EXPERIMENT OF BONE MORPHOGENETIC PROTEIN 2 INDUCED CHONDROGENIC DIFFERENTIATION OF HUMAN Achilles TENDON-DERIVED STEM CELLS IN VITRO_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

RUI Yunfeng ^1,2 , GUO Yonggang ³ , LIN Yucheng ¹ , MA Liangyu ² , CHENG Xinkun ² , CHEN Hui ¹ , WANG Chen ¹

1Department of Orthopedics, Zhongda Hospital, Affiliated to Southeast University, Nanjing Jiangsu, 210009, P.R.China;;
2School of Medicine, Southeast University;;
3Department of Orthopedics, the First People’s Hospital of Horqin District of Tongliao. Corresponding author: RUI Yunfeng, E-mail: ruiyunfeng@126.com;

Corresponding author：

Keywords：

Tendon-derived stem cells; Chondrogenic differentiation; Tendinopathy; Bone morphogenetic protein 2

DOI：

10.7507/1002-1892.20130328

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Objective To investigate the effects of bone morphogenetic protein 2 (BMP-2) on the chondrogenic differentiation of human Achilles tendon-derived stem cells (hATDSCs) in vitro. Methods Achilles tendon was harvested from a voluntary donor with acute Achilles tendon rupture. And nucleated cells were obtained by digesting with collagenase and were cultured to the 3rd passage. The flow cytometry was used to measure the immunophenotyping; and Oil red O staining, alizarin red staining, and Safranin O/fast green staining were used to identify the adipogenic differentiation, osteogenic differentiation, and chondrogenic differentiation, respectively. The hATDSCs pellet was cultured in complete culture medium with (experimental group) or without recombinant human BMP-2 (rhBMP-2) (control grup) for 3 weeks. Chondrogenic differentiation of hATDSCs was evaluated by HE staining, Safranin O/fast green staining, and immunohistochemical staining for collagen type II; and the mRNA expressions of SOX9, collagen type II, and Aggrecan were detected by real-time fluorescence quantitative PCR. Results Primary hATDSCs cultured in vitro showed clonal growth; after cell passage, homogeneous spindle fibroblast-like cells were seen. The cells were positive for CD44, CD90, and CD105, while negative for CD34, CD45, and CD146. The results were positive for Oil red O staining at 3 weeks after adipogenic differentiation, for alizarin red staining at 4 weeks after osteogenic differentiation, and for Safranin O/fast green staining at 3 weeks after chondrogenic differentiation. After hATDSCs were induced with rhBMP-2 for 3 weeks, pellets formed in the experimental group, and the size of pellets was significantly larger than that in the control group; the results of HE staining, Safranin O/fast green staining, and immunohistochemical staining for collagen type II were all positive. The results of real-time fluorescence quantitative PCR showed that the mRNA expressions of SOX9, collagen type II, and Aggrecan in the experimental group were significantly higher than those in the control group (P lt; 0.05). Conclusion BMP-2 can promote proteoglycan deposition and induce chondrogenic differentiation of hATDSCs in vitro. The effect of BMP-2 on hATDSCs might provide a possible explanation for histopathological changes of tendinopathy.

Citation： RUI Yunfeng,GUO Yonggang,LIN Yucheng,MA Liangyu,CHENG Xinkun,CHEN Hui,WANG Chen. EXPERIMENT OF BONE MORPHOGENETIC PROTEIN 2 INDUCED CHONDROGENIC DIFFERENTIATION OF HUMAN Achilles TENDON-DERIVED STEM CELLS IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(12): 1492-1498. doi: 10.7507/1002-1892.20130328 Copy

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2.	Rees JD, Maffulli N, Cook J. Management of tendinopathy. Am J Sports Med, 2009, 37(9): 1855-1867.
3.	Xu Y, Murrell GA. The basic science of tendinopathy. Clin Orthop Relat Res, 2008, 466(7): 1528-1538.
4.	Rui YF, Lui PP, Li G, et al. Isolation and characterization of multipotent rat tendon-derived stem cells. Tissue Eng Part A, 2010, 16(5): 1549-1558.
5.	Rui YF, Lui PP, Chan LS, et al. Does erroneous differentiation of tendon-derived stem cells contribute to the pathogenesis of calcifying tendinopathy? Chin Med J (Engl), 2011, 124(4): 606-610.
6.	Rui YF, Lui PP, Wong YM, et al. Altered fate of tendon-derived stem cells isolated from a failed tendon-healing animal model of tendinopathy. Stem Cells Dev, 2013, 22(7): 1076-1085.
7.	Yee Lui PP, Wong YM, Rui YF, et al. Expression of chondro-osteogenic BMPs in ossified failed tendon healing model of tendinopathy. J Orthop Res, 2011, 29(6): 816-821.
8.	Rui YF, Lui PP, Rolf CG, et al. Expression of chondro-osteogenic BMPs in clinical samples of patellar tendinopathy. Knee Surg Sports Traumatol Arthrosc, 2012, 20(7): 1409-1417.
9.	Hashimoto Y, Yoshida G, Toyoda H, et al. Generation of tendon-to-bone interface “enthesis” with use of recombinant BMP-2 in a rabbit model. J Orthop Res, 2007, 25(11): 1415-1424.
10.	Rui YF, Lui PP, Wong YM, et al. BMP-2 stimulated non-tenogenic differentiation and promoted proteoglycan deposition of tendon-derived stem cells (TDSCs) in vitro. J Orthop Res, 2013, 31(5): 746-753.
11.	Lee WY, Lui PP, Rui YF. Hypoxia-mediated efficient expansion of human tendon-derived stem cells in vitro. Tissue Eng Part A, 2012, 18(5-6): 484-498.
12.	Bi Y, Ehirchiou D, Kilts TM, et al. Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche. Nat Med, 2007, 13(10): 1219-1227.
13.	Zhang J, Wang JH. Characterization of differential properties of rabbit tendon stem cells and tenocytes. BMC Musculoskelet Disord, 2010, 11: 10.
14.	Mienaltowski MJ, Adams SM, Birk DE. Regional differences in stem cell/progenitor cell populations from the mouse achilles tendon. Tissue Eng Part A, 2013, 19(1-2): 199-210.
15.	Yin Z, Chen X, Chen JL, et al. The regulation of tendon stem cell differentiation by the alignment of nanofibers. Biomaterials, 2010, 31(8): 2163-2175.
16.	Tang Y, Tang W, Lin Y, et al. Combination of bone tissue engineering and BMP-2 gene transfection promotes bone healing in osteoporotic rats. Cell Biol Int, 2008, 32(9): 1150-1157.
17.	Chen CH, Liu HW, Tsai CL, et al. Photoencapsulation of bone morphogenetic protein-2 and periosteal progenitor cells improve tendon graft healing in a bone tunnel. Am J Sports Med, 2008, 36(3): 461-473.
18.	Knippenberg M, Helder MN, Zandieh Doulabi B, et al. Osteogenesis versus chondrogenesis by BMP-2 and BMP-7 in adipose stem cells. Biochem Biophys Res Commun, 2006, 342(3): 902-908.
19.	Asahina I, Sampath TK, Hauschka PV. Human osteogenic protein-1 induces chondroblastic, osteoblastic, and/or adipocytic differentiation of clonal murine target cells. Exp Cell Res, 1996, 222(1): 38-47.
20.	Date T, Doiguchi Y, Nobuta M, et al. Bone morphogenetic protein-2 induces differentiation of multipotent C3H10T1/2 cells into osteoblasts, chondrocytes, and adipocytes in vivo and in vitro. J Orthop Sci, 2004, 9(5): 503-508.
21.	Zeiter S, Lezuo P, Ito K. Effect of TGF beta1, BMP-2 and hydraulic pressure on chondrogenic differentiation of bovine bone marrow mesenchymal stromal cells. Biorheology, 2009, 46(1): 45-55.
22.	段智霞, 郑启新, 郭晓东, 等. 骨形成蛋白 2 活性多肽体外定向诱导骨髓间充质干细胞向成骨方向分化的剂量依赖性研究. 中国修复重建外科杂志, 2007, 21(10): 1118-1122.
23.	An C, Cheng Y, Yuan Q, et al. IGF-1 and BMP-2 induces differentiation of adipose-derived mesenchymal stem cells into chondrocytes-like cells. Ann Biomed Eng, 2010, 38(4): 1647-1654.
24.	Qing W, Guang-Xing C, Lin G, et al. The osteogenic study of tissue engineering bone with BMP2 and BMP7 gene-modified rat adipose-derived stem cell. J Biomed Biotechnol, 2012, 2012: 410879.
25.	Rui YF, Lui PP, Lee YW, et al. Higher BMP receptor expression and BMP-2-induced osteogenic differentiation in tendon-derived stem cells compared with bone-marrow-derived mesenchymal stem cells. Int Orthop, 2012, 36(5): 1099-1107.

1. Riley G. Tendinopathy—from basic science to treatment. Nat Clin Pract Rheumatol, 2008, 4(2): 82-89.
2. Rees JD, Maffulli N, Cook J. Management of tendinopathy. Am J Sports Med, 2009, 37(9): 1855-1867.
3. Xu Y, Murrell GA. The basic science of tendinopathy. Clin Orthop Relat Res, 2008, 466(7): 1528-1538.
4. Rui YF, Lui PP, Li G, et al. Isolation and characterization of multipotent rat tendon-derived stem cells. Tissue Eng Part A, 2010, 16(5): 1549-1558.
5. Rui YF, Lui PP, Chan LS, et al. Does erroneous differentiation of tendon-derived stem cells contribute to the pathogenesis of calcifying tendinopathy? Chin Med J (Engl), 2011, 124(4): 606-610.
6. Rui YF, Lui PP, Wong YM, et al. Altered fate of tendon-derived stem cells isolated from a failed tendon-healing animal model of tendinopathy. Stem Cells Dev, 2013, 22(7): 1076-1085.
7. Yee Lui PP, Wong YM, Rui YF, et al. Expression of chondro-osteogenic BMPs in ossified failed tendon healing model of tendinopathy. J Orthop Res, 2011, 29(6): 816-821.
8. Rui YF, Lui PP, Rolf CG, et al. Expression of chondro-osteogenic BMPs in clinical samples of patellar tendinopathy. Knee Surg Sports Traumatol Arthrosc, 2012, 20(7): 1409-1417.
9. Hashimoto Y, Yoshida G, Toyoda H, et al. Generation of tendon-to-bone interface “enthesis” with use of recombinant BMP-2 in a rabbit model. J Orthop Res, 2007, 25(11): 1415-1424.
10. Rui YF, Lui PP, Wong YM, et al. BMP-2 stimulated non-tenogenic differentiation and promoted proteoglycan deposition of tendon-derived stem cells (TDSCs) in vitro. J Orthop Res, 2013, 31(5): 746-753.
11. Lee WY, Lui PP, Rui YF. Hypoxia-mediated efficient expansion of human tendon-derived stem cells in vitro. Tissue Eng Part A, 2012, 18(5-6): 484-498.
12. Bi Y, Ehirchiou D, Kilts TM, et al. Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche. Nat Med, 2007, 13(10): 1219-1227.
13. Zhang J, Wang JH. Characterization of differential properties of rabbit tendon stem cells and tenocytes. BMC Musculoskelet Disord, 2010, 11: 10.
14. Mienaltowski MJ, Adams SM, Birk DE. Regional differences in stem cell/progenitor cell populations from the mouse achilles tendon. Tissue Eng Part A, 2013, 19(1-2): 199-210.
15. Yin Z, Chen X, Chen JL, et al. The regulation of tendon stem cell differentiation by the alignment of nanofibers. Biomaterials, 2010, 31(8): 2163-2175.
16. Tang Y, Tang W, Lin Y, et al. Combination of bone tissue engineering and BMP-2 gene transfection promotes bone healing in osteoporotic rats. Cell Biol Int, 2008, 32(9): 1150-1157.
17. Chen CH, Liu HW, Tsai CL, et al. Photoencapsulation of bone morphogenetic protein-2 and periosteal progenitor cells improve tendon graft healing in a bone tunnel. Am J Sports Med, 2008, 36(3): 461-473.
18. Knippenberg M, Helder MN, Zandieh Doulabi B, et al. Osteogenesis versus chondrogenesis by BMP-2 and BMP-7 in adipose stem cells. Biochem Biophys Res Commun, 2006, 342(3): 902-908.
19. Asahina I, Sampath TK, Hauschka PV. Human osteogenic protein-1 induces chondroblastic, osteoblastic, and/or adipocytic differentiation of clonal murine target cells. Exp Cell Res, 1996, 222(1): 38-47.
20. Date T, Doiguchi Y, Nobuta M, et al. Bone morphogenetic protein-2 induces differentiation of multipotent C3H10T1/2 cells into osteoblasts, chondrocytes, and adipocytes in vivo and in vitro. J Orthop Sci, 2004, 9(5): 503-508.
21. Zeiter S, Lezuo P, Ito K. Effect of TGF beta1, BMP-2 and hydraulic pressure on chondrogenic differentiation of bovine bone marrow mesenchymal stromal cells. Biorheology, 2009, 46(1): 45-55.
22. 段智霞, 郑启新, 郭晓东, 等. 骨形成蛋白 2 活性多肽体外定向诱导骨髓间充质干细胞向成骨方向分化的剂量依赖性研究. 中国修复重建外科杂志, 2007, 21(10): 1118-1122.
23. An C, Cheng Y, Yuan Q, et al. IGF-1 and BMP-2 induces differentiation of adipose-derived mesenchymal stem cells into chondrocytes-like cells. Ann Biomed Eng, 2010, 38(4): 1647-1654.
24. Qing W, Guang-Xing C, Lin G, et al. The osteogenic study of tissue engineering bone with BMP2 and BMP7 gene-modified rat adipose-derived stem cell. J Biomed Biotechnol, 2012, 2012: 410879.
25. Rui YF, Lui PP, Lee YW, et al. Higher BMP receptor expression and BMP-2-induced osteogenic differentiation in tendon-derived stem cells compared with bone-marrow-derived mesenchymal stem cells. Int Orthop, 2012, 36(5): 1099-1107.

Chinese Journal of Reparative and Reconstructive Surgery

EXPERIMENT OF BONE MORPHOGENETIC PROTEIN 2 INDUCED CHONDROGENIC DIFFERENTIATION OF HUMAN Achilles TENDON-DERIVED STEM CELLS IN VITRO

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