培地中のアスコルビン酸濃度が再生軟骨組織の力学特性に与える影響

抄録

The chondrocyte-agarose construct has been employed as an experimental model in the cartilage tissue engineering context. We studied the influence of L-Ascorbic acid (AsA) concentration in culture medium on mechanical property of regenerated cartilage. Cylindrical chondrocyte-agarose constructs with a diameter of 4mm and a height of 2.5mm were prepared as test specimens. Chondrocytes isolated from metacarpal-phalangeal joint of steers were seeded in 1wt% agarose (type VII, Sigma) to give an initial cell density of 1×10^7 cells/mL, and cultured in sterile culture medium (DMEM+20%FBS) within a humidified tissue culture incubator. We cultured by using levels of five AsA concentrations, 0.64, 2.2, 3.2 and 6.4pmol/10^9 cells and without AsA. Culture medium was exchanged every two days. After culture periods of 1, 8, 15 and 22 days, tangent modulus of the cultured constructs and glycosaminoglycan (GAG) biosynthesis were evaluated by the unconfined compression test and the demethylmethylene blue (DMMB) assay, respectively. The structural organization of the elaborated tissue was also examined morphologically by the confocal laser scanning microscope (CLSM). Results indicated that the tangent modulus of the cultured constructs 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 self-assembly of the collagen fiber network and resultant improvement in the mechanical property, since the reduction ability of AsA could enhance the procollagen expression in cultured chondrocytes.