Stable transfection of human fetal chondrocytes with a type II procollagenminigene - Expression of the mutant protein and alterations in the structure of the extracellular matrix in vitro

Objective. To perform stable transfections of human chondrocytes under cond itions that allow the maintenance of the chondrocyte-specific phenotype, an d to examine the effects of the stable transfection of a mutated type II pr ocollagen gene (COL2A1) on the structure of the cartilaginous extracellular matrix produced in vitro. Methods. A type II procollagen minigene that lacks exons 16-27 was stably t ransfected into human fetal epiphyseal chondrocytes in vitro, Expression of the minigene was detected by reverse transcriptase polymerase chain reacti on, and the encoded protein was identified by Western blot with a human typ e II collagen-specific antibody. The cartilaginous extracellular matrix pro duced by the cultured transfected chondrocytes was characterized using hist ochemical staining, polarized light microscopy analysis, and transmission e lectron microscopy, Results. A shortened type II collagen encoded by the transfected minigene w as biosynthesized and produced in the cultures of transfected cells, Histol ogic analyses demonstrated a more dense, negatively charged cartilaginous m atrix in control cultures. In contrast, COL2A1 minigene-transfected culture s were more cellular, were populated with cells of irregular shape and less -chondrocytic appearance, contained abundant intracellular dense granules, and were sur rounded by a less-dense matrix. Polarized light microscopy and transmission electron microscopy revealed a well-organized collagen fibril lar matrix in untransfected, control chondrocyte cultures, while the matrix in the transfected cultures was less birefringent and contained numerous t runcated collagen fibrils, Conclusion. The findings illustrate the feasibility of gene transfer into h uman fetal chondrocytes under conditions that allow the preservation of the ir specific phenotype, and also shed light on the function of type II colla gen in the maintenance of the structural integrity of articular cartilage m atrix.