Aims. Stiffness is a common complication after total knee arthroplasty (TKA). Pathogenesis is not understood, treatment options are limited, and diagnosis is challenging. The aim of this study was to investigate if MRI can be used to visualize intra-articular scarring in patients with stiff, painful knee arthroplasties. Methods. Well-functioning primary TKAs (n = 11), failed non-fibrotic TKAs (n = 5), and patients with a clinical diagnosis of fibrosis. 1. (n = 8) underwent an MRI scan with advanced metal suppression (Slice Encoding for Metal Artefact Correction, SEMAC) with gadolinium contrast. Fibrotic tissue (low intensity on T1 and T2, low-moderate post-contrast enhancement) was quantified (presence and tissue thickness) in six compartments: supra/infrapatella, medial/lateral gutters, and posterior medial/lateral. Results. Fibrotic tissue was identified in all patients studied. However, tissue was significantly thicker in fibrotic patients (4.4 mm ± 0.2 mm) versus non-fibrotic (2.5 mm ± 0.4 mm) and normal TKAs (1.9 mm ± 0.2 mm, p = < 0.05). Significant (> 4 mm thick) tissue was seen in 26/48 (54%) of compartments examined in the fibrotic group, compared with 17/30 (57%) non-fibrotic, and 10/66 (15%) normal TKAs. Although revision surgery did improve range of movement (ROM) in all fibrotic patients, clinically significant restriction remained post-surgery. Conclusion. Stiff TKAs contain intra-articular fibrotic tissue that is identifiable by MRI. Studies should evaluate whether MRI is useful for surgical planning of debridement, and as a non-invasive measurement tool following interventions for stiffness caused by fibrosis. Revision for stiffness can improve ROM, but outcomes are sub-optimal and new treatments are required. Cite this article: Bone Joint J 2020;102-B(10):1331–1340

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. Methods. Eight-week-old wild-type (WT) and type 2 diabetic (db/db) mice were divided into four groups without or with metformin treatment (WT met(-/+), Db met(-/+)). Mice received daily intraperitoneal administration of metformin and were killed at 12 and 14 weeks of age. Fibrosis morphology and its related genes and proteins were evaluated. Fibroblasts were extracted from the capsules of 14-week-old mice, and the expression of fibrosis-related genes in response to glucose and metformin was evaluated in vitro. Results. The expression of all fibrosis-related genes was higher in Db met(-) than in WT met(-) and was suppressed by metformin. Increased levels of fibrosis-related genes, posterior capsule thickness, and collagen density were observed in the capsules of db/db mice compared with those in WT mice; these effects were suppressed by metformin. Glucose addition increased fibrosis-related gene expression in both groups of mice in vitro. When glucose was added, metformin inhibited the expression of fibrosis-related genes other than cellular communication network factor 2 (Ccn2) in WT mouse cells. Conclusion. Hyperglycaemia promotes fibrosis in the mouse knee joint capsule, which is inhibited by metformin. These findings can help inform the development of novel strategies for treating knee joint capsule fibrosis. Cite this article: Bone Joint Res 2024;13(7):321–331

Aims. Dupuytren’s contracture is characterized by increased fibrosis of the palmar aponeurosis, with eventual replacement of the surrounding fatty tissue with palmar fascial fibromatosis. We hypothesized that adipocytokines produced by adipose tissue in contact with the palmar aponeurosis might promote fibrosis of the palmar aponeurosis. Methods. We compared the expression of the adipocytokines adiponectin and leptin in the adipose tissue surrounding the palmar aponeurosis of male patients with Dupuytren’s contracture, and of male patients with carpal tunnel syndrome (CTS) as the control group. We also examined the effects of adiponectin on fibrosis-related genes and proteins expressed by fibroblasts in the palmar aponeurosis of patients with Dupuytren’s contracture. Results. Adiponectin expression in the adipose tissue surrounding the palmar aponeurosis was significantly lower in patients with Dupuytren’s contracture than in those with CTS. The expression of fibrosis-related genes and proteins, such as types 1 and 3 collagen and α-smooth muscle actin, was suppressed in a concentration-dependent manner by adding AdipoRon, an adiponectin receptor agonist. The expression of fibrosis-related genes and proteins was also suppressed by AdipoRon in the in vitro model of Dupuytren’s contracture created by adding TGF-β to normal fibroblasts collected from patients with CTS. Conclusion. Fibrosis of the palmar aponeurosis in Dupuytren’s contracture in males may be associated with adiponectin expression in the adipose tissue surrounding the palmar aponeurosis. Although fibroblasts within the palmar aponeurosis are often the focus of attention when elucidating the pathogenesis of Dupuytren’s contracture, adiponectin expression in adipose tissues warrants closer attention in future research. Cite this article: Bone Joint Res 2023;12(8):486–493

Objectives. Rotator cuff tears are among the most frequent upper extremity injuries. Current treatment strategies do not address the poor quality of the muscle and tendon following chronic rotator cuff tears. Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor that activates many genes that are important in skeletal muscle regeneration. HIF-1α is inhibited under normal physiological conditions by the HIF prolyl 4-hydroxylases (PHDs). In this study, we used a pharmacological PHD inhibitor, GSK1120360A, to enhance the activity of HIF-1α following the repair of a chronic cuff tear, and measured muscle fibre contractility, fibrosis, gene expression, and enthesis mechanics. Methods. Chronic supraspinatus tears were induced in adult rats, and repaired 28 days later. Rats received 0 mg/kg, 3 mg/kg, or 10 mg/kg GSK1120360A daily. Collagen content, contractility, fibre type distribution and size, the expression of genes involved in fibrosis, lipid accumulation, atrophy and inflammation, and the mechanical properties of the enthesis were then assessed two weeks following surgical repair. Results. At two weeks following repair, treatment groups showed increased muscle mass but there was a 15% decrease in force production in the 10 mg/kg group from controls, and no difference between the 0 mg/kg and the 3 mg/kg groups. There was a decrease in the expression of several gene transcripts related to matrix accumulation and fibrosis, and a 50% decrease in collagen content in both treated groups compared with controls. Additionally, the expression of inflammatory genes was reduced in the treated groups compared with controls. Finally, PHD inhibition improved the maximum stress and displacement to failure in repaired tendons. Conclusions. GSK1120360A resulted in improved enthesis mechanics with variable effects on muscle function. PHD inhibition may be beneficial for connective tissue injuries in which muscle atrophy has not occurred. Cite this article: J. P. Gumucio, M. D. Flood, A. Bedi, H. F. Kramer, A. J. Russell, C. L. Mendias. Inhibition of prolyl 4-hydroxylase decreases muscle fibrosis following chronic rotator cuff tear. Bone Joint Res 2017;6:57–65. DOI: 10.1302/2046-3758.61.BJR-2016-0232.R1

Aims. To develop an early implant instability murine model and explore the use of intermittent parathyroid hormone (iPTH) treatment for initially unstable implants. Methods. 3D-printed titanium implants were inserted into an oversized drill-hole in the tibiae of C57Bl/6 mice (n = 54). After implantation, the mice were randomly divided into three treatment groups (phosphate buffered saline (PBS)-control, iPTH, and delayed iPTH). Radiological analysis, micro-CT (µCT), and biomechanical pull-out testing were performed to assess implant loosening, bone formation, and osseointegration. Peri-implant tissue formation and cellular composition were evaluated by histology. Results. iPTH reduced radiological signs of loosening and led to an increase in peri-implant bone formation over the course of four weeks (timepoints: one week, two weeks, and four weeks). Observational histological analysis shows that iPTH prohibits the progression of fibrosis. Delaying iPTH treatment until after onset of peri-implant fibrosis still resulted in enhanced osseointegration and implant stability. Despite initial instability, iPTH increased the mean pull-out strength of the implant from 8.41 N (SD 8.15) in the PBS-control group to 21.49 N (SD 10.45) and 23.68 N (SD 8.99) in the immediate and delayed iPTH groups, respectively. Immediate and delayed iPTH increased mean peri-implant bone volume fraction (BV/TV) to 0.46 (SD 0.07) and 0.34 (SD 0.10), respectively, compared to PBS-control mean BV/TV of 0.23 (SD 0.03) (PBS-control vs immediate iPTH, p < 0.001; PBS-control vs delayed iPTH, p = 0.048; immediate iPTH vs delayed iPTH, p = 0.111). Conclusion. iPTH treatment mediated successful osseointegration and increased bone mechanical strength, despite initial implant instability. Clinically, this suggests that initially unstable implants may be osseointegrated with iPTH treatment. Cite this article: Bone Joint Res 2022;11(5):260–269

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.

Tendon injuries occur frequently in athletes and the general population, with inferior healing leading to deposition of fibrotic scar tissue. New treatments are essential to limit fibrosis and enable tendon regeneration post-injury. In this study, we tested the hypothesis that rapamycin improves tendon repair and limits fibrosis by inhibiting the mTOR pathway. The left hindlimb of female adult Wistar rats was injured by needle puncture and animals were either given daily injections of rapamycin (2mg/kg) or vehicle. Animals were euthanized 1 week or 3 weeks post-injury (n=6/group). Left and right Achilles tendons were harvested, with the right limbs acting as controls. Tendon sections were stained with haematoxylin & eosin, and scored by 2 blinded scorers, assessing alterations in cellularity, cell morphology, vascularity, extracellular matrix (ECM) organization and peritendinous fibrosis. Immunohistochemistry was performed for the tendon pan-vascular marker CD146 and the autophagy marker LC3. Injury resulted in significantly altered ECM organization, cell morphology and cellularity in both rapamycin and vehicle-treated groups, but no alterations in vascularity compared to uninjured tendons. Rapamycin had a limited effect on tendon repair, with a significant reduction in peritendinous fibrosis 3 weeks after injury (p=0.028) but no change in cell morphology, cellularity or ECM organization compared to vehicle treated tendons at either 1 week or 3 weeks post injury. CD146 labelling was increased at the site of injury, but there was no apparent difference in CD146 or LC3 labelling in rapamycin and vehicle treated tendons. The decrease in peritendinous fibrosis post-injury observed in rapamycin treated tendons indicates rapamycin as a potential therapy for tendon adhesions. However, the lack of improvement of other morphological parameters in response to rapamycin treatment indicates that rapamycin is not an effective therapy for injuries to the tendon core. Acknowledgements: This study was funded by Versus Arthritis (22607)

Pericytes are contractile, motile cells that surround the capillary. Recent studies have shown that pericytes promoted joint fibrosis and induced subchondral bone angiogenesis, indicating the role of pericytes in osteoarthritis (OA). However, whether pericytes are involved in regulating inflammatory and catabolic response, as well as fibrotic repair of cartilage is still unclear. Here we used 2D and 3D models to investigate the communication of pericytes and chondrocytes under inflammatory osteoarthritis conditions. CD34-CD146+ pericytes were isolated and sorted from human bone marrow. Human OA chondrocytes were isolated from OA joints. In 2D studies, monolayer cultured chondrocytes were treated +/- pericyte conditioned media, +/- 1ng/ml IL1β for 24h. In 3D studies, pericytes and chondrocytes were cultured within fibrin gel in 3D polyurethane scaffolds, separately or combined for 7 days, followed by treatment of +/- IL1β for another 7 days (Fig 2A). The inflammatory response, catabolic activity and expression of fibrosis markers of chondrocytes and pericytes were measured by ELISA and/or q-rtPCR. Pericytes had weak inflammatory, catabolic and fibrotic response to IL1β (data not shown). The 2D study showed that pericyte conditioned media promoted inflammation, catabolism and fibrosis markers of chondrocytes, in the absence of IL1β treatment (Figure 1). However, study in 3D showed that coculture of chondrocytes and pericytes reduced the inflammatory and catabolic response of chondrocytes to IL1β and induced fibrosis markers in chondrocytes (Figure 2). Pericytes are involved in regulating inflammatory response, catabolic response and fibrosis of chondrocytes. The opposite results from 2D and 3D experiments indicate the variety of the regulatory role of pericytes in the interaction with chondrocytes within different culture models. The underlying mechanism is under evaluation with on-going studies. Acknowledgements. This study was funded by SINPAIN project, from European Union's Horizon Europe research and innovation programme under Grant Agreement NO. 101057778. Funded by the European Union. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union. Neither the European Union nor the granting authority can be held responsible for them. For any figures or tables, please contact the authors directly

Dupuytren's disease (DD) is a fibroproliferative soft tissue disease affecting the palmar fascia of the hand causing permanent and irreversible flexion contracture. Aberrant fibrosis is likely to manifest through a combination of extrinsic, intrinsic, and environmental factors, including genetics and epigenetics. However, the role of epigenetics in soft tissue fibrosis in diseases such as DD is not well established. Therefore, we conducted a comprehensive multi-omic study investigating the epigenetic profiles that influence gene expression in DD pathology. Using control (patients undergoing carpal tunnel release) and diseased fibroblasts (patients undergoing Dupuytren's fasciectomy), we conducted ATAC-seq to assess differential chromatin accessibility between control and diseased fibroblasts. Additionally, ChIP-seq mapped common histone modifications (histone H4; H3K4me3, H3K9me3, H3K27me3, H4K16Ac, H4K20Me3) associated with fibrosis. Furthermore, we extracted RNA from control and DD tissue and performed bulk RNA-seq. ATAC-seq analysis identified 2470 accessible genomic loci significantly more accessible in diseased fibroblasts compared to control. Comparison between diseased and control cells identified numerous significantly different peaks in histone modifications (H4K20me3, H3K27me3, H3K9me3) associated with gene repression in control cells but not in diseased cells. Pathway analysis demonstrated a substantial overlap in genes being de-repressed across these histone modifications (Figure 1). Both, ATAC-seq and ChIP-seq analysis indicated pathways such as cell adhesion, differentiation, and extracellular matrix organisation were dysregulated as a result of epigenetic changes. Moreover, de novo motif enrichment analysis identified transcription factors that possibly contributed to the differential gene expression between control and diseased tissue, including HIC1, NFATC1 and TEAD2. RNA-seq analysis found that these transcription factors were upregulated in DD tissue compared to control tissue. The current epigenetic study provides insights into the aberrant fibrotic processes associated with soft tissue diseases such as DD and indicates that epigenetic-targeted therapies may be an interesting viable treatment option in future. For any figures or tables, please contact the authors directly

In-vitro models of disease are valuable tools for studying disease and analysing response to therapeutics. Recently, advances in patient-derived organoid (PDO) models have been shown to faithfully recapitulate structure, function, and therapeutic response for a wide range of tissues. Frozen shoulder is a rare example of a chronic inflammatory fibrotic disease which is self-limiting, unlike many other soft tissue fibrotic disorders. As no in-vitro 3D models or in-vivo animal models exist for frozen shoulder, establishing an organoid model which recapitulates core diseases features may give insight into fibrosis resolution. Consequently, using biocompatible hydrogels, primary capsular fibroblasts, monocyte-derived macrophages and HUVEC cells, we generated stable PDO cultures which exhibited key disease phenotypes, including vascularization, increased stiffness, and an expanded lining layer over 21 days of culture. Through further investigation of cell-matrix and cell-cell interactions in the organoid model, we intend to unpack the differences between resolving and non-resolving fibrotic disease and uncover clinically relevant therapeutic targets for fibrosis

Stem cell therapy is an effective means to address the repair of large segmental bone defects. However, the intense inflammatory response triggered by the implants severely impairs stem cell differentiation and tissue regeneration. High-dose transforming growth factor β1 (TGF-β1), the most locally expressed cytokine in implants, inhibits osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and promotes tissue fibrosis, severely compromising the efficacy of stem cell therapy. Small molecule inhibitors of TGF-β1 can be used to ameliorate the osteogenic disorders caused by high concentrations of TGF-β1, but systemic inhibition of TGF-β1 function will cause strong adverse effects. How to find safe and reliable molecular targets to antagonize TGF-β1 remains to be elucidated. Orphan nuclear receptor Nr4a1, an endogenous inhibitory molecule of TGF-β1, suppresses tissue fibrosis, but its role in BMSC osteogenesis is unclear. We found that TGF-β1 inhibited Nr4a1 expression through HDAC4. Overexpression of Nr4a1 in BMSCs reversed osteogenic differentiation inhibited by high levels of TGF- β1. Mechanistically, RNA sequencing showed that Nr4a1 activated the ECM-receptor interaction and Hippo signaling pathway, which in turn promoted BMSC osteogenesis. In bone defect repair and fracture healing models, transplantation of Nr4a1-overexpressing BMSCs into C57BL/6J mice or treatment with the Nr4a1 agonist Csn-B significantly ameliorated inflammation-induced bone regeneration disorders. In summary, our findings confirm the endogenous inhibitory effect of Nr4a1 on TGF- β1 and uncover the effectiveness of Nr4a1 agonists as a therapeutic tool to improve bone regeneration, which provides a new solution strategy for the treatment of clinical bone defects and inflammatory skeletal diseases

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

Subacromial bursa fibrosis are linked to rotator cuff lesion with shoulder stiffness; however, the mechanism underlying this shoulder disorder remain elusive. MicroRNA-29s (miR-29s) are emerging fibrosis inhibitor targeting fibrogenic matrices during tissue fibrosis. This study is aimed to investigate clinical relevance and function of miR-29 signalling to subacromial bursa homeostasis in shoulder stiffness. Subacromial bursa in patients with rotator cuff lesion with or without shoulder stiffness who required open acromioplasty were harvested for assessing fibrosis histopathology using Manson's trichrome staining. Expressions of proinflammatory cytokines, fibrotic matrices, and miR-29s were quantified using RT-PCR and in situ hybridization. Range of motion and pain scores of the stiffness group were higher than those of non-stiffness group. Upregulated proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and fibrotic matrices (collagen 1α1, 3α1, and 4α1) but decreased miR-29a and b expression existed in the stiffness group. Affected tissues exhibited severe fibrotic matrix accumulation, synovial hyperangiogenesis, hyperplasia, and strong miR-29a transcripts. In vitro, IL-1β rather than IL-6 and TNF-α decreased miR-29a expression of subacromial bursa fibroblasts. miR-29a knockdown escalated fibrotic matrix expression, whereas forced miR-29a expression alleviated the IL-1β-induced fibrotic matrix expression. Of interest, miR-29a transgenic mice displayed moderate responses to supraspinatus and infraspinatus tenotomy-induce fibrosis and gait irregularity of affected shoulders. Weak miR-29 signalling causes excessive fibrosis and remodelling in subacromial bursa and ultimately increases the prevalence of shoulder stiffness. This study reveals a new mechanistic underlying shoulder stiffness and highlights that sustained miR-29a potentially ameliorates the severity and function of stiff shoulder

Geriatric syndromes could lead individuals to exhibit significant mobility and psychological deficits resulting in significant healthcare costs. Thus, identifying strategies to delay aging, or prevent progressive loss of tissue homeostasis could dramatically restore the function and independence of millions of elderly patients and significantly improve quality of life. One of the fundamental properties of aging is the accumulation of senescent cells and senescence associated secretory phenotypes (SASPs) that needs to be treated in wide range of therapeutics including orthobiologics. Senolytic compounds selectively target and kill senescent cells and inhibit anti-apoptotic pathways that are upregulated in senescent cells thereby inducing apoptotic cell death and abrogating systemic SASP factors. We have also shown that blocking fibrosis with Losartan (TGF-β1 blocker) can improve musculoskeletal healing and cartilage repair by reducing the amount of fibrosis. Thus, we hypothesize that administration of anti-fibrotic agents will enhance the beneficial effects of orthobiologics. The safety and efficacy of several senolytic and anti-fibrotic agents to delay age-related dysfunction and improve the function of orthobiologics have been demonstrated in a variety of animal models (in vivo). Overall, our innovative approaches target senescent cells (inflammation) and TGF-β1 (fibrosis) to enhance the clinical efficacy and use of orthobiologics for musculoskeletal repair. We will also discuss ongoing active clinical trials on orthobiologics to aiming at evaluating the safety and efficacy of senolytic agent (Fisetin) and anti-fibrotic agent (Losartan), used independently or in combination, to enhance the beneficial effects of orthobiologics for patients afflicted with musculoskeletal diseases and conditions

Dual mobility (DM) total hip replacements (THRs) were introduced to reduce dislocation risk, which is the most common cause of early revision. Although DM THRs have shown good overall survivorship and low dislocation rates, the mechanisms which describe how these bearings function in-vivo are not fully understood. Therefore, the study aim was to comprehensively assess retrieved DM polyethylene liners for signs of damage using visual inspection and semi-quantitative geometric assessment methods. Retrieved DM liners (n=18) were visually inspected for the presence of surface damage, whereby the internal and external surfaces were independently assigned a score of one (present) or zero (not present) for seven damage modes. The severity of damage was not assessed. The material composition of embedded debris was characterised using energy-dispersive x-ray analysis (EDX). Additionally, each liner was geometrically assessed for signs of wear/deformation [1]. Scratching and pitting were the most common damage modes on either surface. Additionally, burnishing was observed on 50% of the internal surfaces and embedded debris was identified on 67% of the external surfaces. EDX analysis of the debris identified several materials including titanium, cobalt-chrome, iron, and tantalum. Geometric analysis demonstrated highly variable damage patterns across the liners. The incidence of burnishing was three times greater for the internal surfaces, suggesting that this acts as the primary articulation site. The external surfaces sustained more observable damage as evidenced by a higher incidence of embedded debris, abrasion, delamination, and deformation. In conjunction with the highly variable damage patterns observed, these results suggest that DM kinematics are complex and may be influenced by several factors (e.g., soft tissue fibrosis, patient activities) and thus further investigation is warranted

Vascular inflammation and activation of myofibroblasts are significant contributors to the progression of fibrosis, which can severely impair tissue function. In various tissues, including tendons, Transforming growth factor beta 1 (TGF-β1) has been identified as a critical driver of adhesion and scar formation. Nevertheless, the mechanisms that underlie fibrotic peritendinous adhesions are still not well comprehended, and human microphysiological systems to help identify effective therapies remain scarce. To address this issue, we developed a novel human Tendon-on-a-Chip (hToC), comprised of an endothelialized vascular compartment harboring circulating monocytes and separated by a 5 μm/100 nm dual-scale ultrathin porous membrane from a type I/III collagen hydrogel with primary tendon fibroblasts and tissue-resident macrophages, all under defined serum-free conditions. The hToC models the crosstalk of the various cells in the system leading to the induction of inflammatory and fibrotic pathways including the activation of mTOR signaling. Consistent with phenotypes observed in vivo in mouse models and clinical human samples, we observed myofibroblast differentiation and senescence, tissue contraction, excessive extracellular matrix deposition, and monocytes’ transmigration and macrophages’ secretion of inflammatory cytokines, which were dependent on the presence of the endothelial barrier. This model offers novel insights on the role of vasculature in the pathophysiology of adhesions, which were previously underappreciated. Moreover, in testing whether the hToC could be used to evaluate efficacy of therapeutics, we were able to capture donor-specific variability in the response to Rapamycin treatment, which reduced myofibroblast activation regardless. Thus, our findings demonstrate the value of the hToC as a human microphysiological system for investigating the pathophysiology of fibrotic conditions in the context of peritendinous injury and similar fibrotic conditions, providing an alternative to animal testing

Scar tissue formation secondary to acute muscle injury, surgical wounding and compartment syndrome can result in significant functional impairment and predispose to further injury. The source of fibroblasts, and the molecular mechanisms driving their activation and persistence in skeletal muscle fibrosis are not known. We hypothesized that cells expressing PDGFRβ become fibroblasts in response to injury and that targeting αv integrins in these cells reduces skeletal muscle fibrosis. We used double-fluorescent reporter mice to demonstrate that cells expressing PDGFRβ become activated myofibroblasts in response to cardiotoxin (CTX) induced skeletal muscle injury. Following injury, PDGFRβ+ cells moved from perivascular locations into the interstitium in a distribution characteristic of fibroblasts, and showed marked induction of fibroblastic genes including αSMA and collagen1 (all p<0.0001). To confirm that αv integrins present on PDGFRβ cells critically regulate skeletal muscle fibrosis we used Itgavflox/flox;PDGFRβ-Cre mice (transgenic mice in which αv integrins are ‘knocked-down’ in PDGFRβ+ cells). These mice were significantly protected from CTX induced fibrosis (p<0.01). To demonstrate potential clinical utility of targeting αv integrins, we used a small molecule inhibitor of αv integrins (CWHM12). Treatment with CWHM12 significantly reduced fibrosis when delivered from the time of injury (p<0.01) and when delivered after the fibrotic response had become established (p<0.01). We have identified a core pathway regulating fibrosis in skeletal muscle. Pharmacologic inhibition of αv integrins has potential clinical utility in the treatment and prevention of skeletal muscle fibrosis

Dual Mobility (DM) Total Hip Replacements (THRs) were introduced to reduce dislocation risk, which is the most common cause of early revision. The in-vivo mechanics of these implants is not well understood, despite their increased use in both elective and trauma settings. Therefore, the aim of this study was to comprehensively assess retrieved DM polyethylene liners for signs of damage using visual inspection and semi-quantitative geometric assessment techniques. Retrieved DM liners (n=20) were visually inspected for the presence of seven established modes of polyethylene damage. If embedded debris was identified on the external surface, its material composition was characterised using energy-dispersive x-ray analysis (EDX). Additionally, each liner was geometrically assessed for signs of wear/deformation using a validated methodology. Visual inspection of the liners revealed that scratching and pitting were the most common damage modes on either surface. Burnishing was observed on 50% and 15% of the internal and external surfaces, respectively. In addition, embedded debris was identified on 25% of the internal and 65% of the external surfaces. EDX analysis of the debris identified several materials including iron, titanium, cobalt-chrome, and tantalum. Geometric analysis demonstrated highly variable damage patterns across the liners. The results of this study provide insight into the in-vivo mechanics of DM bearings. For example, the results suggest that the internal bearing (i.e., between the head and liner) acts as the primary articulation site for DM-THRs as evidenced by a higher incidence of burnishing and larger, more concentrated regions of penetration across the liners’ internal surfaces. Furthermore, circumferential, and crescent-shaped damage patterns were identified on the articulating surfaces of the liners thus providing evidence that these components can rotate within the acetabular shell with varying degrees of mobility. The mechanics of DM bearings are complex and may be influenced by several factors (e.g., soft tissue fibrosis, patient activities) and thus further investigation is warranted. Finally, the results of this study suggest that DM liners may be susceptible to ex-vivo surface damage and thus caution is advised when handling and/or assessing these types of components

Common tendon injuries impair healing, leading to debilitation and an increased re-rupture risk. The impact of oxygen-sensing pathways on repair mechanisms, vital in regulating inflammation and fibrosis, remains unclear despite their relevance in tendon pathologies. Recent studies show that pulsed electromagnetic field (PEMF) reduce inflammation in human tendon cells (hTDCs) and in hypoxia-induced inflammation. We investigated the hypoxia's impact (1% and 2% oxygen tension) using magnetic cell sheet constructs (IL-1β-magCSs) primed with IL-1β. IL-1β-magCSs were exposed to low OT (1h, 4h,6h) in a hypoxic chamber. To confirm the role of PEMF (5Hz, 4mT, 50% duty cycle) on hypoxia modulation, IL-1β-magCSs, previously exposed to OT, were 1h-stimulated with PEMF. Our results show a significant increase in HIF- 1a and HIF-2a expression on IL-1β-magCSs after exposure to 2%-OT at all time points, compared to 1%- OT and normoxia. TNFa, IL-6, and IL-8 expression increased after 6 hours of 1%-OT exposure. PEMF stimulation of hypoxic IL-1β-magCSs led to decreased pro-inflammatory genes and increased anti-inflammatory (IL-4,IL-10) expression compared to unstimulated magCSs. IFN-g, TNF-α, and IL-6 release increased after 6 hours, regardless of %-OT, while IL-10 levels tended to rise after PEMF stimulation at 2%-OT. Also, NFkB expression was increased on IL-1β-magCSs exposed to 4 h and 6 h of 2%-OT, suggesting a link between NFkB and the production of pro-inflammatory factors. Moreover, PEMF stimulation showed a significantly decreased NFkB level in IL-1β-magCSs. Overall, low OT enhances expression of hypoxia-associated genes and inflammatory markers in IL-1β-magCSs with the involvement of NFkB. PEMF modulates the response of magCSs, previously conditioned to hypoxia and to inflammatory triggers, favouring expression of anti-inflammatory genes and proteins, supporting PEMF impact in pro-regenerative tendon strategies. Acknowledgements: ERC CoG MagTendon(No.772817), FCT under the Scientific Employment Stimulus-2020.01157.CEECIND. Thanks to Hospital da Prelada for providing tendon tissue samples (Portugal), and TERM. RES Hub (Norte-01-0145-FEDER-022190)

Prosthetic joint infection (PJI) is a complex disease that causes significant damage to the peri-implant tissue. Developing an animal model that is clinically relevant in depicting this disease process is an important step towards developing novel successful therapies. In this study, we have performed a thorough histologic analysis of peri-implant tissue harvested post Staphylococcus aureus (S. aureus) infection of a cemented 3D-printed titanium hip implant in rats. Sprague-Dawley rats underwent left hip cemented 3D-printed titanium hemiarthroplasty via posterior approach under general anesthesia. Four surgeries were performed for the control group and another four for the infected group. The hip joint was inoculated with 5×10. 9. CFU/mL of S. aureus Xen36 prior to capsule closure. The animals were scarified 3 weeks after infection. The femur was harvested and underwent micro-CT and histologic analysis. Hematoxylin and eosin (H&E), as well as Masson's trichrome (MT) stains were performed. Immunohistochemistry (IHC) using rabbit antibody for S. aureus was also used to localize bacterial presence within femur and acetabulum tissue . The histologic analysis revealed strong resemblance to tissue changes in the clinical setting of chronic PJI. IHC demonstrated the extent of bacterial spread within the peri-implant tissue away from the site of infection. The H&E and MT stains showed 5 main features in infected bone: 1) increased PMNs, 2) fibrovascular inflammation, 3) bone necrosis, and 4) increased osteoclasts 5) fibrosis of muscular tissue and cartilage. Micro CT data showed significantly more osteolysis present around the infected prosthesis compared to control (surgery with no infection). This is the first clinically relevant PJI animal model with detailed histologic analysis that strongly resembles the clinical tissue pathology of chronic PJI. This model can provide a better understanding of how various PJI therapies can halt or reverse peri-implant tissue damage caused by infection