Purpose

Recent work has shown that joint contracture severity can be decreased with the mast cell stabilizer ketotifen in association with decreased numbers of myofibroblasts and mast cells in the joint capsule of a rabbit model of post-traumatic contractures. Neuropeptides such as Substance P (SP) can induce mast cells to release growth factors. Using a gel contraction assay, we test the hypothesis that joint capsule cell-mediated contraction of a collagen gel can be enhanced with SP, but the effect is magnified in the presence of mast cells.

Purpose: Elbow osteoarthritis (OA) is characterized by a loss of elbow motion secondary to joint capsular hypertrophy and osteophyte formation. Previous work on joint capsules in post-traumatic (PT) elbow joint contractures has shown that alterations in cell populations (increased number of alpha-SMA positive myofibroblasts), matrix molecule and enzyme, and growth factor mRNA profiles are associated with loss of elbow motion in this condition. The objective of this study was to determine whether alterations in joint capsule parameters were similar or different in two etiologies of human elbow contractures, primary OA and PT.

Method: Posterior elbow joint capsules were obtained from eight male patients with primary elbow OA (age 52±12 yr ), five male patients with chronic (> 1 year) PT (age 47±12 yr ) and four male organ donors free of OA and contractures (age 43±10 yr ). RNA was extracted for subsequent real-time PCR for alpha-SMA, interleu-kin-1beta, MMP-1, MMP-3, collagen type III, biglycan, versican, tenascin C, TIMP-1, MMP-2, iNOS, COX-2, glyceraldehyde – 3 phosphate dehydrogenase (GAPDH) and 18S. 18S was used to normalize gene expression. Statistical comparisons used a oneway ANOVA followed by posthoc Tukey test. Significance was p < 0.05.

Results: The mRNA levels in the OA and PT capsules were increased compared to controls in most cases. This includes the major matrix molecule collagen I and the myofibroblast marker alpha-SMA, the growth factors TGF-beta1 and CTGF plus decorin, the injury response elements (collagen III, biglycan, versican, tenascin C) as well as TIMP-1 and MMP-2. The housekeeping gene GAPDH was similar in all 3 groups as was COX-2, while iNOS was elevated in both groups characterized by contractures. When comparing the two contracture groups, the mRNA levels were similar for some molecules while differences were evident in other instances. In PT, alpha-SMA and collagen I were greater than in OA. Conversely, in the OA group, the growth factors and matrix enzyme systems exhibited higher levels than PT.

Conclusion: In this study of human elbow joint capsules, we have shown that relative mRNA levels for markers of myofibroblasts, major matrix components, injury response elements and selected growth factors are significantly elevated in elbow OA and post-traumatic contractures when compared to age matched organ donor controls free of contractures. When comparing the OA and PT groups, the injury response molecules were elevated to similar relative levels. The OA group had greater increases in the growth factors and many of the matrix enzymes / inhibitors measured, while the PT group had greater increases in the myofibroblast marker alpha-SMA and the major matrix molecule collagen I. Thus in general matrix, growth factor and cellular properties appear to be preferentially altered in the two conditions studied when compared to control tissues, strengthened by the fact that the housekeeping gene GAPDH had similar relative levels in all 3 groups.

Purpose: The presence of hemarthrosis during joint injury is a potential inciting stimulus in the genesis of joint capsule fibrosis. Using a rabbit model of posttraumatic knee joint contracture, our hypothesis was that, bone marrow-derived elements of hemarthrosis rather than simply the presence of blood in the joint, trigger the induction of capsule fibrosis in post-traumatic joint contracture.

Method: 35 Skeletally mature New Zealand White female rabbits (12–18 months old, 5.5 ± 0.5 kg) were randomly assigned to one of five groups: Immobilization-Only (IMO), Immobilization+ Bone Marrow (IMBM), Immobilization+ Peripheral Blood (IMPB), Bone Marrow-Only (BMO), and Controls. Surgeries: Immobilization groups had one knee joint fixed at full flexion with a Kirschner wire drilled through the tibia, passed posterior (extra-articular) to the knee joint and bent around the femur. Bone marrow groups had cortical windows removed from the non-articular cartilage portion of the medial and lateral femoral condyles. In the IMPB group, autologous peripheral venous blood was injected into the immobilized knee joint to recreate a non-traumatic hemarthrosis. The control group did not have any intervention. Joint angle measurements: After 8 weeks, rabbits were euthanized, all muscular tissue was removed and maximum extension angle of the joints with intact capsule was measured using a standard torque applied via a custom made rabbit knee gripping device attached to a MTS TestStar II. Each joint was cycled 5 times (0.2 Nm) and the average of 5 cycles was calculated. Statistical analysis consisted of a one-way ANOVA with posthoc Scheffe test (significance p < 0.05). Data are presented as mean +/ − standard deviation.

Results: The IMBM (n=8) and IMPB (n=7) groups had significantly greater contractures (52 +/ − 12 and 58 +/ − 13 degrees, respectively) when compared to the BMO (n=7) and control (n=6) groups (32 +/ − 10 and 32 +/ − 13 degrees, respectively). The IMO group had average contracture measures of 44 +/ − 15 degrees. There was no statistically significant difference between the IMBM and IMPB groups.

Conclusion: The present study showed differences in the contracture severity of the immobilized knees associated with hemarthrosis compared to other experimental and control groups. There does not appear to be a difference whether the hemarthrosis arose from a fracture (bone marrow) versus peripheral blood in rabbits. Future work will look at reversibility of contractures in the various groups. Studies on the joint capsule will evaluate myofibroblast numbers in concert with mast cell and neuropeptide distribution based on our previous work. Such knowledge will aid the prevention and treatment of the difficult and disabling problem of contracture formation after joint injury.

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.

We have devised a new scoring system using visual analogue scales (VAS) to determine the functional outcome in 15 patients with 20 displaced intra-articular calcaneal fractures, confirmed by CT. The average follow-up was 19 months.

A VAS was completed separately by the patient, the surgeon and an independent assessor. It showed satisfactory agreement between observers and strong correlations with a General Health Survey (SF36), a pain scale (McGill Pain Questionnaire) and a disease-specific, historical scale for calcaneal fractures (the Rowe score).