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Osteogenic differentiation of mesenchymal stem cells promoted by overexpression of connective tissue growth factor

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Abstract

Objective

Large segmental bone defect repair remains a clinical and scientific challenge with increasing interest focusing on combining gene transfection with tissue engineering techniques. The aim of this study is to investigate the effect of connective tissue growth factor (CTGF) on the proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells (MSCs).

Methods

A CTGF-expressing plasmid (pCTGF) was constructed and transfected into MSCs. Then expressions of bone morphogenesis-related genes, proliferation rate, alkaline phosphatase activity, and mineralization were examined to evaluate the osteogenic potential of the CTGF gene-modified MSCs.

Results

Overexpression of CTGF was confirmed in pCTGF-MSCs. pCTGF transfection significantly enhanced the proliferation rates of pCTGF-MSCs (P<0.05). CTGF induced a 7.5-fold increase in cell migration over control (P<0.05). pCTGF transfection enhanced the expression of bone matrix proteins, such as bone sialoprotein, osteocalcin, and collagen type I in MSCs. The levels of alkaline phosphatase (ALP) activities of pCTGF-MSCs at the 1st and 2nd weeks were 4.0- and 3.0-fold higher than those of MSCs cultured in OS-medium, significantly higher than those of mock-MSCs and normal control MSCs (P<0.05). Overexpression of CTGF in MSCs enhanced the capability to form mineralized nodules.

Conclusion

Overexpression of CTGF could improve the osteogenic differentiation ability of MSCs, and the CTGF gene-modified MSCs are potential as novel cell resources of bone tissue engineering.

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References

  • Abreu, J.G., Ketpura, N.I., Reversade, B., de Robertis, E.M., 2002. Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta. Nat. Cell Biol., 4(8):599–604. [doi:10.1038/ncb826]

    PubMed  CAS  Google Scholar 

  • Alden, T.D., Varady, P., Kallmes, D.F., Jane, J.A.Jr., Helm, G.A., 2002. Bone morphogenetic protein gene therapy. Spine, 27(16S):S87–S93. [doi:10.1097/00007632-200208151-00016]

    Article  PubMed  Google Scholar 

  • Arnott, J.A., Nuglozeh, E., Rico, M.C., Arango, H.I., Odgren, P.R., Safadi, F.F., Popoff, S.N., 2007. Connective tissue growth factor (CTGF/CCN2) is a downstream mediator for TGF-beta1-induced extracellular matrix production in osteoblasts. J. Cell. Physiol., 210(3):843–852. [doi:10.1002/jcp.20917]

    Article  PubMed  CAS  Google Scholar 

  • Betz, V.M., Betz, O.B., Harris, M.B., Vrahas, M.S., Evans, C.H., 2008. Bone tissue engineering and repair by gene therapy. Front Biosci., 13(13):833–841. [doi:10.2741/2724]

    Article  PubMed  CAS  Google Scholar 

  • Beyer Nardi, N., da Silva Meirelles, L., 2006. Mesenchymal Stem Cells: Isolation, in Vitro Expansion and Characterization. In: Stem Cells. Handbook of Experimental Pharmacology. Springer Berlin Heidelberg, Vol. 174, p.249–282. [doi:10.1007/978-3-540-77855-4]

    Google Scholar 

  • Brigstock, D.R., 2002. Regulation of angiogenesis and endothelial cell function by connective tissue growth factor (CTGF) and cysteine-rich 61 (CYR61). Angiogenesis, 5(3):153–165. [doi:10.1023/A:1023823803510]

    Article  PubMed  CAS  Google Scholar 

  • Brigstock, D.R., 2003. The CCN family: a new stimulus package. J. Endocrinol., 178(2):169–175. [doi:10.1677/joe.0.1780169]

    Article  PubMed  CAS  Google Scholar 

  • Brigstock, D.R., Goldschmeding, R., Katsube, K.I., Lam, S.C., Lau, L.F., Lyons, K., Naus, C., Perbal, B., Riser, B., Takigawa, M., Yeger, H., 2003. Proposal for a unified CCN nomenclature. Mol. Pathol., 56(2):127–128. [doi:10.1136/mp.56.2.127]

    Article  PubMed  CAS  Google Scholar 

  • Caterson, E., Nesti, L., Danielson, K., Tuan, R., 2002. Human marrow-derived mesenchymal progenitor cells. Mol. Biotechnol., 20(3):245–256. [doi:10.1385/MB:20:3:245]

    Article  PubMed  CAS  Google Scholar 

  • Chan, J., O’Donoghue, K., de la Fuente, J., Roberts, I.A., Kumar, S., Morgan, J.E., Fisk, N.M., 2005. Human fetal mesenchymal stem cells as vehicles for gene delivery. Stem. Cells, 23(1):93–102. [doi:10.1634/stemcells.2004-0138]

    Article  PubMed  CAS  Google Scholar 

  • Cho, H.H., Park, H.T., Kim, Y.J., Bae, Y.C., Suh, K.T., Jung, J.S., 2005. Induction of osteogenic differentiation of human mesenchymal stem cells by histone deacetylase inhibitors. J. Cell. Biochem., 96(3):533–542. [doi:10.1002/jcb.20544]

    Article  PubMed  CAS  Google Scholar 

  • Duan, X., Yang, L., Dong, S., Xin, R., Chen, G., Guo, L., 2008. Characterization of EGFP-labeled mesenchymal stem cells and redistribution of allogeneic cells after subcutaneous implantation. Archives of Orthopaedic and Trauma Surgery, 128(7):751–759. [doi:10.1007/s00402-008-0585-y]

    Article  PubMed  Google Scholar 

  • Edgar, C.M., Chakravarthy, V., Barnes, G., Kakar, S., Gerstenfeld, L.C., Einhorn, T.A., 2007. Autogenous regulation of a network of bone morphogenetic proteins (BMPs) mediates the osteogenic differentiation in murine marrow stromal cells. Bone, 40(5):1389–1398. [doi:10.1016/j.bone.2007.01.001]

    Article  PubMed  CAS  Google Scholar 

  • Gamradt, S.C., Lieberman, J.R., 2004. Genetic modification of stem cells to enhance bone repair. Ann. Biomed. Eng., 32(1):136–147. [doi:10.1023/B:ABME.0000007798.78548.b8]

    Article  PubMed  Google Scholar 

  • Gersbach, C.A., Le Doux, J.M., Guldberg, R.E., Garcia, A.J., 2006. Inducible regulation of Runx2-stimulated osteogenesis. Gene Ther., 13(11):873–882. [doi:10.1038/sj.gt.3302725]

    PubMed  CAS  Google Scholar 

  • Goessler, U.R., Riedel, K., Hormann, K., Riedel, F., 2006. Perspectives of gene therapy in stem cell tissue engineering. Cells Tissues Organs, 183(4):169–179. [doi:10.1159/000096508]

    Article  PubMed  Google Scholar 

  • Heng, E.C., Huang, Y., Black, S.A.Jr., Trackman, P.C., 2006. CCN2, connective tissue growth factor, stimulates collagen deposition by gingival fibroblasts via module 3 and alpha6- and beta1 integrins. J. Cell. Biochem., 98(2):409–420. [doi:10.1002/jcb.20810]

    Article  PubMed  CAS  Google Scholar 

  • Howard, D., Buttery, L.D., Shakesheff, K.M., Roberts, S.J., 2008. Tissue engineering: strategies, stem cells and scaffolds. J. Anat., 213(1):66–72. [doi:10.1111/j.1469-7580.2008.00878.x]

    Article  PubMed  CAS  Google Scholar 

  • Huang, W., Rudkin, G.H., Carlsen, B., Ishida, K., Ghasri, P., Anvar, B., Yamaguchi, D.T., Miller, T.A., 2002. Overexpression of BMP-2 modulates morphology, growth, and gene expression in osteoblastic cells. Exp. Cell Res., 274(2):226–234. [doi:10.1006/excr.2002.5483]

    Article  PubMed  CAS  Google Scholar 

  • Igarashi, M., Kamiya, N., Hasegawa, M., Kasuya, T., Takahashi, T., Takagi, M., 2004. Inductive effects of dexamethasone on the gene expression of Cbfa1, osterix and bone matrix proteins during differentiation of cultured primary rat osteoblasts. J. Mol. Histol., 35(1):3–10. [doi:10.1023/B:HIJO.0000020883.33256.fe]

    Article  PubMed  CAS  Google Scholar 

  • Ivkovic, S., Yoon, B.S., Popoff, S.N., Safadi, F.F., Libuda, D.E., Stephenson, R.C., Daluiski, A., Lyons, K.M., 2003. Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development, 130(12):2779–2791. [doi:10.1242/dev.00505]

    Article  PubMed  CAS  Google Scholar 

  • Jiang, C.Y., Gui, C., He, A.N., Hu, X.Y., Chen, J., Jiang, Y., Wang, J.A., 2008. Optimal time for mesenchymal stem cell transplantation in rats with myocardial infarction. J. Zhejiang Univ. Sci. B, 9(8):630–637. [doi:10.1631/jzus.B0820004]

    Article  PubMed  CAS  Google Scholar 

  • Kanaan, R.A., Aldwaik, M., Al-Hanbali, O.A., 2006. The role of connective tissue growth factor in skeletal growth and development. Med. Sci. Monit., 12(12):RA277–RA281.

    PubMed  CAS  Google Scholar 

  • Kanczler, J.M., Oreffo, R.O., 2008. Osteogenesis and angiogenesis: the potential for engineering bone. Eur. Cell Mater., 15:100–114.

    PubMed  CAS  Google Scholar 

  • Karp, J.M., Tanaka, T.S., Zohar, R., Sodek, J., Shoichet, M.S., Davies, J.E., Stanford, W.L., 2005. Thrombin mediated migration of osteogenic cells. Bone, 37(3):337–348. [doi:10.1016/j.bone.2005.04.022]

    Article  PubMed  CAS  Google Scholar 

  • Kawaki, H., Kubota, S., Suzuki, A., Yamada, T., Matsumura, T., Mandai, T., Yao, M., Maeda, T., Lyons, K.M., Takigawa, M., 2008. Functional requirement of CCN2 for intramembranous bone formation in embryonic mice. Biochem. Biophys. Res. Commun., 366(2):450–456. [doi:10.1016/j.bbrc.2007.11.155]

    Article  PubMed  CAS  Google Scholar 

  • Kofron, M.D., Laurencin, C.T., 2006. Bone tissue engineering by gene delivery. Adv. Drug Deliv. Rev., 58(4):555–576. [doi:10.1016/j.addr.2006.03.008]

    Article  PubMed  CAS  Google Scholar 

  • Kubota, S., Takigawa, M., 2007. Role of CCN2/CTGF/Hcs24 in bone growth. Int. Rev. Cytol., 257:1–41. [doi:10.1016/S0074-7696(07)57001-4]

    Article  PubMed  CAS  Google Scholar 

  • Lee, H.S., Huang, G.T., Chiang, H., Chiou, L.L., Chen, M.H., Hsieh, C.H., Jiang, C.C., 2003. Multipotential mesenchymal ctem cells from femoral cone carrow cear the cite of osteonecrosis. Stem. Cells, 21(2):190–199. [doi:10.1634/stemcells.21-2-190]

    Article  PubMed  CAS  Google Scholar 

  • Lee, R.H., Kim, B., Choi, I., Kim, H., Choi, H.S., Suh, K., Bae, Y.C., Jung, J.S., 2004. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol. Biochem., 14(4–6):311–324. [doi:10.1159/000080341]

    Article  PubMed  CAS  Google Scholar 

  • Lin, Y., Luo, E., Chen, X., Liu, L., Qiao, J., Yan, Z., Li, Z., Tang, W., Zheng, X., Tian, W., 2005. Molecular and cellular characterization during chondrogenic differentiation of adipose tissue-derived stromal cells in vitro and cartilage formation in vivo. J. Cell. Mol. Med., 9(4):929–939. [doi:10.1111/j.1582-4934.2005.tb00389.x]

    Article  PubMed  CAS  Google Scholar 

  • Liu, L.D., Shi, H.J., Jiang, L., Wang, L.C., Ma, S.H., Dong, C.H., Wang, J.J., Zhao, H.L., Liao, Y., Li, Q.H., 2007. The repairing effect of a recombinant human connective-tissue growth factor in a burn-wounded rhesus-monkey (Macaca mulatta) model. Biotechnol. Appl. Biochem., 47(2):105–112. [doi:10.1042/BA20060114]

    Article  PubMed  CAS  Google Scholar 

  • Luft, F.C., 2008. CCN2, the connective tissue growth factor. J. Mol. Med., 86(1):1–3. [doi:10.1007/s00109-007-0287-x]

    Article  PubMed  Google Scholar 

  • Luo, Q., Kang, Q., Si, W., Jiang, W., Park, J.K., Peng, Y., Li, X., Luu, H.H., Luo, J., Montag, A.G., Haydon, R.C., He, T.C., 2004. Connective tissue growth factor (CTGF) is regulated by WNT and bone morphogenetic proteins signaling in osteoblast differentiation of mesenchymal stem cells. J. Biol. Chem., 279(53):55958–55968. [doi:10.1074/jbc.M407810200]

    Article  PubMed  CAS  Google Scholar 

  • Mauney, J.R., Volloch, V., Kaplan, D.L., 2005. Role of adult mesenchymal stem cells in bone tissue engineering applications: current status and future prospects. Tissue Eng., 11(5–6):787–802. [doi:10.1089/ten.2005.11.787]

    Article  PubMed  CAS  Google Scholar 

  • Meijer, G.J., de Bruijn, J.D., Koole, R., van Blitterswijk, C.A., 2007. Cell-based bone tissue engineering. PLoS. Med., 4(2):e9. [doi:10.1371/journal.pmed.0040009]

    Article  PubMed  Google Scholar 

  • Mercurio, S., Latinkic, B., Itasaki, N., Krumlauf, R., Smith, J.C., 2004. Connective-tissue growth factor modulates WNT signalling and interacts with the WNT receptor complex. Development, 131(9):2137–2147. [doi:10.1242/dev.01045]

    Article  PubMed  CAS  Google Scholar 

  • Nishida, T., Nakanishi, T., Asano, M., Shimo, T., Takigawa, M., 2000. Effects of CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, on the proliferation and differentiation of osteoblastic cells in vitro. J. Cell. Physiol., 184(2):197–206. [doi:10.1002/1097-4652(200008)184:2〈197::AID-JCP7〉3.0.CO;2-R]

    Article  PubMed  CAS  Google Scholar 

  • Nishida, T., Kawaki, H., Baxter, R.M., Deyoung, R.A., Takigawa, M., Lyons, K.M., 2007. CCN2 (connective cissue crowth cactor) is essential for extracellular matrix production and integrin signaling in chondrocytes. J. Cell Commun. Signal., 1(1):45–58. [doi:10.1007/s12079-007-0005-z]

    Article  PubMed  Google Scholar 

  • Oakes, D.A., Lieberman, J.R., 2000. Osteoinductive applications of regional gene therapy: ex vivo gene transfer. Clin. Orthop. Relat. Res., 379(Suppl.):S101–S112. [doi:10.1097/00003086-200010001-00014]

    Article  PubMed  Google Scholar 

  • Ono, M., Kubota, S., Fujisawa, T., Sonoyama, W., Kawaki, H., Akiyama, K., Oshima, M., Nishida, T., Yoshida, Y., Suzuki, K., et al., 2007. Promotion of attachment of human bone marrow stromal cells by CCN2. Biochem. Biophys. Res. Commun., 357(1):20–25. [doi:10.1016/j.bbrc.2007.03.052]

    Article  PubMed  CAS  Google Scholar 

  • Ono, M., Kubota, S., Fujisawa, T., Sonoyama, W., Kawaki, H., Akiyama, K., Shimono, K., Oshima, M., Nishida, T., Yoshida, Y., et al., 2008. Promotion of hydroxyapatiteassociated, stem cell-based bone regeneration by CCN2. Cell Transplant., 17(1–2):231–240. [doi:10.3727/000000008783907143]

    Article  PubMed  Google Scholar 

  • Pelled, G.G., Turgeman, G., Aslan, H., Gazit, Z., Gazit, D., 2002. Mesenchymal stem cells for bone gene therapy and tissue engineering. Curr. Pharm. Des., 8(21):1917–1928. [doi:10.2174/1381612023393666]

    Article  PubMed  CAS  Google Scholar 

  • Perbal, B., Brigstock, D.R., Lau, L.F., 2003. Report on the second international workshop on the CCN family of genes. Mol. Pathol., 56(2):80–85. [doi:10.1136/mp.56.2.80]

    Article  PubMed  CAS  Google Scholar 

  • Pountos, I., Corscadden, D., Emery, P., Giannoudis, P.V., 2007. Mesenchymal stem cell tissue engineering: techniques for isolation, expansion and application. Injury, 38(S4):23–33. [doi:10.1016/S0020-1383(08)70006-8]

    Article  Google Scholar 

  • Rachfal, A.W., Brigstock, D.R., 2005. Structural and functional properties of CCN proteins. Vitam. Horm., 70:69–103. [doi:10.1016/S0083-6729(05)70003-0]

    Article  PubMed  CAS  Google Scholar 

  • Rose, F.R., Oreffo, R.O., 2002. Bone tissue engineering: hope vs hype. Biochem. Biophys. Res. Commun., 292(1):1–7. [doi:10.1006/bbrc.2002.6519]

    Article  PubMed  CAS  Google Scholar 

  • Rydziel, S., Stadmeyer, L., Zanotti, S., Durant, D., Smerdel-Ramoya, A., Canalis, E., 2007. Nephroblastoma overexpressed (Nov) inhibits osteoblastogenesis and causes osteopenia. J. Biol. Chem., 282(27):19762–19772. [doi:10.1074/jbc.M700212200]

    Article  PubMed  CAS  Google Scholar 

  • Safadi, F.F., Xu, J., Smock, S.L., Kanaan, R.A., Selim, A.H., Odgren, P.R., Marks, S.C.Jr., Owen, T.A., Popoff, S.N., 2003. Expression of connective tissue growth factor in bone: its role in osteoblast proliferation and differentiation in vitro and bone formation in vivo. J. Cell. Physiol., 196(1):51–62. [doi:10.1002/jcp.10319]

    Article  PubMed  CAS  Google Scholar 

  • Slater, B.J., Kwan, M.D., Gupta, D.M., Panetta, N.J., Longaker, M.T., 2008. Mesenchymal cells for skeletal tissue engineering. Expert. Opin. Biol. Ther., 8(7):885–893. [doi:10.1517/14712598.8.7.885]

    Article  PubMed  CAS  Google Scholar 

  • van Damme, A., van den Driessche, T., Collen, D., Chuah, M.K., 2002. Bone marrow stromal cells as targets for gene therapy. Curr. Gene Ther., 2(2):195–209. [doi:10.2174/1566523024605645]

    Article  PubMed  Google Scholar 

  • Xiang, Y., Zheng, Q., Jia, B.B., Huang, G.P., Xu, Y.L., Wang, J.F., Pan, Z.J., 2007. Ex vivo expansion and pluripotential differentiation of cryopreserved human bone marrow mesenchymal stem cells. J. Zhejiang Univ. Sci. B, 8(2): 136–146. [doi:10.1631/jzus.2007.B0136]

    Article  PubMed  CAS  Google Scholar 

  • Yamaai, T., Nakanishi, T., Asano, M., Nawachi, K., Yoshimichi, G., Ohyama, K., Komori, T., Sugimoto, T., Takigawa, M., 2005. Gene expression of connective tissue growth factor (CTGF/CCN2) in calcifying tissues of normal and cbfa1-null mutant mice in late stage of embryonic development. J. Bone Miner. Metab., 23(4):280–288. [doi:10.1007/s00774-004-0600-5]

    Article  PubMed  CAS  Google Scholar 

  • Yamanaka, O., Saika, S., Ikeda, K., Miyazaki, K., Kitano, A., Ohnishi, Y., 2008. Connective tissue growth factor modulates extracellular matrix production in human subconjunctival fibroblasts and their proliferation and migration in vitro. Jpn. J. Ophthalmol, 52(1):8–15. [doi:10.1007/s10384-007-0497-3]

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Key Laboratory of Transplant Engineering and Immunology of Ministry of Health, West China Hospital, Sichuan University, Chengdu, 610041, China

    Jin-jing Wang, Feng Ye, Li-jia Cheng, Yu-jun Shi, Ji Bao, Huai-qiang Sun, Wei Wang & Peng Zhang

  2. Department of Pathology, West China Hospital, Sichuan University, Chengdu, 610041, China

    Hong Bu

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  1. Jin-jing Wang
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  2. Feng Ye
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  3. Li-jia Cheng
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  9. Hong Bu
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Correspondence to Hong Bu.

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Project supported by the National Basic Research Program (973) of China (No. 2005CB623900)

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Wang, Jj., Ye, F., Cheng, Lj. et al. Osteogenic differentiation of mesenchymal stem cells promoted by overexpression of connective tissue growth factor. J. Zhejiang Univ. Sci. B 10, 355–367 (2009). https://doi.org/10.1631/jzus.B0820252

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