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

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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

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2.	胡琪, 张友乐, 朱伟, 等. 异体肌腱移植基础研究与临床应用的现状. 2011第八届北京骨科年会. 北京: 2011.
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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. 慢性腱病治疗的新策略: 调控肌腱干细胞成肌腱分化促进肌腱愈合. 中国矫形外科杂志, 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, 张国强, 等. 肌腱干细胞与骨髓间充质干细胞促进髌腱愈合的对比研究. 浙江大学学报(医学版), 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.	张发惠, 郑和平, 张朝春, 等. 冷冻温度对大鼠肌腱超微结构及力学性能的影响. 解剖学杂志, 2004, 27(6): 664-667.
16.	刘英杰. 肌腱组织的深低温保存研究. 石家庄: 河北医科大学, 2007.
17.	唐林俊, 程国良, 方光荣, 等. 反复冻融及超深低温处理的异体肌腱移植实验研究. 中国修复重建外科杂志, 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, Fässler 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, Fässler 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, 张友乐, 朱伟, 等. 异体肌腱移植基础研究与临床应用的现状. 2011第八届北京骨科年会. 北京: 2011.
3. 孙燕琨, 张友乐, 孙磊, 等. 深低温冷冻肌腱细胞活性的研究. 中华手外科杂志, 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. 慢性腱病治疗的新策略: 调控肌腱干细胞成肌腱分化促进肌腱愈合. 中国矫形外科杂志, 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, 张国强, 等. 肌腱干细胞与骨髓间充质干细胞促进髌腱愈合的对比研究. 浙江大学学报(医学版), 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. 张发惠, 郑和平, 张朝春, 等. 冷冻温度对大鼠肌腱超微结构及力学性能的影响. 解剖学杂志, 2004, 27(6): 664-667.
16. 刘英杰. 肌腱组织的深低温保存研究. 石家庄: 河北医科大学, 2007.
17. 唐林俊, 程国良, 方光荣, 等. 反复冻融及超深低温处理的异体肌腱移植实验研究. 中国修复重建外科杂志, 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, Fässler 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, Fässler 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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