Differentiation of bone marrow mesenchymal stem cells into fibrochondrocyte phenotype / 中国组织工程研究

ABSTRACT

BACKGROUND: The meniscus has limited ability in repairing itself after being injured. However, tissue engineering provides a new way to meniscus repair after injury. Bone marrow mesenchymal stem cells (BMSCs), which possess the potential of multi-directional differentiations, can be ideal seed cells in meniscus tissue engineering. OBJECTIVE: To investigate the feasibility of differentiation of in vitro cultured porcine BMSCs into fibrochondrocyte phenotypes in inductive medium. METHODS: BMSCs were isolated with whole bone marrow culture method. Then, BMSCs of the third passage were digested and incubated in a medium containing transforming growth factor-β1, insulin-like growth factor-Ⅰ, dexamethasone and ascorbic acid in a 24-well plate at a density of 2.0×10~4/cm~2 in the experimental group. While in the control group, the DMEM-LG complete culture medium containing no inductive factor were used instead. At day 7, 14 and 21 after induction respectively, Toluidine blue staining and immunocytochemical staining were performed to detect differentiation. MAIN OUTCOME MEASURES: ①Population double time (PDT) of BMSCs; ②Morphological changes of BMSCs under light microscope;③Proteoglycan expression;④Collagen type Ⅰ and type Ⅱ expression. RESULTS AND CONCLUSION: ① The PDT of the second passage BMSCs was 2 days, which was the shortest. The PDT prolonged relatively after the fourth passage, which were 5 to 9 days. ② The BMSCs changed from a spindle-like appearance into a polygonal shape after induction. ③ In the experimental group, toluidine blue staining resulted in hyacinthine-stained cytoplasm and the blue was even deeper in the area where cells were dense; The degree of staining increased with the increasing induction time. While in the control group, only nucleus of BMSCs were stained blue. ④ Collagen type Ⅰ immunocytochemical staining was positive in both the experimental and the control group and there was no difference of significance between various induction time. No collagen type Ⅱwas seen expressed in the control group, while in the experimental group it was seen to be expressed steadily after 14 days of induction. It is indicated tlat BMSCs can be induced to synthesize fibrochondrocyte-characterized extracellular matrixes in vitro, which suggests that BMSCs are available as seed cells in meniscus tissue engineering.