[Differentiation of directly co-cultured bone marrow mesenchymal stem cells and ligament fibroblasts into ligament cells after induced by transforming growth factor β1 and basic fibroblast growth factor].

OBJECTIVE: To investigate the effect of transforming growth factor β1 (TGF-β1) and basic fibroblast growth factor 1 (bFGF-1) on the cellular activities, proliferation, and expressions of ligament-specific mRNA and proteins in bone marrow mesenchymal stem cells (BMSCs) and ligament fibroblasts (LFs) after directly co-cultured.

METHODS: BMSCs from 3-month-old Sprague Dawley rats were isolated and cultured using intensity gradient centrifugation. LFs were isolated using collagenase. The cells at passage 3 were divided into 6 groups: non-induced BMSCs group (group A), non-induced LFs group (group B), non-induced co-cultured BMSCs and LFs group (group C), induced BMSCs group (group D), induced LFs group (group E), and induced co-cultured BMSCs and LFs group (group F). The cellular activities and proliferation were examined by inverted contrast microscope and MTT; the concentrations of collagen type I and type III were determined by ELISA; and mRNA expressions of collagen types I and III, fibronectin, tenascin C, and matrix metalloproteinase 2 (MMP-2) were measured by real-time fluorescent quantitative PCR.

RESULTS: A single cell layer formed in the co-cultured cells under inverted contrast microscope. Group F had fastest cell fusion (> 90%). The MTT result indicated that group F showed the highest absorbance (A) value, followed by group D, and group B showed the lowest A value at 9 days after culture, showing significant difference (P < 0.05). Moreover, the result of ELISA showed that group F had the highest concentration of collagen type I and type III (P < 0.05); the concentration of collagen type III in group E was significantly higher than that in group D (P < 0.05), but no significant difference was found in the concentration of collagen type I between 2 groups (P > 0.05). The ratios of collagen type I to type III were 1.17, 1.19, 1.10, 1.25, 1.17, and 1.18 in groups A-F; group D was higher than the other groups. The real-time fluorescent quantitative PCR results revealed that the mRNA expressions of collagen type I and type III and fibronectin were highest in group F; the expression of tenascin C was highest in group D; the expression of MMP-2 was highest in group E; and all differencs were significant (P < 0.05).

CONCLUSION: Directly co-cultured BMSCs and LFs induced by TGF-β1 and bFGF-1 have higher cellular activities, proliferation, and expressions of ligament-specific mRNA and protein, which can be used as a potential source for ligament tissue engineering.