Osteogenic potential of recombinant human bone morphogenetic protein-9/absorbable collagen sponge (rhBMP-9/ACS) in rat critical size calvarial defects



It has been reported that bone morphogenetic protein (BMP)-9 has potent osteoinductive properties among the BMP family by adenovirus-transfection experiments. We very recently reported that absorbable collagen sponge (ACS) as a carrier for recombinant human (rh) BMP-9, compared with chitosan sponge, was suitable for inducing bone healing/regeneration by BMP-9 in a rat calvarial defect model. The aim of this study was to evaluate different doses of rhBMP-9/ACS on new bone formation in rat critical size calvarial defects.

Materials and methods

Bilateral calvarial defects (n = 32) were surgically created in 16 wistar rats and randomly filled with one of the following materials: (1) absorbable collagen sponge (ACS) alone; (2) 1 μg-rhBMP-9/ACS (L-rhBMP-9/ACS); (3) 5 μg-rhBMP-9/ACS (H-rhBMP-9/ACS); and (4) blank defects (control). The animals were sacrificed 8 weeks postsurgery for radiographic and histomorphometric analyses.


Bone volume and defect closure were statistically higher in the rhBMP-9/ACS-implanted (L-rhBMP-9/ACS and H-rhBMP-9/ACS) groups when compared with ACS-alone group (p < 0.05). Furthermore, defects filled with H-rhBMP-9/ACS showed the highest levels of newly formed bone area (NBA) and NBA/total defect area among all groups. No significant differences in any of the radiographic and histometric parameters could be observed between both concentrations of rhBMP-9.


Within the limits of this study, it can be concluded that rhBMP-9/ACS-induced bone formation can be reached with as little as 1 μg/site in rat critical size calvarial defects.

Clinical relevance

RhBMP-9 could be a potential therapeutic growth factor for future bone regenerative procedures.

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  1. 1.

    Deschaseaux F, Sensebe L, Heymann D (2009) Mechanisms of bone repair and regeneration. Trends Mol Med 15:417–429

    Article  PubMed  Google Scholar 

  2. 2.

    Herford AS, Boyne PJ (2008) Reconstruction of mandibular continuity defects with bone morphogenetic protein-2 (rhBMP-2). J Oral Maxillofac Surg 66:616–624

    Article  PubMed  Google Scholar 

  3. 3.

    Dickinson BP, Ashley RK, Wasson KL, O’Hara C, Gabbay J, Heller JB, Bradley JP (2008) Reduced morbidity and improved healing with bone morphogenic protein-2 in older patients with alveolar cleft defects. Plast Reconstr Surg 121:209–217

    Article  PubMed  Google Scholar 

  4. 4.

    McKay WF, Peckham SM, Badura JM (2007) A comprehensive clinical review of recombinant human bone morphogenetic protein-2 (INFUSE bone graft). Int Orthop 31:729–734

    Article  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Kang Q, Sun MH, Cheng H, Peng Y, Montag AG, Deyrup AT, Jiang W, Luu HH, Luo J, Szatkowski JP et al (2004) Characterization of the distinct orthotopic bone-forming activity of 14 B.P. using recombinant adenovirus-mediated gene delivery. Gene Ther 11:1312–1320

    Article  PubMed  Google Scholar 

  6. 6.

    Lamplot JD, Qin J, Nan G, Wang J, Liu X, Yin L, Tomal J, Li R, Shui W, Zhang H et al (2013) BMP9 signaling in stem cell differentiation and osteogenesis. Am J Stem Cells 2:1–21

    PubMed  PubMed Central  Google Scholar 

  7. 7.

    Lopez-Coviella I, Berse B, Krauss R, Thies RS, Blusztajn JK (2000) Induction and maintenance of the neuronal cholinergic phenotype in the central nervous system by BMP-9. Science 289:313–316

    Article  PubMed  Google Scholar 

  8. 8.

    Lord E, Bergeron E, Senta H, Park H, Faucheux N (2010) Effect of BMP-9 and its derived peptide on the differentiation of human white preadipocytes. Growth Factors 28:149–156

    Article  PubMed  Google Scholar 

  9. 9.

    Majumdar MK, Wang E, Morris EA (2001) BMP-2 and BMP-9 promotes chondrogenic differentiation of human multipotential mesenchymal cells and overcomes the inhibitory effect of IL-1. J Cell Physiol 189:275–284

    Article  PubMed  Google Scholar 

  10. 10.

    Chen C, Grzegorzewski KJ, Barash S, Zhao Q, Schneider H, Wang Q, Singh M, Pukac L, Bell AC, Duan R et al (2003) An integrated functional genomics screening program reveals a role for BMP-9 in glucose homeostasis. Nat Biotechnol 21:294–301

    Article  PubMed  Google Scholar 

  11. 11.

    Ricard N, Ciais D, Levet S, Subileau M, Mallet C, Zimmers TA, Lee SJ, Bidart M, Feige JJ, Bailly S (2012) BMP9 and BMP10 are critical for postnatal retinal vascular remodeling. Blood 119:6162–6171

    Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Luu HH, Song WX, Luo X, Manning D, Luo J, Deng ZL, Sharff KA, Montag AG, Haydon RC, He TC (2007) Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells. J Orthop Res 25:665–677

    Article  PubMed  Google Scholar 

  13. 13.

    Cheng H, Jiang W, Phillips FM, Haydon RC, Peng Y, Zhou L, Luu HH, An N, Breyer B, Vanichakarn P et al (2003) Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg Am 85-A:1544–1552

    Article  PubMed  Google Scholar 

  14. 14.

    Wang Y, Hong S, Li M, Zhang J, Bi Y, He Y, Liu X, Nan G, Su Y, Zhu G et al (2013) Noggin resistance contributes to the potent osteogenic capability of BMP9 in mesenchymal stem cells. J Orthop Res 31:1796–1803

    PubMed  Google Scholar 

  15. 15.

    Nakamura T, Shinohara Y, Momozaki S, Yoshimoto T, Noguchi K (2013) Co-stimulation with bone morphogenetic protein-9 and FK506 induces remarkable osteoblastic differentiation in rat dedifferentiated fat cells. Biochem Biophys Res Commun 440:289–294

    Article  PubMed  Google Scholar 

  16. 16.

    Rosen V (2006) BMP and BMP inhibitors in bone. Ann N Y Acad Sci 1068:19–25

    Article  PubMed  Google Scholar 

  17. 17.

    Li JZ, Li H, Sasaki T, Holman D, Beres B, Dumont RJ, Pittman DD, Hankins GR, Helm GA (2003) Osteogenic potential of five different recombinant human bone morphogenetic protein adenoviral vectors in the rat. Gene Ther 10:1735–1743

    Article  PubMed  Google Scholar 

  18. 18.

    Fujioka-Kobayashi M, Sawada K, Kobayashi E, Schaller B, Zhang Y, Miron, RJ (2016) Recombinant human bone morphogenetic protein 9 (rhBMP9) induced osteoblastic behaviour on a collagen membrane compared with rhBMP2. J Periodontol 87:e101–e107

  19. 19.

    Shinohara Y, Nakamura T, Shirakata Y, Noguchi K (2016) Bone healing capabilities of recombinant human bone morphogenetic protein-9 (rhBMP-9) with a chitosan or collagen carrier in rat calvarial defects. Dent Mater J 35:454–460

    Article  PubMed  Google Scholar 

  20. 20.

    Artzi Z, Kozlovsky A, Nemcovsky CE, Moses O, Tal H, Rohrer MD, Prasad HS, Weinreb M (2008) Histomorphometric evaluation of natural mineral combined with a synthetic cell-binding peptide (P-15) in critical-size defects in the rat calvaria. Int J Oral Maxillofac Implants 23:1063–1070

    PubMed  Google Scholar 

  21. 21.

    Pelaez M, Susin C, Lee J, Fiorini T, Bisch FC, Dixon DR, McPherson JC 3rd, Buxton AN, Wikesjo UM (2014) Effect of rhBMP-2 dose on bone formation/maturation in a rat critical-size calvarial defect model. J Clin Periodontol 41:827–836

    Article  PubMed  Google Scholar 

  22. 22.

    Hyun SJ, Han DK, Choi SH, Chai JK, Cho KS, Kim CK, Kim CS (2005) Effect of recombinant human bone morphogenetic protein-2, −4, and −7 on bone formation in rat calvarial defects. J Periodontol 76:1667–1674

    Article  PubMed  Google Scholar 

  23. 23.

    Rothamel D, Schwarz F, Sager M, Herten M, Sculean A, Becker J (2005) Biodegradation of differently cross-linked collagen membranes: an experimental study in the rat. Clin Oral Implants Res 16:369–378

    Article  PubMed  Google Scholar 

  24. 24.

    Shiozaki Y, Kitajima T, Mazaki T, Yoshida A, Tanaka M, Umezawa A, Nakamura M, Yoshida Y, Ito Y, Ozaki T et al (2013) Enhanced in vivo osteogenesis by nanocarrier-fused bone morphogenetic protein-4. Int J Nanomedicine 8:1349–1360

    PubMed  PubMed Central  Google Scholar 

  25. 25.

    Tumialan LM, Rodts GE (2007) Adverse swelling associated with use of rh-BMP-2 in anterior cervical discectomy and fusion. Spine J 7:509–510

    Article  PubMed  Google Scholar 

  26. 26.

    Smucker JD, Rhee JM, Singh K, Yoon ST, Heller JG (2006) Increased swelling complications associated with off-label usage of rhBMP-2 in the anterior cervical spine. Spine 31:2813–2819

    Article  PubMed  Google Scholar 

  27. 27.

    McClellan JW, Mulconrey DS, Forbes RJ, Fullmer N (2006) Vertebral bone resorption after transforaminal lumbar interbody fusion with bone morphogenetic protein (rhBMP-2). J Spinal Disord Tech 19:483–486

    Article  PubMed  Google Scholar 

  28. 28.

    Leblanc E, Trensz F, Haroun S, Drouin G, Bergeron E, Penton CM, Montanaro F, Roux S, Faucheux N, Grenier G (2011) BMP-9-induced muscle heterotopic ossification requires changes to the skeletal muscle microenvironment. J Bone Miner Res 26:1166–1177

    Article  PubMed  Google Scholar 

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Corresponding author

Correspondence to Kazuyuki Noguchi.

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Conflict of interest

The authors declare no conflicts of interests.


This study was supported by Grants-in-Aid for Scientific Research (B) (no. 24792147) and (C) (no. 26462972) from the Japan Society for the Promotion of Science (JSPS).

Ethical approval

All animal experimental protocols and procedures were approved by the Ethical Committee of the Animal Research Center of Kagoshima University, Japan (D14022).

Informed consent

Informed consent was not required in this study.

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Nakamura, T., Shirakata, Y., Shinohara, Y. et al. Osteogenic potential of recombinant human bone morphogenetic protein-9/absorbable collagen sponge (rhBMP-9/ACS) in rat critical size calvarial defects. Clin Oral Invest 21, 1659–1665 (2017). https://doi.org/10.1007/s00784-016-1963-4

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  • Growth factor
  • Bone morphogenetic protein-9
  • Bone formation
  • Collagen
  • Osteoinduction