Influence of ascorbic acid(AsA) concentration in culture medium on mechanical property of regenerated cartilage

A chondrocyte-agarose construct has been employed as an experimental model in the cartilage tissueengineering context. In this study, the influence of L-ascorbic acid (AsA) concentration in culture medium on a mechanical property of the cultured chondrocyte-agarose model of regenerated cartilage was examined. Cylindrical chondrocyteagarose constructs with a diameter of 4 mm and a height of 2.5 mm were prepared as test specimens. Chondrocytes isolated from metacarpal-phalangeal joints of steer were seeded in 1 wt% agarose (type VII, Sigma) to give an initial cell density of 1 × 10⁷ cells/mL and cultured in sterile culture medium (DMEM+20%FBS) within a humidified tissue culture incubator. Four levels of AsA concentrations of culture medium, 0.64, 2.2, 3.2 and 6.4 pmol/10⁹ cell were tested. Culture medium without AsA was also used as a control. All culture mediums were changed every two days. After culture periods of 1, 8, 15 and 22 days, a mechanical property of the cultured constructs, tangent modulus, was evaluated by the unconfined compression test. The three dimensional structure of the elaborated cartilage tissue was also examined morphologically by using the confocal laser scanning microscopy (CLSM). Results indicated that the tangent modulus of the cultured constructs was increased with increasing the AsA concentration in culture medium. In addition, the growth rate of the tangent modulus was proportional to the AsA concentration. The increased amount of AsA would contribute to the accelerated selfassembly of the collagen fiber network and resultant improvement in the mechanical property, since the reducing ability of AsA could enhance the procollagen expression in cultured chondrocytes.