Human Osteoarthritic Joints Research Articles

Dysregulated fibrinolysis and plasmin activation promote the pathogenesis of osteoarthritis.

Joint injury is associated with risk for development of osteoarthritis (OA). Increasing evidence suggests that activation of fibrinolysis is involved in OA pathogenesis. However, the role of the fibrinolytic pathway is not well understood. Here, we showed that the fibrinolytic pathway, which includes plasminogen/plasmin, tissue plasminogen activator, urokinase plasminogen activator (uPA), and the uPA receptor (uPAR), was dysregulated in human OA joints. Pharmacological inhibition of plasmin attenuated OA progression after a destabilization of the medial meniscus in a mouse model whereas genetic deficiency of plasmin activator inhibitor, or injection of plasmin, exacerbated OA. We detected increased uptake of uPA/uPAR in mouse OA joints by microPET/CT imaging. In vitro studies identified that plasmin promotes OA development through multiple mechanisms, including the degradation of lubricin and cartilage proteoglycans and induction of inflammatory and degradative mediators. We showed that uPA and uPAR produced inflammatory and degradative mediators by activating the PI3K, 3'-phosphoinositide-dependent kinase-1, AKT, and ERK signaling cascades and activated matrix metalloproteinases to degrade proteoglycan. Together, we demonstrated that fibrinolysis contributes to the development of OA through multiple mechanisms and suggested that therapeutic targeting of the fibrinolysis pathway can prevent or slow development of OA.

Tribological behavior of shape-specific microplate-enriched synovial fluids on a linear two-axis tribometer

Nano- and micro-particles are being increasingly used to tune interfacial frictional properties in diverse applications, from friction modifiers in industrial lubrication to enhanced biological fluids in human osteoarthritic joints. Here, we assessed the tribological properties of a simulated synovial fluid enriched with non-spherical, poly lactic-co-glycolic acid (PLGA) microparticles (µPL) that have been previously demonstrated for the pharmacological management of osteoarthritis (OA). Three different µPL configurations were fabricated presenting a 20 µm × 20 µm square base and a thickness of 5 µm (thin, 5H µPL), 10 µm (10H µPL), and 20 µm (cubical, 20H µPL). After extensive morphological and physicochemical characterizations, the apparent Young’s modulus of the µPL was quantified under compressive loading returning an average value of ∼ 6 kPa, independently of the particle morphology. Then, using a linear two-axis tribometer, the static (µs) and dynamic (µd) friction coefficients of the µPL-enriched simulated synovial fluid were determined in terms of particle configuration and concentration, varying from 0 (fluid only) to 6µ105 µPL/mL. The particle morphology had a modest influence on friction, possibly because the µPL were fully squeezed between two mating surfaces by a 5.8 N normal load realizing boundary-like lubrication conditions. Differently, friction was observed to depend on the dimensionless parameter Ω, defined as the ratio between the total volume of the µPL enriching the simulated synovial fluid and the volume of the fluid itself. Both coefficients of friction were documented to grow with Ω reaching a plateau of µs ∼ 0.4 and µd ∼ 0.15, already at Ω ∼ 2×10−3. Future investigations will have to systematically analyze the effect of sliding velocity, normal load, and rigidity of the mating surfaces to elucidate in full the tribological behavior of µPL in the context of osteoarthritis.

Mechanical stress and inflammation have opposite effects on Wnt signaling in human chondrocytes.

Dysregulation of Wingless and Int-1 (Wnt) signaling has been strongly associated with development and progression of osteoarthritis (OA). Here, we set out to investigate the independent effects of either mechanical stress (MS) or inflammation on Wnt signaling in human neocartilage pellets, and to relate this Wnt signaling to OA pathophysiology. OA synovium-conditioned media (OAS-CM) was collected after incubating synovium from human end-stage OA joints for 24 h in medium. Cytokine levels in the OAS-CM were determined with a multiplex immunoassay (Luminex). Human neocartilage pellets were exposed to 20% MS, 2% OAS-CM or 1 ng/mL Interleukin-1β(IL-1β). Effects on expression levels of Wnt signaling members were determined by reverse transcription-quantitative polymerase chain reaction. Additionally, the expression of these members in articular cartilage from human OA joints was analyzed in association with joint space narrowing (JSN) and osteophyte scores. Protein levels of IL-1β, IL-6, IL-8, IL-10, tumor necrosis factor α, and granulocyte-macrophage colony-stimulating factor positively correlated with each other. MS increased noncanonical WNT5A and FOS expression. In contrast, these genes were downregulated upon stimulation with OAS-CM or IL-1β. Furthermore, Wnt inhibitors DKK1 and FRZB decreased in response to OAS-CM or IL-1β exposure. Finally, expression of WNT5A in OA articular cartilage was associated with increased JSN scores, but not osteophyte scores. Our results demonstrate that MS and inflammatory stimuli have opposite effects on canonical and noncanonical Wnt signaling in human neocartilage. Considering the extent to which MS and inflammation contribute to OA in individual patients, we hypothesize that targeting specific Wnt pathways offers a more effective, individualized approach.

Inflammation-Mediated Aberrant Glucose Metabolism in Subchondral Bone Induces Osteoarthritis.

Osteoarthritis (OA) is an entire joint disease with pathological alteration in both articular cartilage and subchondral bone. It has been recognized recently the association between metabolic syndrome and OA, particularly glucose metabolism in regulation of articular cartilage homeostasis and joint integrity. Whereas the role of glucose metabolism in subchondral bone sclerosis remains largely unknown during pathogenesis of OA. Consistent with common OA features, we observed subchondral bone sclerosis and abnormal bone remodeling in human OA joints and murine OA joints as reflected by hyperactive bone resorption and overall bone formation which was measured via dynamic histomorphometry. Osx-CreER;tdTomato mice also displayed the similar overall bone formation under injury-induced OA condition. Immunohistochemistry further revealed increased IL-1β expression in human and murine OA subchondral bone. Given the inflammatory environment in joints under OA condition, we treated MC3T3-E1 cell, a pre-osteoblast cell line, with IL-1β in this study and demonstrated that IL-1β treatment could stimulate the cell osteogenic differentiation and meanwhile upregulate glycolysis and oxidative phosphorylation in cell cultures. More importantly, intraperitoneal injection of 2-deoxy-D-glucose (2-DG) and oligomycin (OGM), respectively, suppressed the subchondral bone glycolysis and oxidative phosphorylation in mice. Consequently, 2-DG and OGM treatment attenuated abnormal osteoblast differentiation and protected against aberrant bone formation in subchondral bone and articular cartilage degradation in wildtype mice following with joint injury. Collectively, these data strongly suggest glycolysis and oxidative may serve as important therapeutic targets for OA treatment.

Defective chaperone-mediated autophagy is a hallmark of joint disease in patients with knee osteoarthritis

Defects in autophagy contribute to joint aging and Osteoarthritis (OA). Identifying specific autophagy types could be useful for developing novel treatments for OA. An autophagy-related gene array was performed in blood from non-OA and knee OA subjects from the Prospective Cohort of A Coruña (PROCOAC). The differential expression of candidate genes was confirmed in blood and knee cartilage and a regression analysis was performed adjusting for age and BMI. HSP90A, a chaperone mediated autophagy (CMA) marker was validated in human knee joint tissues, as well as, in mice with aging-related and surgically-induced OA. The consequences of HSP90AA1 deficiency were evaluated on OA pathogenesis. Finally, the contribution of CMA to homeostasis was studied by assessing the capacity to restore proteostasis upon ATG5-mediated macroautophagy deficiency and genetic HSP90AA1 overexpression. 16 autophagy-related genes were significantly down-regulated in blood from knee OA subjects. Validation studies showed that HSP90AA1 was down-regulated in blood and human OA cartilage and correlated with risk incidence of OA. Moreover, HSP90A was reduced in human OA joints tissues and with aging and OA in mice. HSP90AA1 knockdown was linked to defective macroautophagy, inflammation, oxidative stress, senescence and apoptosis. However, macroautophagy deficiency increased CMA, highlighting the CMA-macroautophagy crosstalk. Remarkably, CMA activation was sufficient to protect chondrocytes from damage. We show that HSP90A is a key chaperone for chondrocyte homeostasis, while defective CMA contributes to joint damage. We propose that CMA deficiency is a relevant disease mechanism and could represent a therapeutic target for OA.

β Boswellic Acid Blocks Articular Innate Immune Responses: An In Silico and In Vitro Approach to Traditional Medicine.

Osteoarthritis (OA) is hallmarked as a silent progressive rheumatic disease of the whole joint. The accumulation of inflammatory and catabolic factors such as IL6, TNFα, and COX2 drives the OA pathophysiology into cartilage degradation, synovia inflammation, and bone destruction. There is no clinical available OA treatment. Although traditional ayurvedic medicine has been using Boswellia serrata extracts (BSE) as an antirheumatic treatment for a millennium, none of the BSE components have been clinically approved. Recently, β boswellic acid (BBA) has been shown to reduce in vivo OA-cartilage loss through an unknown mechanism. We used computational pharmacology, proteomics, transcriptomics, and metabolomics to present solid evidence of BBA therapeutic properties in mouse and primary human OA joint cells. Specifically, BBA binds to the innate immune receptor Toll-like Receptor 4 (TLR4) complex and inhibits both TLR4 and Interleukin 1 Receptor (IL1R) signaling in OA chondrocytes, osteoblasts, and synoviocytes. Moreover, BBA inhibition of TLR4/IL1R downregulated reactive oxygen species (ROS) synthesis and MAPK p38/NFκB, NLRP3, IFNαβ, TNF, and ECM-related pathways. Altogether, we present a solid bulk of evidence that BBA blocks OA innate immune responses and could be transferred into the clinic as an alimentary supplement or as a therapeutic tool after clinical trial evaluations.

RNA-seq characterization of histamine-releasing mast cells as potential therapeutic target of osteoarthritis

ObjectiveMast cells in the osteoarthritis (OA) synovium correlate with disease severity. This study aimed to further elucidate the role of mast cells in OA by RNA-Seq analysis and pharmacological blockade of the activity of histamine, a key mast cell mediator, in murine OA. MethodsWe examined OA synovial tissues and fluids by flow cytometry, immunostaining, single-cell and bulk RNA-Seq, qPCR, and ELISA. Cetirizine, a histamine H1 receptor (H1R) antagonist, was used to treat the destabilization of the medial meniscus (DMM) mouse model of OA. ResultsFlow cytometry and immunohistology analysis of OA synovial cells revealed KIT+ FcεRI+ and TPSAB1+ mast cells. Single-cell RNA-Seq of OA synovial cells identified the expression of prototypical mast cell markers KIT, TPSAB1, CPA3 and HDC, as well as distinctive markers HPGD, CAVIN2, IL1RL1, PRG2, and CKLF, confirmed by bulk RNA-Seq and qPCR. A mast cell prototypical marker expression score classified 40 OA patients into three synovial pathotypes: mast cell-high, −medium, and -low. Additionally, we detected mast cell mediators including histamine, tryptase AB1, CPA3, PRG2, CAVIN2, and CKLF in OA synovial fluids. Elevated H1R expression was detected in human OA synovium, and treatment of mice with the H1 receptor antagonist cetirizine reduced the severity and OA-related mediators in DMM. ConclusionBased on differential expression of prototypical and distinct mast cell markers, human OA joints can be stratified into mast cell-high, −medium, and -low synovial tissue pathotypes. Pharmacologic blockade of histamine activity holds the potential to improve OA disease outcome.

The Synovium of Human Osteoarthritic Joints Retains Its Chondrogenic Potential Irrespective of Age.

The autologous synovium is a potential tissue source for local induction of chondrogenesis by tissue engineering approaches to repair articular cartilage defects that occur in osteoarthritis. It was the aim of the present study to ascertain whether the aging of human osteoarthritic patients compromises the chondrogenic potential of their knee-joint synovium and the structural and metabolic stability of the transformed tissue. The patients were allocated to one of the following two age categories: 54-65 years and 66-86 years (n = 7-11 donors per time point and experimental group; total number of donors: 64). Synovial biopsies were induced in vitro to undergo chondrogenesis by exposure to bone morphogenetic protein-2 (BMP-2) alone, transforming growth factor-β1 (TGF-β1) alone, or a combination of the two growth factors, for up to 6 weeks. The differentiated explants were evaluated morphologically and morphometrically for the volume fraction of metachromasia (sulfated proteoglycans), immunohistochemically for type-II collagen, and for the gene expression levels of anabolic chondrogenic markers as well as catabolic factors by a real-time polymerase chain reaction analysis. Quantitative metachromasia revealed that chondrogenic differentiation of human synovial explants was induced to the greatest degree by either BMP-2 alone or the BMP-2/TGF-β1 combination, that is, to a comparable level with each of the two stimulation protocols and within both age categories. The BMP-2/TGF-β1combination protocol resulted in chondrocytes of a physiological size for normal human articular cartilage, unlike the BMP-2-alone stimulation that resulted in cell sizes of terminal hypertrophy. The stable gene expression levels of the anabolic chondrogenic markers confirmed the superiority of these two stimulation protocols and demonstrated the hyaline-like qualities of the generated cartilage matrix. The gene expression levels of the catabolic markers remained extremely low. The data also confirmed the usefulness of experimental in vitro studies with bovine synovial tissue as a paradigm for human synovial investigations. Our data reveal the chondrogenic potential of the human knee-joint synovium of osteoarthritic patients to be uncompromised by aging and catabolic processes. The potential of synovium-based clinical engineering (repair) of cartilage tissue using autologous synovium may thus not be reduced by the age of the human patient. Impact statement Our data reveal that in younger and older age groups alike, synovial explants from osteoarthritic joints can be equally well induced to undergo chondrogenesis in vitro; that is, the chondrogenic potential of the human synovium is not compromised by aging. These findings imply that the autologous synovium represents an adequate tissue source for the repair of articular cartilage in clinical practice by tissue engineering approaches in human patients suffering from osteoarthritis, independent of the patient's age.

Rapid 48-hour inflammation-resolving polarization of monocytes/macrophages is feasible at larger scale and shows anabolic effect in human osteoarthritic joint explant model

Background & Aim Monocytes/macrophages (MΦs, as a heterogeneous population) exist on a pro-inflammatory to pro-resolving spectrum. Endogenous polarization maintains tissue homeostasis but dysfunctionally perpetuates disease states in chronic inflammation. Exogenous cytokines can also polarize MΦs. This has allowed clinical trial manufacturing of MΦ subsets under the pro-resolving umbrella for indications such as liver cirrhosis and renal transplant. These approaches require 5-to-7-day processing. We have developed a pro-resolving MΦ polarization protocol that takes 48 hours and scales to higher culture volumes. We will test the role of polarized MΦs in osteoarthritis (OA). Inflammation is a contributor to OA progression, with a central role for pro-inflammatory MΦs, as we have previously demonstrated. We will evaluate potential therapeutic effects of pro-resolving MΦs on inflammation and cartilage loss in a human joint model that mimics OA processes. Methods, Results & Conclusion CD14+ cells (MΦs) are obtained from peripheral whole blood or leukapheresis via manual or automated immunomagnetic isolation using the CliniMACS Prodigy. MΦs are polarized in either 1 mL in a 24-well plate or 1000 mL in a MACS GMP Cell Differentiation bag using IL-10+TGF-β1 (pro-resolving). Control groups are naive and IFN-γ+LPS (pro-inflammatory) MΦs. MΦs are characterized by surface markers, gene expression, endocytosis, reactive oxygen species production, and Treg induction. Polarized MΦs are then co-cultured with OA cartilage-synovium explants from arthroplasty for 48 hours. Cartilage and culture media are measured for gene expression and secreted proteins. Surface marker expression of pro-resolving MΦs is comparable at plate and bag scales, demonstrating an expected CD163hi/HLA-DRlo/CD86neg profile with gene expression of IL-10. Surface markers are maintained after pro-inflammatory explant co-culture, confirming stability of this approach. Ongoing tests will confirm pro-resolving functional properties. Importantly, pro-resolving MΦs upregulate OA cartilage expression of anabolic matrix genes (such as lubricin and protease inhibitor TIMP1) and secreted proteins. Our rapid pro-resolving MΦ polarization method eases translation into potent, cost-effective cell therapy products. It is functional, scalable, and allows modulation of input; we are currently screening small molecules as an alternative to cytokines. In parallel with our murine OA studies, our results support proof-of-concept towards a feasible MΦ-based therapy for OA.

Human osteoarthritic joint explant model reflects diverse disease-relevant changes in inflammation and degradation

Purpose: Immune cell-mediated inflammation has been implicated in the pathogenesis and progression of osteoarthritis (OA). However, our current understanding of the phenotype and interactions between joint resident and infiltrating immune cells is not comprehensive enough to appropriately guide the design of new inflammation-targeting OA therapeutics. We have developed a human OA joint explant model containing osteoarthritic synovium and cartilage that more accurately represents the human immune condition. The explants contain joint cell types within their endogenous matrix, including resident synovial macrophages and fibroblasts that are drivers of inflammation and fibrosis. The purpose of this study is to optimize a human OA joint explant model to be representative of the human condition by maintaining in vivo morphology, viability, and responsiveness to inflammation-mediated changes in gene expression and extracellular matrix (ECM) dynamics. Methods: Cartilage and synovium are obtained from late-stage OA knee replacement. Full-depth 1.5 mm diameter punch-cut cartilage and minced (fat removed) synovial tissue are randomly distributed by wet weight into 24-wells or transwells inserts for 48 hours before co-culture. Pro-inflammatory cytokine conditions are used as positive controls (oncostatin M + interleukin 1B). Treatment conditions or other cell types can be co-cultured with the joint explants for 2-day or 7-day time points. Explant conditions are evaluated by cartilage, synovium, and conditioned medium harvest: viability (resazurin), tissue gene expression (RT-qPCR), secreted cytokine/protease (immunoassay) and proteoglycan (GAG) content (dimethylmethylene blue), and morphological (H&E) and ECM (Safranin-O) histology. Synovium tissue is digested for flow cytometry of fibroblast and polarized monocyte/macrophage surface markers. Conditions are compared by principal component analysis for gene panels and analysis of variance for single dependent variable assays. Results: 48-hour cartilage gene expression demonstrates distinct clusters between the pro-inflammatory cytokine control and cartilage alone control conditions within a 26-gene panel capturing catabolic, anabolic, and inflammatory/chemotactic genes (N=12; ΔΔCT values relative to cartilage alone control in PCA plot). The pro-inflammatory control upregulates genes such as IL6, CCL2, MMP1, MMP13, ADAMTS4 while downregulating ACAN, PRG4, COL2A1, TIMP1. The synovium-cartilage co-culture cluster overlaps with the control group, but a subset of this condition is shifted towards the pro-inflammatory control cluster. GAG loss into the medium was significantly increased with the pro-inflammatory positive control (N=18, P<0.0001). The mean GAG loss is higher in the synovium-cartilage co-culture but is not statistically significant. These changes are observable by GAG staining of histological sections. Synovium digest reveals presence of CD90+ fibroblasts and CD45+CD14+ monocytes/macrophage before and after explant co-culture. Conclusions: Our human OA joint explant model is viable and demonstrates changes relevant to inflammation and cartilage degradation in response to pro-inflammatory insult. Co-culture of synovium and cartilage as our baseline OA joint condition shows a shift towards the pro-inflammatory response. Increasing the sample size will allow us to annotate patient subsets based on their clinical outcomes to identify subsets with different levels of synovium-derived inflammatory involvement in their disease, and therefore understand the scope of our model. We position our explant model as a useful complement to in vivo animal models in understanding immune contribution to OA progression and for developing new OA treatment strategies ranging from small molecule immunomodulators to cell-based therapies.

Ex vivo polarized pro-inflammatory vs. homeostatic monocytes/macrophages elicit differential responses within a human osteoarthritic joint explant model

Purpose: Infiltrating pro-inflammatory monocytes/macrophages (MΦs, as a heterogenous population) are the most prevalent immune cells within the osteoarthritic knee joint and play a key role in osteoarthritis (OA) pathogenesis. MΦs exist on a functional spectrum from pro-inflammatory to homeostatic to promote inflammation and its subsequent resolution, as well as tissue remodeling in wound healing. This balance becomes dysregulated in disease states such as knee OA. However, the broad functional capacity of MΦs also allows them to be ex vivo polarized towards homeostatic or regulatory phenotypes to promote repair and inflammation resolution; polarized MΦ-based cell therapy has been escalated to early phase clinical trials for indications such as spinal cord injury, liver cirrhosis, and stroke. Our aim is to generate proof-of-concept data for a MΦ-based therapy for knee OA while investigating the disease contribution of pro-inflammatory and homeostatic MΦs in OA progression. Methods: An in-house human explant culture was optimized using full-depth biopsy punch cartilage and minced synovium from late stage knee OA arthroplasties for replicable responses in cartilage degradation and inflammation: qRT-PCR for gene expression, dimethylmethylene blue, Safranin-O, picrosirius red histology for extracellular matrix (ECM) loss, haemotoxylin and eosin histology for synovium structure, and multiplex immunoassays for secreted protein levels of cytokines and matrix proteases. Ex vivo polarized MΦs were generated using 48h cytokine-based protocols (IFN-γ+LPS, pro-inflammatory; IL-10+TGF-β, homeostatic) from healthy donor CD14+ peripheral blood mononuclear cells, validated by flow cytometry for surface markers (CD86/HLA-DR/CD163/CD206) and function (dextran endocytosis). Ex vivo polarized MΦs were then co-cultured with explant cartilage and synovium; tissue and conditioned medium were harvested at 2- and 7-day time points for analysis. Gene expression was analyzed by ΔΔCTand principle component analysis, while protein levels were evaluated by ANOVA. Results: Ex vivo polarized pro-inflammatory MΦs promote pro-inflammatory and catabolic cartilage gene expression, whereas homeostatic MΦs upregulate anabolic ECM gene expression and secretion of protease inhibitors (N=6, Figure 1); notably, PRG4 and TIMP-1 increase with homeostatic MΦ treatment by greater than 2-fold (gene, Figure 2A) or 1.5-fold (protein, Figure 2B). No significant change in proteoglycan loss was observed with MΦ treatment, but histology demonstrates chondrocytes retain physiological morphology over the course of explant culture. Synovium gene expression was variable between patients (N=4), but pro-inflammatory MΦ treatment trended to upregulate pro-inflammatory and chemotactic genes. Ex vivo polarized MΦs maintained their functional phenotype after co-culture with knee OA tissue. Conclusions: Ex vivo polarized pro-inflammatory versus homeostatic MΦs have differential effects on knee OA explant cartilage in co-culture with synovium, where homeostatic MΦs promote anabolic ECM and protease inhibitor genes. Our in-house explant model optimized for evaluating diverse short-term output on inflammation and cartilage degradation will allow further evaluation of MΦ roles in OA, as well as the testing of other novel OA therapies within human conditions. Our concurrent studies with murine knee OA models treated with ex vivo polarized MΦs will confirm our observations and lend credence to developing MΦ-based therapies for OA. View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Differential responses to ex vivo polarized monocytes/macrophages in a human osteoarthritis joint explant co-culture

Differential responses to ex vivo polarized monocytes/macrophages in a human osteoarthritis joint explant co-culture

Ascorbic Acid Attenuates Senescence of Human Osteoarthritic Osteoblasts.

The accumulation of senescent cells is implicated in the pathology of several age-related diseases. While the clearance of senescent cells has been suggested as a therapeutic target for patients with osteoarthritis (OA), cellular senescence of bone-resident osteoblasts (OB) remains poorly explored. Since oxidative stress is a well-known inducer of cellular senescence, we here investigated the effect of antioxidant supplementation on the isolation efficiency, expansion, differentiation potential, and transcriptomic profile of OB from osteoarthritic subchondral bone. Bone chips were harvested from sclerotic and non-sclerotic regions of the subchondral bone of human OA joints. The application of 0.1 mM ascorbic acid-2-phosphate (AA) significantly increased the number of outgrowing cells and their proliferation capacity. This enhanced proliferative capacity showed a negative correlation with the amount of senescent cells and was accompanied by decreased expression of reactive oxygen species (ROS) in cultured OB. Expanded cells continued to express differentiated OB markers independently of AA supplementation and demonstrated no changes in their capacity to osteogenically differentiate. Transcriptomic analyses revealed that apoptotic, cell cycle–proliferation, and catabolic pathways were the main pathways affected in the presence of AA during OB expansion. Supplementation with AA can thus help to expand subchondral bone OB in vitro while maintaining their special cellular characteristics. The clearance of such senescent OB could be envisioned as a potential therapeutic target for the treatment of OA.

Electrospun Nanofibrous Meshes Cultured With Wharton's Jelly Stem Cell: An Alternative for Cartilage Regeneration, Without the Need of Growth Factors.

Many efforts are being directed worldwide to the treatment of OA-focal lesions. The majority of those efforts comprise either the refinement of surgical techniques or combinations of biomaterials with various autologous cells. Herein, we tested electrospun polycaprolactone (PCL) nanofibrous meshes for cartilage tissue engineering. For that, articular chondrocytes (hACs) isolated from human osteoarthritic joints and Wharton's Jelly Stem Cells (hWJSCs) are cultured on electrospun nanofiber meshes, without adding external growth factors. We observed higher glycosaminoglycans production and higher over-expression of cartilage-related genes from hWJSCs cultured with basal medium, when compared to hACs isolated from osteoarthritic joints. Moreover, the presence of sulfated proteoglycans and collagen type II is observed on both types of cell cultures. We believe that this effect is due to either the electrospun nanofibers topography or the intrinsic chondrogenic differentiation potential of hWJSCs. Therefore, we propose the electrospun nanofibrous scaffolds in combination with hWJSCs as a viable alternative to the commercial membranes used in autologous chondrogenic regeneration approaches.

Early cathepsin K degradation of type II collagen in vitro and in vivo in articular cartilage

To characterize the initial events in the cleavage of type II collagen mediated by cathepsin K and demonstrate the presence of the resulting products in human and equine articular osteoarthritic cartilage. Equine type II collagen was digested with cathepsin K and the cleavage products characterized by mass spectrometry. Anti-neoepitope antibodies were raised against the most N-terminal cleavage products and used to investigate the progress of collagen cleavage, invitro, and the presence of cathepsin K-derived products in equine and human osteoarthritic cartilage. Six cathepsin K cleavage sites distributed throughout the triple helical region were identified in equine type II collagen. Most of the cleavages occurred following a hydroxyproline residue. The most N-terminal site was within three residues of the previously identified site in bovine type II collagen. Western blotting using anti-neoepitope antibodies showed that the initial cleavages occurred at the N-terminal sites and this was followed by more extensive degradation resulting in products too small to be resolved by SDS gel electrophoresis. Immunohistochemical staining of cartilage sections from equine or human osteoarthritic joints showed staining in lesional areas which was not observed in non-arthritic sites. Cathepsin K cleaves triple helical collagen by erosion from the N-terminus and with subsequent progressive cleavages. The liberated fragments can be detected in osteoarthritic cartilage and may represent useful biomarkers for disease activity.

Long intergenic non-coding RNAs mediate the inflammatory response in human osteoarthritis joint tissues

Long intergenic non-coding RNAs mediate the inflammatory response in human osteoarthritis joint tissues

TGF-β is a potent inducer of Nerve Growth Factor in articular cartilage via the ALK5-Smad2/3 pathway. Potential role in OA related pain?

Pain is the main problem for patients with osteoarthritis (OA). Pain is linked to inflammation, but in OA a subset of patients suffers from pain without inflammation, indicating an alternative source of pain. Nerve Growth Factor (NGF) inhibition is very efficient in blocking pain during OA, but the source of NGF is unclear. We hypothesize that damaged cartilage in OA releases Transforming Growth Factor-β (TGF-β), which in turn stimulates chondrocytes to produce NGF. Murine and human chondrocyte cell lines, primary bovine and human chondrocytes, and cartilage explants from bovine metacarpal joints and human OA joints were stimulated with TGF-β1 and/or Interleukin-1 (IL-1)β. We analyzed NGF expression on mRNA level with QPCR and stained human OA cartilage for NGF immunohistochemically. Cultures were additionally pre-incubated with inhibitors for TAK1, Smad2/3 or Smad1/5/8 signaling to identify the TGF-β pathway inducing NGF. NGF expression was consistently induced in higher levels by TGF-β than IL-1 in all of our experiments: murine, bovine and human origin, in cell lines, primary chondrocytes and explants cultures. TAK1 inhibition consistently reduced TGF-β-induced NGF whereas it fully blocked IL-1β-induced NGF expression. In contrast, ALK5-Smad2/3 inhibition fully blocked TGF-β-induced NGF expression. Despite the large variation in basal NGF in human OA samples (mRNA and histology), TGF-β exposure led to a consistent high level of NGF induction. We show for the first time that TGF-β induces NGF expression in chondrocytes, in a ALK5-Smad2/3 dependent manner. This reveals a potential alternative non-inflammatory source of pain in OA.

The immunomodulatory properties of mesenchymal stromal cells isolated from the synovial fluid of human osteoarthritic joints

The immunomodulatory properties of mesenchymal stromal cells isolated from the synovial fluid of human osteoarthritic joints

Expression and function of visfatin (Nampt), an adipokine-enzyme involved in inflammatory pathways of osteoarthritis

IntroductionVisfatin is an adipokine that may be involved in intertissular joint communication in osteoarthritis (OA). With a homodimeric conformation, it exerts nicotinamide phosphoribosyltransferase (Nampt) enzymatic activity, essential for nicotinamide adenine dinucleotide biosynthesis. We examined the tissular origin and conformation of visfatin/Nampt in human OA joints and investigated the role of visfatin/Nampt in chondrocytes and osteoblasts by studying Nampt enzymatic activity.MethodsSynovium, cartilage and subchondral bone from human OA joints were used for protein extraction or incubated for 24 hours in serum-free media (conditioned media), and synovial fluid was obtained from OA patients. Visfatin/Nampt expression in tissular extracts and conditioned media was evaluated by western blot and enzyme-linked immunosorbent assay (ELISA), respectively. Nampt activity was assessed in OA synovium by colorimetric assay. Primary cultures of murine chondrocytes and osteoblasts were stimulated with visfatin/Nampt and pretreated or not with APO866, a pharmacologic inhibitor of Nampt activity. The effect on cytokines, chemokines, growth factors and hypertrophic markers expression was examined by quantitative reverse transcriptase polymerase chain reaction and/or ELISA.ResultsIn tissular explants, conditioned media and synovial fluid, visfatin/Nampt was found as a homodimer, corresponding to the enzymatically active conformation. All human OA joint tissues released visfatin/Nampt (synovium: 628 ± 106 ng/g tissue; subchondral bone: 195 ± 26 ng/g tissue; cartilage: 152 ± 46 ng/g tissue), with significantly higher level for synovium (P <0.0005). Nampt activity was identified ex vivo in synovium. In vitro, visfatin/Nampt significantly induced the expression of interleukin 6, keratinocyte chemoattractant and monocyte chemoattractant protein 1 in chondrocytes and osteoblasts. APO866 decreased the mRNA and protein levels of these pro-inflammatory cytokines in the two cell types (up to 94% and 63% inhibition, respectively). Levels of growth factors (vascular endothelial growth factor, transforming growth factor β) and hypertrophic genes were unchanged with treatment.ConclusionVisfatin/Nampt is released by all human OA tissues in a dimeric enzymatically active conformation and mostly by the synovium, which displays Nampt activity. The Nampt activity of visfatin is involved in chondrocyte and osteoblast activation, so targeting this enzymatic activity to disrupt joint tissue interactions may be novel in OA therapy.

The innervation of synovium of human osteoarthritic joints in comparison with normal rat and sheep synovium

To study whether osteoarthritis (OA) in the knee is associated with a change of the innervation pattern in the synovial layer. In synovial tissue from the normal knee joint of rat and sheep we studied the presence of vessels and of nerve fibres using transmission electron microscopy and immunohistochemistry. Synovial material was also obtained from patients who underwent total knee replacement surgery. This material was examined for inflammatory changes, and the presence of vessels and nerve fibres was assessed. The synovium in the parapatellar region of the normal knee joint of rat and sheep exhibited a dense capillary and neuronal network. It was entered by calcitonin gene-related peptide containing sensory fibres and tyrosine hydroxylase-positive sympathetic nerve fibres. Synovial material from patients with knee OA exhibited different degrees of inflammation. Synovial material without inflammation exhibited a similar vascular and neuronal network as the normal knee joint from rat and sheep. However, in synovium with inflammatory changes we found a significant decrease of nerve fibres in depth ranges close to the synovial lining layer depending on the degree of inflammation whereas deeper regions were less affected. Inflammatory changes in the synovium of OA joints are associated with a massive destruction of the capillary and neuronal network which is present in normal synovium. Due to the disappearance of the sensory fibres it is unlikely that OA pain is initiated directly in the synovium. The loss of normally innervated vascularisation may have multiple consequences for the physiological functions of the synovium.