title: [Cartilage-derived morphogenetic protein 1 initiates chondrogenic differentiation of human dermal fibroblasts in vitro].
abstract: OBJECTIVE
  To investigate the feasibility of human dermal fibroblasts in vitro differentiation into chondrogenic phenotype with induction of cartilage-derived morphogenetic protein (CDMP) growth factor.
  
  
  METHODS
  Human dermal fibroblasts were isolated from foreskin and cultured in monolayer ex vivo. Dermal fibroblasts of passage2 was plated at density of 1 x 10(4) cells/cm(2) and induced with CDMP1 (100 ng/ml) in medium of F12 + 10% FBS. After 7 days of induction, morphology of cells was observed under phase-contrast microscopy and the length:width ratio of cells was calculated by Image Plus software analysis. Expression of type I, II, III collagen was detected by immunofluorescence and observed with confocal microscopy. The method of Western-Blot was applied to detect secretion of collagen type II. mRNA expression of chondrogenic related Sox9, Aggrecan as well as collagen type II, IX was detected by RT-PCR. The osteogenic related expression of collagen type X, Alkaline Phosphatase (AKP) was also detected by RT-PCR. Pellet cultured dermal fibroblasts at a density of 2 x 10(7) cells/ml was observed respectively for proteoglycan and collagen type II expression with Alcian blue and immunohistochemistry staining.
  
  
  RESULTS
  With the induction of CDMP1, the morphology of cells changed from spindle fibroblastic appearance to that of typical chondrocyte-like polygon shape. By Image Plus software analysis, it was found that the length/width ratio changed significantly from 7.40 +/- 1.30 of preinduction to 1.40 +/- 0.15 of post-induction (P < 0.05). No significant difference was found between the postinduction and normal chondrocyte (1.29 +/- 0.24). By confocal microscope observation, expression of collagen type II was found intracellularly in CDMP1 treated fibroblasts. Western-Blot detection confirmed collagen type II expression by 7 days induction. RT-PCR gene expression analysis of characteristic chondrogenic related genes, such as Sox9, Aggrecan as well as collagen type II, IX, revealed induction of chondrocytic phenotype in monolayered culture upon stimulation with CDMP1 for 7 days. While osteogenic related gene expression of collagen type X, AKP was not detected by RT-PCR, which indicates that osteogenic differentiation was not initiated by CDMP1 in 7 days culture. Histological staining of proteoglycan with Alcian blue and immunohistochemical staining cartilage specific type II collagen revealed deposition of typical cartilage extracellular matrix deposition in pellet cultured fibroblasts.
  
  
  CONCLUSIONS
  These results suggests human dermal derived fibroblast could be differentiated into chondrogenic phenotype with CDMP1 induction in vitro.
authors.affiliation: Department of Plastic and Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Second Medical University. Shanghai Tissue Engineering Research and Development Center, Shanghai 200011, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Cui
authors.forename: Shuo
authors.initials: S
authors.lastname: Yin
authors.forename: Chen-liang
authors.initials: CL
authors.lastname: Deng
authors.forename: Guang-hui
authors.initials: GH
authors.lastname: Yang
authors.forename: Fu-guo
authors.initials: FG
authors.lastname: Chen
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.forename: De-li
authors.initials: DL
authors.lastname: Liu
authors.forename: Yi-lin
authors.initials: YL
authors.lastname: Cao
mesh_terms: D000293:Adolescent; D000328:Adult; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D002356:Cartilage / Q000166:cytology / Q000254:growth & development; D002454:Cell Differentiation / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002648:Child; D019902:Chondrocytes / Q000166:cytology*; D020405:Dermis / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology*; D055428:Growth Differentiation Factor 5; D006801:Humans; D010641:Phenotype
chemical_list: D019485:Bone Morphogenetic Proteins; D055428:Growth Differentiation Factor 5