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Bone & Joint Research
Vol. 7, Issue 4 | Pages 274 - 281
1 Apr 2018
Collins KH Hart DA Seerattan RA Reimer RA Herzog W

Objectives

Metabolic syndrome and low-grade systemic inflammation are associated with knee osteoarthritis (OA), but the relationships between these factors and OA in other synovial joints are unclear. The aim of this study was to determine if a high-fat/high-sucrose (HFS) diet results in OA-like joint damage in the shoulders, knees, and hips of rats after induction of obesity, and to identify potential joint-specific risks for OA-like changes.

Methods

A total of 16 male Sprague-Dawley rats were allocated to either the diet-induced obesity group (DIO, 40% fat, 45% sucrose, n = 9) or a chow control diet (n = 7) for 12 weeks. At sacrifice, histological assessments of the shoulder, hip, and knee joints were performed. Serum inflammatory mediators and body composition were also evaluated. The total Mankin score for each animal was assessed by adding together the individual Modified Mankin scores across all three joints. Linear regression modelling was conducted to evaluate predictive relationships between serum mediators and total joint damage.


Bone & Joint Research
Vol. 4, Issue 3 | Pages 38 - 44
1 Mar 2015
Thornton GM Reno CR Achari Y Morck DW Hart DA

Objectives

Ligaments which heal spontaneously have a healing process that is similar to skin wound healing. Menopause impairs skin wound healing and may likewise impair ligament healing. Our purpose in this study was to investigate the effect of surgical menopause on ligament healing in a rabbit medial collateral ligament model.

Methods

Surgical menopause was induced with ovariohysterectomy surgery in adult female rabbits. Ligament injury was created by making a surgical gap in the midsubstance of the medial collateral ligament. Ligaments were allowed to heal for six or 14 weeks in the presence or absence of oestrogen before being compared with uninjured ligaments. Molecular assessment examined the messenger ribonucleic acid levels for collagens, proteoglycans, proteinases, hormone receptors, growth factors and inflammatory mediators. Mechanical assessments examined ligament laxity, total creep strain and failure stress.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_IV | Pages 555 - 556
1 Nov 2011
Hildebrand KA Hart DA
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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.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_IV | Pages 551 - 551
1 Nov 2011
Hildebrand KA Zhang M Salo PT Hart DA
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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.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_III | Pages 243 - 243
1 Jul 2011
Monument M Hart DA Befus AD Salo PT Hildebrand K
Full Access

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.