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F the scaffold fibers (Figure 6B, black double arrowheads), while chondrocytes PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/15957913 showed no staining (Figure 6B, black arrowhead). In general, the expression level of osteocalcin was low. The mean expression level in native ovine articular cartilage was 16 of the expression level found for the housekeeping gene GAPDH. Expanded chondrocytes showed levels between 0.06 and 1.9 , while chondrocytes embedded in PGA-fibrin scaffolds showed osteocalcin expression levels between 0.8 and 1.6 . There is no evidence that ovine chondrocytes differentiate intobone or osteogenic cells, when expanded or cultured in vitro in PGA-fibrin scaffolds.Discussion In the present study, we used an ovine in vitro model for scaffold-assisted cartilage grafts Methyl 3-fluoro-2-thiophenecarboxylate and showed that extensively expanded chondrocytes lose their chondrocyte phenotype, but re-differentiate in resorbable polyglycolic acid-fibrin polymer scaffolds in vitro as assessed by gene expression analysis and immune-histochemical staining of type II collagen. In addition, our data suggest that the extracellular matrix molecules aggrecan and type II collagen may be suitable markers for chondrocyte identity and potency, regarding the use of the ovine model for preclinical animal studies and quality assurance of humanEndres et al. Journal of Orthopaedic Surgery and Research 2012, 7:37 http://www.josr-online.com/content/7/1/Page 9 ofFigure 5 Semi-quantitative real-time gene expression analysis of ovine chondrocytes in three-dimensional scaffold-assisted cartilage grafts derived from 5th passage chondrocytes. Chondrocyte dedifferentiation and re-differentiation was analyzed by gene expression analysis of the typical chondrocytic marker genes aggrecan and type II1 collagen. Matrix remodeling was assessed by gene expression analysis of the matrix metalloproteinases (MMP)-1, -2 and -13 as well as the tissue inhibitors of metalloproteinases (TIMP)-1, -2 and -3. The expression level was calculated as the percentage of the expression level of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The mean (n = 3 donors) is plotted and the error bars represent SD. (*) significant (p<0.05) difference compared to expression level in passage PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/8627573 5; (+) significant (p<0.05) difference Phenyl (4-chloro-3-fluorophenyl)carbamate compared to expression level in 3D grafts cultured for 1 week; (#) significant (p<0.05) difference compared to expression level in 3D grafts cultured for 2 weeks.cell-based medicinal products for cartilage repair. With respect to secure fixation of scaffold-assisted cartilage grafts in cartilage defects, biomechanical characterization of the scaffolds should comprise testing of tensile strengths. A variety of studies have documented that chondrocytes dedifferentiate during culture and expansion in monolayer. The cells lose their typical macroscopical and molecular phenotype which is accompanied by the decrease of the cartilage-specific type II collagen and the increase of fibroblast-related type I collagen [28-30]. Three-dimensional re-arrangement of cells initiates cartilaginous re-differentiation of the dedifferentiated chondrocytes, characterized by the re-expression of cartilage marker molecules like aggrecan and type II collagen.However, the in vitro expression levels of chondrocyte marker genes are at least a magnitude lower than found in native articular cartilage. This may indicate that three-dimensional assembly of expanded chondrocytes in PGA-fibrin scaffolds activates the chondrogenic redifferentiation process in v.