Effects of cryopreservation on biological characteristics of tendon-derived stem cells in rat patellar tendon_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

DAI Guangchun ^1,2 , LI Yingjuan ³ , XU Hongliang ⁴ , LIN Yucheng ¹ , LIU Junyan ^1,2 , XU Lin ⁴ ,  RUI Yunfeng ^1,2,4

1. Department of Orthpaedics, Zhongda Hospital, School of Medicine, Southeast University, Nanjing Jiangsu, 210000, P.R.China;
2. School of Medicine, Southeast University, Nanjing Jiangsu, 210000, P.R.China;
3. Department of Geriatrics, Zhongda Hospital, School of Medicine, Southeast University, Nanjing Jiangsu, 210000, P.R.China;
4. Department of Orthopaedics, Wuxi Branch, Zhongda Hospital, School of Medicine, Southeast University, Wuxi Jiangsu, 214000, P.R.China;

Corresponding?author：

RUI Yunfeng, Email: ruiyunfeng@126.com

Keywords：

Cryopreservation; tendon-derived stem cells; tendon defect; allogeneic tendon transplantation; tenogenic differentiation

DOI：

10.7507/1002-1892.201703033

Video：

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ObjectiveTo explore the effects of cryopreservation on the cell survival rate, cell viability, early apoptosis, migration ability, and tendon-related marker expression of tendon-derived stem cells (TDSCs) in rat patellar tendons.MethodsThe patellar tendon tissues were harvested from 12 4-month-old male Sprague Dawley rats; 12 patellar tendon tissues from 6 rats were cryopreserved (the experimental group), and the other 12 patellar tendon tissues were not treated (the control group). The patellar tendons were digested with 0.3% type I collagenase to obtain nucleated cells. The survival rate of nucleated cells was detected by trypan blue exclusion assay, and colony-forming ability by crystal violet staining. TDSCs were isolated and cultured to passage 3 (P3). The cell viability of TDSCs was detected by Alamar Blue method, the early apoptosis by Annexin V-FITC/PI assay, the cell migration ability by Transwell method, and the mRNA expressions of tendon-related markers [collagen type I (Col1α1), scleraxis (Scx), and tenomodulin (Tnmd)] by real-time quantitative PCR.ResultsThe survival rate of nucleated cells was 91.00%±3.63% in the control group, and was 61.65%±4.76% in the experimental group, showing significant difference (t=12.010, P=0.000). The formation of the primary nucleated cell clones was observed in 2 groups. At 12 days, the number of colonies forming of the experimental group [(8.41±0.33)/1 000 nucleated cells] was significantly lower than that of the control group [(15.19±0.47)/1 000 nucleated cells] (t=28.910, P=0.000). The percentage of TDSCs in the active nucleated cells in the experimental group (1.37%±0.09%) was significantly lower than that in the control group (1.67%±0.10%) (t=5.508, P=0.003). The growth trend of TDSCs (P3) in the 2 groups was consistent within 14 days. There was no significant difference in absorbance (A) value between 2 groups at each time point (P>0.05). The early apoptotic rate of TDSCs was 1.67%±0.06% in the experimental group and was 1.63%±0.06% in the control group, showing no significant difference (t=0.707, P=0.519). Under microscope, TDSCs adhered to the lower chamber of the Transwell chamber; the number of cells was 445.00±9.70 in the experimental group and was 451.50±12.66 in the control group, showing no significant difference (t=0.998, P=0.342). The relative mRNA expressions of Col1α1, Scx, and Tnmd were 3.498±0.065, 0.062±0.002, and (4.211±0.211)×10^–5 in the experimental group and were 3.499±0.113, 0.062±0.001, and (4.341±0.274)×10^–5 in the con-trol group, showing no significant difference (t=0.013, P=0.991; t=0.042, P=0.969; t=0.653, P=0.549).ConclusionThe survival rate of nucleated cells in cryopreserved rat tendon tissues is lower, but a large number of active TDSCs, and its cell viability, early apoptosis rate, migration ability in vitro, and cell tenogenic differentiation ability are remained.

Citation： DAI Guangchun, LI Yingjuan, XU Hongliang, LIN Yucheng, LIU Junyan, XU Lin, RUI Yunfeng. Effects of cryopreservation on biological characteristics of tendon-derived stem cells in rat patellar tendon. Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(7): 845-852. doi: 10.7507/1002-1892.201703033 Copy

1.	Sharma P, Maffulli N. Tendon injury and tendinopathy: healing and repair. J Bone Joint Surg (Am), 2005, 87(1): 187-202.
2.	胡琪, 張友樂, 朱偉, 等. 異體肌腱移植基礎研究與臨床應用的現狀. 2011第八屆北京骨科年會. 北京: 2011.
3.	孫燕琨, 張友樂, 孫磊, 等. 深低溫冷凍肌腱細胞活性的研究. 中華手外科雜志, 2006, 22(3): 133-136.
4.	Strickland JW. Development of flexor tendon surgery: twenty-five years of progress. J Hand Surg (Am), 2000, 25(2): 214-235.
5.	Li L, Clevers H. Coexistence of quiescent and active adult stem cells in mammals. Science, 2010, 327(5965): 542-545.
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.	林禹丞, 王宸, 芮云峰. 慢性腱病治療的新策略: 調控肌腱干細胞成肌腱分化促進肌腱愈合. 中國矯形外科雜志, 2013, 21(21): 2169-2176.
8.	Lui PP, Rui YF, Ni M, et al. Tenogenic differentiation of stem cells for tendon repair-what is the current evidence? J Tissue Eng Regen Med, 2011, 5(8): e144-e163.
9.	孔祥朋, 倪明, 張國強, 等. 肌腱干細胞與骨髓間充質干細胞促進髕腱愈合的對比研究. 浙江大學學報(醫學版), 2016, 45(2): 112-119.
10.	Zeng YC, Tang HR, Zeng LP, et al. Assessment of the effect of different vitrification solutions on human ovarian tissue after short-term xenotransplantation onto the chick embryo chorioallantoic membrane. Mol Reprod Dev, 2016, 83(4): 359-369.
11.	Iancu CV, Tivol WF, Schooler JB, et al. Electron cryotomography sample preparation using the Vitrobot. Nat Protoc, 2006, 1(6): 2813-2819.
12.	Lucci CM, Kacinskis MA, Lopes LH, et al. Effect of different cryoprotectants on the structural preservation of follicles in frozen zebu bovine (Bos indicus) ovarian tissue. Theriogenology, 2004, 61(6): 1101-1114.
13.	Oosthuyzen W, Sime NE, Ivy JR, et al. Quantification of human urinary exosomes by nanoparticle tracking analysis. J Physiol, 2013, 591(23): 5833-5842.
14.	Dumont F, Marechal PA, Gervais P. Cell size and water permeability as determining factors for cell viability after freezing at different cooling rates. Appl Environ Microbiol, 2004, 70(1): 268-272.
15.	張發惠, 鄭和平, 張朝春, 等. 冷凍溫度對大鼠肌腱超微結構及力學性能的影響. 解剖學雜志, 2004, 27(6): 664-667.
16.	劉英杰. 肌腱組織的深低溫保存研究. 石家莊: 河北醫科大學, 2007.
17.	唐林俊, 程國良, 方光榮, 等. 反復凍融及超深低溫處理的異體肌腱移植實驗研究. 中國修復重建外科雜志, 1998, 12(4): 241-245.
18.	Tanaka M, Sakai D, Hiyama A, et al. Effect of cryopreservation on canine and human activated nucleus pulposus cells: a feasibility study for cell therapy of the intervertebral disc. Biores Open Access, 2013, 2(4): 273-282.
19.	Chan SC, Lam S, Leung VY, et al. Minimizing cryopreservation-induced loss of disc cell activity for storage of whole intervertebral discs. Eur Cell Mater, 2010, 19: 273-277.
20.	Bravo D, Rigley TH, Gibran N, et al. Effect of storage and preservation methods on viability in transplantable human skin allografts. Burns, 2000, 26(4): 367-378.
21.	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.
22.	Zhang J, Wang JH. Characterization of differential properties of rabbit tendon stem cells and tenocytes. BMC Musculoskelet Disord, 2010, 11: 10.
23.	Muthyala S, Safley S, Gordan K, et al. The effect of hypoxia on free and encapsulated adult porcine islets-an in vitro study. Xenotransplantation, 2017, 24(1): e12275.
24.	Kohler J, Popov C, Klotz B, et al. Uncovering the cellular and molecular changes in tendon stem/progenitor cells attributed to tendon aging and degeneration. Aging Cell, 2013, 12(6): 988-999.
25.	Rottner K, Stradal TE. Actin dynamics and turnover in cell motility. Curr Opin Cell Biol, 2011, 23(5): 569-578.
26.	Yin Z, Hu JJ, Yang L, et al. Single-cell analysis reveals a nestin+ tendon stem/progenitor cell population with strong tenogenic potentiality. Sci Adv, 2016, 2(11): e1600874.
27.	Kovacevic D, Rodeo SA. Biological augmentation of rotator cuff tendon repair. Clin Orthop Relat Res, 2008, 466(3): 622-633.
28.	Shukunami C, Takimoto A, Oro M, et al. Scleraxis positively regulates the expression of tenomodulin, a differentiation marker of tenocytes. Dev Biol, 2006, 298(1): 234-247.
29.	Docheva D, Hunziker EB, F?ssler R, et al. Tenomodulin is necessary for tenocyte proliferation and tendon maturation. Mol Cell Biol, 2005, 25(2): 699-705.
30.	Brandau O, Meindl A, F?ssler R, et al. A novel gene, tendin, is strongly expressed in tendons and ligaments and shows high homology with chondromodulin-I. Dev Dyn, 2001, 221(1): 72-80.

1. Sharma P, Maffulli N. Tendon injury and tendinopathy: healing and repair. J Bone Joint Surg (Am), 2005, 87(1): 187-202.
2. 胡琪, 張友樂, 朱偉, 等. 異體肌腱移植基礎研究與臨床應用的現狀. 2011第八屆北京骨科年會. 北京: 2011.
3. 孫燕琨, 張友樂, 孫磊, 等. 深低溫冷凍肌腱細胞活性的研究. 中華手外科雜志, 2006, 22(3): 133-136.
4. Strickland JW. Development of flexor tendon surgery: twenty-five years of progress. J Hand Surg (Am), 2000, 25(2): 214-235.
5. Li L, Clevers H. Coexistence of quiescent and active adult stem cells in mammals. Science, 2010, 327(5965): 542-545.
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. 林禹丞, 王宸, 芮云峰. 慢性腱病治療的新策略: 調控肌腱干細胞成肌腱分化促進肌腱愈合. 中國矯形外科雜志, 2013, 21(21): 2169-2176.
8. Lui PP, Rui YF, Ni M, et al. Tenogenic differentiation of stem cells for tendon repair-what is the current evidence? J Tissue Eng Regen Med, 2011, 5(8): e144-e163.
9. 孔祥朋, 倪明, 張國強, 等. 肌腱干細胞與骨髓間充質干細胞促進髕腱愈合的對比研究. 浙江大學學報(醫學版), 2016, 45(2): 112-119.
10. Zeng YC, Tang HR, Zeng LP, et al. Assessment of the effect of different vitrification solutions on human ovarian tissue after short-term xenotransplantation onto the chick embryo chorioallantoic membrane. Mol Reprod Dev, 2016, 83(4): 359-369.
11. Iancu CV, Tivol WF, Schooler JB, et al. Electron cryotomography sample preparation using the Vitrobot. Nat Protoc, 2006, 1(6): 2813-2819.
12. Lucci CM, Kacinskis MA, Lopes LH, et al. Effect of different cryoprotectants on the structural preservation of follicles in frozen zebu bovine (Bos indicus) ovarian tissue. Theriogenology, 2004, 61(6): 1101-1114.
13. Oosthuyzen W, Sime NE, Ivy JR, et al. Quantification of human urinary exosomes by nanoparticle tracking analysis. J Physiol, 2013, 591(23): 5833-5842.
14. Dumont F, Marechal PA, Gervais P. Cell size and water permeability as determining factors for cell viability after freezing at different cooling rates. Appl Environ Microbiol, 2004, 70(1): 268-272.
15. 張發惠, 鄭和平, 張朝春, 等. 冷凍溫度對大鼠肌腱超微結構及力學性能的影響. 解剖學雜志, 2004, 27(6): 664-667.
16. 劉英杰. 肌腱組織的深低溫保存研究. 石家莊: 河北醫科大學, 2007.
17. 唐林俊, 程國良, 方光榮, 等. 反復凍融及超深低溫處理的異體肌腱移植實驗研究. 中國修復重建外科雜志, 1998, 12(4): 241-245.
18. Tanaka M, Sakai D, Hiyama A, et al. Effect of cryopreservation on canine and human activated nucleus pulposus cells: a feasibility study for cell therapy of the intervertebral disc. Biores Open Access, 2013, 2(4): 273-282.
19. Chan SC, Lam S, Leung VY, et al. Minimizing cryopreservation-induced loss of disc cell activity for storage of whole intervertebral discs. Eur Cell Mater, 2010, 19: 273-277.
20. Bravo D, Rigley TH, Gibran N, et al. Effect of storage and preservation methods on viability in transplantable human skin allografts. Burns, 2000, 26(4): 367-378.
21. 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.
22. Zhang J, Wang JH. Characterization of differential properties of rabbit tendon stem cells and tenocytes. BMC Musculoskelet Disord, 2010, 11: 10.
23. Muthyala S, Safley S, Gordan K, et al. The effect of hypoxia on free and encapsulated adult porcine islets-an in vitro study. Xenotransplantation, 2017, 24(1): e12275.
24. Kohler J, Popov C, Klotz B, et al. Uncovering the cellular and molecular changes in tendon stem/progenitor cells attributed to tendon aging and degeneration. Aging Cell, 2013, 12(6): 988-999.
25. Rottner K, Stradal TE. Actin dynamics and turnover in cell motility. Curr Opin Cell Biol, 2011, 23(5): 569-578.
26. Yin Z, Hu JJ, Yang L, et al. Single-cell analysis reveals a nestin+ tendon stem/progenitor cell population with strong tenogenic potentiality. Sci Adv, 2016, 2(11): e1600874.
27. Kovacevic D, Rodeo SA. Biological augmentation of rotator cuff tendon repair. Clin Orthop Relat Res, 2008, 466(3): 622-633.
28. Shukunami C, Takimoto A, Oro M, et al. Scleraxis positively regulates the expression of tenomodulin, a differentiation marker of tenocytes. Dev Biol, 2006, 298(1): 234-247.
29. Docheva D, Hunziker EB, F?ssler R, et al. Tenomodulin is necessary for tenocyte proliferation and tendon maturation. Mol Cell Biol, 2005, 25(2): 699-705.
30. Brandau O, Meindl A, F?ssler R, et al. A novel gene, tendin, is strongly expressed in tendons and ligaments and shows high homology with chondromodulin-I. Dev Dyn, 2001, 221(1): 72-80.

Chinese Journal of Reparative and Reconstructive Surgery

Effects of cryopreservation on biological characteristics of tendon-derived stem cells in rat patellar tendon

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