In Vitro Differentiation and Matrix Vesicle Biogenesis in Primary Cultures of Rat Growth Plate Chondrocytes

摘要

During endochondral ossification cartilage is replaced by bone. Chondrocytes of growth plate undergo proliferation, maturation, hypertrophy, matrix vesicle (MV) biogenesis and programmed cell death (apoptosis). It has been suggested that not all chondrocytes are destined to die. Some may become bone-forming cells, and secrete bone matrix within existing chondrocytic lacunae. The in vitro system presented here provides a potential experimental model for studying in vitro differentiation and MV biogenesis in chondrocyte cultures. Chondrocytes were obtained from collagenase digested tibial and femoral growth plate cartilage of 4 week old rachitic rats. The isolated chondrocytes were plated as monolayers at a density of 0.5 x 106 cellsbeta5mm plate and grown for 17 days in BGJb medium supplemented with 10 % fetal bovine serum, 50 Jig/ml ascorbic acid. Light microscopy revealed sirius red-positive, apparent bone matrix in layers at the surfaces of cartilaginous nodules that developed in the cultures. The central matrix was largely alcian blue staining thus resembling cartilage matrix. Electron microscopy revealed superficial areas of bone like matrix with large banded collagen fibrils, consistent with type I collagen. Most of the central matrix was cartilaginous, with small fibrils, randomly arranged consistent with type II collagen. The presence of peripheral Type-I and central Type-II collagen was confirmed by Immunohisto chemical staining. Immunohisto chemistry with anti-Bone morphogenetic proteins 2, 4 and 6 showed that BMP expression is associated with maturing hypertrophic central chondrocytes, many of which were TUNEL positive and undergoing cell death with plasma membrane breaks, hydropic swelling and cell fragmentation. During early mineralization small radial clusters of hydroxyapatite-like mineral were associated with matrix vesicles. Collagenase digestion-released MVs from the cultures showed a high specific activity for alkaline phosphatase and demonstrated a pattern of AMP-stimuIated non-radioactive CaPO_4 deposition similar to that observed with native MVs. These studies confirm that primary cultures of rat growth plate chondrocytes are a reasonable in vitro model of growth plate histotype, MV biogenesis and programmed cell death.