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

Tendinopathy is the most common form of chronic tendon disorders, accounting for up 30% of all musculoskeletal clinic visits [1]. In tendon disease, the largely avascular tendon tissue often becomes hypervascularized and fibrotic [2]. As blood vessel growth and angiogenic signaling molecules are often induced by the lack of adequate nutrients and oxygen, hypoxic signaling is speculated to be a root cause of tendon neovascularization and tendinopathy [3,4,5]. However, how the vascular switch is initiated in tendons, and how vascularization contributes to tendon pathology remains unknown. In this talk, we provide evidence that HIF-1α is implicated in tendon disease and HIF-1α stabilization in human tendon cells induces vascular recruitment of endothelial cells via VEGFa secretion. More interesting, HIF-1α stabilization in tendon cells in vivo, seems to recapitulate all main features of fibrotic human tendon disease, including vascular ingrowth, matrix disorganization, changes in tissue mechanics, cell proliferation and innervation. Surprisingly, in vivo knock-out of VEGFa rescued angiogenesis in the tendon core but it did not affect tendon mechanical properties and tissue pathophysiological changes, suggesting that blood vessels ingrowth might not be a primary cause but a consequence of HIF-1α activation.

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.

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.

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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.

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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

Aims

Lumbar spinal stenosis (LSS) is a common skeletal system disease that has been partly attributed to genetic variation. However, the correlation between genetic variation and pathological changes in LSS is insufficient, and it is difficult to provide a reference for the early diagnosis and treatment of the disease.

Aims

As has been shown in larger animal models, knee immobilization can lead to arthrofibrotic phenotypes. Our study included 168 C57BL/6J female mice, with 24 serving as controls, and 144 undergoing a knee procedure to induce a contracture without osteoarthritis (OA).

Aims

Aseptic loosening is a leading cause of uncemented arthroplasty failure, often accompanied by fibrotic tissue at the bone-implant interface. A biological target, neutrophil extracellular traps (NETs), was investigated as a crucial connection between the innate immune system’s response to injury, fibrotic tissue development, and proper bone healing. Prevalence of NETs in peri-implant fibrotic tissue from aseptic loosening patients was assessed. A murine model of osseointegration failure was used to test the hypothesis that inhibition (through Pad4^-/- mice that display defects in peptidyl arginine deiminase 4 (PAD4), an essential protein required for NETs) or resolution (via DNase 1 treatment, an enzyme that degrades the cytotoxic DNA matrix) of NETs can prevent osseointegration failure and formation of peri-implant fibrotic tissue.

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.

Introduction. Patients with aseptic loosening, a cause of failure in uncemented total joint arthroplasty (TJA), often present with fibrous tissue at the bone-implant interface. 1. In this study, we characterize the presence of neutrophil extracellular traps (NETs) in the intramedullary fibrotic membrane of aseptic loosening patients. We further explore the role of NETs, mediated by peptidyl arginine deiminase (PAD4), in peri-implant fibrosis and osseointegration failure through a murine model of unstable tibial implantation. 2–4. Methods. Peri-implant membrane was retrieved from five patients during total hip revision surgery and analyzed for the presence of NETs (citH3+ with extracellular DNA) via immunofluorescence. A Ti-6Al-4V implant was inserted in an oversized drill-hole in the right proximal tibia of 8-week-old C57BL/6J and PAD4 knockout mice (n=3 per group). Fourteen days later, all mice were euthanized, and implanted tibias were dissected. Fibrosis and osseointegration at the bone-implant interface were assessed by micro-computed tomography (microCT) and hematoxylin and eosin (H&E) staining. H&E samples were scored blindly by the investigator and another observer for signs of poor (score=0) to excellent osseointegration (score=3) using a rubric established in our lab. Results. NETs were found in peri-implant membrane collected from aseptic loosening patients (Figure 1a) and at the bone-implant interface in a murine model (Figure 1b). Unstable implants in wild type mice failed to osseointegrate, indicated by presence of fibroblast-like cells (dashed arrow), immature bone matrix (Figure 1c), low bone volume fraction (BV/TV) and bone surface area (BS) (Figure 1e). Unstable implants in PAD4. −/−. mice showed signs of good osseointegration such as mature trabeculae (solid arrow) (Figure 1d), higher BV/TV (p<0.10) and BS (p<0.05) (Figure 1f). Histological osseointegration scoring indicated wildtype mice exhibited an average score of 0.83 and PAD4. −/−. exhibited an average score of 2.5 (p<0.05, weighted Cohen's kappa = 0.714) (Figure 1g). Conclusion. NETs were characterized in fibrotic tissue in both aseptic loosening patients and in a murine model of unstable tibial implantation. NET inhibition was able to successfully prevent peri-implant fibrosis and osseointegration failure, leading the way for a potential novel non-invasive therapeutic approach for the treatment of aseptic loosening. For any figures, tables, or references, please contact the authors directly

Introduction

Initial post-operative implant instability leads to impaired osseointegration, one of the most common reasons for aseptic loosening and revision surgery. In this study, we developed a novel murine model of implant instability and demonstrated the anabolic effect of immediate and delayed intermittent Parathyroid Hormone (iPTH) treatment in the setting of instability-induced osseointegration failure.

Tungsten has been increasing in demand for use in manufacturing and recently, medical devices, as it imparts flexibility, strength, and conductance of metal alloys. Given the surge in tungsten use, our population may be subjected to elevated exposures. For instance, embolism coils made of tungsten have been shown to degrade in some patients. In a cohort of breast cancer patients who received tungsten-based shielding for intraoperative radiotherapy, urinary tungsten levels remained over tenfold higher 20 months post-surgery. In vivo models have demonstrated that tungsten exposure increases tumor metastasis and enhances the adipogenesis of bone marrow-derived mesenchymal stem cells while inhibiting osteogenesis. We recently determined that when mice are exposed to tungsten [15 ppm] in their drinking water, it bioaccumulates in the intervertebral disc tissue and vertebrae. This study was performed to determine the toxicity of tungsten on intervertebral disc.

Bovine nucleus pulposus (bNP) and annulus fibrosus (bAF) cells were isolated from bovine caudal tails. Cells were expanded in flasks then prepared for 3D culturing in alginate beads at a density of 1×10^∧6 cells/mL. Beads were cultured in medium supplemented with increasing tungsten concentrations in the form of sodium tungstate [0, 0.5, 5, 15 ug/mL] for 12 days. A modified GAG assay was performed on the beads to determine proteoglycan content and Western blotting for type II collagen (Col II) synthesis. Cell viability was determined by counting live and dead cells in the beads following incubation with the Live/Dead Viability Assay kit (Thermo Fisher Scientific). Cell numbers in beads at the end of the incubation period was determined using Quant-iT dsDNA Assay Kit (Thermo Fisher Scientific)

Tungsten dose-dependently decreased the synthesis of proteoglycan in IVD cells, however, the effect was significant at the highest dose of 15 ug/mL. (n=3). Furthermore, although tungsten decreased the synthesis of Col II in IVD cells, it significantly increased the synthesis of Col I. Upregulation of catabolic enzymes ADAMTS4 and −5 were also observed in IVD cells treated with tungsten (n=3). Upon histological examination of spines from mice treated with tungsten [15 ug/mL] in their drinking water for 30 days, disc heights were diminished and Col I upregulation was observed (n=4). Cell viability was not markedly affected by tungsten in both bNP and bAF cells, but proliferation of bNP cells decreased at higher concentration. Surprisingly, histological examination of IVDs and gene expression analysis demonstrated upregulation of NGF expression in both NP and AF cells. In addition, endplate capillaries showed increases in CGRP and PGP9.5 expression as determined on histological sections of mouse IVDs, suggesting the development of sensory neuron invasion of the disc.

We provide evidence that prolonged tungsten exposure can induce disc fibrosis and increase the expression of markers associated with pain. Tungsten toxicity may play a role in disc degeneration disease.

Aims

CERAMENT|G is an absorbable gentamicin-loaded biocomposite used as an on-site vehicle of antimicrobials for the treatment of chronic osteomyelitis. The purpose of the present study was to investigate the sole effect of CERAMENT|G, i.e. without additional systemic antimicrobial therapy, in relation to a limited or extensive debridement of osteomyelitis lesions in a porcine model.

Aims

Complex total hip arthroplasty (THA) with subtrochanteric shortening osteotomy is necessary in conditions other than developmental dysplasia of the hip (DDH) and septic arthritis sequelae with significant proximal femur migration. Our aim was to evaluate the hip centre restoration with THAs in these hips.

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.

Aims

Early evidence has emerged suggesting that ceramic-on-ceramic articulations induce a different tissue reaction to ceramic-on-polyethylene and metal-on-metal bearings. Therefore, the aim of this study was to investigate the tissue reaction and cellular response to ceramic total hip arthroplasty (THA) materials in vitro, as well as the tissue reaction in capsular tissue after revision surgery of ceramic-on-ceramic THAs.

Objectives

The objectives of this study were: 1) to examine osteophyte formation, subchondral bone advance, and bone marrow lesions (BMLs) in osteoarthritis (OA)-prone Hartley guinea pigs; and 2) to assess the disease-modifying activity of an orally administered phosphocitrate ‘analogue’, Carolinas Molecule-01 (CM-01).

Aims

The number of patients undergoing arthroscopic surgery of the hip has increased significantly during the past decade. It has now become an established technique for the treatment of many intra- and extra-articular conditions affecting the hip. However, it has a steep learning curve and is not without the risk of complications. The purpose of this systematic review was to determine the prevalence of complications during and following this procedure.

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.