Cellular characterisation of advanced osteoarthritis knee synovium.

OBJECTIVES: Osteoarthritis (OA) is increasingly recognised as a whole joint disease, with an important role for synovium. However, the repertoire of immune cells and fibroblasts that constitute OA synovium remains understudied. This study aims to characterise the cellular composition of advanced OA synovium and to explore potential correlations between different cell types and patient demographics or clinical scores. METHODS: Synovium, collected from 10 patients with advanced OA during total knee replacement surgery, was collagenase-digested, and cells were stained for flow cytometry analysis. Formalin-fixed paraffin-embedded synovium was sectioned, stained with immunofluorescence, and imaged using the multiplex Cell DIVE platform. Patient demographics and clinical scores were also collected. RESULTS: The proportion of immune cells in OA synovium varied between patients (8-38% of all cells). Macrophages and T cells were the dominant immune cell populations, together representing 76% of immune cells. Age positively correlated with the proportion of macrophages, and negatively correlated with T cells. CCR6+ T cells were found in 6/10 patients; these patients had a higher mean Kellgren-Lawrence grade across the three knee compartments. Immunofluorescence staining showed that macrophages were present in the lining as well as distributed throughout the sublining, while T and B cells were mainly localised near vessels in the sublining. Fibroblast subsets (CD45-PDPN+) based on the expression of CD34/CD90 or FAP/CD90 were identified in all patient samples, and some populations correlate with the percentage of immune cells or clinical scores. Immunofluorescence staining showed that FAP expression was particularly strong in the lining layer, but also present throughout the sublining layer. CD90 expression was exclusively found around vessels in the sublining, while CD34 was mostly found in the sublining but also occasionally in the lining layer. CONCLUSIONS: There are significant differences in the relative proportions and subsets of immune cells in OA synovium; exploratory correlative analyses suggest that these differences might be correlated with age, clinical scores, or fibroblast subsets. Additional studies are required to understand how different cell types affect OA pathobiology, and if the presence or proportion of cell subsets relates to disease phenotypes.

Loss of mutual protection between human osteoclasts and chondrocytes in damaged joints initiates osteoclast-mediated cartilage degradation by MMPs.

Osteoclasts are multinucleated, bone-resorbing cells. However, they also digest cartilage during skeletal maintenance, development and in degradative conditions including osteoarthritis, rheumatoid arthritis and primary bone sarcoma. This study explores the mechanisms behind the osteoclast-cartilage interaction. Human osteoclasts differentiated on acellular human cartilage expressed osteoclast marker genes (e.g. CTSK, MMP9) and proteins (TRAP, VNR), visibly damaged the cartilage surface and released glycosaminoglycan in a contact-dependent manner. Direct co-culture with chondrocytes during differentiation increased large osteoclast formation (p < 0.0001) except when co-cultured on dentine, when osteoclast formation was inhibited (p = 0.0002). Osteoclasts cultured on dentine inhibited basal cartilage degradation (p = 0.012). RNA-seq identified MMP8 overexpression in osteoclasts differentiated on cartilage versus dentine (8.89-fold, p = 0.0133), while MMP9 was the most highly expressed MMP. Both MMP8 and MMP9 were produced by osteoclasts in osteosarcoma tissue. This study suggests that bone-resident osteoclasts and chondrocytes exert mutually protective effects on their 'native' tissue. However, when osteoclasts contact non-native cartilage they cause degradation via MMPs. Understanding the role of osteoclasts in cartilage maintenance and degradation might identify new therapeutic approaches for pathologies characterized by cartilage degeneration.

Host-biomaterial interactions in mesh complications after pelvic floor reconstructive surgery.

Polypropylene (PPL) mesh is widely used in pelvic floor reconstructive surgery for prolapse and stress urinary incontinence. However, some women, particularly those treated using transvaginal PPL mesh placement for prolapse, experience intractable pain and mesh exposure or extrusion. Explanted tissue from patients with complications following transvaginal implantation of mesh is typified by a dense fibrous capsule with an immune cell-rich infiltrate, suggesting that the host immune response has a role in transvaginal PPL mesh complications through the separate contributions of the host (patient), the biological niche within which the material is implanted and biomaterial properties of the mesh. This immune response might be strongly influenced by both the baseline inflammatory status of the patient, surgical technique and experience, and the unique hormonal, immune and microbial tissue niche of the vagina. Mesh porosity, surface area and stiffness also might have an effect on the immune and tissue response to transvaginal mesh placement. Thus, a regulatory pathway is needed for mesh development that recognizes the roles of host and biological factors in driving the immune response to mesh, as well as mandatory mesh registries and the longitudinal surveillance of patients.

Interleukin-17A Causes Osteoarthritis-Like Transcriptional Changes in Human Osteoarthritis-Derived Chondrocytes and Synovial Fibroblasts In Vitro.

Increased interleukin (IL)-17A has been identified in joints affected by osteoarthritis (OA), but it is unclear how IL-17A, and its family members IL-17AF and IL-17F, can contribute to human OA pathophysiology. Therefore, we aimed to evaluate the gene expression and signalling pathway activation effects of the different IL-17 family members in chondrocytes and synovial fibroblasts derived from cartilage and synovium of patients with end-stage knee OA. Immunohistochemistry staining confirmed that IL-17 receptor A (IL-17RA) and IL-17RC are expressed in end-stage OA-derived cartilage and synovium. Chondrocytes and synovial fibroblasts derived from end-stage OA patients were treated with IL-17A, IL-17AF, or IL-17F, and gene expression was assessed with bulk RNA-Seq. Hallmark pathway analysis showed that IL-17 cytokines regulated several OA pathophysiology-related pathways including immune-, angiogenesis-, and complement-pathways in both chondrocytes and synovial fibroblasts derived from end-stage OA patients. While overall IL-17A induced the strongest transcriptional response, followed by IL-17AF and IL-17F, not all genes followed this pattern. Disease-Gene Network analysis revealed that IL-17A-related changes in gene expression in these cells are associated with experimental arthritis, knee arthritis, and musculoskeletal disease gene-sets. Western blot analysis confirmed that IL-17A significantly activates p38 and p65 NF-κB. Incubation of chondrocytes and synovial fibroblasts with anti-IL-17A monoclonal antibody secukinumab significantly inhibited IL-17A-induced gene expression. In conclusion, the association of IL-17-induced transcriptional changes with arthritic gene-sets supports a role for IL-17A in OA pathophysiology. Future studies should further investigate the role of IL-17A in the OA joint to establish whether anti-IL-17 treatment could be a potential therapeutic option in OA patients with an inflammatory phenotype.

Histopathological and immunohistochemical evaluation of cellular response to a woven and electrospun polydioxanone (PDO) and polycaprolactone (PCL) patch for tendon repair.

We investigated endogenous tissue response to a woven and electrospun polydioxanone (PDO) and polycaprolactone (PCL) patch intended for tendon repair. A sheep tendon injury model characterised by a natural history of consistent failure of healing was chosen to assess the biological potential of woven and aligned electrospun fibres to induce a reparative response. Patches were implanted into 8 female adult English Mule sheep. Significant infiltration of tendon fibroblasts was observed within the electrospun component of the patch but not within the woven component. The cellular infiltrate into the electrospun fibres was accompanied by an extensive network of new blood vessel formation. Tendon fibroblasts were the most abundant scaffold-populating cell type. CD45+, CD4+ and CD14+ cells were also present, with few foreign body giant cells. There were no local or systemic signs of excessive inflammation with normal hematology and serology for inflammatory markers three months after scaffold implantation. In conclusion, we demonstrate that an endogenous healing response can be safely induced in tendon by means of biophysical cues using a woven and electrospun patch.

Characterizing the macro and micro mechanical properties of scaffolds for rotator cuff repair.

BACKGROUND: Retearing after rotator cuff surgery is a major clinical problem. Numerous scaffolds are being used to try to reduce retear rates. However, few have demonstrated clinical efficacy. We hypothesize that this lack of efficacy is due to insufficient mechanical properties. Therefore, we compared the macro and nano/micro mechanical properties of 7 commercially available scaffolds to those of the human supraspinatus tendons, whose function they seek to restore. METHODS: The clinically approved scaffolds tested were X-Repair, LARS ligament, Poly-Tape, BioFiber, GraftJacket, Permacol, and Conexa. Fresh frozen cadaveric human supraspinatus tendon samples were used. Macro mechanical properties were determined through tensile testing and rheometry. Scanning probe microscopy and scanning electron microscopy were performed to assess properties of materials at the nano/microscale (morphology, Young modulus, loss tangent). RESULTS: None of the scaffolds tested adequately approximated both the macro and micro mechanical properties of human supraspinatus tendon. Macroscale mechanical properties were insufficient to restore load-bearing function. The best-performing scaffolds on the macroscale (X-Repair, LARS ligament) had poor nano/microscale properties. Scaffolds approximating tendon properties on the nano/microscale (BioFiber, biologic scaffolds) had poor macroscale properties. CONCLUSION: Existing scaffolds failed to adequately approximate the mechanical properties of human supraspinatus tendons. Combining the macroscopic mechanical properties of a synthetic scaffold with the micro mechanical properties of biologic scaffold could better achieve this goal. Future work should focus on advancing techniques to create new scaffolds with more desirable mechanical properties. This may help improve outcomes for rotator cuff surgery patients.

Differential expression of alarmins-S100A9, IL-33, HMGB1 and HIF-1α in supraspinatus tendinopathy before and after treatment.

BACKGROUND: Alarmins, endogenous molecules released on tissue damage have been shown to play an important role in inflammatory musculoskeletal conditions including fracture repair andrheumatoid arthritis. However, the contribution of alarmins to the pathogenesis of tendon disease is not fully understood. METHODS: We investigated expression of alarmin proteins (S100A9, high-mobility group box 1 (HMGB1) and interleukin-33 (IL-33) and hypoxia-inducible factor 1α (HIF-1α), a subunit of an oxygen sensitive transcription factor, in three cohorts of human supraspinatus tissues: healthy (n=6), painful diseased (n=13) and post-treatment pain-free tendon samples (n=5). Tissue samples were collected during shoulder stabilisation surgery (healthy) or by biopsy needle (diseased/treated). Immunohistochemistry was used to investigate the protein expression of these factors in these healthy, diseased and treated tendons. Kruskal-Wallis with pairwise post hoc Mann-Whitney U tests were used to test for differences in immunopositive staining between these tissue cohorts. Additionally, costaining was performed to identify the cell types expressing HIF-1α, S100A9, IL-33 and HMGB1 in diseased tendons. RESULTS: Immunostaining showed HIF-1α and S100A9 were increased in diseased compared with healthy and post-treatment pain-free tendons (p<0.05). IL-33 was reduced in diseased compared with healthy tendons (p=0.0006). HMGB1 was increased in post-treatment pain-free compared with healthy and diseased tendons (p<0.01). Costaining of diseased tendon samples revealed that HIF-1α, S100A9 and IL-33 were expressed by CD68+ and CD68- cells, whereas HMGB1 was predominantly expressed by CD68- cells. CONCLUSIONS: This study provides insight into the pathways contributing to the progressionand resolution of tendon disease. We found potential pro-inflammatory and pathogenic roles for HIF-1α and S100A9, a protective role fornuclear IL-33 and a potentially reparative function for HMGB1 in diseased supraspinatus tendons.

Identifying the optimum source of mesenchymal stem cells for use in knee surgery.

Single sitting procedures where the mononuclear cell fraction is extracted from bone marrow and implanted directly into cartilage and bone defects are becoming more popular as novel treatments for cartilage defects which have, until now had few treatment options. This is on the basis that the mesenchymal stem cells (MSCs) contained within will repair the damaged tissue. This study sought to determine if the femur and tibia could provide equivalent amounts of mesenchymal stem cells, with equivalent viability and proliferative capacity, to that obtained from the gold standard of the pelvis in order to potentially reduce the morbidity associated with these procedures. Bone marrow was extracted from the pelvis, femur, and tibia of human subjects. The mononuclear cell fraction was extracted and cultured in the laboratory. Mesenchymal stem cell populations were assessed using a colony forming unit count. Viability was assessed using a PrestoBlue viability assay. Population doubling number was calculated between the end of passage 0 and passage three to determine the proliferative abilities of the different populations. Finally, the cell surface phenotype of the cells was determined by flow cytometry. The results showed that the pelvis was superior to the femur and tibia in terms of the number of stem cells isolated. There was no statistically significant difference in the phenotype of the cells isolated from different locations. This work shows that when undertaking single sitting procedures, the pelvis remains the optimum source for obtaining MSCs, despite the morbidity associated with bone marrow collection from the pelvis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1868-1875, 2017.

H3K27me3 demethylases regulate in vitro chondrogenesis and chondrocyte activity in osteoarthritis.

BACKGROUND: Epigenetic changes (i.e., chromatin modifications) occur during chondrogenesis and in osteoarthritis (OA). We investigated the effect of H3K27me3 demethylase inhibition on chondrogenesis and assessed its utility in cartilage tissue engineering and in understanding cartilage destruction in OA. METHODS: We used a high-content screen to assess the effect of epigenetic modifying compounds on collagen output during chondrogenesis of monolayer human mesenchymal stem cells (MSCs). The impact of GSK-J4 on gene expression, glycosaminoglycan output and collagen formation during differentiation of MSCs into cartilage discs was investigated. Expression of lysine (K)-specific demethylase 6A (UTX) and Jumonji domain-containing 3 (JMJD3), the HEK27Me3 demethylases targeted by GSK-J4, was measured in damaged and undamaged cartilage from patients with OA. The impact of GSK-J4 on ex vivo cartilage destruction and expression of OA-related genes in human articular chondrocytes (HACs) was assessed. H3K27Me3 demethylase regulation of transforming growth factor (TGF)-ß-induced gene expression was measured in MSCs and HACs. RESULTS: Treatment of chondrogenic MSCs with the H3K27me3 demethylase inhibitor GSK-J4, which targets JMJD3 and UTX, inhibited collagen output; expression of chondrogenic genes, including SOX9 and COL2A1; and disrupted glycosaminoglycan and collagen synthesis. JMJD3 but not UTX expression was increased during chondrogenesis and in damaged OA cartilage, suggesting a predominant role of JMJD3 in chondrogenesis and OA. GSK-J4 prevented ex vivo cartilage destruction and expression of the OA-related genes MMP13 and PTGS2. TGF-ß is a key regulator of chondrogenesis and articular cartilage homeostasis, and TGF-ß-induced gene expression was inhibited by GSK-J4 treatment of both chondrogenic MSCs and HACs. CONCLUSIONS: Overall, we show that H3K27me3 demethylases modulate chondrogenesis and that enhancing this activity may improve production of tissue-engineered cartilage. In contrast, targeted inhibition of H3K27me3 demethylases could provide a novel approach in OA therapeutics.

A comparative evaluation of the effect of polymer chemistry and fiber orientation on mesenchymal stem cell differentiation.

Bioengineered tissue scaffolds in combination with cells hold great promise for tissue regeneration. The aim of this study was to determine how the chemistry and fiber orientation of engineered scaffolds affect the differentiation of mesenchymal stem cells (MSCs). Adipogenic, chondrogenic, and osteogenic differentiation on aligned and randomly orientated electrospun scaffolds of Poly (lactic-co-glycolic) acid (PLGA) and Polydioxanone (PDO) were compared. MSCs were seeded onto scaffolds and cultured for 14 days under adipogenic-, chondrogenic-, or osteogenic-inducing conditions. Cell viability was assessed by alamarBlue metabolic activity assays and gene expression was determined by qRT-PCR. Cell-scaffold interactions were visualized using fluorescence and scanning electron microscopy. Cells grew in response to scaffold fiber orientation and cell viability, cell coverage, and gene expression analysis showed that PDO supports greater multilineage differentiation of MSCs. An aligned PDO scaffold supports highest adipogenic and osteogenic differentiation whereas fiber orientation did not have a consistent effect on chondrogenesis. Electrospun scaffolds, selected on the basis of fiber chemistry and alignment parameters could provide great therapeutic potential for restoration of fat, cartilage, and bone tissue. This study supports the continued investigation of an electrospun PDO scaffold for tissue repair and regeneration and highlights the potential of optimizing fiber orientation for improved utility. © 2016 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2843-2853, 2016.

Comparison of transforming growth factor beta expression in healthy and diseased human tendon.

BACKGROUND: Diseased tendons are characterised by fibrotic scar tissue, which adversely affects tendon structure and function and increases the likelihood of re-injury. The mechanisms and expression profiles of fibrosis in diseased tendon is understudied compared to pulmonary and renal tissues, where transforming growth factor (TGF)ß and its associated superfamily are known to be key drivers of fibrosis and modulate extracellular matrix homeostasis. We hypothesised that differential expression of TGFß superfamily members would exist between samples of human rotator cuff tendons with established disease compared to healthy control tendons. METHODS: Healthy and diseased rotator cuff tendons were collected from patients presenting to an orthopaedic referral centre. Diseased tendinopathic (intact) and healthy rotator cuff tendons were collected via ultrasound-guided biopsy and torn tendons were collected during routine surgical debridement. Immunohistochemistry and quantitative real-time polymerase chain reaction were used to investigate the protein and gene expression profiles of TGFß superfamily members in these healthy and diseased tendons. RESULTS: TGFß superfamily members were dysregulated in diseased compared to healthy tendons. Specifically, TGFß-1, TGFß receptor (R)1 and TGFß R2 proteins were reduced (p < 0.01) in diseased compared to healthy tendons. At the mRNA level, TGFß R1 was significantly reduced in samples of diseased tendons, whereas TGFß R2 was increased (p < 0.01). BMP-2, BMP-7 and CTGF mRNA remained unchanged with tendon disease. CONCLUSIONS: We propose that downregulation of TGFß pathways in established tendon disease may be a protective response to limit disease-associated fibrosis. The disruption of the TGFß axis with disease suggests associated downstream pathways may be important for maintaining healthy tendon homeostasis. The findings from our study suggest that patients with established tendon disease would be unlikely to benefit from therapeutic TGFß blockade, which has been investigated as a treatment strategy in several animal models. Future studies should investigate the expression profile of fibrotic mediators in earlier stages of tendon disease to improve understanding of the targetable mechanisms underpinning tendon fibrosis.

Differences in glutamate receptors and inflammatory cell numbers are associated with the resolution of pain in human rotator cuff tendinopathy.

INTRODUCTION: The relationship between peripheral tissue characteristics and pain symptoms in soft tissue inflammation is poorly understood. The primary aim of this study was to determine immunohistochemical differences in tissue obtained from patients with persistent pain and patients who had become pain-free after surgical treatment for rotator cuff tendinopathy. The secondary aim was to investigate whether there would be differences in glutaminergic and inflammatory gene expression between disease-derived and healthy control cells in vitro. METHODS: Supraspinatus tendon biopsies were obtained from nine patients with tendon pain before shoulder surgery and from nine further patients whose pain had resolved completely following shoulder surgery. Histological markers relating to the basic tendon characteristics, inflammation and glutaminergic signalling were quantified by immunohistochemical analysis. Gene expression of glutaminergic and inflammatory markers was determined in tenocyte explants derived from painful rotator cuff tendon tears in a separate cohort of patients and compared to that of explants from healthy control tendons. Dual labelling was performed to identify cell types expressing nociceptive neuromodulators. RESULTS: Tendon samples from patients with persistent pain demonstrated increased levels of metabotropic glutamate receptor 2 (mGluR2), kainate receptor 1 (KA1), protein gene product 9.5 (PGP9.5), CD206 (macrophage marker) and CD45 (pan-leucocyte marker) versus pain-free controls (p <0.05). NMDAR1 co-localised with CD206-positive cells, whereas PGP9.5 and glutamate were predominantly expressed by resident tendon cells. These results were validated by in vitro increases in the expression of mGluR2, N-methyl-D-aspartate receptor (NMDAR1), KA1, CD45, CD206 and tumour necrosis factor alpha (TNF-α) genes (p <0.05) in disease-derived versus control cells. CONCLUSIONS: We conclude that differences in glutamate receptors and inflammatory cell numbers are associated with the resolution of shoulder pain in rotator cuff tendinopathy, and that disease-derived cells exhibit a distinctly different neuro-inflammatory gene expression profile to healthy control cells.

Glucocorticoids induce senescence in primary human tenocytes by inhibition of sirtuin 1 and activation of the p53/p21 pathway: in vivo and in vitro evidence.

UNLABELLED: Cellular senescence is an irreversible side effect of some pharmaceuticals which can contribute to tissue degeneration. OBJECTIVE: To determine whether pharmaceutical glucocorticoids induce senescence in tenocytes. METHODS: Features of senescence (ß-galactosidase activity at pH 6 (SA-ß-gal) and active mammalian/mechanistic target of rapamycin (mTOR) in cell cycle arrest) as well as the activity of the two main pathways leading to cell senescence were examined in glucocorticoid-treated primary human tenocytes. Evidence of senescence-inducing pathway induction in vivo was obtained using immunohistochemistry on tendon biopsy specimens taken before and 7 weeks after subacromial Depo-Medrone injection. RESULTS: Dexamethasone treatment of tenocytes resulted in an increased percentage of SA-ßgal-positive cells. Levels of phosphorylated p70S6K did not decrease with glucocorticoid treatment indicating mTOR remained active. Increased levels of acetylated p53 as well as increased RNA levels of its pro-senescence effector p21 were evident in dexamethasone-treated tenocytes. Levels of the p53 deacetylase sirtuin 1 were lower in dexamethasone-treated cells compared with controls. Knockdown of p53 or inhibition of p53 activity prevented dexamethasone-induced senescence. Activation of sirtuin 1 either by exogenous overexpression or by treatment with resveratrol or low glucose prevented dexamethasone-induced senescence. Immunohistochemical analysis of tendon biopsies taken before and after glucocorticoid injection revealed a significant increase in the percentage of p53-positive cells (p=0.03). The percentage of p21-positive cells also tended to be higher post-injection (p=0.06) suggesting glucocorticoids activate the p53/p21 senescence-inducing pathway in vivo as well as in vitro. CONCLUSION: As cell senescence is irreversible in vivo, glucocorticoid-induced senescence may result in long-term degenerative changes in tendon tissue.

BMP5 activates multiple signaling pathways and promotes chondrogenic differentiation in the ATDC5 growth plate model.

The bone morphogenetic protein 5 (BMP5) participates in skeletal development but its direct effects on the function of growth plate chondrocytes during chondrogenesis have not been explored. We have investigated the signaling pathways activated by BMP5 and its effect on chondrogenic differentiation in the ATDC5 growth plate chondrocyte model. BMP5 transiently activated p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase signaling after 10 days of differentiation; sustained Smad and p38 MAPK signaling were seen after 15 days differentiation. All three pathways were activated by BMP5 in human adult articular chondrocytes. BMP5 alone and in combination with the chondrogenic enhancer, insulin, induced proteoglycan synthesis, aggrecan core protein 1 expression, and alkaline phosphatase activity. Upregulation of hypertrophic markers parathyroid receptor 1 and collagen type X alpha 1 occurred in BMP5-treated ATDC5 cultures. BMP5 is clearly chondrogenic and exhibits stage-specific regulation of multiple signaling pathways in this growth plate model. In particular, BMP5 accelerates expression of hypertrophy markers which is of relevance in both development and diseases such as osteoarthritis.