Differential Chondrocyte Gene Expression Subsequent to In Vitro Shear and Axial Impactions in an Articular Cartilage Injury Model of Osteoarthritis.

摘要

Osteoarthritis (OA) is a degenerative disease of the articular joints. OA leads to fibrillation, degradation, and lesions of the articular joint surfaces, resulting in pain and decreased mobility. A prior joint injury is a known risk factor for the development of OA. Therefore, this study used in vitro impact injuries to porcine patellae to model the early degenerative process. The goal was to examine gene expression levels in a panel of 18 OA associated genes related to: the cartilage matrix, degradative enzymes and their inhibitors, inflammatory response and signaling, and cell apoptosis and proliferation. Gene expression changes were evaluated by comparing axial or shear impact specimens to non-impacted control specimens. Identifying the gene expression changes following an injury may help identify potential targets for future intervention to slow disease progression.;Seventy-two patellae were randomly assigned to one of three treatments: axial impaction (2000 N compressive load), shear impaction (500N compressive load by 10mm tangential displacement), or a non-impacted control. Impactions were conducted in a hydraulic load frame with a stainless steel impactor. After the impaction, the patellae were placed in organ culture and full-thickness cartilage tissue specimens were harvested at 0, 3, 7, or 14 days post-injury. Total RNA was extracted from each specimen, and expression levels were measured using quantitative real-time PCR. Differential gene expression was then evaluated by computing fold changes and evaluating statistical differences with a mixed model.;Using both Best Keeper and geNorm software, four reference genes were identified as having the most stable expression in porcine articular cartilage specimens exposed to these loading scenarios and culture conditions : actb, gapdh, sdha, and ppia. The geometric mean of these four genes was used to normalize the results for the 18 genes of interest.;Both the relative rise in col2a1 at day 3 in shear vs. axial specimens, and the higher expression of agc and sox-9 (a transcription factor for agc and col2a1) at the later time points may signify that the shear specimens are mounting a stronger repair attempt. However, the relative increase in col1a1, a collagen not normally expressed in articular cartilage, may indicate a less effective repair by the shear impaction specimens. The higher initial mmp levels in both axial and shear specimens as compared to control specimens suggests that the initial matrix breakdown following injury tapers off by the later time points. Increased expression of timp-2, an mmp inhibitor, indicates that cells in the shear specimens make a stronger attempt, relative to axial specimens, to limit matrix breakdown. The lowered expression levels of the genes associated with inflammatory response and signaling at the later time points may imply less inflammation for both shear and axial treatments. Increased expression of casp-8 may indicate that apoptosis is initiated at the later time points in the shear specimens.;The higher expression levels of cartilage matrix components in shear specimens compared to axial specimens signifies a more aggressive repair effort underway in the shear specimens, although the expression of col1a1 indicates this repair effort may not be correct. The decreased levels of degradative enzymes in shear specimens at the later time points denote an attempt to preserve the cartilage matrix. The shear impaction model provides an in vitro method of studying the early degenerative changes associated with OA progression.