Brian P. Rooney

Hypoxia: a critical regulator of early human tendinopathy

Objectives To seek evidence for the role of hypoxia in early human tendinopathy, and thereafter to explore mechanisms whereby tissue hypoxia may regulate apoptosis, inflammatory mediator expression and matrix regulation in human tenocytes. Methods Fifteen torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing ‘early pathology’) biopsies were collected from patients undergoing arthroscopic shoulder surgery. Control samples of the subscapularis tendon were collected from 10 patients undergoing arthroscopic stabilisation surgery. Markers of hypoxia were quantified by immunohistochemical methods. Human tendon-derived primary cells were derived from hamstring tendon tissue obtained during hamstring tendon anterior cruciate ligament reconstruction. The impact of hypoxia upon tenocyte biology ex vivo was measured using quantitative real-time PCR, multiplex cytokine assays, apoptotic proteomic profiling, immunohistochemistry and annexin V fluorescence-activated cell sorter staining. Results Increased expression of hypoxia-inducible factor 1α, Bcl-2 and clusterin was detected in subscapularis tendon samples compared with both matched torn samples and non-matched control samples (p<0.01). Hypoxic tenocytes exhibited increased production of proinflammatory cytokines (p<0.001), altered matrix regulation (p<0.01) with increased production of collagen type III operating through a mitogen-activated protein kinase-dependent pathway. Finally, hypoxia increased the expression of several mediators of apoptosis and thereby promoted tenocyte apoptosis. Conclusion Hypoxia promotes the expression of proinflammatory cytokines, key apoptotic mediators and drives matrix component synthesis towards a collagen type III profile by human tenocytes. The authors propose hypoxic cell injury as a critical pathophysiological mechanism in early tendinopathy offering novel therapeutic opportunities in the management of tendon disorders.

Does Degree of Trochlear Dysplasia and Position of Femoral Tunnel Influence Outcome After Medial Patellofemoral Ligament Reconstruction

Background: The medial patellofemoral ligament (MPFL) is the main restraining force against lateral patellar displacement. It is disrupted after patellar subluxation or dislocation. Reconstruction of the MPFL is frequently performed when nonoperative management fails and the patient experiences recurrent patellar dislocation. Purpose: To determine the relationship between the degree of trochlear dysplasia and femoral tunnel position and outcome after MPFL reconstruction. Study Design: Case series; Level of evidence, 4. Methods: A total of 68 patients (72 knees) with recurrent dislocation of the patella underwent MPFL reconstruction. The mean follow-up was 31.3 months (range, 13-72 months). Clinical and functional outcomes were recorded using the Kujala, Lysholm, and Tegner scores. Postoperative complications, participation in sporting activity, and overall patient satisfaction were determined. Radiographs were analyzed to evaluate congruence angle, lateral patellofemoral angle, patellar height, trochlear dysplasia, trochlear boss height, and position of the femoral tunnel. Results: The mean Kujala, Lysholm, and Tegner scores postoperatively were 76.2, 73.8, and 3.6, respectively (n = 61). The mean congruence angle (n = 30) improved from 22.5° to 1.0° postoperatively (P = .000038), the lateral patellofemoral angle (n = 30) improved from 7.4° to 7.8° postoperatively (P = .048), and the patellar height (n = 46) using the Caton-Deschamps method improved from 1.1 to 1.0 postoperatively (P = .000016). Mild trochlear dysplasia grade A/B was found in 89% of patients (n = 54), and 11% of patients (n = 7) had severe grade C/D dysplasia. The mean distance from the anatomic insertion of the MPFL to the center of the tunnel was 9.3 mm (range, 0.5-28.2 mm), with 71.7% thought to be within 10 mm of the anatomic position defined by Schottle (n = 46). When patients with high-grade trochlear dysplasia were excluded, anatomically placed femoral tunnels demonstrated significantly better clinical scores than did tunnels not placed anatomically (Kujala score, P = .028; Lysholm score, P = .012). A multivariate logistic regression analysis also demonstrated that the distance of the femoral tunnel from the anatomic position predicted clinical outcome (Kujala score, P = .043; Lysholm score, P = .028). All of the patients with severe trochlear dysplasia (n = 7) suffered from recurrent dislocations postoperatively, compared with only 9.3% of patients (n = 5) with mild trochlear dysplasia (P = .0001). Four patients had patellar fractures postoperatively. Of patients with mild dysplasia, 83% were either very satisfied or satisfied with the outcome of their surgery compared with only 57% with severe dysplasia (P = .05). Of patients with mild trochlear dysplasia, 56% returned to sport postoperatively compared with only 43% of patients with severe trochlear dysplasia (P = .526). Conclusion: This study demonstrates the importance of restoration of the anatomic insertion point of the MPFL when performing MPFL reconstruction and proposes that this procedure should not be performed in isolation in patients with high-grade trochlear dysplasia.

IL-17A mediates inflammatory and tissue remodelling events in early human tendinopathy

Increasingly, inflammatory mediators are considered crucial to the onset and perpetuation of tendinopathy. We sought evidence of interleukin 17A (IL-17A) expression in early human tendinopathy and thereafter, explored mechanisms whereby IL-17A mediated inflammation and tissue remodeling in human tenocytes. Torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing ‘early pathology’) along with control biopsies were collected from patients undergoing shoulder surgery. Markers of inflammation and IL-17A were quantified by RT-PCR and immunohistochemistry. Human tendon cells were derived from hamstring tendon obtained during ACL reconstruction. In vitro effects of IL-17A upon tenocytes were measured using RT-PCR, multiplex cytokine assays, apoptotic proteomic profiling, immunohistochemistry and annexin V FACS staining. Increased expression of IL-17A was detected in ‘early tendinopathy’ compared to both matched samples and non-matched control samples (p < 0.01) by RT-PCR and immunostaining. Double immunofluoresence staining revealed IL-17A expression in leukocyte subsets including mast cells, macrophages and T cells. IL-17A treated tenocytes exhibited increased production of proinflammatory cytokines (p < 0.001), altered matrix regulation (p < 0.01) with increased Collagen type III and increased expression of several apoptosis related factors. We propose IL-17A as an inflammatory mediator within the early tendinopathy processes thus providing novel therapeutic approaches in the management of tendon disorders.

IL-21 Receptor Expression in Human Tendinopathy

The pathogenetic mechanisms underlying tendinopathy remain unclear, with much debate as to whether inflammation or degradation has the prominent role. Increasing evidence points toward an early inflammatory infiltrate and associated inflammatory cytokine production in human and animal models of tendon disease. The IL-21/IL-21R axis is a proinflammatory cytokine complex that has been associated with chronic inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease. This project aimed to investigate the role and expression of the cytokine/receptor pair IL-21/IL-21R in human tendinopathy. We found significantly elevated expression of IL-21 receptor message and protein in human tendon samples but found no convincing evidence of the presence of IL-21 at message or protein level. The level of expression of IL-21R message/protein in human tenocytes was significantly upregulated by proinflammatory cytokines (TNFα/IL-1β) in vitro. These findings demonstrate that IL-21R is present in early human tendinopathy mainly expressed by tenocytes and macrophages. Despite a lack of IL-21 expression, these data again suggest that early tendinopathy has an inflammatory/cytokine phenotype, which may provide novel translational targets in the treatment of tendinopathy.

67 The Interleukin 17/mast Cell Axis In Early Human Tendinopathy

Background Increasingly, inflammatory mediators are considered crucial to the onset and perpetuation of tendinopathy.1 We have previously reported increased expression of key cytokines in human and rodent models of tendinopathy.2 Interleukin-17 is the founding member of a group of cytokines called the IL-17 family and induces the production of IL-1, IL-6, TNF-α, inducible NO synthase, matrix metalloproteinases (MMPs), and chemokines by fibroblasts, macrophages, and endothelial cells.3 Objectives To seek evidence of interleukin 17 (IL-17) expression in early human tendinopathy and thereafter, to explore mechanisms whereby IL-17 may regulate apoptosis, inflammatory mediators and matrix regulation in human tenocytes. Methods Fifteen torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing ‘early pathology’) biopsies were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from 10 patients undergoing arthroscopic stabilisation surgery. Human tendon-derived primary cells were derived from hamstring tendon tissue obtained during hamstring tendon ACL reconstruction. The Human mast cells were generated from CD133+ cells from buffy coat preparations. Markers of inflammation and IL-17 were quantified by immunohistochemical methods. The impact of IL-17 upon tenocyte biology in vitro was measured using quantitative RT-PCR, multiplex cytokine assays, apoptotic proteomic profiling, immunohistochemistry and annexin V FACS staining. Results Increased expression of IL-17A was detected in subscapularis tendon samples compared to both matched from samples and non-matched control samples (p < 0.01). Double immunofluoresence staining revealed several IL-17A expressing lineages, but particularly mast cells were the predominant source of IL-17A in biopsy samples. IL-17 treated tenocytes exhibited increased production of proinflammatory cytokines (p < 0.001), altered matrix regulation (p < 0.01) with increased production of Collagen type III and increased expression of several apoptosis is related factors to thereby promote tenocyte apoptosis. Cytokine stimulation with IL-1β and Il-23 resulted in significant (p < 0.01) production of IL-17A from human derived mast cells in vitro. Abstract 67 Figure 1 (A) IL-17A mRNA levels in control torn and early tendinopathic tendon. (B) Mast cell positive /IL-17A positive cells in tendon biopsies

68 Interleukin 33 Is A Key Tissue Alarmin In Tendinopathy

Background Increasingly, inflammatory mediators are considered crucial to the onset and perpetuation of tendinopathy.1 We have previously reported increased expression of key cytokines in human and rodent models of tendinopathy.2 IL-33 and its receptor ST2 have become increasingly associated with musculoskeletal pathologies over the past few years and are purported as critical endogenous tissue danger signals.3 There remains a significant unmet clinical need in the understanding of tendon disorders, largely owing to a lack of in-depth interrogation of the biological mechanisms underpinning the disease process. Abstract 68 Figure 1 (A) Col 1/3 message levels in human tenocytes + rhIL-33 (B) IL-33 messge levels in WT and ST2-/- mice (C) Biomechanical strength in WT and ST2-/- mice + rhIL-33 Objectives To seek evidence of IL-33 expression in early human tendinopathy and thereafter, to explore mechanisms whereby IL-33 may regulate inflammatory mediators and matrix regulation in human tenocytes and a murine tendon model. Methods Fifteen torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing ‘early pathology’) biopsies were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from 10 patients undergoing arthroscopic stabilisation surgery. Human tendon-derived primary cells were derived from hamstring tendon tissue obtained during hamstring tendon ACL reconstruction. The impact of IL-33 upon tenocyte biology ex vivo was measured using quantitative RT-PCR, collagen I and III ELISAs and luminex cytokine multiplexes. In vivo work composed of experiments utilising a murine patellar tendon injury model in WT and ST2-/-. Results IL-33, soluble and membrane bound ST2 transcripts were significantly upregulated in early tendinopathy compared to control or torn tendon biopsies. IL-33 induced dose and time dependent upregulation of total collagen protein accounted for by increased expression of type I but particularly type III collagen mRNA and protein. Following array analysis and consistent with reported IL-33. IL-33 mRNA was elevated on days 1 and 3 post tendon injury in WT mice. This was significantly reduced in injured ST2-/- mice suggesting autocrine regulation. Analysis of collagen synthesis revealed significantly greater expression of collagen 3 at all time points post injury in WT mice compared to uninjured controls or injured ST2-/- mice Importantly injury of WT mice tendons resulted in a significant decrease in biomechanical strength at Day 1 post injury compared to ST2-/- that recovered by days 7 and 21. These data suggest altered collagen matrix synthesis in ST2-/- mice implicating IL-33/ST2 as an early modulator of collagen changes in tendon injury that has biomechanical significance. Administration of rhIL-33 did not affect Collagen 1 synthesis but did significantly increase Collagen 3 synthesis particularly in injured tendons. Moreover, rhIL33 administration significantly reduced ultimate tendon strength at all time points post injection in WT mice suggesting that such changes were of functional impact. Finally we directly targeted IL-33 in vivo. Neutralising antibodies to IL-33 attenuated the collagen I to III switch at days 1 and 3 post injury in WT injured mice resulting in a significant increase in biomechanical strength at day 1 post injury WT mice tendons. Conclusion We herein provide new evidence for a role of IL-33 in the initial steps that lead to the important clinical entity of tendinopathy. Our data implicate IL-33 as an alarmin in early tendinopathy, and importantly, our biomechanical data suggest such expression has a pathogenically relevant role. References 1 Dourte LM, et al. J Orthop Res. 2008;26(10):1297–305 2 Millar NL, et al. J Bone Joint Surg Br. 2009;91(3):417–24 3 Liew FY. Ann Rheum Dis. Apr 2012 Suppl