Effect of high-density cell culture on the synthesis of glycosaminoglycan in rabbit articular chondrocytes
10.3969/j.issn.1673-8225.2009.33.041
- VernacularTitle:高密度细胞培养对兔关节软骨细胞糖胺多糖合成的作用
- Author:
Kai LI
;
Xiaochun WEI
;
Gang XU
;
Yuefeng SHAO
;
Pengcui LI
;
Juan DING
;
Shuhua YANG
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2009;13(33):6589-6592
- CountryChina
- Language:Chinese
-
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.MATERIALS:Five 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.
