Chinese Journal of Tissue Engineering Research ›› 2019, Vol. 23 ›› Issue (29): 4644-4650.doi: 10.3969/j.issn.2095-4344.1803

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Scleraxis combined with basic fibroblast growth factor promotes the differentiation of human amniotic mesenchymal stem cells into human ligament fibroblasts in vitro

Sang Peng, Liu Yi   

  1. First Department of Orthopedics, Affiliated Hospital of Zunyi Medical University, Zunyi 563000, Guizhou Province, China
  • Revised:2019-04-17 Online:2019-10-18 Published:2019-10-18
  • Contact: Liu Yi, Professor, Master’s supervisor, First Department of Orthopedics, Affiliated Hospital of Zunyi Medical University, Zunyi 563000, Guizhou Province, China
  • About author:Sang Peng, Master, Attending physician, First Department of Orthopedics, Affiliated Hospital of Zunyi Medical University, Zunyi 563000, Guizhou Province, China

Abstract:

BACKGROUND: Human amniotic mesenchymal stem cells have multi-directional differentiation ability. Studies have shown that both Scleraxis and basic fibroblast growth factor can promote the differentiation of human amniotic mesenchymal stem cells into human ligament fibroblasts.
OBJECTIVE: To explore whether Scleraxis combined with basic fibroblast growth factor can promote the differentiation of human amniotic mesenchymal stem cells into human ligament fibroblasts and to observe their differentiation effects.
METHODS: The study protocol was approved by the ethic committee of the Affiliated Hospital of Zunyi Medical University, and written informed consent was obtained from each puerpera. The amniotic membrane from the full-term placenta was separated, and human amniotic mesenchymal stem cells were isolated by a two-step enzyme digestion. The morphology of the cells was observed by inverted phase contrast microscope. The structure of fresh human amniotic membrane was observed by hematoxylin-eosin staining. The third generation of human amniotic mesenchymal stem cells were cultured in four groups: (1) normally cultured human amnion mesenchymal stem cell culture (simple culture group); (2) human amniotic mesenchymal stem cells infected with Scleraxis gene lentivirus (Scleraxis group); (3) human amniotic mesenchymal stem cells induced by basic fibroblast growth factor (basic fibroblast growth factor group); (4) human amniotic mesenchymal stem cells induced by Scleraxis and basic fibroblast growth factor (combined induction group). The proliferation ability of the cells in each group was detected by cell counting kit-8 method at 3 days of cell culture. Real-time quantitative PCR was performed to evaluate the cell differentiation into human ligament fibroblasts in each group at 14 days of culture.
RESULTS AND CONCLUSION: (1) Under the inverted phase contrast microscope, the third-generation human amniotic mesenchymal stem cells were long-fusiform and exhibited vortex-like adherent growth. (2) Under the inverted phase contrast microscope, the fresh human amniotic membrane had an obvious layered structure, which was divided into five layers: epithelial layer, basal layer, dense layer, fibroblast layer and sponge layer. (3) Human amniotic mesenchymal stem cells strongly and stably expressed green fluorescence at 24 hours after infection with Scleraxis gene lentivirus. The morphology of human amniotic mesenchymal stem cells induced by basic fibroblast growth factor was changed, and after 14 days of induction its morphology was similar to that of human ligament fibroblasts. (4) The results of cell counting kit-8 showed that the four groups of cells showed a S-type growth. The Scleraxis group, basic fibroblast growth factor group, combined induction group had stronger proliferation ability than the simple culture group, but there was no significant difference in the proliferative capacity between the three groups. (5) Real-time fluorescent quantitative PCR showed that the mRNA expression levels of ligament-related genes type I collagen, type III collagen, Fibronectin, Tenascin-C and TNMD in the Scleraxis group, basic fibroblast growth factor group, and combined induction group were significantly higher than those in the simple culture group (P < 0.05). The mRNA expression levels of ligament-related genes in the combined induction group were significantly higher than those in the Slckleris group and basic fibroblast growth factor group (P < 0.05). These results indicate that the combination of Scleraxis gene and basic fibroblast growth factor can promote the differentiation of human amniotic mesenchymal stem cells into human ligament fibroblasts, providing a new idea for the treatment of ligament injury.

Key words: human amniotic mesenchymal stem cells, Scleraxis, basic fibroblast growth factor, directed differentiation, human ligament fibroblasts, lentivirus infection, tissue engineering

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