Jay, Anthony G.

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.

Cardiac troponin I (cTnI) is a key regulator of cardiomyocyte contraction. Phosphorylation of cTnI at Ser22/23 is mediated by protein kinase A, C, and G, and is associated with reduced Ca 2+ sensitivity of myocardial contraction. In previous in vivo and in vitro studies, we showed that hypothermia/rewarming (rewarming shock) reduces the Ca 2+ sensitivity of myocardial contraction due to cTnI Ser22/23 phosphorylation. Rewarming shock was also found in isolated cardiomyocytes; however, demonstrating a relationship with cTnI phosphorylation is complicated by varying cardiomyocyte viability after isolation. Using a modified Langendorff technique, the cardiomyocyte viability gradually declines over a 4‐h period from ~80% to ~40%. This decline in cell viability is not accounted for in typical Western blot measurements and, therefore, assessments of cTnI phosphorylation are highly subject to misrepresenting actual physiological changes in cTnI phosphorylation. To address this issue, we developed a novel quantitative technique to measure cTnI phosphorylation at Ser22/23 in viable cardiomyocytes using confocal microscopy. We demonstrated that cardiomyocytes with a rod‐shaped morphology and uniform striation patterns display robust transient contractile and cytosolic Ca 2+ responses to electrical stimulation. All of these rod‐shaped cardiomyocytes exclude trypan blue, while a disproportionate fraction of non‐rod‐shaped cardiomyocytes are non‐viable. Separately, using 2D gel electrophoresis, we validated the specificity of an antibody that targets phosphorylated cTnI. Using this antibody, we verified that the extent of cTnI phosphorylation in rod‐shaped cardiomyocytes increased with isoproterenol in a dose dependent fashion. This novel methodology allows a more accurate measurement of cTnI phosphorylation in isolated primary cardiomyocytes that is not affected as cell viability declines over time. Support or Funding Information This publication was made possible by Grant Number T32 HL 105355 from the National Heart, Lung and Blood Institute (NHLBI), a component of the National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official view of NIH.

AbstractThe dense formation of abnormal scar tissue after total knee arthroplasty results in arthrofibrosis, an unfortunate sequela of inflammation. The purpose of this study was to use a validated rabbit model to assess the effects on surgically‐induced knee joint contractures of two combined pharmacological interventions: celecoxib (CXB) loaded on an implanted collagen membrane, and subcutaneously (SQ) injected ketotifen. Thirty rabbits were randomly divided into five groups. The first group received no intervention after the index surgery. The remaining four groups underwent intra‐articular implantation of collagen membranes loaded with or without CXB at the time of the index surgery; two of which were also treated with SQ ketotifen. Biomechanical joint contracture data were collected at 8, 10, 16, and 24 weeks. At the time of necropsy (24 weeks), posterior capsule tissue was collected for messenger RNA and histopathologic analyses. At 24 weeks, there was a statistically significant increase in passive extension among rabbits in all groups treated with CXB and/or ketotifen compared to those in the contracture control group. There was a statistically significant decrease in COL3A1, COL6A1, and ACTA2 gene expression in the treatment groups compared to the contracture control group (P < .001). Histopathologic data also demonstrated a trend towards decreased fibrous tissue density in the CXB membrane group compared to the vehicle membrane group. The present data suggest that intra‐articular placement of a treated collagen membrane blunts the severity of contracture development in a rabbit model of arthrofibrosis, and that ketotifen and CXB may independently contribute to the prevention of arthrofibrosis. Statement of clinical significance: Current literature has demonstrated that arthrofibrosis may affect up to 5% of primary total knee arthroplasty patients. For that reason, novel pharmacologic prophylaxis and treatment modalities are critical to mitigating reoperations and revisions while improving the quality of life for patients with this debilitating condition.

ABSTRACTArthrofibrosis is a common complication following total knee arthroplasty caused by pathologic fibroblast activation and excessive collagen deposition around a synovial joint leading to debilitating loss of motion. Treatment options are limited because the pathologic mechanisms remain to be characterized. Dysregulation of the inflammatory cascade may lead to communication between myofibroblasts and immune cells triggering tissue metaplasia, and excessive collagen deposition described clinically as arthrofibrosis. We explored the novel use of celecoxib (selective cyclooxygenase‐2 [COX‐2] inhibitor) to disrupt the downstream effects of the post‐traumatic inflammatory cascade and inhibit scar tissue formation in a validated rabbit model of arthrofibrosis combined with new parameters for quantifying the stiffness of the posterior capsule. Biomechanical and molecular analyses, of contracted rabbit knee posterior capsule tissue after COX‐2 inhibition revealed increased maximal passive extension and down‐regulation of collagen messenger RNA compared with controls. Histopathologic examination suggested a trend of decreased quantities of dense fibrous connective tissue with COX‐2 inhibition. These data may suggest that inhibiting the inflammatory cascade could potentially reduce pathologic myofibroblast activation, thereby reducing scar tissue formation and increasing the range of motion in arthrofibrotic joints. Implementing a multi‐modal pharmacologic approach may simultaneously target numerous cellular components contributing to the complex process of arthrofibrogenesis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2609–2620, 2019