Tissue engineering has become a promising option toward the treatment of osteoarthritis, allowing researchers to produce functional replacements for diseased cartilage. The mechanical behavior of articular cartilage depends on an extracellular matrix (ECM), i.e., type II collagen and proteoglycans. The collagen fibers play an important role to establish the mechanical strength in the cartilage tissue. Thus we focused the influence of L-ascorbic acid (AsA, as an acidic form) and ascorbic acid 2-phosphate (A2P, as a non-acidic form), as different concentrations in culture medium on mechanical property of the regenerated cartilage tissue because the collagen synthesis needs AsA. By increasing AsA concentration in culture medium, the result indicated that the tangent modulus of the cultured constructs was significantly increased and the synthesized collagen fibrils were spread out spatially in the immunofluorescence images. This study strongly suggests, therefore, that mechanical property of regenerated-cartilage tissue depended on interconnecting among cells by the ECMs, mainly by the collagen network. In contrast, significant damage by cellular toxicity was found in the high AsA concentration and the tangent modulus did not increase. Although damage of the construct was not shown by dose of A2P into culture medium as a consequence of its low reactivity, the tangent modulus did not statistically increase as collagen network had connected among chondrocytes.