title: [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].
abstract: 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).
  
  
  CONCLUSIONS
  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.
authors: Yi
authors: Y
authors: Liu
authors: Chenghao
authors: C
authors: Zhang
authors: Qinghong
authors: Q
authors: Fan
authors: Pengpeng
authors: P
authors: Sun
authors: Shuhong
authors: S
authors: Wu
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods*; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D024061:Collagen Type III / Q000235:genetics / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D005347:Fibroblasts; D006412:Hematopoietic Stem Cells; D008022:Ligaments; D020778:Matrix Metalloproteinase 2; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D023822:Tissue Engineering; D053773:Transforming Growth Factor beta1
chemical_list: D024042:Collagen Type I; D024061:Collagen Type III; D012333:RNA, Messenger; D053773:Transforming Growth Factor beta1; D016222:Fibroblast Growth Factor 2; D020778:Matrix Metalloproteinase 2