ABSTRACT
INTRODUCTION: This study was designed to quantify the role of the pericellular matrix (PCM) in chondrocyte apoptosis using chondrons, which are a cartilage functional unit including a chondrocyte and its associated PCM. METHODS: Chondrocytes and chondrons were enzymatically isolated from human articular cartilage and exposed to monosodium iodoacetate (MIA) and staurosporine for apoptosis induction. Chondrons were defined by the presence of type VI collagen, a basic component of the PCM. Apoptosis of chondrocytes and chondrons was measured with annexin V binding by flow cytometry and verified with terminal dUTP nick end-labeling staining. In a separate experiment, isolated chondrocytes were treated with soluble type VI collagen, before or after apoptosis induction with MIA, and cell death was measured by the activity of LDH and terminal dUTP nick end-labeling staining. RESULTS: Chondrocytes treated with MIA incurred 27% cell death, compared with 12% in chondrons. On treating with MIA, 9% of chondrocytes underwent apoptosis, compared with only 1.6% of chondrons. Similarly, staurosporine induced 13% apoptosis in chondrocytes, whereas it was 3% in chondrons. Preincubation of type VI collagen effectively prevented chondrocytes from MIA-induced cell death. After apoptosis was induced with MIA, however, treatment with type VI collagen failed to rescue chondrocytes from death. CONCLUSION: The PCM, a native microenvironment of chondrocytes, protects chondrocytes from apoptosis. Type VI collagen is a functional component of the PCM that contributes to the survival of chondrocytes.
Subject(s)
Apoptosis , Chondrocytes/cytology , Extracellular Matrix/metabolism , Protective Agents/metabolism , Annexin A5/metabolism , Apoptosis/drug effects , Cell Separation , Cell Survival/drug effects , Chondrocytes/drug effects , Chondrocytes/metabolism , Collagen Type VI/pharmacology , Extracellular Matrix/drug effects , Humans , In Situ Nick-End Labeling , Iodoacetates/pharmacology , Solubility/drug effects , Staining and Labeling , Staurosporine/pharmacologyABSTRACT
One of the major obstacles hindering cartilage repair is the integration of the reparative cartilage with the recipient cartilage. The purpose of this study was to develop an in vitro model that can be conveniently applied to simulate and improve the integration of tissue engineered cartilage with native articular cartilage. This model, a cartilage integration construct, consists of a cartilage explant and isolated chondrocytes. The explant was anchored to agarose gel on a culture plate as agarose gelation at 4 degrees C to seal the gap between the bottom of the explant and culture plate surface. Isolated chondrocytes were added and confined in the defect created in the center of the explant. After 4 weeks of culture, neocartilage containing proteoglycans and type II collagen was formed. Minimal integration occurred between the neocartilage and the cartilage explant, resembling the failure of cartilage integration manifested in experimental and clinical cartilage repair. In this model, agarose gel anchors the explant onto culture plate by altering temperatures and effectively prevents "leakage" of the isolated chondrocytes from the defect of the explant. This model provides a convenient simulation of the cartilage integration process in vitro and has applications in studies of cartilage integration and cartilage tissue engineering.