Cartilage degeneration following an injury is a poorly understood process and symptoms may take years to progress to a state where treatment is sought. While the final stages of the disease process are quite well documented, the early tissue changes that start the tissue down the degenerative pathway are poorly understood. We propose to use an in vitro porcine patellae impact injury model to document changes in chondrocyte gene expression and tissue damage that may initiate the degenerative processes. We will examine different types of injury (axial and shear) and the physical extent over which damage occurs. We will examine three different loading cases;an axial impaction (loading normal to the surface), a shear impaction (loading perpendicular to the surface), and a non impacted control. Patella will be impacted twice, once on each facet and load cases will be paired on left and right patellae. Following impaction intact patellae will be maintained in organ culture for 0, 3, 7, or 14 days. After remove from culture full thickness cartilage strips will be harvested from the patellae. Tissue will be taken from two locations, directly below the impaction and adjacent to the impaction. A 2 mm slice of tissue from beneath the impaction will be taken for histological analysis and the rest will be flash frozen for gene expression analysis. Total RNA will be extracted from the tissue and reverse transcribed for real-time PCR assays. We propose to examine eighteen genes related to chondrocyte apoptosis, extracellular matrix components, and matrix degenerative enzymes production using real-time PCR. Histological analysis will include staining for chondrocyte necrosis and apoptosis, aggrecan loss, and immunohistochemistry for damage to type II collagen. Knowing the damage that occurs and the changes in chondrocyte gene expression following an impact injury will provide insights into the progression of early degenerative changes in the cartilage tissue. Are chondrocytes capable of orchestrating a successful repair or are their efforts doomed to failure with the decrease in cell number due to necrosis and apoptosis, the production of increased degenerative enzymes, and damaged, defective, or insufficient replacement matrix products? Understanding the early degenerative changes that precede advanced joint degeneration and loss of cartilage function will provide potential targets for therapeutic intervention and potential mechanisms to intervene with the ultimate goal of mitigating the disease process or preventing the debilitating events that occur altogether.
| Ashwell, Melissa S; Gonda, Michael G; Gray, Kent et al. (2013) Changes in chondrocyte gene expression following in vitro impaction of porcine articular cartilage in an impact injury model. J Orthop Res 31:385-91 |
