A genome-wide transcriptomic analysis of articular cartilage during normal maturation in pigs

Abstract

ObjectiveThe articular cartilage undergoes dramatic changes in structure and composition during post-natal maturation, but the associated transcriptional changes are not well characterized. Compared to a mature stage, the immature articular cartilage shows developmental features such as increased thickness, presence of blood vessels, and the presence of a deep layer of growth cartilage which undergoes endochondral ossification. These features decrease during normal development. Following maturation, the articular cartilage is known to undergo few minor modifications. Since mature articular cartilage has poor regenerative and repair capacity compared to the immature articular cartilage, a better understanding of the molecular changes during the normal postnatal articular cartilage development might reveal insights on the molecular adaptation. It may also provide new therapeutic strategies. The purpose of this study was to determine the differential expression of genes in the femoral head articular cartilage of 6-weeks old and 6-months old pigs using a genome-wide transcriptomic analysis. MethodsThe articular cartilage of the femoral head of 6-weeks and 6-months old normal pigs was assessed for thickness and vascularity (number of vascular canals) using Safranin O/Fast Green staining of paraffin sections (n=4 pigs/age group). The measurements were determined using Image J software. RNA was isolated from the femoral head articular cartilage from 6-weeks and 6-months old pigs (n=8 pigs/age group). A microarray analysis was performed using an Affymetrix Porcine GeneChip Array. A gene enrichment analysis and a functional clustering analysis were performed by DAVID and STRING software, respectively. The differential expression of selected genes was confirmed by a quantitative RTPCR analysis. ResultsThe femoral head articular cartilage showed a significant decrease in thickness and number of vascular canals in 6-months old compared to 6-weeks old pigs. A microarray analysis revealed a differential gene expression of 576 genes, with 206 genes that were significantly upregulated and 370 genes that were significantly downregulated (>2-fold change, p<0.05) at 6-months compared to 6-weeks of age. Among the upregulated genes, DAVID analysis revealed that a significant number of genes represented the biological processes of responses to external stimuli, and wounding and inflammation at 6-months of age. These processes involved genes representing secretory and signaling proteins such as MMP-1, MMP-3, IL-8 and STAT3 suggesting increased inflammatory activity. In addition, an assessment of the downregulated genes indicated a decrease in the expression of genes representing the biological processes of developmental processes (e.g. BMPR1A, BMPR2, ACVR2, periostin, SFRP2, COL5A3) and regulation of blood vessel size (e.g. alpha adrenergic receptor 1B, alpha-SMA) at 6-months of age. A real-time qRTPCR analysis of selected upregulated genes, fibronectin, MMP-3, IL-8 and downregulated genes, BMPR2, PECAM, CCL2, TLR4 confirmed the differential gene expression in the microarray analysis. ConclusionDuring the process of articular cartilage maturation from 6-weeks to 6-months of age in normal pigs, genes associated with inflammatory responses to injury were upregulated and genes involved in the development and vascular responses were downregulated. These findings suggest that during articular cartilage maturation, the transcriptional changes might increase the susceptibility of cartilage to inflammatory damage and decrease the regenerative capacity.