Purpose: The objective of the present study was to determine whether human mast cells can modify behavior of human elbow contracture capsule cells in an in vitro collagen gel contraction assay.

Method: Posterior elbow joint capsule was obtained from a 38 year old man with a chronic (> 1 year) post-traumatic joint contracture. Joint capsule cells were isolated and suspended at a density of 2.5 x 105 cells/ml, and mixed with neutralized Collagen solution composed with 58% Vitrogen 100 purified collagen. Aliquots of collagen gel without cells, with only the human mast cell line, HMC-1 (2.5× 105), human capsule cells (2.5 × 105), human capsule cells (2.5 × 105) and an equal number of mast cells (1:1), or human capsule cells (2.5× 105) and 7.5× 105 mast cells (1:3) were then cast into wells tissue culture plate. The gels were maintained with 0.5 ml DMEM composed with 2% BSA and incubated at 37°C for 12 h for gelation to occur. After 12 hr initial culture, the gels were detached from the wall and the bottom of culture plate wells, and gel area was determined at 0h, 2h, 4h, 6h, 24h, 48h, and 72h Gel contraction studies were carried out on passage 6 and done in triplicate. The blocking assay to inhibit mast cell – joint capsule cell interaction employed antibodies to Stem Cell Factor (SCF) and c-kit. SCF (0.5, 1 or 10 microg/ml) and/or c-kit (0.05, 0.1 or1 microg/ml) were added individually or in combination (SCF 10 microg/ ml and c-kit 1 microg/ml only) to cells/collagen gel mixture before gel casting. The ratio of human capsule cells and HMC-1 were kept constant at 1:3 throughout the experiment. The inhibitory effect of SCF and c-kit antibodies on collagen gel contraction induced by human capsule cells and HMC-1 was expressed in percentage of gel areas at 24h post release. Inhibition effect (%) = 100% – [(gel size – c-kit or SCF gel size)/(blank gel size – JC:M gel size)x 100%]. Statistical analysis involved an ANOVA with posthoc Bonferroni correction. P < 0.001 was significant. Data are mean ± standard deviation.

Results: Joint capsule cells were able to contract collagen gels in a time-dependent manner. This contraction was significantly enhanced in the presence of the HMC-1 cells in a dose dependent fashion (p < 0.001). HMC-1 cells were unable to contract the collagen gels by themselves. Experiments with antibodies to the mast cell – fibroblast direct cell-cell communication determinants SCF or c-kit showed inhibition of the enhanced contraction at 24 hours between 43 – 72%. Combining the highest dose of SCF and c-kit led to 82% inhibition.

Conclusion: This study has shown that cells isolated from human elbow joint contracture capsules respond to mast cells in a collagen gel assay in a dose dependent manner. This study is consistent with our previous work which has shown that ketotifen, a mast cell stabilizer that prevents mast cell degranulation and liberation of factors, can reduce contracture severity in a rabbit model of post-traumatic joint contractures.

Purpose: To determine if mast cell activity is vital to the induction of joint capsule fibrosis and contracture formation in a rabbit model of posttraumatic joint contracture.

Method: To reproducibly induce joint contractures, we used a model of surgical injury and immobilization of the knee in skeletally mature New Zealand white rabbits. Four animals groups were studied: a non-operative control group (CON), an operative contracture group (ORC) and two-operative groups treated with a mast cell stabilizer, Ketotifen fumarate at doses of 0.5mg/kg (KF0.5) and 1.0mg/kg (KF1.0) twice daily subcutaneously, respectively. Animals were sacrificed after 8 weeks of immobilization. Flexion contractures (biomechanics), cellular counts of myofibroblasts and mast cells within the joint capsule (immunohistochemistry) and the joint capsule protein expression of TGF-β1, collagen I and III were quantified (western blots). Biomechanical data was interpreted using a linear regression analysis of repeated measures and an ANOVA analysis of variance was used for molecular data. Significance was defined at p< 0.05 for all statistical tests.

Results: Flexion contractures were most severe in the ORC group and treatment with Ketotifen (both KF0.5 and KF1.0) significantly reduced contracture severity by 52% and 42%, respectively (p< 0.03). Joint capsule myofibroblast and mast cell hyperplasia was a prominent feature of the more severely contracted ORC group and myofibroblast and mast cell numbers were dramatically reduced in both Ketotifen groups (p< 0.001). The expression of TGF-β1 and collagen I was also increased in the ORC group and significantly reduced in both Ketotifen groups (p< 0.01).

Conclusion: Joint capsule fibrosis, characterized by hyperplasia of myofibroblasts and mast cells and enhanced collagen deposition, is a prominent feature of posttraumatic joint contractures in this animal model. Treatment with a mast cell stabilizer reduced the molecular markers of joint capsule fibrosis and the resultant biomechanical severity of contracture formation. These results suggest mast cell activity may be an important process in the development of posttraumatic contractures and future work is needed to determine if pharmacological inhibition of mast cell activity has a preventative or therapeutic role in humans.