This study was to investigate the effects of insulin-transferrin-selenium (ITS) on the proliferation and quantitative gene expression of adult human nasal septum chondrocytes in monolayer culture expansion and the formation of tissue engineered hyaline cartilage. Effects of ITS on human nasal septum chondrocytes monolayer culture expansion and gene expression were evaluated in various culture media either added with 2% fetal bovine serum (FBS) or 1 ng/mL basic fibroblast growth factor plus 1 ng/mL transforming growth factor or both serum and growth factors supplementation in comparison with medium added with 10%FBS. Chondrocytes cultured in medium added with 2% fetal bovine serum and growth factors either supplemented with or without ITS were then mixed with pluronic F-127 hydrogel for in vivo tissue engineered cartilage formation in nude mice model. Engineered tissues were removed after 8 weeks of implantation and evaluated with histological staining, immunohistochemistry, transmission electron microscopy and quantitative gene expression analysis. ITS promoted human chondrocytes proliferation and reduced chondrocytes dedifferentiation in media supplemented with serum and growth factors. ITS with 2% FBS and growth factors provided 15-fold increased in chondrocytes number by the end of the culture period compared to the standard culture medium used in chondrocytes culture (medium added with 10% FBS). Engineered tissue resulted from ITS supplementation demonstrated higher quality of cartilage formation. In conclusion, our study has demonstrated the benefits of ITS supplementation in human chondrocytes monolayer culture and tissue engineering cartilage formation.
Our preliminary results indicated that fibrin and poly(lactic-co-glycolic acid) (PLGA) hybrid scaffold promoted early chondrogenesis of articular cartilage constructs in vitro. The aim of this study was to evaluate in vivo cartilaginous tissue formation by chondrocyte-seeded fibrin/PLGA hybrid scaffolds. PLGA scaffolds were soaked carefully, in chondrocyte-fibrin suspension, and polymerized by dropping thrombin-calcium chloride (CaCl2) solution. PLGA-seeded chondrocytes were used as a control. Resulting constructs were implanted subcutaneously, at the dorsum of nude mice, for 4 weeks. Macroscopic observation, histological evaluation, gene expression and sulphated-glycosaminoglycan (sGAG) analyses were performed at each time point of 1, 2 and 4 weeks post-implantation. Cartilaginous tissue formation in fibrin/PLGA hybrid construct was confirmed by the presence of lacunae and cartilage-isolated cells embedded within basophilic ground substance. Presence of proteoglycan and glycosaminoglycan (GAG) in fibrin/PLGA hybrid constructs was confirmed by positive Safranin O and Alcian Blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrices. Chondrogenic properties were further demonstrated by the expression of gene encoded cartilage-specific markers, collagen type II and aggrecan core protein. The sGAG production in fibrin/PLGA hybrid constructs was higher than in the PLGA group. In conclusion, fibrin/PLGA hybrid scaffold promotes cartilaginous tissue formation in vivo and may serve as a potential cell delivery vehicle and a structural basis for articular cartilage tissue-engineering.
The effects of Glucosamine Sulphate (GS) and Chondroitin Sulphate (CS) on the healing of damaged and repaired articular cartilage were investigated. This study was conducted using 18 New Zealand white rabbits as experimental models. Focal cartilage defects, surgically created in the medial femoral condyle, were either treated by means of autologous chondrocyte implantation (ACI) or left untreated as controls. Rabbits were then divided into groups which received either GS+/-CS or no pharmacotherapy. Three rabbits from each group were sacrificed at 12 and 24 weeks post-surgery. Knees dissected from rabbits were then evaluated using gross quantification of repair tissue, glycosaminoglycan (GAG) assays, immunoassays and histological assessments. It was observed that, in contrast to untreated sites, surfaces of the ACI-repaired sites appeared smooth and continuous with the surrounding native cartilage. Histological examination demonstrated a typical hyaline cartilage structure; with proteoglycans, type II collagen and GAGs being highly expressed in repair areas. The improved regeneration of these repair sites was also noted to be significant over time (6 months vs. 3 months) and in GS and GS+CS groups compared to the untreated (without pharmacotherapy) group. Combination of ACI and pharmacotherapy (with glucosamine sulphate alone/ or with chondroitin sulphate) may prove beneficial for healing of damaged cartilage, particularly in relation to focal cartilage defects.