE stain, original magnification ⫻50.) (M, muscle of the host; BM, bone marrow; NB, newly formed bone; OB, osteoblast; CL, atelopeptide type I collagen.)
bridge and deletion of the N-terminal amino group (12). In most studies, potent inhibition by calcitonin of bone resorption induced by the experimental models was observed, resulting in an increase in bone mass. Histomorphometric and biochemical studies indicate maintenance of skeletal integrity, and it can, therefore, be concluded that the reduction in resorptive activity and increase in bone mass act together to reduce bone fragility and subsequent fracture risk (18). However, calcitonin accelerated the proliferation of osteoblasts and enhanced their function (19). Hori et al. (20) also reported that when beagle dogs were administered a low dose (1 U/kg) or a high dose (5 U/kg) of elcatonin for 13 consecutive weeks, both bone resorption and bone formation were suppressed in the high dose group, but bone resorption was reduced and bone formation was increased in the low dose group. On the other hand, it was reported that short-term treatment with calcitonin did not stimulate osteoblast activity, on the contrary, it exerted a negative effect on osteoblastic bone formation
and mineralization (21). In the radiologic findings, the area of the oval shadow depended on the dosage of elcatonin. In the histological findings, the amount of trabecular bone and the number of osteoblasts depended on the dose of elcatonin. There appeared to be little difference in these findings between CG and LEG, and between MEG and HEG. However, it was suspected that there is a marked difference between LEG and MEG. The values of ALP activity and Ca content, which can be thought of as indices of osteogenesis, showed a dose-dependent fashion of elcatonin. In particular, ALP activity in MEG and HEG, which can be thought of as an index of osteoblast activity, was significantly higher than that in CG. The biochemical results almost paralleled the histological and radiological findings. BMP-2 promotes the differentiation of immature mesenchymal cells into osteoblastic precursor cells (22). BMP-2 has high osteoinducive activity and maintains the vertebrate skeleton. The results of this study
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FIG. 3. Values of ALP activity/total protein (A) and Ca content/tissue weight (B) 21 days after the implantation. Data are means ⫾ SD. *, p ⬍ 0.05; **, p ⬍ 0.01.
suggested that immature mesenchymal cells at the intramuscular site are differentiated to osteoblasts by rhBMP-2, that the proliferation of the osteoblasts and their function are enhanced by elcatonin and that the effect of elcatonin shows a dose-dependent fashion within the range of the dosages used in this study. Primarily, osteoinducive activity at higher concentrations should be evaluated. However, the kinetics of calcitonin fibrillation showed a linear dependence of the logarithm of fibrillation time versus that of concentration (23). Aggregation and fibrillation were minimized by avoiding the use of highly concentrated calcitonin solution (24). In our data, aggregation occurred at 500 U/ml of elcatonin. These results suggested that elcatonin is effective in enhancing osteoinduction by rhBMP-2, and that elcatonin has an anabolic effect on osteoblasts in addition to an anti-resorptive effect. ACKNOWLEDGMENT The authors thank Asahi Chemical Industry Co., Ltd. (Tokyo, Japan) for providing Elcatonin (Elcitonin).
REFERENCES 1. Wozney, J. M., Rosen, V., Celeste, A. J., Mitsock, L. M., Whitters, M. J., Kriz, R. W., Hewick, R. M., and Wang, E. A. (1988) Science 242, 1528 –1534. 2. Reddi, A. H. (1997) Cytokine Growth Factor. Rev. 8, 11–20.
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