Sox9 Potentiates BMP2-Induced Chondrogenic Differentiation and Inhibits BMP2-Induced Osteogenic Differentiation

  • Junyi Liao
  • Ning Hu
  • Nian Zhou
  • Chen Zhao
  • Xi Liang
  • Hong Chen
  • Wei Xu
  • Cheng Chen
  • Qiang Cheng
  • Wei HuangEmail author


Bone morphogenetic protein 2 (BMP2) is one of the key chondrogenic growth factors involved in the cartilage regeneration. However, it also exhibits osteogenic abilities and triggers endochondral ossification. Effective chondrogenesis and inhibition of BMP2-induced osteogenesis and endochondral ossification can be achieved by directing the mesenchymal stem cells (MSCs) towards chondrocyte lineage with chondrogenic factors, such as Sox9. Here we investigated the effects of Sox9 on BMP2-induced chondrogenic and osteogenic differentiation of MSCs. We found that exogenous overexpression of Sox9 enhanced the BMP2-induced chondrogenic differentiation of MSCs in vitro. Also, it inhibited early and late osteogenic differentiation of MSCs in vitro. Subcutaneous stem cell implantation demonstrated Sox9-potentiated BMP2-induced cartilage formation and inhibited endochondral ossification. Mouse limb cultures indicated that BMP2 and Sox9 acted synergistically to stimulate chondrocyte proliferation, and Sox9 inhibited BMP2-induced chondrocyte hypertrophy and ossification. This study strongly suggests that Sox9 potentiates BMP2-induced MSC chondrogenic differentiation and cartilage formation, and inhibits BMP2-induced MSC osteogenic differentiation and endochondral ossification. Thus, exogenous overexpression of Sox9 in BMP2-induced mesenchymal stem cell differentiation may be a new strategy for cartilage tissue engineering.


Sox9 Bmp2 Chondogenic growth factors MSC 



We are grateful for the Department of Clinical Hematology, Third Military Medical University, and all persons in this department. We greatly thank Pro. Tong-Chuan He (Molecular Oncology Laboratory, Department of Surgery, University of Chicago Medical Center) for the use of AdBMP2, AdSox9, and AdGFP.


  1. 1.
    Steinert AF, Noth U, Tuan RS. Concepts in gene therapy for cartilage repair. Injury. 2008;39(Suppl 1):S97–113.PubMedPubMedCentralCrossRefGoogle Scholar
  2. 2.
    Benthien JP, Schwaninger M, Behrens P. We do not have evidence based methods for the treatment of cartilage defects in the knee. Knee Surg Sports Traumatol Arthrosc. 2011;19:543–52.PubMedCrossRefGoogle Scholar
  3. 3.
    Rodriguez-Merchan EC. Regeneration of articular cartilage of the knee. Rheumatol Int. 2013;33(4):837–45.PubMedCrossRefGoogle Scholar
  4. 4.
    Kao YJ, Ho J, Allen CR. Evaluation and management of osteochondral lesions of the knee. Phys Sportsmed. 2011;39:60–9.PubMedCrossRefGoogle Scholar
  5. 5.
    Chen H, Sun J, Hoemann CD, Lascau-Coman V, Ouyang W, McKee MD, Shive MS, Buschmann MD. Drilling and microfracture lead to different bone structure and necrosis during bone-marrow stimulation for cartilage repair. J Orthop Res. 2009;27:1432–8.PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Dhinsa BS, Adesida AB. Current clinical therapies for cartilage repair, their limitation and the role of stem cells. Curr Stem Cell Res Ther. 2012;7:143–8.PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Matricali GA, Dereymaeker GP, Luyten FP. Donor site morbidity after articular cartilage repair procedures: a review. Acta Orthop Belg. 2010;76:669–74.PubMedGoogle Scholar
  8. 8.
    Longo UG, Petrillo S, Franceschetti E, Berton A, Maffulli N, Denaro V. Stem cells and gene therapy for cartilage repair. Stem Cells Int. 2012;2012:168385.PubMedPubMedCentralGoogle Scholar
  9. 9.
    Deng ZL, Sharff KA, Tang N, Song WX, Luo J, Chen J, Bennett E, Reid R, Manning D, Xue A, Montag AG, Luu HH, Haydon RC, He TC. Regulation of osteogenic differentiation during skeletal development. Front Biosci. 2008;13:2001–21.PubMedCrossRefGoogle Scholar
  10. 10.
    Pelttari K, Steck E, Richter W. The use of mesenchymal stem cells for chondrogenesis. Injury. 2008;39(Suppl 1):S58–65.PubMedCrossRefGoogle Scholar
  11. 11.
    Augello A, Kurth TB, De Bari C. Mesenchymal stem cells: a perspective from in vitro cultures to in vivo migration and niches. Eur Cell Mater. 2010;20:121–33.PubMedCrossRefGoogle Scholar
  12. 12.
    Yoon BS, Lyons KM. Multiple functions of BMPs in chondrogenesis. J Cell Biochem. 2004;93:93–103.PubMedCrossRefGoogle Scholar
  13. 13.
    Cucchiarini M, Ekici M, Schetting S, Kohn D, Madry H. Metabolic activities and chondrogenic differentiation of human mesenchymal stem cells following recombinant adeno-associated virus-mediated gene transfer and overexpression of fibroblast growth factor 2. Tissue Eng Part A. 2011;7:1921–33.CrossRefGoogle Scholar
  14. 14.
    Handorf AM, Li WJ. Fibroblast growth factor-2 primes human mesenchymal stem cells for enhanced chondrogenesis. PLoS One. 2011;6(7):e22887.PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    An C, Cheng Y, Yuan Q, Li J. IGF-1 and BMP-2 induces differentiation of adipose-derived mesenchymal stem cells into chondrocytes-like cells. Ann Biomed Eng. 2010;38:1647–54.PubMedCrossRefGoogle Scholar
  16. 16.
    Shintani N, Siebenrock KA, Hunziker EB. TGF-β1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants and arrests downstream differentiation at an early stage of hypertrophy. PLoS One. 2013;8(1):e53086.PubMedPubMedCentralCrossRefGoogle Scholar
  17. 17.
    Keller B, Yang T, Chen Y, Munivez E, Bertin T, Zabel B, Lee B. Interaction of TGFbeta and BMP signaling pathways during chondrogenesis. PLoS One. 2011;6(1):e16421.PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Danisovic L, Varga I, Polak S. Growth factors and chondrogenic differentiation of mesenchymal stem cells. Tissue Cell. 2012;44:69–73.PubMedCrossRefGoogle Scholar
  19. 19.
    Pan Q, Yu Y, Chen Q, Li C, Wu H, Wan Y, Ma J, Sun F. Sox9, a key transcription factor of bone morphogenetic protein-2-induced chondrogenesis, is activated through BMP pathway and a CCAAT box in the proximal promoter. J Cell Physiol. 2008;217:228–41.PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Mahmoudifar N, Doran PM. Chondrogenic differentiation of human adipose-derived stem cells in polyglycolic acid mesh scaffolds under dynamic culture conditions. Biomaterials. 2010;31:3858–67.PubMedCrossRefGoogle Scholar
  21. 21.
    Kurth T, Hedbom E, Shintani N, Sugimoto M, Chen FH, Haspl M, Martinovic S, Hunziker EB. Chondrogenic potential of human synovial mesenchymal stem cells in alginate. Osteoarthr Cartil. 2007;15:1178–89.PubMedCrossRefGoogle Scholar
  22. 22.
    Lian JB, Stein GS, Javed A, van Wijnen AJ, Stein JL, Montecino M, Hassan MQ, Gaur T, Lengner CJ, Young DW. Networks and hubs for the transcriptional control of osteoblastogenesis. Rev Endocr Metab Disord. 2006;7:1–16.PubMedCrossRefGoogle Scholar
  23. 23.
    Nishimura R, Hata K, Ono K, Amano K, Takigawa Y, Wakabayashi M, Takashima R, Yoneda T. Regulation of endochondral ossification by transcription factors. Front Biosci (Landmark Ed). 2012;17:2657–66.CrossRefGoogle Scholar
  24. 24.
    Lefebvre V, Dumitriu B, Penzo-Mendez A, Han Y, Pallavi B. Control of cell fate and differentiation by Sry-related high-mobility-group box (Sox) transcription factors. Int J Biochem Cell Biol. 2007;39:2195–214.PubMedPubMedCentralCrossRefGoogle Scholar
  25. 25.
    Akiyama H. Control of chondrogenesis by the transcription factor Sox9. Mod Rheumatol. 2008;18:213–9.CrossRefGoogle Scholar
  26. 26.
    Guerit D, Philipot D, Chuchana P, Toupet K, Brondello JM, Mathieu M, Jorgensen C, Noël D. Sox9-regulated miRNA-574-3p inhibits chondrogenic differentiation of mesenchymal stem cells. PLoS One. 2013;8(4):e62582.PubMedPubMedCentralCrossRefGoogle Scholar
  27. 27.
    Cairns DM, Liu R, Sen M, Canner JP, Schindeler A, Little DG, Zeng L. Interplay of Nkx3.2, Sox9 and Pax3 regulates chondrogenic differentiation of muscle progenitor cells. PLoS One. 2012;7(7):e39642.PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Cheng A, Genever PG. SOX9 determines RUNX2 transactivity by directing intracellular degradation. J Bone Miner Res. 2010;25:2680–9.PubMedCrossRefGoogle Scholar
  29. 29.
    Yamashita S, Andoh M, Ueno-Kudoh H, Sato T, Miyaki S, Asahara H. Sox9 directly promotes Bapx1 gene expression to repress Runx2 in chondrocytes. Exp Cell Res. 2009;315:2231–40.PubMedPubMedCentralCrossRefGoogle Scholar
  30. 30.
    Ding M, Lu Y, Abbassi S, Li F, Li X, Song Y, Geoffroy V, Im HJ, Zheng Q. Targeting Runx2 expression in hypertrophic chondrocytes impairs endochondral ossification during early skeletal development. J Cell Physiol. 2012;227:3446–56.PubMedPubMedCentralCrossRefGoogle Scholar
  31. 31.
    He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A. 1998;95:2509–14.PubMedCentralCrossRefPubMedGoogle Scholar
  32. 32.
    Luo J, Deng ZL, Luo X, Tang N, Song WX, et al. A protocol for rapid generation of recombinant adenoviruses using the AdEasy system. Nat Protoc. 2007;2:1236–47.PubMedCrossRefGoogle Scholar
  33. 33.
    Cheng H, Jiang W, Phillips FM, Haydon RC, Peng Y, Zhou L, Luu HH, An N, Breyer B, Vanichakarn P, Szatkowski JP, Park JY, He TC. Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg Am. 2003;85-A(8):1544–52.CrossRefGoogle Scholar
  34. 34.
    Kang Q, Sun MH, Cheng H, Peng Y, Montag AG, Deyrup AT, Jiang W, Luu HH, Luo J, Szatkowski JP, Vanichakarn P, Park JY, Li Y, Haydon RC, He TC. Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery. Gene Ther. 2004;11:1312–20.PubMedCrossRefGoogle Scholar
  35. 35.
    Sharff KA, Song WX, Luo X, Tang N, Luo J, Chen J, Bi Y, He BC, Huang J, Li X, Jiang W, Zhu GH, Su Y, He Y, Shen J, Wang Y, Chen L, Zuo GW, Liu B, Pan X, Reid RR, Luu HH, Haydon RC, He TC. Hey1 basic helix-loop-helix protein plays an important role in mediating BMP9-induced osteogenic differentiation of mesenchymal progenitor cells. J Biol Chem. 2009;284:649–59.PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    Chen L, Jiang W, Huang J, He BC, Zuo GW, Zhang W, Luo Q, Shi Q, Zhang BQ, Wagner ER, Luo J, Tang M, Wietholt C, Luo X, Bi Y, Su Y, Liu B, et al. Insulin-like growth factor 2 (IGF-2) potentiates BMP-9-induced osteogenic differentiation and bone formation. J Bone Miner Res. 2010;25:2447–59.PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Wang JH, Liu YZ, Yin LJ, Chen L, Huang J, Liu Y, Zhang RX, Zhou LY, Yang QJ, Luo JY, Zuo GW, Deng ZL, He BC. BMP9 and COX-2 form an important regulatory loop in BMP9-induced osteogenic differentiation of mesenchymal stem cells. Bone. 2013;57:311–21.PubMedCrossRefGoogle Scholar
  38. 38.
    Lengner CJ, Lepper C, van Wijnen AJ, Stein JL, Stein GS, Lian JB. Primary mouse embryonic fibroblasts: a model of mesenchymal cartilage formation. J Cell Physiol. 2004;200:327–33.PubMedCrossRefGoogle Scholar
  39. 39.
    Hu N, Jiang D, Huang E, Liu X, Li R, Liang X, Kim SH, Chen X, Gao JL, Zhang H, Zhang W, Kong YH, Zhang J, Wang J, Shui W, Luo X, Liu B, et al. BMP9-regulated angiogenic signaling plays an important role in the osteogenic differentiation of mesenchymal progenitor cells. J Cell Sci. 2013;126:532–41.PubMedPubMedCentralCrossRefGoogle Scholar
  40. 40.
    Hollander AP, Dickinson SC, Kafienah W. Stem cells and cartilage development: complexities of a simple tissue. Stem Cells. 2010;28:1992–6.PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Prockop DJ, Kota DJ, Bazhanov N, Reger RL. Evolving paradigms for repair of tissues by adult stem/progenitor cells (MSCs). J Cell Mol Med. 2010;14:2190–9.PubMedPubMedCentralCrossRefGoogle Scholar
  42. 42.
    Woo EJ. Adverse events after recombinant human BMP2 in nonspinal orthopaedic procedures. Clin Orthop Relat Res. 2013;471:1707–11.PubMedCrossRefGoogle Scholar
  43. 43.
    Jang WG, Kim EJ, Kim DK, Ryoo HM, Lee KB, Kim SH, Choi HS, Koh JT. BMP2 protein regulates osteocalcin expression via Runx2-mediated Atf6 gene transcription. J Biol Chem. 2012;287:905–15.PubMedCrossRefPubMedCentralGoogle Scholar
  44. 44.
    Yu YY, Lieu S, Lu C, Colnot C. Bone morphogenetic protein 2 stimulates endochondral ossification by regulating periosteal cell fate during bone repair. Bone. 2010;47:65–73.PubMedPubMedCentralCrossRefGoogle Scholar
  45. 45.
    Wang Y, Zheng Y, Chen D, Chen Y. Enhanced BMP signaling prevents degeneration and leads to endochondral ossification of Meckel’s cartilage in mice. Dev Biol. 2013;381:301–11.PubMedPubMedCentralCrossRefGoogle Scholar
  46. 46.
    Yu L, Han M, Yan M, Lee J, Muneoka K. BMP2 induces segment-specific skeletal regeneration from digit and limb amputations by establishing a new endochondral ossification center. Dev Biol. 2012;372:263–73.PubMedPubMedCentralCrossRefGoogle Scholar
  47. 47.
    Hu N, Wang C, Liang X, Yin L, Luo X, Liu B, Zhang H, Shui W, Nan G, Wang N, Wu N, Chen X, He Y, Wen S, Deng F, Zhang H, Liao Z, Luu HH, Haydon RC, He TC, Huang W. Inhibition of histone deacetylases potentiates BMP9-induced osteogenic signaling in mouse mesenchymal stem cells. Cell Physiol Biochem. 2013;32:486–98.PubMedCrossRefGoogle Scholar
  48. 48.
    Dy P, Wang W, Bhattaram P, Wang Q, Wang L, Ballock RT, Lefebvre V. Sox9 directs hypertrophic maturation and blocks osteoblast differentiation of growth plate chondrocytes. Dev Cell. 2012;22:597–609.PubMedPubMedCentralCrossRefGoogle Scholar
  49. 49.
    Yang HN, Park JS, Woo DG, Jeon SY, Do HJ, Lim HY, Kim SW, Kim JH, Park KH. Chondrogenesis of mesenchymal stem cells and dedifferentiated chondrocytes by transfection with SOX trio genes. Biomaterials. 2011;32:7695–704.PubMedCrossRefGoogle Scholar
  50. 50.
    Ikeda T, Kamekura S, Mabuchi A, Kou I, Seki S, Takato T, Nakamura K, Kawaguchi H, Ikegawa S, Chung UI. The combination of SOX5, SOX6, and SOX9 (the SOX trio) provides signals sufficient for induction of permanent cartilage. Arthritis Rheum. 2004;50:3561–73.PubMedCrossRefGoogle Scholar
  51. 51.
    Venkatesan JK, Ekici M, Madry H, Schmitt G, Kohn D, Cucchiarini M. SOX9 gene transfer via safe, stable, replication-defective recombinant adeno-associated virus vectors as a novel, powerful tool to enhance the chondrogenic potential of human mesenchymal stem cells. Stem Cell Res Ther. 2012;3:22.PubMedPubMedCentralCrossRefGoogle Scholar
  52. 52.
    Cucchiarini M, Orth P, Madry H. Direct rAAV SOX9 administration for durable articular cartilage repair with delayed terminal differentiation and hypertrophy in vivo. J Mol Med (Berl). 2012;91(5):625–36.CrossRefGoogle Scholar
  53. 53.
    Cao L, Yang F, Liu G, Yu D, Li H, Fan Q, Gan Y, Tang T, Dai K. The promotion of cartilage defect repair using adenovirus mediated Sox9 gene transfer of rabbit bone marrow mesenchymal stem cells. Biomaterials. 2011;32:3910–20.PubMedCrossRefGoogle Scholar
  54. 54.
    Park JS, Yang HN, Woo DG, Jeon SY, Do HJ, Lim HY, Kim JH, Park KH. Chondrogenesis of human mesenchymal stem cells mediated by the combination of SOX trio SOX5, 6, and 9 genes complexed with PEI-modified PLGA nanoparticles. Biomaterials. 2011;32:3679–88.PubMedCrossRefGoogle Scholar
  55. 55.
    Garza-Veloz I, Romero-Diaz VJ, Martinez-Fierro ML, Marino-Martinez IA, Gonzalez-Rodriguez M, Martinez-Rodriguez HG, Espinoza-Juarez MA, Bernal-Garza DA, Ortiz-Lopez R, Rojas-Martinez A. Analyses of chondrogenic induction of adipose mesenchymal stem cells by combined co-stimulation mediated by adenoviral gene transfer. Arthritis Res Ther. 2013;15:R80.PubMedPubMedCentralCrossRefGoogle Scholar
  56. 56.
    Nishimura R, Hata K, Matsubara T, Wakabayashi M, Yoneda T. Regulation of bone and cartilage development by network between BMP signalling and transcription factors. J Biochem. 2012;151:247–54.PubMedCrossRefGoogle Scholar
  57. 57.
    Kawato Y, Hirao M, Ebina K, Tamai N, Shi K, Hashimoto J, Yoshikawa H, Myoui A. Nkx3.2-induced suppression of Runx2 is a crucial mediator of hypoxia-dependent maintenance of chondrocyte phenotypes. Biochem Biophys Res Commun. 2011;416:205–10.PubMedCrossRefGoogle Scholar
  58. 58.
    Lin L, Shen Q, Leng H, Duan X, Fu X, Yu C. Synergistic inhibition of endochondral bone formation by silencing Hif1alpha and Runx2 in trauma-induced heterotopic ossification. Mol Ther. 2011;19:1426–32.PubMedPubMedCentralCrossRefGoogle Scholar
  59. 59.
    Yoshida CA, Yamamoto H, Fujita T, Furuichi T, Ito K, Inoue K, Yamana K, Zanma A, Takada K, Ito Y, Komori T. Runx2 and Runx3 are essential for chondrocyte maturation, and Runx2 regulates limb growth through induction of Indian hedgehog. Genes Dev. 2004;18:952–63.PubMedPubMedCentralCrossRefGoogle Scholar
  60. 60.
    Kim EJ, Cho SW, Shin JO, Lee MJ, Kim KS, Jung HS. Ihh and Runx2/Runx3 signaling interact to coordinate early chondrogenesis: a mouse model. PLoS One. 2013;8(2):e55296.PubMedPubMedCentralCrossRefGoogle Scholar
  61. 61.
    Chun JS, Oh H, Yang S, Park M. Wnt signaling in cartilage development and degeneration. BMB Rep. 2008;41:485–94.PubMedCrossRefGoogle Scholar
  62. 62.
    Bakker AC, van de Loo FA, van Beuningen HM, Sime P, van Lent PL, van der Kraan PM, Richards CD, van den Berg WB. Overexpression of active TGF-beta-1 in the murine knee joint: evidence for synovial-layer-dependent chondro-osteophyte formation. Osteoarthr Cartil. 2001;9:128–36.PubMedCrossRefGoogle Scholar
  63. 63.
    Mi Z, Ghivizzani SC, Lechman E, Glorioso JC, Evans CH, Robbins PD. Adverse effects of adenovirus-mediated gene transfer of human transforming growth factor beta 1 into rabbit knees. Arthritis Res Ther. 2003;5:R132–9.PubMedPubMedCentralCrossRefGoogle Scholar
  64. 64.
    Steinert AF, Ghivizzani SC, Rethwilm A, Tuan RS, Evans CH, Nöth U. Major biological obstacles for persistent cell-based regeneration of articular cartilage. Arthritis Res Ther. 2007;9:213.PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Junyi Liao
    • 1
  • Ning Hu
    • 1
  • Nian Zhou
    • 1
  • Chen Zhao
    • 1
  • Xi Liang
    • 1
  • Hong Chen
    • 1
  • Wei Xu
    • 1
  • Cheng Chen
    • 1
  • Qiang Cheng
    • 1
  • Wei Huang
    • 1
    Email author
  1. 1.Department of Orthopaedic SurgeryThe First Affiliated Hospital of Chongqing Medical UniversityChongqingChina

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