EXPERIMENTAL RESEARCH OF DIFFERENTIATION OF HUM AN AMNIOTIC MESENCHYM AL STEM CELLS INTO LIGAMENT CELLS IN VITRO_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

JINYing ¹ , LIYuwan ¹ , ZHANGChenghao ² , WUShuhong ¹ , CHENGDaixiong ³ ,  LIUYi ¹

1. Department of Orthopaedics, the Affiliated Hospital of Zunyi Medical College, Zunyi Guizhou, 563000, P. R. China;
2. Department of Orthopaedics, West China Hospital, Sichuan University;
3. Key Laboratory Cell Engineering of Guizhou Province, the Affiliated Hospital of Zunyi Medical College, Zunyi Guizhou, 563000, P. R. China;

Corresponding author：

LIUYi, Email: 13308529536@163.com

Keywords：

Human amniotic mesenchymal stem cells; Ligament tissue engineering; Transforming growth factor β₁; Basic fibroblast growth factor; Hyaluronic acid

DOI：

10.7507/1002-1892.20160048

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Objective To discuss whether human amniotic mesenchymal stem cells (hAMSCs) possesses the characteristic of mesenchymal stem cells, and could differentiate into ligament cells in vitro after induction. Methods The hAMSCs were separated through enzyme digestion, and the phenotypic characteristics of hAMSCs were tested through flow cytometry. The cells at passage 3 were cultured with L-DMEM/F12 medium containing transforming growth factor β₁ (TGF-β₁)+basic fibroblast growth factor (bFGF) (group A), containing hyaluronic acid (HA) (group B), containing TGF-β₁+bFGF+HA (group C), and simple L-DMEM/F12 medium (group D) as control group. The morphology changes of cells in each group were observed by inverted phase contrast microscope at 21 days after induction; the cellular activities and proliferation were examined by sulforhodamine (SRB) colorimetric method; and specific mRNA and protein expressions of ligament including collagen type I, collagen type III, and tenascin C (TNC) were measured by real-time fluorescence quantitative PCR and immunohistochemical staining. Results The flow cytometry result indicated that hAMSCs expressed mesenchymal stem cell phenotype. After 21 days of induction, the cells in groups A, B, and C grew like spindle-shaped fibroblasts under inverted phase contrast microscope, and cells showed single shape, obvious directivity, and compact arrangement in group C. The SRB result indicated that the cells in each group reached the peak of growth curve at 6 days; the cellular activities of groups A, B, and C were significantly higher than that of group D at 6 days after induction. Also, the immunohistochemical staining results showed that no expressions of TNC were detected in 4 groups at 7 days; expressions of collagen type I in groups A, B, and C were significantly higher than that in group D at 7, 14, and 21 days (P<0.001); the expressions of collagen type III in groups A, B, and C were significantly higher than that in group D at 14 and 21 days (P<0.001). There was an increasing tendency with time in collagen type I of group B, in collagen type III and TNC of groups A and C, showing significant difference among different time points (P<0.001). The real-time fluorescence quantitative PCR results revealed that the mRNA expressions of collagen type I and TNC in group C were significantly higher than those in groups A and B (P<0.05), and the mRNA expression of collagen type III in group B were significantly higher than that in groups A and C at 21 days (P<0.05). The mRNA expressions of collagen type I and TNC in groups A and C and mRNA expression of collagen type III in group C had an increasing tendency with time, showing significant difference among different time points (P<0.001). Conclusion The hAMSCs possesses the characteristics of mesenchymal stem cells and excellent proliferation capacity. After in vitro induction, the expressions of ligament specific genes can be up-regulated and the synthesis of ligament specific proteins can be also strengthened. As a result, it can be used as one of ligament tissue engineering seed cell sources.

Citation： JINYing, LIYuwan, ZHANGChenghao, WUShuhong, CHENGDaixiong, LIUYi. EXPERIMENTAL RESEARCH OF DIFFERENTIATION OF HUM AN AMNIOTIC MESENCHYM AL STEM CELLS INTO LIGAMENT CELLS IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(2): 237-244. doi: 10.7507/1002-1892.20160048 Copy

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2.	Van Eijk F, Saris DB, Riesle J, et al. Tissue engineering of ligaments:a comparison of bone marrow stromal cells, anterior cruciate ligament, and skin fibroblasts as cell source. Tissue Eng, 2004, 10(5-6):893-903.
3.	Niwa H, Masui S, Chambers I, et al. Phenotypic complementation establishes requirements for specific POU domain and generic transactivation function of Oct-3/4 in embryonic stem cells. Mol Cell Biol, 2002, 22(5):1526-1536.
4.	方宁, 张路, 宋秀军, 等. 人羊膜间充质干细胞的分离、培养及鉴定. 遵义医学院学报, 2009, 32(3):234-236.
5.	Bilic G, Zeisberger SM, Mallik AS, et al. Comparative characterization of cultured human term amnion epithelial and mesenchymal stromal cells for application in cell therapy. Cell Transplant, 2008, 17(8):955-968.
6.	Ilancheran S, Moodley Y, Manuelpillai U. Human fetal membranes:a source of stem cells for tissue regeneration and repair. Placenta, 2009, 30(1):2-10.
7.	Insausti CL, Blanquer M, García-Hernández AM, et al. Amniotic membrane-derived stem cells:immunomodulatory properties and potential clinical application. Stem Cells Cloning, 2014, 7:53-63.
8.	Parolini O, Caruso M. Review:Preclinical studies on placenta-derived cells and amniotic membrane:an update. Placenta, 2011, 32 Suppl 2:186-195.
9.	Eppley BL, Woodell JE, Higgins J. Platelet quantification and growth factor analysis from platelet-rich plasma:implications for wound healing. Plast Reconstr Surg, 2004, 114(6):1502-1508.
10.	Augello A, Kurth TB, De Bari C. Mesenchymal stem cells:a perspective from in vitro cultures to in vivo migration and niches. Eur Cell Mater, 2010, 20:121-133.
11.	Sahoo S, Teh TKh, He P, et al. Interface tissue engineering:next phase in musculoskeletal tissue repair. Ann Acad Med Singapore, 2011, 40(5):245-251.
12.	Kondo E, Yasuda K, Yamanaka M, et al. Effects of administration of exogenous growth factors on biomechanical properties of the elongation-type anterior cruciate ligament injury with partial laceration. Am J Sports Med, 2005, 3(2):188-196.
13.	Kobayashi D, Kurosaka M, Yoshiya S, et al. Effect of basic fibroblast growth factor on the healing of defects in the canine anterior cruciate ligament. Knee Surg Sports Traumatol Arthrosc, 1997, 5(3):189-194.
14.	周盛源, 朱巍, 陈雄生, 等. 转基因骨髓间充质干细胞修复严重韧带损伤的实验观察. 中华医学杂志, 2014, 94(19):1494-1498.
15.	刘毅, 张承昊, 范青洪, 等. TGF-β₁和bFGF-1对单层共培养 BMSCs 和韧带成纤维细胞韧带特异性的影响. 中国修复重建外科杂志, 2014, 28(11):1406-1412.
16.	Wiig ME, Amiel D, VandeBerg J, et al. The early effect of high molecular weight hyaluronan (hyaluronic acid) on anterior cruciate ligament healing:an experimental study in rabbits. J Orthop Res, 1990, 8(3):425-434.
17.	Pruksakorn D, Leuvitoonvechakij S, Pothacharoen P, et al. Diurnal variation of serum chondroitin sulfate WF6 and hyaluronic acid in the healthy, traumatic knee and the osteoarthritic knee. J Med Assoc Thai, 2015, 98(1):45-52.

1. Yates EW, Rupani A, Foley GT, et al. Ligament tissue engineering and its potential role in anterior cruciate ligament reconstruction. Stem Cells Int, 2012, 2012:438125.
2. Van Eijk F, Saris DB, Riesle J, et al. Tissue engineering of ligaments:a comparison of bone marrow stromal cells, anterior cruciate ligament, and skin fibroblasts as cell source. Tissue Eng, 2004, 10(5-6):893-903.
3. Niwa H, Masui S, Chambers I, et al. Phenotypic complementation establishes requirements for specific POU domain and generic transactivation function of Oct-3/4 in embryonic stem cells. Mol Cell Biol, 2002, 22(5):1526-1536.
4. 方宁, 张路, 宋秀军, 等. 人羊膜间充质干细胞的分离、培养及鉴定. 遵义医学院学报, 2009, 32(3):234-236.
5. Bilic G, Zeisberger SM, Mallik AS, et al. Comparative characterization of cultured human term amnion epithelial and mesenchymal stromal cells for application in cell therapy. Cell Transplant, 2008, 17(8):955-968.
6. Ilancheran S, Moodley Y, Manuelpillai U. Human fetal membranes:a source of stem cells for tissue regeneration and repair. Placenta, 2009, 30(1):2-10.
7. Insausti CL, Blanquer M, García-Hernández AM, et al. Amniotic membrane-derived stem cells:immunomodulatory properties and potential clinical application. Stem Cells Cloning, 2014, 7:53-63.
8. Parolini O, Caruso M. Review:Preclinical studies on placenta-derived cells and amniotic membrane:an update. Placenta, 2011, 32 Suppl 2:186-195.
9. Eppley BL, Woodell JE, Higgins J. Platelet quantification and growth factor analysis from platelet-rich plasma:implications for wound healing. Plast Reconstr Surg, 2004, 114(6):1502-1508.
10. Augello A, Kurth TB, De Bari C. Mesenchymal stem cells:a perspective from in vitro cultures to in vivo migration and niches. Eur Cell Mater, 2010, 20:121-133.
11. Sahoo S, Teh TKh, He P, et al. Interface tissue engineering:next phase in musculoskeletal tissue repair. Ann Acad Med Singapore, 2011, 40(5):245-251.
12. Kondo E, Yasuda K, Yamanaka M, et al. Effects of administration of exogenous growth factors on biomechanical properties of the elongation-type anterior cruciate ligament injury with partial laceration. Am J Sports Med, 2005, 3(2):188-196.
13. Kobayashi D, Kurosaka M, Yoshiya S, et al. Effect of basic fibroblast growth factor on the healing of defects in the canine anterior cruciate ligament. Knee Surg Sports Traumatol Arthrosc, 1997, 5(3):189-194.
14. 周盛源, 朱巍, 陈雄生, 等. 转基因骨髓间充质干细胞修复严重韧带损伤的实验观察. 中华医学杂志, 2014, 94(19):1494-1498.
15. 刘毅, 张承昊, 范青洪, 等. TGF-β₁和bFGF-1对单层共培养 BMSCs 和韧带成纤维细胞韧带特异性的影响. 中国修复重建外科杂志, 2014, 28(11):1406-1412.
16. Wiig ME, Amiel D, VandeBerg J, et al. The early effect of high molecular weight hyaluronan (hyaluronic acid) on anterior cruciate ligament healing:an experimental study in rabbits. J Orthop Res, 1990, 8(3):425-434.
17. Pruksakorn D, Leuvitoonvechakij S, Pothacharoen P, et al. Diurnal variation of serum chondroitin sulfate WF6 and hyaluronic acid in the healthy, traumatic knee and the osteoarthritic knee. J Med Assoc Thai, 2015, 98(1):45-52.

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Chinese Journal of Reparative and Reconstructive Surgery

EXPERIMENTAL RESEARCH OF DIFFERENTIATION OF HUM AN AMNIOTIC MESENCHYM AL STEM CELLS INTO LIGAMENT CELLS IN VITRO

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