The Effect of Uniaxial Cyclic Tensile Load on Gene Expression in Canine Cranial Cruciate Ligamentocytes

Abstract

To determine the effects of uniaxial cyclic tensile load amplitude and duration on gene expression in cranial cruciate ligamentocytes cultured in monolayer. In vitro experimental study. Adult dogs (n=9) weighing 20-35 kg. Cranial cruciate ligaments (CCL, n=18) were aseptically collected, diced, digested using clostridial collagenase, and primary monolayer cultures were established. CCL cells were seeded at a concentration of 3 x 10(5) cells/mL onto a specialized collagen membrane. After 24 hours to allow attachment, ligamentocytes were subjected to 0%, 4%, or 8% uniaxial strain for 24 or 48 hours using a sinusoidal strain profile at 0.5 Hz. At the end of each time point, the ligamentocytes were harvested and analyzed for collagen 1 (COL1), collagen 3 (COL3), and matrix metalloproteinase-3 (MMP-3) gene expression using reverse transcriptase real-time polymerase chain reaction. Approximately 33% of CCL processed for this study yielded viable cell cultures compared with 100% of the medial collateral ligaments processed. For CCL cells under uniaxial strain, gene expression for COL1 was variable, but higher strains and longer time in culture resulted in increased COL1 expression. There were no significant differences found for COL3 at any time point or between strain regimens. In general, MMP-3 gene expression was increased early in tissue culture and at higher strains. COL1 and MMP-3 gene expression can be influenced by amplitude and duration of strain on CCL cells in monolayer culture. These data have implications for modeling and understanding canine cruciate ligament pathophysiology. In particular, MMP-3 could serve as a potential preventative or therapeutic target in cruciate disease.