Summary. In contrast to the current literature, myofibroblasts are not present in chronic posttraumatic elbow contractures. However, myofibroblasts are present in the acute phase after an elbow fracture and/or dislocation. This suggests a physiological role in normal capsule healing and a potential role in the early phase of posttraumatic contracture formation. Introduction. Elbow stiffness is a common complication after elbow trauma. The elbow capsule is often thickened, fibrotic and contracted upon surgical release. The limited studies available suggest that the capsule is contracted because of fibroblast to myofibroblast differentiation. However, the timeline is controversial and data on human capsules are scarce. We hypothesise that myofibroblasts are absent in normal capsules and early after acute trauma and elevated in patients with posttraumatic elbow contracture. Patients & Methods. We obtained twenty-one human elbow joint capsules within fourteen days after an elbow fracture and/or dislocation and thirty-four capsules from thirty-four patients who had operative release of posttraumatic contractures greater than five months after injury. Myofibroblasts in the joint capsules were quantified using immunohistochemistry. Alpha-smooth muscle actin (α-SMA) was used as a marker for myofibroblasts. Samples were characterised and scored by an independent pathologist blinded for clinical data. Results. Eleven capsules were associated with the acute phase after trauma (hours to 7 days), and staining for α-SMA was negative in all eleven specimens. Ten specimens were associated with a later phase post trauma with myofibroblasts staining positive for α-SMA in all but two. All, but two, thirty-four long standing contractures showed a histological pattern consistent with chronic stages of fibrosis, characterised by increased fibroblast-like cell proliferation and higher cellular density of fibroblast-like cells with highly unstructured collagen. There was no staining of α-SMA in fibroblast-like cells in, all but two of these longstanding contractures suggesting absence of myofibroblasts. Conclusions. This study present ‘negative results’ on the hypothesis that myofibroblast numbers are elevated in longstanding (> 5 months) human posttraumatic elbow capsules. This is in contrast to all studies on human tissue in the literature to date. One recent animal study is in agreement withy our data. We did find some myofibroblasts in elbow capsules in the late-phase posttrauma (between 7 and 14 days) suggesting a potential role in early phase of posttraumatic contracture formation

Aims

Developmental dysplasia of the hip (DDH) is a complex musculoskeletal disease that occurs mostly in children. This study aimed to investigate the molecular changes in the hip joint capsule of patients with DDH.

Tendon injuries present a major clinical challenge, as they necessitate surgical intervention and are prone to fibrotic progression. Despite advances in physical therapy and surgical technique, tendons fail to return to full native functioning, underlining the need for a biological therapeutic to improve tendon healing. Myofibroblasts are activated fibroblasts that participate in the proliferative and remodeling phases of wound healing, and while these matrix-producing cells are essential for proper healing, they are also linked to fibrotic initiation. A subset of tenocytes has been shown to give rise to the myofibroblast fate, and potentially contribute to fibrotic tendon healing. A viable anti-fibrotic therapy in other tissues has been reprogramming the fibroblast-myofibroblast differentiation route, avoiding a more pro-fibrotic myofibroblast phenotype. Thus, defining the molecular programs that underlie both physiological and pathological tendon healing is critical for the development of potential pharmacologic treatments. Towards that end, we have taken advantage of spatial transcriptomics, using the tenocyte marker Scleraxis as a tool, and have outlined three major spatiotemporally distinct tenocyte differentiation trajectories (synthetic, proliferative, and reactive) following acute tendon injury in mouse FDL. We have further outlined key transcriptional controls that may be manipulated to alter the differentiation process and influence the resulting myofibroblast phenotype, thereby promoting regenerative tendon healing

The August 2014 Shoulder & Elbow Roundup. 360 . looks at: Myofibroblasts perhaps not implicated in post-traumatic elbow stiffness; olecranon tip biomechanically sound for coranoid reconstruction; obesity and elbow replacement don’t mix; single column plating successful for extra-articular distal humeral fractures; satisfaction not predictable in frozen shoulder; tenodesis and repair both acceptable in Grade II SLAP tears; glenoid bone grafting is effective and glenohumeral articular lesions best seen with an arthroscope

Introduction: The rotator cuff is subject to constant pressure from the head of the humerus. This tends to ‘wring out’ the blood supply resulting in a functionally avascular critical zone, although microvessels can be identified. This zone is the site of degeneration and tears. Damage repair under these conditions would be difficult. Myofibroblasts are characteristic of the contractile phase of wound healing. We have examined their distribution in both healthy resected and torn, degenerating rotator cuff tissue and correlated their presence with vascularity and hypoxia in the surrounding tissue. Methods: Rotator cuff tissue was obtained from ten patients undergoing surgical repair. The size of tear was 1–4.5cm, Immunohistochemical staining with commercial monoclonal antibodies to HIF-1α (Hypoxia inducible factor), vimentin, smooth muscle actin (SMA), CD31 and VEGF was performed on formalin fixed paraffin embedded tissues. Visualisation used standard DAB chromagen technique. Results: Focal myofibroblast positivity (SMA+/VIM+) was detected, areas of positivity were found at the interface between torn and degenerating tissues adjacent to the tear. Myofibroblasts were absent in degenerating tissue. The areas of myofibroblast positivity were well vascularized, with strong VEGF positivity. Nuclear HIF-1α positivity was identified in the adjacent endothelial cell population and sporadically in fibroblast population, although not in the myofibroblasts. Conclusion: Evidence of an ongoing wound healing response was found in tissue from torn rotator cuffs. However, it was patchy and infrequent

Background. There are currently no effective treatments for skeletal muscle fibrosis. Myofibroblasts are the major cellular effectors of fibrosis but their origin in muscle is unknown. We report that PDGFRβ (platelet derived growth factor receptor beta) Cre inactivates genes in murine PDGFRβ+ cells and myofibroblasts in muscle with high efficiency. We used this system to delete the integrin αv subunit because of the suggested role of multiple αv integrins as central mediators of fibrosis in multiple organs. Methods. Muscle fibrosis was induced by intramuscular cardiotoxin (CTX) injection. The contribution of PDGFRβ+ cells to fibrosis was assessed in double-flourescent reporter (mTmG) mice under PDGFRβ-Cre control. Itgavflox/flox;PDGFRβ-Cre mice were used to investigate whether loss of αv integrins on PDGFRβ+ cells influences fibrosis development. A small-molecule inhibitor of αv integrins (CWHM12) was used to determine whether pharmacological blockade of αv integrins could attenuate fibrosis. Results. At 21 days following injury PDGFRβ+ cells in mTmG;PDGFRβ-Cre mice were distributed in a manner characteristic of myofibroblasts. PDGFRβ+ cells sorted from injured muscles of mTmG;PDGFRβ-Cre mice showed induction of genes associated with myofibroblastic transition. Itgavflox/flox;PDGFRβ-Cre mice were protected from CTX induced fibrosis, as determined by picrosirius red staining for collagen (p<0.01). Sorted and culture activated αv-null PDGFRβ+ cells demonstrated significant reduction in collagen1 over controls (p<0.05). CWHM12 significantly reduced muscle fibrosis when delivered from the time of injury (prophylactic model: p<0.01) and from day 10 post injury (therapeutic model: p<0.01). Furthermore, CWHM12 inhibited collagen1 expression by PDGFRβ+ cells ex-vivo (p<0.05). Conclusions. PDGFRβ-Cre labels profibrotic cells in skeletal muscle and depletion of αv integrins in these cells reduces muscle fibrosis. Most importantly from a treatment standpoint, pharmacologic inhibition of αv integrins using a small molecule inhibitor may have utility in the prevention and treatment of skeletal muscle fibrosis. Level of Evidence. Basic Science

Purpose: To evaluate the role of myofibroblasts in post-traumatic contractures, studies were performed on the myofibroblast marker & #945;-SMA and myofibroblast up-regulators TGF-& #946;1 and the ED-A domain of fibronectin (ED-A) in joint capsules during early stages of post-traumatic contractures. Our hypotheses are mRNA expression of & #945;-SMA, TGF-& #946;1, and ED-A, and myofibroblast numbers, would increase in joint capsules of post-traumatic contractures when compared to contralateral and normal capsule. Methods: Post-traumatic joint contractures were stimulated in right knees of 24 skeletally mature female rabbits by injury and immobilization. They were equally divided based on time of immobilization: 0-weeks, 2-weeks, 4-weeks, or 6-weeks. Contralateral limbs served as unoperated controls. Normal knee capsules were obtained from three age and gender matched rabbits. Posterior joint capsules were collected for semi-quantitative RT-PCR and mRNA levels of & #945;-SMA, TGF-& #946;1, and ED-A were evaluated in all four groups. Primers were normalized to GAPDH. Myofibroblasts were counted in the 4-weeks immobilization group. Immunohistochemistry was employed using a double labeling technique: monoclonal antibodies to & #945;-SMA and affinity purified antibodies to laminin. DAPI was applied to label nuclei. Statistical analysis was completed. Paired t-tests examining intragroup comparisons and ANOVA with posthoc tukey analyzing changes over time were used (significant if p& #8804;0.05). Results: There was a significant increase in & #945;-SMA and TGF-& #946;1 mRNA expression in the posterior joint capsule of contracture knees when compared to contralateral control knees in all four groups. The mRNA levels for ED-A were significantly increased in the contracture group compared to the control group at 0-weeks. At 4-weeks immobilization, myofibroblasts were present in control and contracture tissue. Absolute myofibroblast numbers and percentage of myofibroblasts to total cells were significantly increased in contracture tissue compared to control tissue. There was no difference between total cells obtained from contracture and control knees. Conclusions: Immediately upon injury (0-weeks), mRNA expression of & #945;-SMA, TGF-& #946;1, and ED-A increased in contracture knees compared to control knees. Myofibroblast numbers and percentage of myofibroblasts were elevated in contracture tissue compared to control tissue. It would appear mRNA changes occur immediately and are associated with increased numbers of myofibroblasts at 4-weeks. Funding: Other Education Grant. Funding Parties: Alberta Heritage Foundation for Medical Research, Health Research Foundation, and Canadian Institutes of Health Research

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.

Objectives

This study aimed to investigate time-dependent gene expression of injured human anterior cruciate ligament (ACL), and to evaluate the histological changes of the ACL remnant in terms of cellular characterisation.

We treated 22 patients with a diagnosis of primary frozen shoulder resistant to conservative treatment by manipulation under anaesthetic and arthroscopic release of the rotator interval, at a mean time from onset of 15 months (3 to 36). Biopsies were taken from this site and histological and immunocytochemical analysis was performed to identify the types of cell present. The tissue was characterised by the presence of fibroblasts, proliferating fibroblasts and chronic inflammatory cells. The infiltrate of chronic inflammatory cells was predominantly made up of mast cells, with T cells, B cells and macrophages also present.

The pathology of frozen shoulder includes a chronic inflammatory response with fibroblastic proliferation which may be immunomodulated.