Objectives. The goal of this study was to determine whether intra-articular administration of the potentially anti-fibrotic agent decorin influences the expression of genes involved in the fibrotic cascade, and ultimately leads to less contracture, in an animal model. Methods. A total of 18 rabbits underwent an operation on their right knees to form contractures. Six limbs in group 1 received four intra-articular injections of decorin; six limbs in group 2 received four intra-articular injections of bovine serum albumin (BSA) over eight days; six limbs in group 3 received no injections. The contracted limbs of rabbits in group 1 were biomechanically and genetically compared with the contracted limbs of rabbits in groups 2 and 3, with the use of a calibrated joint measuring device and custom microarray, respectively. Results. There was no statistical difference in the flexion contracture angles between those limbs that received intra-articular decorin versus those that received intra-articular BSA (66° vs 69°; p = 0.41). Likewise, there was no statistical difference between those limbs that received intra-articular decorin versus those who had no injection (66° vs 72°; p = 0.27). When compared with BSA, decorin led to a statistically significant increase in the mRNA expression of 12 genes (p < 0.01). In addition, there was a statistical change in the mRNA expression of three genes, when compared with those without injection. . Conclusions. In this model, when administered intra-articularly at eight weeks, 2 mg of decorin had no significant effect on joint contractures. However, our genetic analysis revealed a significant alteration in several fibrotic genes. Cite this article: Bone Joint Res 2014;3:82–8

Summary. Based upon genetic analysis, decorin is an exciting pharmacologic agent of potential anti-fibrogenic effect on arthrofibrosis in our animal model. Introduction. While the pathophysiology of arthrofibrosis is not fully understood, some anti-fibrotic molecules such as decorin could potentially be used for the prevention or treatment of joint stiffness. The goal of this study was to determine whether intra-articular administration of decorin influences the expression of genes involved in the fibrotic cascade ultimately leading to less contracture in an animal model. Material and Methods. Eighteen rabbits had their right knees operated on to form contractures. The left knees served as controls. The 6 right limbs in the experimental group (Group 1) received four 500 ug/ml intra-articular injections of decorin over 8 days starting at 8 week, for a total of 2 mg. The 6 right limbs in the first control group (Group 2) received four intra-articular injections of bovine serum albumin (BSA) over 8 days starting at 8 weeks as well. The 6 six right limbs in the second control group (Group 3) received no injections. The contracted limbs of rabbits in Group 1 were biomechanically and genetically compared to the contracted limbs of rabbits in Groups 2 and 3 with the use of a calibrated joint measuring device and custom microarray, respectively. Results. There was no statistical difference in the flexion contracture angles between those right limbs that received intra-articular decorin versus those that received intra-articular BSA (66° vs. 69°; p = 0.41). Likewise, there was no statistical difference between those right limbs that received intra-articular decorin as opposed to those who had no injection (66° vs. 72°; p = 0.27). The lack of significance remained when the control left limbs were taken into account (p > 0.40). When compared to bovine serum albumin (BSA), decorin led to a statistically significant increase in the mRNA expression of 5 genes: substance P, neuropeptide γ, and neurokinin A, cyclin E2, and MMP-9 (p < 0.001). In addition, there was a statistically significant decrease in fibroblast growth factor receptor-2 (FGFR-2), rho-associated coiled-coil containing protein kinase-1 (ROCK-1), and vascular cell adhesion molecule-1 (VCAM-1) genes when intra-articular decorin was compared to no injection (p < 0.001). Conclusions. In this model, when administered intra-articularly at 8 weeks, 2 mg of decorin had no significant effect on joint contractures. However, our genetic analysis revealed a significant alteration in the expression of several fibrotic genes. Further studies investigating the route of administration, dosing, frequency, and timing are required before definitive conclusions may be drawn on the effects of decorin on joint contractures

Our objective was to determine the plasma levels of substance P (SP) in patients with postoperative stiffness after arthroscopic rotator cuff repair.

Plasma samples were obtained at 15 months from surgery from 2 groups of patients who underwent arthroscopic repair of a rotator cuff tear. In Group 1, 30 subjects (14 men and 16 women, mean age: 64.6 years, range 47 to 78) with shoulder stiffness 15 months after arthroscopic rotator cuff repair were recruited. In Group 2, 30 patients (11 men and 19 women, mean age: 57.8 years, range 45 to 77) were evaluated 15 months after successful arthroscopic rotator cuff repair. Immunoassays were performed with commercially available assay kits to detect the plasma levels of SP.

The mean plasma levels of SP in patients with postoperative stiffness were significantly greater than those in the control group (81.06 ± 27.76 versus 23.49 ± 5.64, P < 0.05).

The plasma concentrations of substance P in patients with shoulder stiffness after arthroscopic rotator cuff repair are higher compared to plasma levels of SP in patients with a good postoperative outcome. The neuronal upregulation of SP shown in the plasma of patients with post operative shoulder stiffness may underlay not only the symptoms of adhesive capsulitis, but also its development.

Postoperative stiffness (POS) of the shoulder may occur after an apparently successful reconstruction of a rotator cuff tear.

The role of the peripheral nervous system in tissue healing has only recently been recognized.

We determined the plasma levels of SP in patients with postoperative stiffness after arthroscopic repair of a rotator cuff tear, and compared them with those in patients with a good outcome after arthroscopic rotator cuff repair.

Plasma samples were obtained at 15 months from surgery from 2 groups of patients who underwent arthroscopic repair of a rotator cuff tear. In Group 1, 30 subjects (14 men and 16 women, mean age: 64.6 years, range 47 to 78) with shoulder stiffness 15 months after arthroscopic rotator cuff repair were recruited. In Group 2, 30 patients (11 men and 19 women, mean age: 57.8 years, range 45 to 77) were evaluated 15 months after successful arthroscopic rotator cuff repair. Immunoassays were performed with commercially available assay kits to detect the plasma levels of SP.

Statistical analysis were performed with Wilcoxon Sign Rank test. Significance was set at P< 0.05

The concentrations of the neuropeptide SP in sera were measurable in all patients. Patients with postoperative stiffness had statistically significant greater plasma levels of SP than patients in whom arthroscopic repair of rotator cuff tears had resulted in a good outcome (P < 0.05)

Postoperative stiffness (POS) of the shoulder may occur after an apparently successful reconstruction of a rotator cuff tear.

An increased amount of SP in the subacromial bursa has been correlated with the pain caused by rotator cuff disease.

SP stimulates DNA synthesis in fibroblasts, which are the cellular components of the adhesive capsulitis of the shoulder. Also, SP is a pain transmitter peptide, and pain may cause a secondary muscular and/or capsular contracture.

Our results show that the plasma concentrations of substance P in patients with shoulder stiffness after arthroscopic rotator cuff repair are higher compared to plasma levels of SP in patients with a good postoperative outcome.

We cannot determine the cause of POS in our patients, but the findings of this study suggest a possible neuronal role in the pathophysiology of POS after arthroscopic repair of rotator cuff tears. The knowledge of the pathophysiological role of sensory nerve peptides in tissue repair in these patients could open new therapeutic options to manage conditions of the musculo-skeletal system with impaired tissue-nervous system interaction.

Objectives. Sustained intra-articular delivery of pharmacological agents is an attractive modality but requires use of a safe carrier that would not induce cartilage damage or fibrosis. Collagen scaffolds are widely available and could be used intra-articularly, but no investigation has looked at the safety of collagen scaffolds within synovial joints. The aim of this study was to determine the safety of collagen scaffold implantation in a validated in vivo animal model of knee arthrofibrosis. Materials and Methods. A total of 96 rabbits were randomly and equally assigned to four different groups: arthrotomy alone; arthrotomy and collagen scaffold placement; contracture surgery; and contracture surgery and collagen scaffold placement. Animals were killed in equal numbers at 72 hours, two weeks, eight weeks, and 24 weeks. Joint contracture was measured, and cartilage and synovial samples underwent histological analysis. Results. Animals that underwent arthrotomy had equivalent joint contractures regardless of scaffold implantation (-13.9° versus -10.9°, equivalence limit 15°). Animals that underwent surgery to induce contracture did not demonstrate equivalent joint contractures with (41.8°) or without (53.9°) collagen scaffold implantation. Chondral damage occurred in similar rates with (11 of 48) and without (nine of 48) scaffold implantation. No significant difference in synovitis was noted between groups. Absorption of the collagen scaffold occurred within eight weeks in all animals. Conclusion. Our data suggest that intra-articular implantation of a collagen sponge does not induce synovitis or cartilage damage. Implantation in a native joint does not seem to induce contracture. Implantation of the collagen sponge in a rabbit knee model of contracture may decrease the severity of the contracture. Cite this article: J. A. Walker, T. J. Ewald, E. Lewallen, A. Van Wijnen, A. D. Hanssen, B. F. Morrey, M. E. Morrey, M. P. Abdel, J. Sanchez-Sotelo. Intra-articular implantation of collagen scaffold carriers is safe in both native and arthrofibrotic rabbit knee joints. Bone Joint Res 2016;6:162–171. DOI: 10.1302/2046-3758.63.BJR-2016-0193

Distal arthrogryposis (DA) is a collection of rare developmental disorders characterized by congenital joint contractures. Most arthrogryposis mutations are in muscle- and joint-related genes, and the anatomical defects originate cell-autonomously within the musculoskeletal tissues. However, gain-of-function (GOF) mutations in PIEZO2, a principal mechanosensor in somatosensation, cause DA subtype 5 via unknown mechanisms. We show that expression of a GOF PIEZO2 mutation in proprioceptive sensory neurons mainly innervating muscle spindles and tendons is sufficient to induce DA5-like phenotypes in mice. Overactive PIEZO2 causes anatomical defects via increased activity within the peripheral nervous system during postnatal development. Surprisingly, overactive PIEZO2 is likely to cause joint abnormalities via increased exocytosis from sensory neuron endings without involving motor circuitry. This reveals a role for somatosensory neurons: excessive mechanosensation within these neurons disrupts musculoskeletal development. We also present proof-of-concept that Botox injection or dietary treatment can counteract the effect of overactive PIEZO2 function to evade DA-like phenotypes in mice when applied during a developmental critical period. These approaches might have clinical applications. Beyond this, our findings call attention to the importance of considering sensory mechanotransduction when diagnosing and treating other musculoskeletal disorders. Acknowledgements: Our work is supported by National Institutes of Health grant (R35 NS105067, R01 DE022358, R25 SC3GM127195, R25 GM07138, R01GM133845, intramural) and Howard Hughes Medical Institute

Abstract. Introduction. Total knee replacement (TKR) in patients with skeletal dysplasia is technically challenging surgery due to deformity, joint contracture, and associated co-morbidities. The aim of this study is to follow up patients with skeletal dysplasia following a TKR. Methodology. We retrospectively reviewed 22 patients with skeletal dysplasia who underwent 31 TKRs at our institution between 2006 and 2022. Clinical notes, operative records and radiographic data were reviewed. Results. Achondroplasia was the most common skeletal dysplasia (8), followed by Chondrodysplasia punctata (7) and Spondyloepiphyseal dysplasia (5). There were fourteen men and eight women with mean age of 51 years (28 to 73). The average height of patients was 1.4 metres (1.16–1.75) and the mean weight was 64.8 Kg (34.3–100). The mean follow up duration was 68.32 months (1–161). Three patients died during follow up. Custom implants were required in twelve patients (38.71%). Custom jigs were utilised in six patients and two patients underwent robotic assisted surgery. Hinged TKR was used in seventeen patients (54.84%), posterior stabilised TKR in nine patients (29.03%), and cruciate retaining TKR in five patients (16.13%). One patient underwent a patella resurfacing for persistent anterior knee pain and another had an intra-operative medial tibial plateau fracture which was managed with fixation. No revisions occurred during the follow up period. Conclusion. Despite the technical challenges and complexity of TKR within this unique patient group, we demonstrate good implant survivorship during the study period. Cross sectional imaging is recommended preoperatively for precise planning and templating

Aims. Arthrofibrosis is a relatively common complication after joint injuries and surgery, particularly in the knee. The present study used a previously described and validated rabbit model to assess the biomechanical, histopathological, and molecular effects of the mast cell stabilizer ketotifen on surgically induced knee joint contractures in female rabbits. Methods. A group of 12 skeletally mature rabbits were randomly divided into two groups. One group received subcutaneous (SQ) saline, and a second group received SQ ketotifen injections. Biomechanical data were collected at eight, ten, 16, and 24 weeks. At the time of necropsy, posterior capsule tissue was collected for histopathological and gene expression analyses (messenger RNA (mRNA) and protein). Results. At the 24-week timepoint, there was a statistically significant increase in passive extension among rabbits treated with ketotifen compared to those treated with saline (p = 0.03). However, no difference in capsular stiffness was detected. Histopathological data failed to demonstrate a decrease in the density of fibrous tissue or a decrease in α-smooth muscle actin (α-SMA) staining with ketotifen treatment. In contrast, tryptase and α-SMA protein expression in the ketotifen group were decreased when compared to saline controls (p = 0.007 and p = 0.01, respectively). Furthermore, there was a significant decrease in α-SMA (ACTA2) gene expression in the ketotifen group compared to the control group (p < 0.001). Conclusion. Collectively, these data suggest that ketotifen mitigates the severity of contracture formation in a rabbit model of arthrofibrosis. Cite this article: Bone Joint Res 2020;9(6):302–310

Objectives. The aims of this study were to determine whether the administration of anti-inflammatory and antifibrotic agents affect the proliferation, viability, and expression of markers involved in the fibrotic development of the fibroblasts obtained from arthrofibrotic tissue in vitro, and to evaluate the effect of the agents on arthrofibrosis prevention in vivo. Methods. Dexamethasone, diclofenac, and decorin, in different concentrations, were employed to treat fibroblasts from arthrofibrotic tissue (AFib). Cell proliferation was measured by DNA quantitation, and viability was analyzed by Live/Dead staining. The levels of procollagen type I N-terminal propeptide (PINP) and procollagen type III N-terminal propeptide (PIIINP) were evaluated with enzyme-linked immunosorbent assay (ELISA) kits. In addition, the expressions of fibrotic markers were detected by real-time polymerase chain reaction (PCR). Fibroblasts isolated from healthy tissue (Fib) served as control. Further, a rabbit model of joint contracture was used to evaluate the antifibrotic effect of the three different agents. Results. Dexamethasone maintained the viability and promoted the proliferation of AFib. Diclofenac decreased the viability and inhibited the cell proliferation during the first week of cultivation. However, decorin inhibited AFib proliferation and downregulated the expressions of fibrotic markers. Additionally, decorin could improve the flexion contracture angle and inhibit the deposition of interstitial matrix components in the rabbit joint model. Conclusion. Decorin decreased the expression of myofibroblast markers in AFib, inhibited the proliferation of AFib, and prevented the initial procedure of arthrofibrosis in vivo, suggesting that decorin could be a promising treatment to inhibit the development of arthrofibrosis. Cite this article: X. Tang, S. Teng, M. Petri, C. Krettek, C. Liu, M. Jagodzinski. The effect of anti-inflammatory and antifibrotic agents on fibroblasts obtained from arthrofibrotic tissue: An in vitro and in vivo study. Bone Joint Res 2018;7:213–222. DOI: 10.1302/2046-3758.73.BJR-2017-0219.R2

We describe the natural history of a rabbit knee model of permanent post-traumatic joint contractures. Twenty-four skeletally mature female NZW rabbits had five-mm-squares of cortical bone removed from both femoral condyles. An extra-articular K-wire immobilized the knee joint in flexion. The K-wire was removed eight weeks later and the rabbits were divided into four groups depending on the remobilization time. The average extension loss for the experimental knees in the zero, eight, sixteen and thirty-two weeks remobilization groups was thirty-eight, thirty-two, twenty-one and twenty degrees, respectively. The motion loss stabilized in the later time intervals suggesting permanent contractures had developed. The contralateral unoperated knees average extension loss was nine degrees. The purpose of this study was to develop a rabbit knee model of post-traumatic contractures. A simulated intra-articular fracture plus eight weeks of immobilization leads to a permanent joint contracture even after thirty-two weeks of remobilization. This animal model of human post-traumatic joint contractures will allow further studies investigating mechanisms underlying the process. Twenty-four skeletally mature female NZW rabbits had 5 mm squares of cortical bone removed from both femoral condyles. An extra-articular Kirschner wire (K-wire) immobilized the knee in flexion. A second operation was performed eight weeks later to remove the K-wire. The rabbits were divided into four groups. Hind limbs were dissected, preserving the joint capsule. A device allowing six degrees-of-freedom coupled to a material testing system which applied a 0.2 Nm torque measured joint angles. Statistical analysis was performed using ANOVA with a posthoc Student-Newman-Keuls test. Data are presented as mean ± SD. The loss of extension for the experimental knees in the zero and eight weeks remobilization groups was significantly greater than the values of all contralateral unoperated knees. The loss of extension for the experimental knees in the sixteen and thirty-two weeks remobilization groups was also greater than the contralateral knees, although it was not statistically significant (p = 0.07). With this model, the severity of the contracture decreased with time of remobilization. However, the degree of contracture stabilized between sixteen and thirty-two weeks of remobilization, suggesting that the joints had developed a permanent contracture. This mimics the human scenario of permanent post-traumatic joint contractures. Funding: has not been received from a commercial party. This work was supported by The Alberta Heritage Foundation for Medical Research. Please contact author for tables and/or graphs

The objective of this report was to evaluate myofibroblast numbers in human elbow anterior joint capsules. Joint capsules were obtained from six patients with post-traumatic contractures and from six elbow joints of age-matched organ donors. Frozen sections were labeled with α-smooth muscle actin (α-SMA), a marker of myofibroblasts. Myofibroblasts were identified in both experimental and control tissues. Myofibroblast numbers and percentage of total cells were significantly elevated in the capsules of patients (919 ± 187; 36 ± 0.04%) when compared to organ donor control tissue (485 ± 335; 9 ± 0.04%). Future work will look at the expression of myofibroblast modulators in human elbow joint contractures. The purpose of this study was to determine whether myofibroblasts are associated with human elbow joint contractures. Myofibroblast numbers and percentage of myofibroblasts to total cells were significantly increased in anterior elbow joint capsules of patients with post-traumatic contractures. Methods to alter myofibroblast expression may be strategies to prevent or treat post-traumatic elbow joint contractures. Joint capsules were obtained from six patients (age 33±13 yrs, preoperative flexion-extension arc range of motion 58°±15°) and from six elbow joints of organ donors free of contractures (age 26±15 yrs). Frozen sections were double labeled using monoclonal antibodies to α-smooth muscle actin (α-SMA) with peroxidase conjugated secondary antibodies, and affinity purified antibodies to laminin with Elexa Fluor 488 conjugated secondary antibodies. The laminin antibodies label components of blood vessels, to differentiate between α-SMA expression associated with blood vessels or myofibroblasts. Endogenous peroxidases were quenched and 10% normal goat serum was used as a blocking agent. DAB/peroxide substrate was added for thirteen minutes. DAPI was applied to label nuclei. Cell nuclei associated with α-SMA and not with laminin were counted as myofibroblasts. Myofibroblast numbers and percentage of total cells were significantly increased (t-test, p < 0.05) in the joint capsules of the patients when compared to organ donor control tissue. Total cell numbers were not significantly different in the patient and control tissue. Modulators of α-SMA expression and myofibroblast formation include growth factors and matrix molecule components. Future work will look at the expression of these modulators in human elbow joint contractures. Funding: Funding has not been received from a commercial party. This work was supported by The Alberta Heritage Foundation for Medical Research. Please contact author for tables and/or diagrams

Aims. Animal models have been developed that allow simulation of post-traumatic joint contracture. One such model involves contracture-forming surgery followed by surgical capsular release. This model allows testing of antifibrotic agents, such as rosiglitazone. Methods. A total of 20 rabbits underwent contracture-forming surgery. Eight weeks later, the animals underwent a surgical capsular release. Ten animals received rosiglitazone (intramuscular initially, then orally). The animals were sacrificed following 16 weeks of free cage mobilisation. The joints were tested biomechanically, and the posterior capsule was assessed histologically and via genetic microarray analysis. Results. There was no significant difference in post-traumatic contracture between the rosiglitazone and control groups (33° (standard deviation (. sd. ) 11) vs 37° (. sd. 14), respectively; p = 0.4). There was no difference in number or percentage of myofibroblasts. Importantly, there were ten genes and 17 pathways that were significantly modulated by rosiglitazone in the posterior capsule. Discussion. Rosiglitazone significantly altered the genetic expression of the posterior capsular tissue in a rabbit model, with ten genes and 17 pathways demonstrating significant modulation. However, there was no significant effect on biomechanical or histological properties. Cite this article: M. P. Abdel. Effectiveness of rosiglitazone in reducing flexion contracture in a rabbit model of arthrofibrosis with surgical capsular release: A biomechanical, histological, and genetic analysis. Bone Joint Res 2016;5:11–17. doi: 10.1302/2046-3758.51.2000593

Summary. Previous work in a rabbit model of post-traumatic joint contractures shows that the mast cell stabilizer ketotifen decreases contracture severity. We show here that ketotifen decreases collagen gel contraction mediated by rabbit joint capsule fibroblasts when mast cells are present. Introduction. Ketotifen was shown to decrease contracture severity and associated joint capsule fibrosis in an animal model of post-traumatic joint contractures. Ketotifen prevents the release of profibrotic growth factors from mast cells (MC). An in vitro collagen gel contraction assay is used to examine the effect of ketotifen on joint capsule fibroblasts obtained from this animal model. Methods. Six New Zealand White rabbits had a standardised procedure to induce post-traumatic joint contractures and the joint capsule was harvested 4 weeks later. The capsules were minced, placed into T75 culture flasks and incubated at 37. 0. C in a humidified atmosphere containing 5% CO. 2. The Joint Capsule fibroblasts (JC, 2.5 × 10. 5. cells/mL) were mixed with neutralised collagen solution composed of 59% neutralised PureCol collagen I, serum free DMEM/F12 with 1x serum replacement and 1x antibiotic-antimycotic. Aliquots of solution were then cast into wells of a tissue culture plate. Gelation occurred over 3h at 37°C in a humidified incubator. The collagen gel/cells were maintained with DMEM/F-12 plus 1% serum replacement and 1% antibiotic-antimycotic and incubated at 37°C for 12 h. The gels were released and gel area was calculated up to 72h post-release. Different experiments were conducted with various combinations of a human mast cell line (HMC-1, 7.5 × 10. 5. cells/mL), the neuropeptide Substance P (SP, 10. −6. M) and Ketotifen fumurate at 10. −4. , 10. −6. , 10. −8. and 10. −10. M. The various interventions were combined with the JC and collagen gel during the gelation step. Statistical comparisons used a two way ANOVA with a Posthoc Tukey test. Significance was set at p < 0.05. Results. The JC contracted the collagen gels in all conditions, with statistically significant differences between time intervals from 6 h to 72 h. When ketotifen alone was added to JC, there was no effect on collagen gel contraction in the range of doses tested. Adding MC to JC led to a significantly increased rate of gel contraction that was inhibited by ketotifen in a dose-dependent manner. The effect was maximal with a concentration of 10. −4. M while the effect was absent by the dose of 10. −10. M. There were statistically significant differences amongst different doses except for comparisons between doses closest to each other (10. −4. vs 10. −6. , 10. −6. vs 10. −8. , 10. −8. vs 10. −10. M). Including SP with MC and JC further increased the rate of gel contraction, which was also significantly inhibited by ketotifen in a similar dose-dependent fashion. Discussion/Conclusion. Fibroblasts from rabbit joint capsules contract collagen gels with the effect enhanced by the addition of mast cells. Ketotifen prevents the release of mediators by mast cells, and ketotifen modified the collagen gel assay. It appears that the inhibition of the gel contraction by the fibroblasts is via mast cell stabilization since ketotifen had no direct affect on the fibroblasts in the concentrations evaluated. Ketotifen is a medication used in the chronic treatment of asthma. It has a wide safety profile, it is already approved for human use and it is available in oral preparations

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

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. Method. Anterior elbow joint capsules were obtained at the time of surgical release from 2 men (age 34 and 54) and 1 woman (age 40) with chronic (> 1 year) post-traumatic joint contractures. The human mast cell line HMC-1 (Mayo Clinic, Rochester), SP and the NK1 receptor antagonist RP67580 (Sigma, Oakville, ON) were used. NK1 is the SP receptor. Neutralized Collagen solution composed with 58% Vitrogen 100 purified collagen mixed with HMC-1 cells only (7.5 105), human capsule cells (2.5 105), or human capsule cells (2.5 105) and 7.5 105 mast cells (1:3) were cast into 24- well tissue culture plates. In some experiments, SP (1 × 10. −5. M) +/− RP67580 (0.5 mM) were added. The gels were maintained with 0.5 ml DMEM composed with 2% BSA and incubated at 37C for 12 h for gelation to occur. The gels were then detached from the wall and the bottom of culture plate wells, and photographed at regular intervals up to 72 hours. Gel contraction studies were carried out on passage 4 and done in triplicate for each patient. The average value of each patients triplicate was combined to give a mean contraction at each time point. Statistical analysis involved an ANOVA with posthoc Bonferroni correction. P < 0.001 was significant. Results. Mast cells alone or with SP were unable to contract collagen gels. Joint capsule cells were able to contract the collagen gels and this was enhanced in the presence of SP, although not statistically significant. Joint capsule cells combined with mast cells enhanced the gel contraction more than joint capsule cells alone or with SP (p<0.001). The addition of SP accelerated the joint capsule cell-mediated gel contraction in the presence of mast cells the greatest (p<0.001 over all other conditions). The inhibitor RP67580 completely abolished the collagen gel contraction of the joint capsule cells in all conditions. Conclusion. The in vitro experiment shows that joint capsule cell function, in the form of collagen gel contraction, is modified by the presence of mast cells and neuropeptides. These findings are significant as they strengthen the hypothesis that a myofibroblast mast cell neuropeptide fibrosis axis may be contributing to the joint capsule changes underling the loss of motion in post-traumatic joint contractures. In vivo studies with the rabbit model of post-traumatic contractures will be performed using the compounds examined in the current study

Ligaments, menisci and joint capsules were obtained from experimental knees with post-traumatic joint contractures and their unoperated contralateral controls in 6 rabbits. Relative mRNA expression was altered for six of seven matrix molecules, growth factors and _-SMA (myofibroblast marker) in the joint capsule, four of seven molecules in the ACL, and two of seven molecules in the MCL and medial meniscus. The joint capsule had the most molecules with altered expression corresponding to it’s acknowledged key role in joint contracture development. Changes in molecular expression of several joint structures in post-traumatic contractures is similar to changes seen following ligament injury. To evaluate alteration of mRNA expression in ligaments, meniscus and joint capsules in post-traumatic contractures. mRNA expression was altered most frequently in the joint capsule. The mRNA expression alterations in the joint capsule reflect it’s significant contribution to contractures. The right knee had a stable intraarticular fracture coupled with Kirschner wire immobilization while the left knee was not surgically manipulated. The rabbits (n=6) were sacrificed two weeks later, and the ACL, MCL, posterior joint capsule and medial meniscus were obtained from both knees. Semiquantitative RT-PCR was used to evaluate relative mRNA expression of selected matrix molecules, growth factors and _-smooth muscle actin (_-SMA), a myofibroblast marker. Glyc-eraldehyde-3-phosphate dehydrogenase, a housekeeping gene, served as a normalization. Optical density measures of the gels were used for analysis. Statistical comparisons were made with a paired t-test. Statistical significance was p< 0.05. Relative mRNA expression was altered for six of seven molecules in the joint capsule, four of seven molecules in the ACL, and two of seven molecules for the MCL and meniscus. For the joint capsule, relative mRNA expression in the contracture capsule was 2-4x greater than the expression in the control capsules, except for TIMP one where the expression in the contracture capsule was 1/3 of the control capsules. As has been noted with other joint injuries (ligament instability), several structures in the joint display altered molecular expression as was found in this model of joint injury, post-traumatic joint contractures. Please contact author for tables and/or graphs

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

Aims

The antidiabetic agent metformin inhibits fibrosis in various organs. This study aims to elucidate the effects of hyperglycaemia and metformin on knee joint capsule fibrosis in mice.

Introduction and Aims: With a great progress in bone regeneration, muscle is currently regarded as a largest limiting factor for successful limb lengthening leading to joint contractures and fractures of distraction regenerate. The purpose of this study was to evaluate muscle architectural changes and potential mechanisms of joint contractures during limb lengthening. Method: Nine mature goats underwent 20% unilateral tibial lengthening (0.25 mm x 3/day) and were sacrificed immediately upon completion of distraction. With the stifle (knee) and hock (ankle) joints fixed at similar angles, both limbs were disarticulated at the hip joint and submerged into 10% buffered formalin. Following tissue fixation, all tibial muscles were sequentially dissected and changes in muscle origin-to-incretion length, belly length, tendon length, myofibers length, and sarcomere length were analysed relative to the muscle measurements on the contralateral limb and bone lengthening. Muscle fiber length was assessed under stereoscopic magnification and sarcomere analysis was performed using laser diffraction. Results: Thirteen muscles were identified for each limb. Anterior compartment consisted of two longitudinal and four pennate muscles, whereas posterior compartment had one longitudinal and six pennate muscles. Origin-to-insertion length measurements showed disproportion between the amounts of muscle and bone length increase with muscle-to-bone lengthening ratio ranging from 0.2 to 1.0. When assessed separately, muscle belly stretched more substantially (range, 11–24%) than muscle tendon (range, 3–14%). Longitudinal muscles showed better compliance to limb lengthening than pennate muscles. Origin-to-insertion, muscle belly, and tendon length increase for longitudinal muscles averaged 15%, 21%, and 11%, respectively, whereas for pennate muscles these parameters averaged 10%, 15%, and 6%, respectively. Although anterior pennate muscles showed higher proportion of muscle length increase than posterior pennate muscles, this difference was not significant. Lengthening of muscle fibers varied greatly, ranging from 0% to 88%. Fiber length of posterior muscles increased tremendously (average, 42%). This was associated with comparable increase in sarcomere length (average, 39%). Anterior muscles showed only 10% lengthening of the fibers. However, 12% reduction in sarcomere length indicated addition of new sarcomeres in series to accommodate increase in fiber length. Conclusion: Different response of anterior and posterior muscles to distraction contributed greatly to the development of joint contractures. Posterior tibial muscles were predominantly pennate, larger in volume, and thus showed higher resistance to lengthening. Moreover, posterior muscle fibers incurred lengthening by sarcomere stretching, whereas anterior muscle fibers showed evidence of neosarcomerogenesis

The hypothesis is that cells isolated from capsules of joints with contractures will contract collagen gels at a faster rate when compared to cells obtained from capsules of joints free of contractures. Post-traumatic joint contractures were produced by removing cortical bone windows from the femoral condyles of three skeletally mature rabbits and immobilizing the knees for four weeks with a K-wire. The contralateral knees served as an unoperated control. At sacrifice, the posterior capsules were immediately placed in medium and the tissue was minced. Upon confluence, cells were trypsinised and gel contraction studies were carried out on passage four cells. Five x 105 cells/ml were mixed with 58% neutralised bovine collagen solution and five hundred microlitres of collagen gel/cells solution were then cast into wells of a tissue culture plate. Gelation occurred overnight at 37C in a humidified incubator containing 5% CO2. At cultured day zero, day one, day three, the gels were released from the well walls. The areas of the gel were measured using an image analyzer immediately after release (zero hour), and one hour, two hour, three hour and four hour post-release. The amount the collagen gels were contracted depended on the time of preincubation of cells and collagen before release and the source of the joint capsule cells. In general, increasing the time of preincubation heightened the contractile response of the cells. The collagen gel contraction was small for the day zero groups over the first four hours, but for the day three groups the rate of contraction was markedly increased. In all cases the collagen gel contraction was larger for the contracture capsule cells when compared to the control capsule cells. The patterns of the contraction over the four hours post release were similar for contracture and control groups. Cells from capsules of joints with post-traumatic contractures have intrinsically heightened in vitro contractile properties when compared to normal cells. Future work will determine whether the response is exaggerated to fibrotic stimuli such as TGF-beta1 in these capsule cells from post-traumatic joint contractures