MATERIALS AND METHODS: Adipose stromal cells were cultured in three different types of medium: (i) F12/DMEM (FD) supplemented with 10% FBS from passage 0 (P0) to P6; (ii) FD supplemented with 2% FBS at P6; and (iii) FD supplemented with 2% FBS plus 50 ng/ml of VEGF at P6. Morphological changes and growth rate of ASCs were recorded. Changes in stemness, angiogenic and endogenic genes' expressions were analysed using Real-Time PCR.

RESULTS: Adipose stromal cells changed from fibroblast-like shape when cultured in 10% FBS medium to polygonal when cultured in 2% FBS plus VEGF-supplemented medium. Their growth rate was lower in 2% FBS medium, but increased with addition of VEGF. Real-Time PCR showed that ASCs maintained most of their stemness and angiogenic genes' expression in 10% FBS at P1, P5 and P6, but this increased significantly in 2% FBS at P6. Endogenic genes expression such as PECAM-1, VE chaderin and VEGFR-2 decreased after serial passage in 10% FBS, but increased significantly at P6 in 2% FBS. Addition of VEGF did not cause any significant change in gene expression level.

MATERIALS AND METHODS: Chondrocyte/agarose constructs were treated with varying concentrations of TNFα (0.1-100 ng/ml) and cultured at 5 and 21 % oxygen tension for 48 h. In separate experiments, constructs were subjected to dynamic compression (15 %) and treated with TNFα (10 ng/ml) and/or L-NIO (1 mM) at 5 and 21 % oxygen tension using an ex vivo bioreactor for 48 h. Markers for catabolic activity (NO, PGE2) and tissue remodelling (GAG, MMPs) were quantified by biochemical assay. ADAMTS-5 and MMP-13 expression were examined by real-time qPCR. 2-way ANOVA and a post hoc Bonferroni-corrected t test were used to analyse data.

METHOD: The co-cultured cells of 2:1 ADSCs-to-NCs ratio were encapsulated in alginate constructs in one of three concentrations (1.0%, 1.2% and 1.5%) and cultured under serum free conditions for 7 days. Cell viability, cell proliferation, immunohistochemical, gycosaminogylycans (GAG) synthesis, and gene expression were examined.

RESULTS: Overall, the 1.2% alginate concentration group was relatively effective in chondrocytic differentiation in comparable to other groups. The cell morphology, cell viability, and cell proliferation revealed initial chondrogenic differentiation by the formation of cell clusters as well as the high permeability for exchange of solutes. The formation of newly synthesis cartilage-specific extracellular matrix in 1.2% group was demonstrated by positive immunohistochemical staining of collagen type II. The co-cultured cells in 1.2% group highly expressed COL II, ACP and SOX-9, compared to 1.0% and 1.5% groups, denote the retention of cartilaginous-specific phenotype by suppressing the undifferentiation stem cell markers of SOX-2 and OCT-4. The study showed 1.2% group was less likely to differentiate towards osteogenesis by downregulating hyperthrophy chondrocytic gene of COL X and osseous marker genes of OSC and OSP.