Abstract Micro-computed tomography (µCT) is widely used to assess trabecular bone microarchitecture, with trabecular separation (Tb.Sp) among the core parameters recommended for reporting. Tb.Sp is typically expressed as a single volume-weighted mean derived from maximal sphere fitting, although the underlying distribution of local separation values is rarely examined. Here, we show that Tb.Sp distributions in metaphyseal trabecular bone are frequently non-Gaussian and bimodal or multimodal. Using µCT datasets from three established models of osteoporosis, spinal cord injury (SCI), ovariectomy (OVX), and ageing, we demonstrate that this behaviour is most evident in metaphyseal trabecular bone and is less apparent in epiphyseal trabecular bone or trabecular thickness distributions. We further show that multimodal metaphyseal Tb.Sp distributions correspond to two spatially distinct contributions within the marrow space: lower-diameter local separation within the residual trabecular network, and higher-diameter regions associated with larger contiguous marrow cavities. Based on this observation, we introduce a simple extension to standard morphometric analysis in which Tb.Sp is decomposed into local trabecular separation (Tb.Sp L ) and marrow cavity separation (Tb.Sp M ). Tb.Sp decomposition revealed model-specific patterns of trabecular deterioration. SCI was characterised predominantly by increased Tb.Sp M , consistent with expansion of larger marrow cavities, whereas OVX showed a more subtle or distributed alteration. Ageing showed changes in both Tb.Sp L and Tb.Sp M , with the higher-diameter component becoming most prominent in older animals. Together, these findings demonstrate that mean Tb.Sp can mask structurally distinct forms of metaphyseal marrow-space organisation and support reporting distributional descriptors, and where appropriate Tb.Sp L and Tb.Sp M , alongside conventional Tb.Sp.

Abstract Background Small extracellular vesicles (sEV) derived from synovial fibroblasts (SF) represent a novel molecular mechanism regulating cartilage erosion in osteoarthritis (OA). However, a comprehensive evaluation using disease relevant cells has not been undertaken. The aim of this study was to isolate and characterise sEV from OA SF and to look at their ability to regulate OA chondrocyte effector responses relevant to disease. Profiling of micro (mi) RNA signatures in sEV and parental OA SF cells was performed. Methods SF and chondrocytes were isolated from OA synovial membrane and cartilage respectively (n = 9). sEV were isolated from OA SF (± IL-1β) conditioned media by ultracentrifugation and characterised using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Particle size was confirmed by nanoparticle tracking analysis (NTA). sEV regulation of OA chondrocyte and cartilage effector response was evaluated using qPCR, ELISA and sulphated glycosaminoglycan assay (sGAG). RNA-sequencing was used to establish miRNA signatures in isolated sEV from OA SF. Results OA SF derived sEV were readily taken up by OA chondrocytes, with increased expression of the catabolic gene MMP 13 (p < 0.01) and decreased expression of the anabolic genes aggrecan and COL2A1 (p < 0.01) observed. Treatment with sEV derived from IL-1β stimulated OA SF significantly decreased expression of aggrecan and COL2A1 (p < 0.001) and increased SOX 9 gene expression (p < 0.05). OA chondrocytes cultured with sEV from either non-stimulated or IL-1β treated OA SF, resulted in a significant increase in the secretion of IL-6, IL-8 and MMP-3 (p < 0.01). Cartilage explants cultured with sEV from SF (± IL-1β) had a significant increase in the release of sGAG (p < 0.01). miRNA signatures differed between parental SF cells and isolated sEV. The recently identified osteoclastogenic regulator miR182, along with miR4472-2, miR1302-3, miR6720, miR6087 and miR4532 were enriched in sEV compared to parental cells, p < 0.01. Signatures were similar in sEVs derived from non-stimulated or IL-1β stimulated SF. Conclusions OA SF sEV regulate chondrocyte inflammatory and remodelling responses. OA SF sEV have unique signatures compared to parental cells which do not alter with IL-1β stimulation. This study provides insight into a novel regulatory mechanism within the OA joint which could inform future targeted therapy.

Recent evidence indicates that microbial biofilm aggregates inhabit the lungs of COPD patients and actively contribute towards chronic colonization and repeat infections. However, there are no contextually relevant complex biofilm models for COPD research. In this study, a meta‐analysis of the lung microbiome in COPD was used to inform development of an optimized biofilm model composed of genera highly associated with COPD. Bioinformatic analysis showed that although diversity matrices of COPD microbiomes were similar to healthy controls, and internal compositions made it possible to accurately differentiate between these cohorts (AUC = 0.939). Genera that best defined these patients included Haemophilus , Moraxella and Streptococcus. Many studies fail to account for fungi; therefore, Candida albicans was included in the creation of an interkingdom biofilm model. These organisms formed a biofilm capable of tolerating high concentrations of antimicrobial therapies with no significant reductions in viability. However, combined therapies of antibiotics and an antifungal resulted in significant reductions in viable cells throughout the biofilm (p < 0.05). This biofilm model is representative of the COPD lung microbiome and results from in vitro antimicrobial challenge experiments indicate that targeting both bacteria and fungi in these interkingdom communities will be required for more positive clinical outcomes.

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are used to mimic many aspects of the disease. The present protocol describes the destabilization and cartilage scratch model (DCS) of post-traumatic osteoarthritis. Based on the widely used destabilization of the medial meniscus (DMM) model, DCS introduces three scratches on the cartilage surface. The current article highlights the steps to destabilize the knee by transecting the medial meniscotibial ligament followed by three intentional superficial scratches on the articular cartilage. The possible analysis methods by dynamic weight-bearing, microcomputed tomography, and histology are also demonstrated. While the DCS model is not recommended for studies that focus on the effect of osteoarthritis on the cartilage, it enables the study of osteoarthritis development in a shorter time window, with special focus on (1) osteophyte formation, (2) osteoarthritic and injury pain, and (3) the effect of cartilage damage in the whole joint.

Trypsin-like proteases (TLPs) belong to a family of serine enzymes with primary substrate specificities for the basic residues, lysine and arginine, in the P1 position. Whilst initially perceived as soluble enzymes that are extracellularly secreted, a number of novel TLPs that are anchored in the cell membrane have since been discovered. Muco-obstructive lung diseases (MucOLDs) are characterised by the accumulation of hyper-concentrated mucus in the small airways, leading to persistent inflammation, infection and dysregulated protease activity. Although neutrophilic serine proteases, particularly neutrophil elastase, have been implicated in the propagation of inflammation and local tissue destruction, it is likely that the serine TLPs also contribute to various disease-relevant processes given the roles that a number of these enzymes play in the activation of both the epithelial sodium channel (ENaC) and protease-activated receptor 2 (PAR2). More recently, significant attention has focused on the activation of viruses such as SARS-CoV-2 by host TLPs. The purpose of this review was to highlight key TLPs linked to the activation of ENaC and PAR2 and their association with airway dehydration and inflammatory signalling pathways, respectively. The role of TLPs in viral infectivity will also be discussed in the context of the inhibition of TLP activities and the potential of these proteases as therapeutic targets.

Objectives The aim of this study was to determine the presence of protease-activated receptor 2 (PAR2) and matriptase proteins and quantify PAR2 and matriptase mRNA expression in the articular cartilage and synovial membrane of cats with and without osteoarthritis (OA). Methods A total of 28 articular cartilage samples from adult cats (14 OA and 14 normal), 10 synovial membranes from adult cats (five OA and five normal) and three cartilage samples from 9-week-old fetal cats were used. The presence of PAR2 and matriptase in the cartilage and synovial membrane of the adult samples was detected by immunohistochemical (IHC) staining, while real-time PCR was used for mRNA expression analyses in all samples. Results PAR2 was detected in all OA and normal articular cartilage and synovial membrane samples but confined to only a few superficial chondrocytes in the normal samples. Matriptase was only detected in OA articular cartilage and synovial membrane samples. PAR2 and matriptase mRNA expression were, however, detected in all cartilage and synovial membrane samples. PAR2 and matriptase mRNA expression levels in OA articular cartilage were five ( P <0.001) and 3.3 ( P <0.001) times higher than that of the healthy group, respectively. There was no significant difference ( P = 0.05) in the OA synovial membrane PAR2 and matriptase mRNA expression compared with the normal samples. Conclusions and relevance Detection of PAR2 and matriptase proteins and gene expression in feline articular tissues is a novel and important finding, and supports the hypothesis that serine proteases are involved in the pathogenesis of feline OA. The consistent presence of PAR2 and matriptase protein in the cytoplasm of OA chondrocytes suggests a possible involvement of proteases in cartilage degradation. Further investigations into the PAR2 and matriptase pathobiology could enhance our understanding of the proteolytic cascades in feline OA, which might lead to the development of novel therapeutic strategies.

Angiotensin II-type 1 receptor stimulation is recognised to promote inflammation, a state central to the development and maintenance of rheumatoid arthritis. Herein we examined the use of losartan, an angiotensin II-type 1 receptor antagonist, on vascular reactivity, knee joint diameter and behavioural assessment of pain in a Freund's complete adjuvant (FCA) mouse model of joint inflammation. Monoarthritis was induced via FCA in the presence or absence of losartan with naive mice serving as controls. Knee joint swelling, joint pain (assessed by dynamic weight bearing of limb use), knee joint artery reactivity (assessed ex vivo) and blood perfusion of the knee joint (assessed in vivo) were determined. FCA mediated a significant increase in knee joint diameter and reduced weight-bearing (a surrogate for pain sensation) of the affected limb. Notably, these phenomena were substantially reduced when mice were prophylactically treated with losartan. Assessment of arterial relaxation and blood perfusion with acetylcholine stimulation revealed that FCA resulted in significant vascular dysfunction, which was resolved to naïve levels with losartan treatment. Through the actions of losartan, these findings indicate that the angiotensin II-type 1 receptor is a likely therapeutic target of importance in the development of the physical changes, pain sensation and vascular dysfunction found in inflammatory arthritis.

Chronic obstructive pulmonary disease (COPD) is a debilitating heterogeneous disease characterised by unregulated proteolytic destruction of lung tissue mediated via a protease-antiprotease imbalance. In COPD, the relationship between the neutrophil serine protease, neutrophil elastase, and its endogenous inhibitor, alpha-1-antitrypsin (AAT) is the best characterised. AAT belongs to a superfamily of serine protease inhibitors known as serpins. Advances in screening technologies have, however, resulted in many members of the serpin superfamily being identified as having differential expression across a multitude of chronic lung diseases compared to healthy individuals. Serpins exhibit a unique suicide-substrate mechanism of inhibition during which they undergo a dramatic conformational change to a more stable form. A limitation is that this also renders them susceptible to disease-causing mutations. Identification of the extent of their physiological/pathological role in the airways would allow further expansion of knowledge regarding the complexity of protease regulation in the lung and may provide wider opportunity for their use as therapeutics to aid the management of COPD and other chronic airways diseases.

Osteoarthritis (OA) is the most prevalent of the musculoskeletal conditions and represents a significant public health burden. While degeneration of articular cartilage is a key feature, it is now increasingly recognized as a complex condition affecting the whole joint, with synovial inflammation present in a significant proportion of patients. As a secretory tissue, the OA synovium is a rich source of both soluble inflammatory mediators and extracellular vesicles, including exosomes, which have been implicated in cell-cell communication. Exosome cargo has been found to include proteins, lipids and various RNA subtypes such as mRNA and miRNA, potentially capable of regulating gene expression in target cells and tissues. Profiling of exosome cargo and understanding effects on cartilage could elucidate novel regulatory mechanisms within the joint, providing insight for targeted treatment. The aim of this article is to review current literature on exosome biology, highlighting the relevance and application for OA pathogenesis.

Cigarette smoke stimulates an inflammatory response and produces oxidants that cause oxidative stress in the lung, which promotes pathophysiological changes related to chronic obstructive pulmonary disease (COPD) (Kirkham, P. Pharmacol Ther 2006; 111: 476-94). Hydrogen peroxide (H2O2) is one of the oxidants detected in the breath condensate of COPD patients (Montuschi, P. Clin Chim Acta 2005; 35: 22-34). We aim to understand how chronic exposure to H2O2 alone or in combination with other inflammatory mediators influences epithelial cell responses relevant to COPD lung pathologenesis. BEAS-2B cells were exposed to H2O2 (2 h/d for 3 days) at different concentrations, alone or in combination with TGF-β (10 ng/ml) or LPS (500 ng/ml). Cell viability was assessed. IL-8 and IL-6 were measured by ELISA. Data was analysed using Multiple Comparison Test. Cells tolerated a repeated exposure of H2O2 (up to 15 μM) ± TGF-β or LPS without significant loss of viability. H2O2 stimulated modest release of IL-8 (mean ± SEM; 38±2 pg/ml) and IL-6 (84±13 pg/ml). However, repeated 15 μM H2O2 exposure significantly enhanced TGF-β induced IL-8 (TGF-β, 194±13 vs. TGF-β+ H2O2, 279±10 pg/ml; p<0.0001) but not IL-6 (TGF-β, 431±22 vs. TGF-β+ H2O2, 449±2 pg/ml). H2O2 enhanced LPS secretion of both IL-8 (LPS, 2487±21 vs. LPS+ H2O2, 2898±109 pg/ml; p<0.0001), and IL-6 (LPS, 2469±72 vs. LPS+ H2O2, 3277±62 pg/ml; p<0.0001). Repeated exposure of BEAS-2B cells to H2O2 induced minimal inflammatory response, but enhanced the effect of TGF-β and LPS on cytokine production. These data suggest such combined exposure models may be useful to study the effects of epithelial cell challenge relevant to COPD pathology.

Lungs from patients with chronic obstructive pulmonary disease (COPD) display hallmarks of premature ageing, including dysregulated autophagy, leading to cellular senescence. The underlying mechanisms remain unclear. Proteinase activated receptor 2 (PAR2) is a potential therapeutic target for inflammatory conditions, with documented roles in lung pathology. A role for this receptor in lung ageing is yet unexplored. Autophagic markers LC3 and ATG7 were examined in C57BL/6 wild type and PAR2-/- knock out lung tissue using immunohistochemistry. Autophagic flux was quantified through Marfluorescent imaging (CYTO-ID detection kit) in human bronchial epithelial cell line BEAS-2B and primary human bronchial epithelial cells from healthy (HBEC) and COPD patient donors (DHBEC), after PAR2 stimulation with SLIGKV agonist (cf. VKGILS control). ATG7 (p<0.005) and LC3 (p<0.05) positive cells were significantly upregulated in PAR2-deficient lungs (Figure 1). PAR2 was present on epithelial cultures, with redistribution upon stimulation. PAR2 stimulation in BEAS-2B resulted in a significant reduction of autophagic vesicles cf. VKGILS (p<0.001). Whilst similar behaviour was observed in HBEC, DHBEC exhibited autophagic flux dysregulation. This study provides the first data describing a role for PAR2 in the regulation of autophagy in airway epithelia, suggesting a potential mechanism that may underpin premature lung ageing in conditions such as COPD.

Hyper-reactivity, inflammation and hyperplasia/hypertrophy of airway smooth muscle (ASM) limit airflow and are key features of chronic obstructive pulmonary disease (COPD). Proteinase activated receptor 2 (PAR2) is a critical modulator of inflammatory responses in respiratory disease such as asthma, yet is reported to promote ASM relaxation. However, the role of ASM PAR2 in COPD is not well understood (Sokolova & Reiser, Pharmacol Ther. 2007; 115(1):70-83). Our aim was to determine the presence and role of PAR2 in murine lung and ASM subjected to an oxidative stressor environment using both immunohistochemistry (IHC) and wire myography. PAR2 was detected using APR-32 antibody (Alomone, Israel) on both murine airway and lung tissue, with clean isotype. Data presented as mean ± SEM. An oxidative stressor environment increased trypsin-induced ASM relaxation in both bronchial (Oxidative 54.5±8.5 % vs. Control 35.1±3.7 %; n=6; p<0.05; paired t-test) and tracheal tissue (Oxidative 56.7±10.5 % vs. Control 30.3±2.9 %; n=6; p=0.052; paired t-test). This was PAR2 dependent, as relaxation to trypsin was virtually abolished in PAR2-/- compared with WT tracheal (WT 56.7±10.5 % vs. PAR2-/- 2.5±0.7 %; n=3-6; p<0.01; unpaired t-test) and bronchial tissue (WT 54.5±8.5 % vs. PAR2-/- 2.3± 0.9 %; n=3-6; p<0.01; unpaired t-test). Further confirmation of PAR2 dependent relaxation was achieved using a PAR2 specific peptide (2-FLIGRLO-NH2). In conclusion, this study confirms a functional PAR2 role in murine airway tissue; importantly, the role of PAR2 in mediating ASM relaxation appears to be enhanced in oxidative environments such as found in COPD. This may have important implications for future potential therapies.

Purpose: Osteoarthritis (OA) is no longer considered to be just a disease of the cartilage but often exhibits low-grade inflammation and disruption of synovial tissue homeostasis. Recent work would suggest that 70% of OA patients present with synovial inflammation, which significantly correlates with pain and cartilage damage. Disruption of joint tissue homeostasis results in an altered secretory profile from the synovial membrane, with extracellular vesicles such as exosomes contributing to this secretome. Importantly, exosomes have been implicated in cell communication through their ability to carry and transfer a range of potentially modulatory/regulatory cargo, including microRNAs. Protease activated receptor 2 (PAR2) has been identified as a critical regulatory molecule for inflammatory joint disease and OA pathophysiology. The aim of this study was to characterise and compare the PAR2 driven secretome of OA tissues and cells, including the exosome content. Methods: Equal weights of human OA synovial membrane and infrapatellar white adipose tissue (WAT) explants (n=50 collected by arthroplasty) were cultured in the presence/absence of IL-1β, PAR2 peptide agonist (SLIGKV-NH2) or a reverse peptide (RP) control and conditioned media harvested after 48h and used for exosome preparation. Parallel experiments were run with synovial fibroblasts. Dead cells and debris were eliminated by differential centrifugation, with exosomes isolated by ultracentrifuge at 100,000g. Exosomes were characterised by western blot, scanning electron microscopy (SEM) and Nanoparticle Tracking Analysis (NTA). Following characterisation, exosomes were labelled with Exo-Red (staining RNA) or Exo-Green (protein) and uptake by primary OA articular chondrocytes (n=6) evaluated using immunofluorescence imaging. Gene expression and cytokine changes 6h post exosome uptake by chondrocytes (n=9) was analysed with real time qPCR and ELISA. RNA was isolated from non-stimulated, IL-1β, SLIGKV-NH2 and RP derived synovial membrane exosomes as well as non-stimulated/IL-1β synovial fibroblast derived exosomes and their parental cells. Once enriched for small RNA, libraries were prepared, sequenced and bioinformatics analysis undertaken. Results: Exosome preparations from synovial fibroblasts, synovial membrane and WAT explants were evaluated and presence of exosome-associated markers CD9, CD81, HSP70 and CD63 confirmed. Furthermore, NTA and SEM demonstrated that within preparations, 80-90% of microvesicles were of exosome size; ranging from 30 to 150nm. Exosomes carried both protein and RNA cargo and were taken up by 90% of primary chondrocytes after 4h exposure, regardless of source. Uptake of non-stimulated synovial membrane and WAT derived exosomes increased IL-6 (p<0.001, p<0.01 respectively) and IL-8 (p<0.05, p>0.01 respectively) protein secretion. Only synovial-derived exosomes increased MMP-3 (p<0.001) and TNFα (p<0.001) protein secretion. Regardless of source, exosomes had no significant effect on IL-10 (p<0.1) protein release from primary chondrocytes. Interestingly, exosomes derived from IL-1β stimulated WAT increased MMP-3 and TNFα (p<0.01, p<0.01) protein secretion compared to non-stimulated controls. Primary chondrocytes that had taken up exosomes (regardless of source) also showed significant transcript expression decreases in COL2A1 (p<0.001) and ACAN (p<0.001) and increased expression of MMP1 (p<0.01). Conclusions: These findings suggest that tissues within the synovial compartment have the potential to impact cartilage destruction through exosomes release, by increasing the release of inflammatory cytokine mediators from chondrocytes and regulating expression of catabolic and anabolic genes. Further analysis of small RNA cargo of exosomes derived from different sources could give insight into novel regulatory mechanisms within the OA joint.

Purpose: Injuries involving damage to the articular cartilage can be both physically limiting and painful. Articular cartilage injuries can result from an isolated injury, or in combination with other joint damage. Cartilage has a poor intrinsic regenerative capability, and therefore once damaged, presents a major clinical challenge. Joint injury is known to be a well-established precursor for the development of the osteoarthritis (OA). Pain is one of the most common, and arguably physically limiting, symptoms of OA. However, the origin of pain in OA is poorly understood. Several murine models have been used to study OA (e.g., destabilisation of the medial meniscus (DMM) and anterior cruciate ligament transection (ACLT)). However neither of these models combines simultaneous cartilage injury with joint destabilisation. Therefore, the principle aim of this study was to investigate if the combination of cartilage damage and DMM accelerates the onset of OA-like symptoms when compared with DMM and cartilage damage alone. Methods: Three models of OA-like joint instability and damage were induced in wild-type (WT) mice. For biomechanical instability, DMM surgery induced medial compartment OA following transection of the medial meniscotibial ligament. For cartilage damage, a micro precision blade was used to make controlled "scratches" (Scratch model) on the cartilage of the tibia under the femoral medial condyle. To combine biomechanical instability with cartilage damage, both DMM and cartilage scratch (Combined model) was performed. Microcomputered tomography (μCT) was used to monitor bone changes, and as an indirect measurement of pain, dynamic weight bearing was assessed in mice 14 days after surgery. Results: At 14 days post-surgery, osteophytes are present in all 3 groups. However, between groups, osteophytes differed in both appearance and number. In DMM, 6/8 mice developed protruding osteophytes with an arboreal structure (the initial phase of osteophyte formation). Osteophytes were present in all mice undergoing cartilage scratch surgery, however, they had a larger more calcified appearance, suggesting cartilage could play a role in the acceleration of OA osteophyte pathology. 8/8 WT mice in the Combined model were observed to exhibit 2 or more large, protruding, calcified osteophytes. Osteophytes observed in this Combined model encompassed a larger area of the subchondral bone when compared with DMM or Scratch model alone (14.12 ± 0.31 versus 12.4 ± 0.62, 12.68 ± 0.54, p<0.01). Compared to the osteophytes in the DMM model , it is clear that addition of cartilage damage accelerates the calcification of osteophytes. μCT analysis of the subchondral bone shows both DMM, and Combined models have an increase in subchondral osteosclerosis compared to cartilage damage alone. Further, analysis of subchondral trabecular bone in the Combined model exhibited a significantly higher bone volume over tissue volume compared with the other models used in this study (p=0.0017, two-way ANOVA).Using dynamic weight bearing 14 days post-surgery to assess OA-related pain, we found the Combined model group have a significant increase in load on their front paws when compared with the other 2 groups, suggesting an increase in OA-like pain. Conclusions: OA is a multi-factorial disease encompassing various different tissues within the joint. Accordingly, a Combined model of OA as presented here allows a more accurate representation of human OA. This study concludes that a combining DMM with cartilage damage provides a more robust and reproducible model for OA due to the increased osteophytogenesis. This model also incorporates OA-related pain, which is arguably one of the most problematic and physically limiting symptoms of OA.

Protease-activated receptor-2 (PAR2) is one member of a small family of transmembrane, G-protein-coupled receptors. These receptors are activated via cleavage of their N terminus by serine proteases (e.g., tryptase), unveiling an N terminus tethered ligand which binds to the second extracellular loop of the receptor. Increasing evidence has emerged identifying key pathophysiological roles for PAR2 in both rheumatoid arthritis (RA) and osteoarthritis (OA). Importantly, this includes both pro-inflammatory and destructive roles. For example, in murine models of RA, the associated synovitis, cartilage degradation, and subsequent bone erosion are all significantly reduced in the absence of PAR2. Similarly, in experimental models of OA, PAR2 disruption confers protection against cartilage degradation, subchondral bone osteosclerosis, and osteophyte formation. This review focuses on the role of PAR2 in rheumatic disease and its potential as an important therapeutic target for treating pain and joint degradation.

Osteoarthritis (OA) is no longer considered a cartilage-centric disease with remodelling of other joint tissues now recognized. While understudied, entheseal pathology is considered a secondary OA feature. A pivotal role for proteinase-activated receptor 2 (PAR2) in OA has been demonstrated previously in cartilage and subchondral bone at early time points, however the entheseal role of PAR2 has not been reported.OA was induced by destabilization of the medial meniscus (DMM) in wild type (WT) and PAR2 deficient (KO) animals. At 4 weeks and one year post surgery, knee joints were harvested for histological analysis. Medial collateral ligament (MCL) width was measured by 2D planimetry analysis. Immunohistochemistry was used to characterize the MCL and anterior cruciate ligament (ACL). Data were expressed as mean±SEM (n=4–6/group) and analysed using Student's t-test, with p<0.05 as the criterion of significance.MCL width increased between 4 weeks and 1 year in WT DMM (0.24 ±0.07 vs 0.40 ±0.008mm respectively,...