Abstract
Purpose: To evaluate the role of myofibroblasts in post-traumatic contractures, studies were performed on the myofibroblast marker & #945;-SMA and myofibroblast up-regulators TGF-& #946;1 and the ED-A domain of fibronectin (ED-A) in joint capsules during early stages of post-traumatic contractures. Our hypotheses are mRNA expression of & #945;-SMA, TGF-& #946;1, and ED-A, and myofibroblast numbers, would increase in joint capsules of post-traumatic contractures when compared to contralateral and normal capsule.
Methods: Post-traumatic joint contractures were stimulated in right knees of 24 skeletally mature female rabbits by injury and immobilization. They were equally divided based on time of immobilization: 0-weeks, 2-weeks, 4-weeks, or 6-weeks. Contralateral limbs served as unoperated controls. Normal knee capsules were obtained from three age and gender matched rabbits. Posterior joint capsules were collected for semi-quantitative RT-PCR and mRNA levels of & #945;-SMA, TGF-& #946;1, and ED-A were evaluated in all four groups. Primers were normalized to GAPDH. Myofibroblasts were counted in the 4-weeks immobilization group. Immunohistochemistry was employed using a double labeling technique: monoclonal antibodies to & #945;-SMA and affinity purified antibodies to laminin. DAPI was applied to label nuclei. Statistical analysis was completed. Paired t-tests examining intragroup comparisons and ANOVA with posthoc tukey analyzing changes over time were used (significant if p& #8804;0.05).
Results: There was a significant increase in & #945;-SMA and TGF-& #946;1 mRNA expression in the posterior joint capsule of contracture knees when compared to contralateral control knees in all four groups. The mRNA levels for ED-A were significantly increased in the contracture group compared to the control group at 0-weeks. At 4-weeks immobilization, myofibroblasts were present in control and contracture tissue. Absolute myofibroblast numbers and percentage of myofibroblasts to total cells were significantly increased in contracture tissue compared to control tissue. There was no difference between total cells obtained from contracture and control knees.
Conclusions: Immediately upon injury (0-weeks), mRNA expression of & #945;-SMA, TGF-& #946;1, and ED-A increased in contracture knees compared to control knees. Myofibroblast numbers and percentage of myofibroblasts were elevated in contracture tissue compared to control tissue. It would appear mRNA changes occur immediately and are associated with increased numbers of myofibroblasts at 4-weeks.
Funding : Other Education Grant
Funding Parties : Alberta Heritage Foundation for Medical Research, Health Research Foundation, and Canadian Institutes of Health Research.
Correspondence should be addressed to Cynthia Vezina, Communications Manager, COA, 4150-360 Ste. Catherine St. West, Westmount, QC H3Z 2Y5, Canada
