The three-dimensional scaffolds play a very important role in regulating cell adhesion and the production of extracellular matrix molecules in in vitro regeneration of cartilage. This study evaluates how the three-dimensional structure and physicochemical properties of the polymeric scaffolds influence in vitro regeneration of cartilage tissue. A synthetic poly(vinyl alcohol)-poly(caprolactone) semi-interpenetrating polymer network (IPN) scaffold and gelatin-albumin, made of natural polymers, are used for the study. The polymers in the semi-IPN synthetic scaffold mimic the properties of collagen and glycosaminoglycans present in native cartilage. Its appropriate swelling and pore structure enabled cell-cell and cell-matrix interactions. This helped the chondrocytes to retain its spherical morphology and resulted in enhanced secretion of extracellular matrix components. In contrast, the biomimetic structure in gelatin-albumin scaffold induced chondrocytes to loose its phenotype by spreading and becoming fibroblastic in morphology. Its high swelling and the large pore size failed to recreate an appropriate microenvironment for chondrogenesis that resulted in less secretion of cartilage-specific molecules. Mesenchymal stem cell differentiation to chondrocytes in the presence of growth factors is also enhanced in the synthetic semi-IPN scaffold. The study thus indicates that the chemical composition and the physicochemical properties of the scaffolds play a very important role in providing appropriate niche in in vitro tissue regeneration.