EFFECTS OF TRANSFORMING GROWTH FACTOR β<sub>3</sub> AT DIFFERENT CONCENTRATIONS ON DIFFERENTIATION OF TEENDON STEM CELLS IN VITRO_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

GUOYupeng ¹ ,  TANGKanglai ¹ , ZHANGJiqiang ² , CHENBo ¹ , SHIYouxing ¹

1. Department of Orthopedics, Orthopedic Center of Chinese PLA, Southwest Hospital, Third Military Medical University, Chongqing, 400038, P. R. China;
2. Department of Neurobiology, Third Military Medical University;

Corresponding author：

TANGKanglai, Email: tangkanglai@hotmail.com

Keywords：

Tendon stem cells; Differentiation; Transforming growth factor β₃; Tendinopathy; Rat

DOI：

10.7507/1002-1892.20150133

Video：

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Objective To investigate the effect of transforming growth factor β₃ (TGF-β₃) at different concentrations on the differentiation of rat Achilles-derived tendon stem cells (TSCs) in vitro. Methods TSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged 3 weeks) by enzymatic digestion method and cultured for 3 passages. The TSCs were stimulated with TGF-β₃ at the concentrations of 5.0, 2.5, 1.0, and 0 ng/mL. At 1, 3, and 5 days, the mRNA expressions of tendogenic differentiation related genes[collagen type Ⅰ,tenascin C (TNC), tenomodulin (TNMD), scleraxis (Scx)], osteogenic differentiation related genes[Runt related transcription factor 2 (Runx2) and alkaline phosphatase (ALP)], chondrogenic differentiation genes (Sox9 and collagen type Ⅱ),and adipogenic differentiation genes[AP2 and peroxisome proliferator-activated receptor γ (PPARγ)] were measured by real-time quantitative PCR (qRT-PCR). Results TSCs could differentiated in different directions after treated with TGF-β₃ at different concentrations at different time points. TGF-β₃ was able to induce TSCs differentiated into tenocytes, which was related to the concentration and time of duration, and the two factors have interaction. Stimulation of TGF-β₃ at low concentration and for short time could inhibit non-tendogenic differentiation of TSCs, but at high concentration and for long time, TGF-β₃ enhanced TSCs differented into osteocytes or chondrocytes. Conclusion Effects of TGF-β₃ on TSCs differentiation are complicated and depend on the concentration and time of duration, which may be a key factor between tendogenic and non-tendogenic differentiations of TSCs.

Citation： GUOYupeng, TANGKanglai, ZHANGJiqiang, CHENBo, SHIYouxing. EFFECTS OF TRANSFORMING GROWTH FACTOR β₃ AT DIFFERENT CONCENTRATIONS ON DIFFERENTIATION OF TEENDON STEM CELLS IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(5): 620-625. doi: 10.7507/1002-1892.20150133 Copy

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2.	Wang JH, Iosifidis MI, Fu FH. Biomechanical basis for tendinopathy. Clin Orthop Relat Res, 2006, 443:320-332.
3.	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.
4.	Zhang J, Wang JH. Characterization of differential properties of rabbit tendon stem cells and tenocytes. BMC Musculoskelet Disord, 2010, 11:10.
5.	Zhou Z, Akinbiyi T, Xu L, et al. Tendon-derived stem/progenitor cell aging:defective self-renewal and altered fate. Aging Cell, 2010, 9(5):911-915.
6.	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.
7.	胡超, 唐康来, 陈万, 等. 大鼠跟腱来源肌腱干细胞的分离培养及鉴定. 第三军医大学学报, 2013, 35(11):1097-1101.
8.	Piek E, Heldin CH, Ten P Dijke. Specificity, diversity, and regulation in TGF-beta superfamily signaling. FASEB J, 1999, 13(15):2105-2124.
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11.	Kamiya Y, Miyazono K, Miyazawa K. Specificity of the inhibitory effects of Dad on TGF-beta family type Ⅰ receptors, Thickveins, Saxophone, and Baboon in Drosophila. FEBS Lett, 2008, 582(17):2496-2500.
12.	邹淑娟, 胡静. 机械张力对人成骨样细胞TGF-β表达的影响. 现代口腔医学杂志, 2002, 16(3):214-215.
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18.	Dennler S, Itoh S, Vivien D, et al. Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J, 1998, 17(11):3091-3100.
19.	Gao S, Steffen J, Laughon A. Dpp-responsive silencers are bound by a trimeric Mad-Medea complex. J Biol Chem, 2005, 280(43):36158-36164.
20.	Shi Y, Wang YF, Jayaraman L, et al. Crystal structure of a Smad MH1 domain bound to DNA:insights on DNA binding in TGF-beta signaling. Cell, 1998, 94(5):585-594.
21.	Choi ME, Ding Y, Kim SI. TGF-β signaling via TAK1 pathway:role in kidney fibrosis. Semin Nephrol, 2012, 32(3):244-252.
22.	Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signaling. Nature, 2003, 425(6958):577-584.
23.	Robbins JR, Evanko SP, Vogel KG. Mechanical loading and TGF-beta regulate proteoglycan synthesis in tendon. Arch Biochem Biophys, 1997, 342(2):203-211.
24.	Li Z, Kupcsik L, Yao SJ, et al. Mechanical load modulates chondrogenesis of human mesenchymal stem cells through the TGF-beta pathway. J Cell Mol Med, 2010, 14(6A):1338-1346.
25.	Balooch G, Balooch M, Nalla RK, et al. TGF-beta regulates the mechanical properties and composition of bone matrix. Proc Natl Acad Sci U S A, 2005, 102(52):18813-18818.
26.	Schmierer B, Hill CS. TGFbeta-SMAD signal transduction:molecular specificity and functional flexibility. Nat Rev Mol Cell Biol, 2007, 8(12):970-982.
27.	Schoeberl B, Eichler-Jonsson C, Gilles ED, et al. Computational modeling of the dynamics of the MAP kinase cascade activated by surface and internalized EGF receptors. Nat Biotechnol, 2002, 20(4):370-375.
28.	Mendias CL, Gumucio JP, Lynch EB. Mechanical loading and TGF-β change the expression of multiple miRNAs in tendon fibroblasts. J Appl Physiol (1985), 2012, 113(1):56-62.
29.	Podos SD, Hanson KK, Wang YC, et al. The DSmurf ubiquitin-protein ligase restricts BMP signaling spatially and temporally during Drosophila embryogenesis. Dev Cell, 2001, 1(4):567-578.

1. Kannus P, Józsa L. Histopathological changes preceding spontaneous rupture of a tendon. A controlled study of 891 patients. J Bone Joint Surg (Am), 1991, 73(10):1507-1525.
2. Wang JH, Iosifidis MI, Fu FH. Biomechanical basis for tendinopathy. Clin Orthop Relat Res, 2006, 443:320-332.
3. 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.
4. Zhang J, Wang JH. Characterization of differential properties of rabbit tendon stem cells and tenocytes. BMC Musculoskelet Disord, 2010, 11:10.
5. Zhou Z, Akinbiyi T, Xu L, et al. Tendon-derived stem/progenitor cell aging:defective self-renewal and altered fate. Aging Cell, 2010, 9(5):911-915.
6. 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.
7. 胡超, 唐康来, 陈万, 等. 大鼠跟腱来源肌腱干细胞的分离培养及鉴定. 第三军医大学学报, 2013, 35(11):1097-1101.
8. Piek E, Heldin CH, Ten P Dijke. Specificity, diversity, and regulation in TGF-beta superfamily signaling. FASEB J, 1999, 13(15):2105-2124.
9. Rojas A, Padidam M, Cress D, et al. TGF-beta receptor levels regulate the specificity of signaling pathway activation and biological effects of TGF-beta. Biochim Biophys Acta, 2009, 1793(7):1165-1173.
10. Karamichos D, Hutcheon AE, Zieske JD. Reversal of fibrosis by TGF-beta3 in a 3D in vitro model. Exp Eye Res, 2014, 124:31-36.
11. Kamiya Y, Miyazono K, Miyazawa K. Specificity of the inhibitory effects of Dad on TGF-beta family type Ⅰ receptors, Thickveins, Saxophone, and Baboon in Drosophila. FEBS Lett, 2008, 582(17):2496-2500.
12. 邹淑娟, 胡静. 机械张力对人成骨样细胞TGF-β表达的影响. 现代口腔医学杂志, 2002, 16(3):214-215.
13. Maeda T, Sakabe T, Sunaga A, et al. Conversion of mechanical force into TGF-β-mediated biochemical signals. Curr Biol, 2011, 21(11):933-941.
14. Zhao B, Chen YG. Regulation of TGF-β Signal Transduction. Scientifica (Cairo), 2014, 2014:874065.
15. Massagué J, Blain SW, Lo RS. TGFbeta signaling in growth control, cancer, and heritable disorders. Cell, 2000, 103(2):295-309.
16. Ross S, Cheung E, Petrakis TG, et al. Smads orchestrate specific histone modifications and chromatin remodeling to activate transcription. EMBO J, 2006, 25(19):4490-4502.
17. Feng XH, Derynck R. Specificity and versatility in tgf-beta signaling through Smads. Annu Rev Cell Dev Biol, 2005, 21:659-693.
18. Dennler S, Itoh S, Vivien D, et al. Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J, 1998, 17(11):3091-3100.
19. Gao S, Steffen J, Laughon A. Dpp-responsive silencers are bound by a trimeric Mad-Medea complex. J Biol Chem, 2005, 280(43):36158-36164.
20. Shi Y, Wang YF, Jayaraman L, et al. Crystal structure of a Smad MH1 domain bound to DNA:insights on DNA binding in TGF-beta signaling. Cell, 1998, 94(5):585-594.
21. Choi ME, Ding Y, Kim SI. TGF-β signaling via TAK1 pathway:role in kidney fibrosis. Semin Nephrol, 2012, 32(3):244-252.
22. Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signaling. Nature, 2003, 425(6958):577-584.
23. Robbins JR, Evanko SP, Vogel KG. Mechanical loading and TGF-beta regulate proteoglycan synthesis in tendon. Arch Biochem Biophys, 1997, 342(2):203-211.
24. Li Z, Kupcsik L, Yao SJ, et al. Mechanical load modulates chondrogenesis of human mesenchymal stem cells through the TGF-beta pathway. J Cell Mol Med, 2010, 14(6A):1338-1346.
25. Balooch G, Balooch M, Nalla RK, et al. TGF-beta regulates the mechanical properties and composition of bone matrix. Proc Natl Acad Sci U S A, 2005, 102(52):18813-18818.
26. Schmierer B, Hill CS. TGFbeta-SMAD signal transduction:molecular specificity and functional flexibility. Nat Rev Mol Cell Biol, 2007, 8(12):970-982.
27. Schoeberl B, Eichler-Jonsson C, Gilles ED, et al. Computational modeling of the dynamics of the MAP kinase cascade activated by surface and internalized EGF receptors. Nat Biotechnol, 2002, 20(4):370-375.
28. Mendias CL, Gumucio JP, Lynch EB. Mechanical loading and TGF-β change the expression of multiple miRNAs in tendon fibroblasts. J Appl Physiol (1985), 2012, 113(1):56-62.
29. Podos SD, Hanson KK, Wang YC, et al. The DSmurf ubiquitin-protein ligase restricts BMP signaling spatially and temporally during Drosophila embryogenesis. Dev Cell, 2001, 1(4):567-578.

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Chinese Journal of Reparative and Reconstructive Surgery

EFFECTS OF TRANSFORMING GROWTH FACTOR β₃ AT DIFFERENT CONCENTRATIONS ON DIFFERENTIATION OF TEENDON STEM CELLS IN VITRO

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EFFECTS OF TRANSFORMING GROWTH FACTOR β3 AT DIFFERENT CONCENTRATIONS ON DIFFERENTIATION OF TEENDON STEM CELLS IN VITRO

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EFFECTS OF TRANSFORMING GROWTH FACTOR β₃ AT DIFFERENT CONCENTRATIONS ON DIFFERENTIATION OF TEENDON STEM CELLS IN VITRO