Effects of different mechanical stretch conditions on differentiation of rat tendon stem cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LI Pao ¹ , GAO Shang ¹ , ZHOU Mei ¹ , TANG Hong ¹ , MU Miduo ¹ , ZHANG Jiqiang ² ,  TANG Kanglai 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, Chongqing, 400038, P.R.China;

Corresponding?author：

TANG Kanglai, Email: tangkanglai@hotmail.com

Keywords：

Mechanical stretch; tendon stem cells; differentiation; rat

DOI：

10.7507/1002-1892.201611102

Video：

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Objective To investigate the effects of different mechanical stretch conditions on the differentiation of rat tendon stem cells (TSCs), to find the best uniaxial cyclic stretching for TSCs tenogenic differentiation, osteogenic differentiation, and adipogenic differentiation. Methods TSCs were isolated from the Achilles tendons of 8-week-old male Sprague Dawley rats by enzymatic digestion method and cultured. The TSCs at passage 3 were randomly divided into 5 groups: group A (stretch strength of 4% and frequency of 1 Hz), group B (stretch strength of 4% and frequency of 2 Hz), group C (stretch strength of 8% and frequency of 1 Hz), group D (stretch strength of 8% and frequency of 2 Hz), and group E (static culture). At 12, 24, and 48 hours after mechanical stretch, the mRNA expressions of the tenogenic differentiation related genes [Scleraxis (SCX) and Tenascin C (TNC)], the osteogenic differentiation related genes [runt related transcription factor 2 (RUNX2) and distal-less homeobox 5 (DLX5)], and the adipogenic differentiation related genes [CCAAT-enhancer-binding protein-α (CEBPα) and lipoprteinlipase (LPL)] were detected by real-time fluorescent quantitative PCR and the protein expressions of TNC, CEBPα, and RUNX2 were detected by Western blot. Results The mRNA expressions of SCX and TNC in group B were significantly higher than those in groups A, C, D, and E at 24 hours after mechanical stretch (P<0.05). The mRNA expressions of CEBPα and LPL in group D were significantly higher than those in groups A, B, C, and E at 48 hours after mechanical stretch (P<0.05). The mRNA expressions of RUNX2 and DLX5 in group C were significantly higher than those in groups A, B, D, and E at 24 hours after mechanical stretch (P<0.05). Western blot detection showed that higher protein expression of TNC in group B than group E at each time point after mechanical stretch (P<0.05), and the protein expression of CEBPα was significantly inhibited when compared with group E at 24 hours after mechanical stretch (P<0.05). At 24 hours after mechanical stretch, the protein expression of RUNX2 in group C was significantly higher than that in group E (P<0.05); and the protein expression of TNC was significantly lower than that in group E at 24 and 48 hours after mechanical stretch (P<0.05). At 48 hours after mechanical stretch, the protein expression of CEBPα was significantly increased and the protein expression of TNC was significantly decreased in group D when compared with group E (P<0.05), but no significant difference was found in the protein expression of RUNX2 between groups D and E (P>0.05). Conclusion The mechanical strain could promote differentiation of TSCs, and different parameter of stretch will lead to different differentiation. The best stretch condition for tenogenic differentiation is 4% strength and 2 Hz frequency for 24 hours; the best stretch condition for osteogenic differentiation is 8% strength and 1 Hz frequency for 24 hours; and the best stretch condition for adipogenic differentiation is 8% strength and 2 Hz frequency for 48 hours.

Citation： LI Pao, GAO Shang, ZHOU Mei, TANG Hong, MU Miduo, ZHANG Jiqiang, TANG Kanglai. Effects of different mechanical stretch conditions on differentiation of rat tendon stem cells. Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(4): 481-488. doi: 10.7507/1002-1892.201611102 Copy

1.	?Wren T, Yerby SA, Beaupré GS, et al. Mechanical properties of the human achilles tendon. Clin Biomech (Bristol Avon), 2001, 16(3): 245-251.
2.	魏傳付, 王予彬, 王惠芳. 肌腱腱病發病機制的分子生物學研究進展. 中國康復醫學雜志, 2011, 26(1): 90-93.
3.	Kvist M. Achilles tendon injuries in athletes. Sports Med, 1994, 18(3): 173-201.
4.	Magnan B, Bondi M, Pierantoni S, et al. The pathogenesis of Achilles tendinopathy: a systematic review. Foot Ankle Surg, 2014, 20(3): 154-159.
5.	Lopez RG, Jung HG. Achilles tendinosis: treatment options. Clin Orthop Surg, 2015, 7(1): 1-7.
6.	唐康來, 陳磊. 跟腱病的外科治療策略. 中國骨與關節外科, 2012, 5(4): 306-309.
7.	Riley G. The pathogenesis of tendinopathy. A molecular perspective. Rheumatology (Oxford), 2004, 43(2): 131-142.
8.	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.
9.	Chen L, Dong SW, Liu JP, et al. Synergy of tendon stem cells and platelet-rich plasma in tendon healing. J Orthop Res, 2012, 30(6): 991-997.
10.	Zhang J, Wang JH. BMP-2 mediates PGE(2)-induced reduction of proliferation and osteogenic differentiation of human tendon stem cells. J Orthop Res, 2012, 30(1): 47-52.
11.	Wang J, Wang CD, Zhang N, et al. Mechanical stimulation orchestrates the osteogenic differentiation of human bone marrow stromal cells by regulating HDAC1. Cell Death Dis, 2016, 7: e2221.
12.	Parvizi M, Bolhuis-Versteeg LA, Poot AA, et al. Efficient generation of smooth muscle cells from adipose-derived stromal cells by 3D mechanical stimulation can substitute the use of growth factors in vascular tissue engineering. Biotechnol J, 2016, 11(7): 932-944.
13.	Sun L, Qu L, Zhu R, et al. Effects of Mechanical Stretch on Cell Proliferation and Matrix Formation of Mesenchymal Stem Cell and Anterior Cruciate Ligament Fibroblast. Stem Cells Int, 2016, 2016: 9842075.
14.	Lin X, Shi Y, Cao Y, et al. Recent progress in stem cell differentiation directed by material and mechanical cues. Biomed Mater, 2016, 11(1): 014109.
15.	Zhang J, Wang JH. Characterization of differential properties of rabbit tendon stem cells and tenocytes. BMC Musculoskelet Disord, 2010, 11: 10.
16.	Liu X, Chen W, Zhou Y, et al. Mechanical Tension Promotes the Osteogenic Differentiation of Rat Tendon-derived Stem Cells Through the Wnt5a/Wnt5b/JNK Signaling Pathway. Cell Physiol Biochem, 2015, 36(2): 517-530.
17.	Li R, Liang L, Dou Y, et al. Mechanical strain regulates osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells. Biomed Res Int, 2015, 2015: 873251.
18.	胡超, 唐康來, 陳萬, 等. 大鼠跟腱來源肌腱干細胞的分離培養及鑒定. 第三軍醫大學學報, 2013, 35(11): 1097-1101.
19.	曹洪輝, 唐康來, 陳磊, 等. 新型肌腱細胞循環牽伸載荷系統的研制及運行效果觀察. 第三軍醫大學學報, 2010(06): 507-510.
20.	Connizzo BK, Yannascoli SM, Soslowsky LJ. Structure-function relationships of postnatal tendon development: a parallel to healing. Matrix Biol, 2013, 32(2): 106-116.
21.	胡超, 李旭. 基于力學生物學的跟腱愈合研究現狀. 中國康復醫學雜志, 2015, 30(10): 1078-1081.
22.	Thorpe CT, Screen HR. Tendon Structure and Composition. Adv Exp Med Biol, 2016, 920: 3-10.
23.	Esquisatto MA, Joazeiro PP, Pimentel ER, et al. The effect of age on the structure and composition of rat tendon fibrocartilage. Cell Biol Int, 2007, 31(6): 570-577.
24.	Dado D, Sagi M, Levenberg S, et al. Mechanical control of stem cell differentiation. Regen Med, 2012, 7(1): 101-116.
25.	Wang JH, Komatsu I. Tendon Stem Cells: Mechanobiology and Development of Tendinopathy. Adv Exp Med Biol, 2016, 920: 53-62.
26.	Zhang J, Wang JH. Mechanobiological response of tendon stem cells: implications of tendon homeostasis and pathogenesis of tendinopathy. J Orthop Res, 2010, 28(5): 639-643.
27.	Zhang J, Yuan T, Wang JH. Moderate treadmill running exercise prior to tendon injury enhances wound healing in aging rats. Oncotarget, 2016, 7(8): 8498-8512.
28.	李國華, 韓亞軍, 伊力哈木·托合提, 等. 過度運動對大鼠岡上肌腱損傷和腱細胞凋亡的影響. 新疆醫科大學學報, 2011, 34(11): 1222-1227.
29.	Zhang J, Wang JH. Moderate Exercise Mitigates the Detrimental Effects of Aging on Tendon Stem Cells. PLoS One, 2015, 10(6): e0130454.
30.	Sharma P, Maffulli N. Biology of tendon injury: healing, modeling and remodeling. J Musculoskelet Neuronal Interact, 2006, 6(2): 181-190.
31.	Shi Y, Fu Y, Tong W, et al. Uniaxial mechanical tension promoted osteogenic differentiation of rat tendon-derived stem cells (rTDSCs) via the Wnt5a-RhoA pathway. J Cell Biochem, 2012, 113(10): 3133-3142.

1. ?Wren T, Yerby SA, Beaupré GS, et al. Mechanical properties of the human achilles tendon. Clin Biomech (Bristol Avon), 2001, 16(3): 245-251.
2. 魏傳付, 王予彬, 王惠芳. 肌腱腱病發病機制的分子生物學研究進展. 中國康復醫學雜志, 2011, 26(1): 90-93.
3. Kvist M. Achilles tendon injuries in athletes. Sports Med, 1994, 18(3): 173-201.
4. Magnan B, Bondi M, Pierantoni S, et al. The pathogenesis of Achilles tendinopathy: a systematic review. Foot Ankle Surg, 2014, 20(3): 154-159.
5. Lopez RG, Jung HG. Achilles tendinosis: treatment options. Clin Orthop Surg, 2015, 7(1): 1-7.
6. 唐康來, 陳磊. 跟腱病的外科治療策略. 中國骨與關節外科, 2012, 5(4): 306-309.
7. Riley G. The pathogenesis of tendinopathy. A molecular perspective. Rheumatology (Oxford), 2004, 43(2): 131-142.
8. 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.
9. Chen L, Dong SW, Liu JP, et al. Synergy of tendon stem cells and platelet-rich plasma in tendon healing. J Orthop Res, 2012, 30(6): 991-997.
10. Zhang J, Wang JH. BMP-2 mediates PGE(2)-induced reduction of proliferation and osteogenic differentiation of human tendon stem cells. J Orthop Res, 2012, 30(1): 47-52.
11. Wang J, Wang CD, Zhang N, et al. Mechanical stimulation orchestrates the osteogenic differentiation of human bone marrow stromal cells by regulating HDAC1. Cell Death Dis, 2016, 7: e2221.
12. Parvizi M, Bolhuis-Versteeg LA, Poot AA, et al. Efficient generation of smooth muscle cells from adipose-derived stromal cells by 3D mechanical stimulation can substitute the use of growth factors in vascular tissue engineering. Biotechnol J, 2016, 11(7): 932-944.
13. Sun L, Qu L, Zhu R, et al. Effects of Mechanical Stretch on Cell Proliferation and Matrix Formation of Mesenchymal Stem Cell and Anterior Cruciate Ligament Fibroblast. Stem Cells Int, 2016, 2016: 9842075.
14. Lin X, Shi Y, Cao Y, et al. Recent progress in stem cell differentiation directed by material and mechanical cues. Biomed Mater, 2016, 11(1): 014109.
15. Zhang J, Wang JH. Characterization of differential properties of rabbit tendon stem cells and tenocytes. BMC Musculoskelet Disord, 2010, 11: 10.
16. Liu X, Chen W, Zhou Y, et al. Mechanical Tension Promotes the Osteogenic Differentiation of Rat Tendon-derived Stem Cells Through the Wnt5a/Wnt5b/JNK Signaling Pathway. Cell Physiol Biochem, 2015, 36(2): 517-530.
17. Li R, Liang L, Dou Y, et al. Mechanical strain regulates osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells. Biomed Res Int, 2015, 2015: 873251.
18. 胡超, 唐康來, 陳萬, 等. 大鼠跟腱來源肌腱干細胞的分離培養及鑒定. 第三軍醫大學學報, 2013, 35(11): 1097-1101.
19. 曹洪輝, 唐康來, 陳磊, 等. 新型肌腱細胞循環牽伸載荷系統的研制及運行效果觀察. 第三軍醫大學學報, 2010(06): 507-510.
20. Connizzo BK, Yannascoli SM, Soslowsky LJ. Structure-function relationships of postnatal tendon development: a parallel to healing. Matrix Biol, 2013, 32(2): 106-116.
21. 胡超, 李旭. 基于力學生物學的跟腱愈合研究現狀. 中國康復醫學雜志, 2015, 30(10): 1078-1081.
22. Thorpe CT, Screen HR. Tendon Structure and Composition. Adv Exp Med Biol, 2016, 920: 3-10.
23. Esquisatto MA, Joazeiro PP, Pimentel ER, et al. The effect of age on the structure and composition of rat tendon fibrocartilage. Cell Biol Int, 2007, 31(6): 570-577.
24. Dado D, Sagi M, Levenberg S, et al. Mechanical control of stem cell differentiation. Regen Med, 2012, 7(1): 101-116.
25. Wang JH, Komatsu I. Tendon Stem Cells: Mechanobiology and Development of Tendinopathy. Adv Exp Med Biol, 2016, 920: 53-62.
26. Zhang J, Wang JH. Mechanobiological response of tendon stem cells: implications of tendon homeostasis and pathogenesis of tendinopathy. J Orthop Res, 2010, 28(5): 639-643.
27. Zhang J, Yuan T, Wang JH. Moderate treadmill running exercise prior to tendon injury enhances wound healing in aging rats. Oncotarget, 2016, 7(8): 8498-8512.
28. 李國華, 韓亞軍, 伊力哈木·托合提, 等. 過度運動對大鼠岡上肌腱損傷和腱細胞凋亡的影響. 新疆醫科大學學報, 2011, 34(11): 1222-1227.
29. Zhang J, Wang JH. Moderate Exercise Mitigates the Detrimental Effects of Aging on Tendon Stem Cells. PLoS One, 2015, 10(6): e0130454.
30. Sharma P, Maffulli N. Biology of tendon injury: healing, modeling and remodeling. J Musculoskelet Neuronal Interact, 2006, 6(2): 181-190.
31. Shi Y, Fu Y, Tong W, et al. Uniaxial mechanical tension promoted osteogenic differentiation of rat tendon-derived stem cells (rTDSCs) via the Wnt5a-RhoA pathway. J Cell Biochem, 2012, 113(10): 3133-3142.

Chinese Journal of Reparative and Reconstructive Surgery

Effects of different mechanical stretch conditions on differentiation of rat tendon stem cells

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