Effect of high-density cell culture on the synthesis of glycosaminoglycan in rabbit articular chondrocytes / 中国组织工程研究

ABSTRACT

BACKGROUND:Chondrocytes may dedifferentiate when they are cultured in vitro,and the capacity of synthetizing glycosaminoglycan (GAG)is also reduced,how to delay the dedifferentiation of chondrocytes is a crucial topic in the field of tissue engineering.OBJECTIVE:To observe the performance of chondrocytes synthetizing GAG at different inoculum densities.DESIGN,TIME AND SETTING:A controlled cellular experiment was performed at the laboratory of Department of Orthopaedics in the Second Hospital of Shanxi Medical University between January 2007 and May 2007.MATERIALSFive New Zealand rabbits of one month old were used in this study.METHODS:Articular chondrocytes were isolated from both knees and digested using 0.4% pronase enzyme and 0.025% Ⅱ type collagenase.The chondrocytes harvested from the same rabbit were divided into two sets,one was seeded at a constant density of 2×104/cm2 in primary and subculture,the other was cultured at a reduced density of 2×103/cm2 following cellular adhesion.Cellular morphology and proliferation were observed under inverted microscope.The culture media were renewed after the primary cells and passage 1 cells were confluent.MAIN OUTCOME MEASURES:GAG concentration was determined using the modified precipitation method with Alcian blue at 12,24,36,48 and 60 hours following the renewal of culture media.RESULTS:Articular chondrocytes in the primary high-density culture group were polygonal with clear boundaries,they have shown to form colony at 3-4 days.Cells around colonies were more slender than those in the center of colonies,shaping as long polygon.There was no obvious change observed in the morphology of passage 1 cells.In the low-density culture group,cells scattered at early stage and formed colonies at 7 days,cellular morphology showed no significant differences in comparison with high-density culture group.The time of primary cells becoming confluent in the low-density culture group was prolonged compared with high-density culture group.The GAG concentration in supernatants in the primary cells of low-density culture group was significantly lower than that in primary cells and passage 1 cells of high-density culture group (P＜0.001,P＜0.05).The GAG concentration showed a greater difference along with the prolonging of culture time.CONCLUSION:High-density culture is better then low-density culture to enhance the performance of chondrocytes synthetizing GAG and to retard the velocity of chondrocytes dedifferentiation,which suggests high-density culture contributes to maintain the chondrocytes phenotype and can be considered as a good way of plate culture.