ISOLATION OF RAT PATELLAR TENDON STEM CELLS AND EFFECT OF MECHANICAL STRETCHING ON Sox-9 EXPRESSION_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

QINShengnan ¹ ,  WANGWen ¹ , FUShiquan ² , CHENGYushan ² , CHENHonghui ¹ , DONGFei ¹ , CHENQiming ² , LIAiguo ¹

1. Guangzhou Institute of Traumatic Surgery, Department of Orthopaedics, Guangzhou Red Cross Hospital, the 4th Affiliated Hospital of Jinan University, Guangzhou Guangdong, 510220, P. R. China;
2. Department of Orthopaedics & Traumatology, Prince of Wales Hospital, the Chinese University of Hong Kong;

Corresponding author：

WANGWen, Email: wangwarren88@hotmail.com

Keywords：

Tendon stem cells; Sox-9; Mechnical stretching; Rat

DOI：

10.7507/1002-1892.20150190

Video：

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Objective To isolate the tendon stem cells (TSCs) from rat patellar tendon and to investigate the effect of mechanical stretching on the expression of Sox-9. Methods TSCs were isolated from Sprague Dawley rat (12 weeks old) patellar tendon by collagenase digestion and low density culture. The cell colony morphology and number were observed by crystal violet staining;the cell morphology was observed by inverted phase contrast microscope, and the immunophenotypes of mesenchymal stem cells (MSCs) were determined by flow cytometry. The TSCs at passage 3 was given the mechanical stretching at 4%, 0.17 Hz for 4 hours and 24 hours in the experimental group, and cells without stretching was used as control. The Sox-9 gene and protein expressions were detected by real-time fluorescence quantitative PCR and Western blot. Results Primary cells showed clonal growth and star shape;after subculture, cells at passage 1 showed fibroblast-like shape. The cells formed cell colonies after 7 days;the expressions were positive for CD29, CD44, and CD90 and negative for CD45. The result of real-time fluorescence quantitative PCR showed that Sox-9 gene was down-regulated at 4 hours after mechanical stretching compared with control (P<0.05), and up-regulated at 24 hours after mechanical stretching when compared with control group (P<0.05). The result of Western blot showed that Sox-9 protein expression was lower at 4 hours after stretching, but higher at 24 hours after mechanical stretching than that in control group (P<0.05). Conclusion The rat patellar TSCs can be isolated successfully, and mechanical stretching inhibits the Sox-9 expression, but the inhibited effect might stimulate the Sox-9 expression after the mechanical stretching effect disappears.

Citation： QINShengnan, WANGWen, FUShiquan, CHENGYushan, CHENHonghui, DONGFei, CHENQiming, LIAiguo. ISOLATION OF RAT PATELLAR TENDON STEM CELLS AND EFFECT OF MECHANICAL STRETCHING ON Sox-9 EXPRESSION. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(7): 884-888. doi: 10.7507/1002-1892.20150190 Copy

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12.	Burgess KE,Graham-Smith P,Pearson SJ.Effect of acute tensile loading on gender-specific tendon structural and mechanical properties.J Orthop Res,2009,27(4):510-516.
13.	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.
14.	Yang Z,Huang CY,Candiotti KA,et al.Sox-9 facilitates differentiation of adipose tissue-derived stem cells into a chondrocyte-like phenotype in vitro.J Orthop Res,2011,29(8):1291-1297.
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1. Wang RY,Chan RC,Tsai MW.Functional electrical stimulation on chronic and acute hemiplegic shoulder subluxation.Am J Phys Med Rehabil,2000,79(4):385-390.
2. Friedenstein AJ.Precursor cells of mechanocytes.Int Rev Cytol,1976,47:327-359.
3. Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Science,1999,284(5411):143-147.
4. Tan Q,Lui PP,Rui YF.Effect of in vitro passaging on the stem cell-related properties of tendon-derived stem cells-implications in tissue engineering.Stem Cells Dev,2012,21(5):790-800.
5. Elkasrawy M,Fulzele S,Bowser M,et al.Myostatin (GDF-8) inhibits chondrogenesis and chondrocyte proliferation in vitro by suppressing Sox-9 expression.Growth Factors,2011,29(6):253-262.
6. Yang HN,Park JS,Woo DG,et al.Chondrogenesis of mesenchymal stem cells and dedifferentiated chondrocytes by transfection with SOX Trio genes.Biomaterials,2011,32(30):7695-7704.
7. Akiyama H.Sox family regulate chondrogenesis.Clin Calcium,2006,16(2):368-372.
8. 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.
9. Rui YF,Lui PP,Ni M,et al.Mechanical loading increased BMP-2 expression which promoted osteogenic differentiation of tendon-derived stem cells.J Orthop Res,2011,29(3):390-396.
10. 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.
11. Bayer ML,Schjerling P,Herchenhan A,et al.Release of tensile strain on engineered human tendon tissue disturbs cell adhesions,changes matrix architecture,and induces an inflammatory phenotype.PLoS One,2014,9(1):e86078.
12. Burgess KE,Graham-Smith P,Pearson SJ.Effect of acute tensile loading on gender-specific tendon structural and mechanical properties.J Orthop Res,2009,27(4):510-516.
13. 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.
14. Yang Z,Huang CY,Candiotti KA,et al.Sox-9 facilitates differentiation of adipose tissue-derived stem cells into a chondrocyte-like phenotype in vitro.J Orthop Res,2011,29(8):1291-1297.
15. Kelly DJ,Jacobs CR.The role of mechanical signals in regulating chondrogenesis and osteogenesis of mesenchymal stem cells.Birth Defects Res C Embryo Today,2010,90(1):75-85.

Chinese Journal of Reparative and Reconstructive Surgery

ISOLATION OF RAT PATELLAR TENDON STEM CELLS AND EFFECT OF MECHANICAL STRETCHING ON Sox-9 EXPRESSION

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