Abstract
Recombinant human bone morphogenetic protein-2 (rhBMP-2) and basic fibroblast growth factor (bFGF) are the focus of research pertaining to the stimulation of bone formation. We ascertained the effects of different concentrations rhBMP-2 on proliferation and differentiation of bone marrow stromal cells (BMSCs) in vitro and on ectopic bone formation in rats. BMSCs were obtained from beagle dogs and cultured in medium containing different concentrations rhBMP-2 and bFGF (0, 25, 50, 100, or 200 ng/mL). In a separate experiment, BMSCs were treated with different ratios (1:1, 2:1, 4:1, or 8:1) of rhBMP to bFGF (in each case the concentration of rhBMP was 100 ng/mL and the bFGF concentrations 100, 50, 25, or 12.5 ng/mL). Proliferation and differentiation of BMSCs were quantified by assessing methyl thiazole tetrazolium (MTT) and alkaline phosphatase (ALP) over 6 consecutive days. Von Kossa staining was performed on day 6. For the in vivo tests, porous calcium phosphate cement (CPC) was seeded with BMSCs (5 × 104) in medium containing 100 ng/mL rhBMP-2, 50 ng/mL bFGF or combined 100 ng/mL rhBMP-2 and 50 ng/mL bFGF. These cells were then subcutaneously implanted in four sites in nude rats. Bone formation was detected by histology at weeks 4 and 12 and quantified using a KS400 computer based image analysis system. It was determined that combined rhBMP-2 and bFGF at a ratio of 2:1 (100:50 ng/mL) promoted significantly increased BMSC proliferation and differentiation of BMSCs compared to rhBMP-2 or bFGF alone (p < 0.05). CPC with combined 100 ng/mL rhBMP-2 and 50 ng/mL bFGF stimulated more bone formation than either 100 ng/mL rhBMP-2 or 100 ng/mL bFGF (p < 0.05). These results show that a combination of rhBMP-2 and bFGF effectively induces early BMSC proliferation and differentiation in vitro. When combined, rhBMP-2 and bFGF synergistically promote new bone formation.
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Akita, S., M. Fukui, H. Nakagawa, T. Fujii, and K. Akino. Cranial bone defect healing is accelerated by mesenchymal stem cells induced by coadministration of bone morphogenetic protein-2 and basic fibroblast growth factor. Wound. Repair Regen. 12:252–259, 2004.
Alam, S., K. Ueki, K. Marukawa, T. Ohara, T. Hase, D. Takazakura, and K. Nakagawa. Expression of bone morphogenetic protein 2 and fibroblast growth factor 2 during bone regeneration using different implant materials as an onlay bone graft in rabbit mandibles. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 103:16–26, 2007.
Asplin, I. R., S. M. Wu, S. Mathew, G. Bhattacharjee, and S. V. Pizzo. Differential regulation of the fibroblast growth factor (FGF) family by alpha(2)-macroglobulin: evidence for selective modulation of FGF-2-induced angiogenesis. Blood 97:3450–3457, 2001.
Calafiori, A. R., M. G. Di, G. Martino, and M. Marotta. Preparation and characterization of calcium phosphate biomaterials. J. Mater. Sci. Mater. Med. 18:2331–2338, 2007.
Carmeliet, P., and R. K. Jain. Angiogenesis in cancer and other diseases. Nature 407:249–257, 2000.
Chaudhary, L. R., A. M. Hofmeister, and K. A. Hruska. Differential growth factor control of bone formation through osteoprogenitor differentiation. Bone 34:402–411, 2004.
Collin, P., J. R. Nefussi, A. Wetterwald, V. Nicolas, M. L. Boy-Lefevre, H. Fleisch, and N. Forest. Expression of collagen, osteocalcin, and bone alkaline phosphatase in a mineralizing rat osteoblastic cell culture. Calcif. Tissue Int. 50:175–183, 1992.
Fei, Z., Y. Hu, D. Wu, H. Wu, R. Lu, J. Bai, and H. Song. Preparation and property of a novel bone graft composite consisting of rhBMP-2 loaded PLGA microspheres and calcium phosphate cement. J. Mater. Sci. Mater. Med. 19:1109–1116, 2008.
Franke, S. V., C. B. Johansson, T. Sawase, Y. Yamasaki, and S. Oida. FGF-4 and titanium implants: a pilot study in rabbit bone. Clin. Oral. Implants Res. 14:363–368, 2003.
Gospodarowicz, D., N. Ferrara, L. Schweigerer, and G. Neufeld. Structural characterization and biological functions of fibroblast growth factor. Endocr. Rev. 8:95–114, 1987.
Hanada, K., J. E. Dennis, and A. I. Caplan. Stimulatory effects of basic fibroblast growth factor and bone morphogenetic protein-2 on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells. J. Bone Miner. Res. 12:1606–1614, 1997.
Hay, E., M. Hott, A. M. Graulet, A. Lomri, and P. J. Marie. Effects of bone morphogenetic protein-2 on human neonatal calvaria cell differentiation. J. Cell. Biochem. 72:81–93, 1999.
Hosokawa, R., K. Kikuzaki, T. Kimoto, T. Matsuura, D. Chiba, M. Wadamoto, Y. Sato, M. Maeda, A. Sano, and Y. Akagawa. Controlled local application of basic fibroblast growth factor (FGF-2) accelerates the healing of GBR. An experimental study in beagle dogs. Clin. Oral Implants Res. 11:345–353, 2000.
Huang, Z., E. R. Nelson, R. L. Smith, and S. B. Goodman. The sequential expression profiles of growth factors from osteoprogenitors [correction of osteroprogenitors] to osteoblasts in vitro. Tissue Eng. 13:2311–2320, 2007.
Jager, M., J. Fischer, W. Dohrn, X. Li, D. C. Ayers, A. Czibere, W. C. Prall, S. Lensing-Hohn, and R. Krauspe. Dexamethasone modulates BMP-2 effects on mesenchymal stem cells in vitro. J. Orthop. Res. 26:1440–1448, 2008.
Khairoun, I., D. Magne, O. Gauthier, J. M. Bouler, E. Aguado, G. Daculsi, and P. Weiss. In vitro characterization and in vivo properties of a carbonated apatite bone cement. J. Biomed. Mater. Res. 60:633–642, 2002.
Kotev-Emeth, S., S. Pitaru, S. Pri-Chen, and N. Savion. Establishment of a rat long-term culture expressing the osteogenic phenotype: dependence on dexamethasone and FGF-2. Connect. Tissue Res. 43:606–612, 2002.
Lan, J., Z. Wang, Y. Wang, J. Wang, and X. Cheng. The effect of combination of recombinant human bone morphogenetic protein-2 and basic fibroblast growth factor or insulin-like growth factor-I on dental implant osseointegration by confocal laser scanning microscopy. J. Periodontol. 77:357–363, 2006.
Lane, N. E., J. Kumer, W. Yao, T. Breunig, T. Wronski, G. Modin, and J. H. Kinney. Basic fibroblast growth factor forms new trabeculae that physically connect with pre-existing trabeculae, and this new bone is maintained with an anti-resorptive agent and enhanced with an anabolic agent in an osteopenic rat model. Osteoporos. Int. 14:374–382, 2003.
Lecanda, F., L. V. Avioli, and S. L. Cheng. Regulation of bone matrix protein expression and induction of differentiation of human osteoblasts and human bone marrow stromal cells by bone morphogenetic protein-2. J. Cell. Biochem. 67:386–396, 1997.
Lieberman, J. R., A. Daluiski, and T. A. Einhorn. The role of growth factors in the repair of bone. Biology and clinical applications. J. Bone Joint Surg. Am. 84-A:1032–1044, 2002.
Maegawa, N., K. Kawamura, M. Hirose, H. Yajima, Y. Takakura, and H. Ohgushi. Enhancement of osteoblastic differentiation of mesenchymal stromal cells cultured by selective combination of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2). J. Tissue Eng. Regen. Med. 1:306–313, 2007.
Manolagas, S. C., D. W. Burton, and L. J. Deftos. 1,25-Dihydroxyvitamin D3 stimulates the alkaline phosphatase activity of osteoblast-like cells. J. Biol. Chem. 256:7115–7117, 1981.
Mathew, S., S. Arandjelovic, W. F. Beyer, S. L. Gonias, and S. V. Pizzo. Characterization of the interaction between alpha2-macroglobulin and fibroblast growth factor-2: the role of hydrophobic interactions. Biochem. J. 374:123–129, 2003.
Morley, P., J. F. Whitfield, and G. E. Willick. Parathyroid hormone: an anabolic treatment for osteoporosis. Curr. Pharm. Des. 7:671–687, 2001.
Naganawa, T., L. Xiao, J. D. Coffin, T. Doetschman, M. G. Sabbieti, D. Agas, and M. M. Hurley. Reduced expression and function of bone morphogenetic protein-2 in bones of Fgf2 null mice. J. Cell. Biochem. 103:1975–1988, 2008.
Pan, Z., and P. Jiang. Assessment of the suitability of a new composite as a bone defect filler in a rabbit model. J. Tissue Eng. Regen. Med. 2:347–353, 2008.
Presta, M., P. Dell’Era, S. Mitola, E. Moroni, R. Ronca, and M. Rusnati. Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis. Cytokine Growth Factor Rev. 16:159–178, 2005.
Reddi, A. H. Bone morphogenetic proteins: an unconventional approach to isolation of first mammalian morphogens. Cytokine Growth Factor Rev. 8:11–20, 1997.
Reddi, A. H., and N. S. Cunningham. Initiation and promotion of bone differentiation by bone morphogenetic proteins. J. Bone Miner. Res. 8(Suppl 2):S499–S502, 1993.
Rice, D. P., T. Aberg, Y. Chan, Z. Tang, P. J. Kettunen, L. Pakarinen, R. E. Maxson, and I. Thesleff. Integration of FGF and TWIST in calvarial bone and suture development. Development 127:1845–1855, 2000.
Rifkin, D. B., and D. Moscatelli. Recent developments in the cell biology of basic fibroblast growth factor. J. Cell Biol. 109:1–6, 1989.
Rubin, M. R., and J. P. Bilezikian. New anabolic therapies in osteoporosis. Curr. Opin. Rheumatol. 14:433–440, 2002.
Ruhe, P. Q., H. C. Kroese-Deutman, J. G. Wolke, P. H. Spauwen, and J. A. Jansen. Bone inductive properties of rhBMP-2 loaded porous calcium phosphate cement implants in cranial defects in rabbits. Biomaterials 25:2123–2132, 2004.
Shen, Q. G., C. H. Lin, and X. Zhen. Practical Biomedical Materials. Shanghai: Shanghai Science and Technology Press, 2005.
Sieron, A. L., N. Louneva, and A. Fertala. Site-specific interaction of bone morphogenetic protein 2 with procollagen II. Cytokine 18:214–221, 2002.
Singhatanadgit, W., V. Salih, and I. Olsen. Up-regulation of bone morphogenetic protein receptor IB by growth factors enhances BMP-2-induced human bone cell functions. J. Cell. Physiol. 209:912–922, 2006.
Sorensen, R. G., U. M. Wikesjo, A. Kinoshita, and J. M. Wozney. Periodontal repair in dogs: evaluation of a bioresorbable calcium phosphate cement (Ceredex) as a carrier for rhBMP-2. J. Clin. Periodontol. 31:796–804, 2004.
Strauss, E. J., B. Pahk, F. J. Kummer, and K. Egol. Calcium phosphate cement augmentation of the femoral neck defect created after dynamic hip screw removal. J. Orthop. Trauma 21:295–300, 2007.
Sun, X. J., Z. Y. Zhang, S. Y. Wang, S. A. Gittens, X. Q. Jiang, and L. L. Chou. Maxillary sinus floor elevation using a tissue-engineered bone complex with OsteoBone and bMSCs in rabbits. Clin. Oral Implants Res. 19:804–813, 2008.
Tabata, Y. Tissue regeneration based on growth factor release. Tissue Eng. 9(Suppl 1):S5–S15, 2003.
Tadic, D., F. Beckmann, K. Schwarz, and M. Epple. A novel method to produce hydroxyapatite objects with interconnecting porosity that avoids sintering. Biomaterials 25:3335–3340, 2004.
Tanaka, E., Y. Ishino, A. Sasaki, T. Hasegawa, M. Watanabe, D. A. la-Bona, E. Yamano, T. M. van Eijden, and K. Tanne. Fibroblast growth factor-2 augments recombinant human bone morphogenetic protein-2-induced osteoinductive activity. Ann. Biomed. Eng. 34:717–725, 2006.
Tanaka, H., A. Wakisaka, H. Ogasa, S. Kawai, and C. T. Liang. Effects of basic fibroblast growth factor on osteoblast-related gene expression in the process of medullary bone formation induced in rat femur. J. Bone Miner. Metab. 21:74–79, 2003.
Tsutsumi, S., A. Shimazu, K. Miyazaki, H. Pan, C. Koike, E. Yoshida, K. Takagishi, and Y. Kato. Retention of multilineage differentiation potential of mesenchymal cells during proliferation in response to FGF. Biochem. Biophys. Res. Commun. 288:413–419, 2001.
Turgeman, G., Y. Zilberman, S. Zhou, P. Kelly, I. K. Moutsatsos, Y. P. Kharode, L. E. Borella, F. J. Bex, B. S. Komm, P. V. Bodine, and D. Gazit. Systemically administered rhBMP-2 promotes MSC activity and reverses bone and cartilage loss in osteopenic mice. J. Cell. Biochem. 86:461–474, 2002.
Ueda, H., L. Hong, M. Yamamoto, K. Shigeno, M. Inoue, T. Toba, M. Yoshitani, T. Nakamura, Y. Tabata, and Y. Shimizu. Use of collagen sponge incorporating transforming growth factor-beta1 to promote bone repair in skull defects in rabbits. Biomaterials 23:1003–1010, 2002.
Varkey, M., C. Kucharski, T. Haque, W. Sebald, and H. Uludag. In vitro osteogenic response of rat bone marrow cells to bFGF and BMP-2 treatments. Clin. Orthop. Relat. Res. 443:113–123, 2006.
Yamagiwa, H., N. Endo, K. Tokunaga, T. Hayami, H. Hatano, and H. E. Takahashi. In vivo bone-forming capacity of human bone marrow-derived stromal cells is stimulated by recombinant human bone morphogenetic protein-2. J. Bone Miner. Metab. 19:20–28, 2001.
Zellin, G., and A. Linde. Effects of recombinant human fibroblast growth factor-2 on osteogenic cell populations during orthopic osteogenesis in vivo. Bone 26:161–168, 2000.
Zhang, Y., B. Shi, C. Li, Y. Wang, Y. Chen, W. Zhang, T. Luo, and X. Cheng. The synergetic bone-forming effects of combinations of growth factors expressed by adenovirus vectors on chitosan/collagen scaffolds. J. Control. Release 136:172–178, 2009.
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Wang, L., Huang, Y., Pan, K. et al. Osteogenic Responses to Different Concentrations/Ratios of BMP-2 and bFGF in Bone Formation. Ann Biomed Eng 38, 77–87 (2010). https://doi.org/10.1007/s10439-009-9841-8
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DOI: https://doi.org/10.1007/s10439-009-9841-8