Dedifferentiation of Human Chondrocyte in Monolayer Culture and its Reversibility in Pellet Culture

Min, Byoung Hyun; Woo, Jeong Im; Park, So Ra; Park, Heon Joo; Kang, Shin Young

Abstract

Articular cartilage is a unique tissue devoid of blood and nerve tissue and so its regeneration is very limited. Recently a clinical trial on transplantation using autologous chondrocyte with periosteal flap has drawn a great deal of attention. Chondrocytes cultured in a plastic flask in monolayer can rapidly dedifferentiate appearing fibroblastic, and exhibit a change in matrix gene expression characterized by a decrease in type II collagen synthesis. It is uncertain whether phenotypic change of dedifferentiated chondrocytes in vitro can be reversible to their original status alter long term culture. It is important to verify tile maintenance of the phenotype and determine the optimum period for culturing chondrocytes to be used in autologous chondrocyte transplantation. This study will be set up to confirm the reversibility of once-dedifferentiated chondrocytes with matrix-producing capability. The phenotype of cultured human chondrocyte is analysed by Northern blot and Western blot analysis for collagen type I and II. Chondrocytes appeared fibroblast right after adhering to the flask buttom at first week of culture. The proliferating rates of chondrocyte in a monolayer culture were maximum at 3rd and 4th week of culture. And thereafter, proliferation rate flowed down or stops as confluence rose. On Northern and Western blot analysis, collagen type II was well expressed by 3th to 4th week culture, thereafter progressively decreased its density with time. On the other hand, collagen type I m-RNA has not expressed until 3rd week of the culture, showing progressive increment of density thereafter. On Northern blot analysis in pellet culture, type II collagen m-RNA is apparantly reexpressed. This study indicates chat in the monolayor culture, the chondrocytic phenotype was lost with regards to morphology and mRNA expression and cartilage specific protein. However, these cells seemed to haute the potential to redifferentiate to well-differentiated chondrocytes in densely packed culture, such as pellet.