PLOD2 gene expression in infrapatellar fat pad is correlated with fat mass in obese patients with end-stage knee osteoarthritis.

Objective: To investigate associations between obesity-linked systemic factors and gene expression indicative for the inflammatory and fibrotic processes in the infrapatellar fat pad (IFP), in a population of obese patients with end-stage knee osteoarthritis (KOA). Methods: We collected human IFPs from 48 patients with a mean body mass index (BMI) of 35.44 â€‹kg/m2 during total knee replacement procedures. These patients were part of a randomized controlled trial and met the criteria of having OA and a BMI of ≥30 â€‹kg/m2. Blood samples were collected to assess serum levels of glucose, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, and leptin. Total body composition was measured using dual-energy X-ray absorptiometry. Gene expressions of IL6, TNFA, COL1A1, IL1B, ASMA, PLOD2 in the IFP were analyzed. Results: Univariate analysis resulted in a positive correlation between BMI and procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2) expression (r2 â€‹= â€‹0.13). In univariate analyses of obesity-linked systemic factors and PLOD2, significant correlations were found for lean mass (r2 â€‹= â€‹0.20), fat mass (r2 â€‹= â€‹0.20), serum cholesterol (r2 â€‹= â€‹0.17), serum triglycerides (r2 â€‹= â€‹0.19) and serum leptin (r2 â€‹= â€‹0.10). A multiple linear regression model indicated fat mass to be a strong predictor of PLOD2 production in the IFP (r2 â€‹= â€‹0.22, P â€‹= â€‹0.003). Conclusion: Our study demonstrates the positive association between fat mass and PLOD2 expression in the IFP of obese end-stage knee OA patients. This may indicate that within this patient population the fibrotic process in the IFP is influenced by systemic adipose tissue, next to local inflammatory processes.

Synovial membrane-derived mesenchymal progenitor cells from osteoarthritic joints in dogs possess lower chondrogenic-, and higher osteogenic capacity compared to normal joints.

BACKGROUND: Synovial membrane-derived mesenchymal progenitor cells (SM-MPCs) are a promising candidate for the cell-based treatment of osteoarthritis (OA) considering their in vitro and in vivo capacity for cartilage repair. However, the OA environment may adversely impact their regenerative capacity. There are no studies for canine (c)SM-MPCs that compare normal to OA SM-MPCs, even though dogs are considered a relevant animal model for OA. Therefore, this study compared cSM-MPCs from normal and OA synovial membrane tissue to elucidate the effect of the OA environment on MPC numbers, indicated by CD marker profile and colony-forming unit (CFU) capacity, and the impact of the OA niche on tri-lineage differentiation. METHODS: Normal and OA synovial membrane were collected from the knee joints of healthy dogs and dogs with rupture of the cruciate ligaments. The synovium was assessed by histopathological OARSI scoring and by RT-qPCR for inflammation/synovitis-related markers. The presence of cSM-MPCs in the native tissue was further characterized with flow cytometry, RT-qPCR, and immunohistochemistry, using the MPC markers; CD90, CD73, CD44, CD271, and CD34. Furthermore, cells isolated upon enzymatic digestion were characterized by CFU capacity, and a population doublings assay. cSM-MPCs were selected based on plastic adherence, expanded to passage 2, and evaluated for the expression of MPC-related surface markers and tri-lineage differentiation capacity. RESULTS: Synovial tissue collected from the OA joints had a significantly higher OARSI score compared to normal joints, and significantly upregulated inflammation/synovitis markers S100A8/9, IL6, IL8, and CCL2. Both normal and OA synovial membrane contained cells displaying MPC properties, including a fibroblast-like morphology, CFU capacity, and maintained MPC marker expression over time during expansion. However, OA cSM-MPCs were unable to differentiate towards the chondrogenic lineage and had low adipogenic capacity in contrast to normal cSM-MPCs, whereas they possessed a higher osteogenic capacity. Furthermore, the OA synovial membrane contained significantly lower percentages of CD90+, CD44+, CD34+, and CD271+ cells. CONCLUSIONS: The OA environment had adverse effects on the regenerative potential of cSM-MPCs, corroborated by decreased CFU, population doubling, and chondrogenic capacity compared to normal cSM-MPCs. OA cSM-MPCs may be a less optimal candidate for the cell-based treatment of OA than normal cSM-MPCs.

A new semi-orthotopic bone defect model for cell and biomaterial testing in regenerative medicine.

In recent decades, an increasing number of tissue engineered bone grafts have been developed. However, expensive and laborious screenings in vivo are necessary to assess the safety and efficacy of their formulations. Rodents are the first choice for initial in vivo screens but their size limits the dimensions and number of the bone grafts that can be tested in orthotopic locations. Here, we report the development of a refined murine subcutaneous model for semi-orthotopic bone formation that allows the testing of up to four grafts per mouse one order of magnitude greater in volume than currently possible in mice. Crucially, these defects are also "critical size" and unable to heal within the timeframe of the study without intervention. The model is based on four bovine bone implants, ring-shaped, where the bone healing potential of distinct grafts can be evaluated in vivo. In this study we demonstrate that promotion and prevention of ossification can be assessed in our model. For this, we used a semi-automatic algorithm for longitudinal micro-CT image registration followed by histological analyses. Taken together, our data supports that this model is suitable as a platform for the real-time screening of bone formation, and provides the possibility to study bone resorption, osseointegration and vascularisation.

Tyrosine kinases regulate chondrocyte hypertrophy: promising drug targets for Osteoarthritis.

Osteoarthritis (OA) is a major health problem worldwide that affects the joints and causes severe disability. It is characterized by pain and low-grade inflammation. However, the exact pathogenesis remains unknown and the therapeutic options are limited. In OA articular chondrocytes undergo a phenotypic transition becoming hypertrophic, which leads to cartilage damage, aggravating the disease. Therefore, a therapeutic agent inhibiting hypertrophy would be a promising disease-modifying drug. The therapeutic use of tyrosine kinase inhibitors has been mainly focused on oncology, but the Food and Drug Administration (FDA) approval of the Janus kinase inhibitor Tofacitinib in Rheumatoid Arthritis has broadened the applicability of these compounds to other diseases. Interestingly, tyrosine kinases have been associated with chondrocyte hypertrophy. In this review, we discuss the experimental evidence that implicates specific tyrosine kinases in signaling pathways promoting chondrocyte hypertrophy, highlighting their potential as therapeutic targets for OA.

Spatiotemporal distribution of thrombospondin-4 and -5 in cartilage during endochondral bone formation and repair.

During skeletal development most bones are first formed as cartilage templates, which are gradually replaced by bone. If later in life those bones break, temporary cartilage structures emerge to bridge the fractured ends, guiding the regenerative process. This bone formation process, known as endochondral ossification (EO), has been widely studied for its potential to reveal factors that might be used to treat patients with large bone defects. The extracellular matrix of cartilage consists of different types of collagens, proteoglycans and a variety of non-collagenous proteins that organise the collagen fibers in complex networks. Thrombospondin-5, also known as cartilage oligomeric matrix protein (TSP-5/COMP) is abundant in cartilage, where it has been described to enhance collagen fibrillogenesis and to interact with a variety of growth factors, matrix proteins and cellular receptors. However, very little is known about the skeletal distribution of its homologue thrombospondin-4 (TSP-4). In our study, we compared the spatiotemporal expression of TSP-5 and TSP-4 during postnatal bone formation and fracture healing. Our results indicate that in both these settings, TSP-5 distributes across all layers of the transient cartilage, while the localisation of TSP-4 is restricted to the population of hypertrophic chondrocytes. Furthermore, in fractured bones we observed TSP-4 sparsely distributed in the periosteum, while TSP-5 was absent. Last, we analysed the chemoattractant effects of the two proteins on endothelial cells and bone marrow stem cells and hypothesised that, of the two thrombospondins, only TSP-4 might promote blood vessel invasion during ossification. We conclude that TSP-4 is a novel factor involved in bone formation. These findings reveal TSP-4 as an attractive candidate to be evaluated for bone tissue engineering purposes.

The lower in vitro chondrogenic potential of canine adipose tissue-derived mesenchymal stromal cells (MSC) compared to bone marrow-derived MSC is not improved by BMP-2 or BMP-6.

Mesenchymal stromal cells (MSC) are used for cell-based treatment for canine osteoarthritis (OA). Compared with human MSCs, detailed information on the functional characterisation of canine MSCs is limited. In particular, the chondrogenic differentiation of canine adipose tissue-derived MSCs (cAT-MSCs) is challenging. In this study, we aimed to compare cAT-MSCs with bone marrow-derived MSCs (cBM-MSCs), focusing specifically on their in vitro chondrogenic potential, with or without bone morphogenetic proteins (BMP). cBM-MSCs and cAT-MSCs were characterised using flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The chondrogenic differentiation potential of all cMSC preparations in the presence of TGF-ß1 alone or when supplemented with 10, 100, or 250 ng/mL BMP-2 or BMP-6 was investigated using RT-qPCR, and biochemical, histochemical and immunohistological analyses. Both cBM-MSCs and cAT-MSCs expressed the surface markers CD90, CD73, and CD29, and were negative for CD45 and CD34, although the expression of CD73 and CD271 varied with donor and tissue origin. Interestingly, expression of ACAN and SOX9 was higher in cBM-MSCs than cAT-MSCs. In contrast with cBM-MSCs, cAT-MSCs could not differentiate toward the chondrogenic lineage without BMP-2/-6, and their in vitro chondrogenesis was inferior to cBM-MSCs with BMP-2/-6. Thus, cAT-MSCs have lower in vitro chondrogenic capacity than cBM-MSC under the studied culture conditions with 10, 100, or 250 ng/mL BMP-2 or BMP-6. Therefore, further characterisation is necessary to explore the potential of cAT-MSCs for cell-based OA treatments.

Evaluation of biomimetic hyaluronic-based hydrogels with enhanced endogenous cell recruitment and cartilage matrix formation.

Biomaterials play a pivotal role in cell-free cartilage repair approaches, where cells must migrate through the scaffold, fill the defect, and then proliferate and differentiate facilitating tissue remodeling. Here we used multiple assays to test the influence of chemokines and growth factors on cell migration and cartilage repair in two different hyaluronan (HA)-based hydrogels. We first investigated bone marrow Mesenchymal Stromal Cells (BMSC) migration in vitro, in response to different concentrations of platelet-derived growth factor-BB (PDGF-BB), chemokine ligand 5 (CCL5/RANTES) and stromal cell-derived factor 1 (SDF-1), using a 3D spheroid-based assay. PDGF-BB was selected as most favourable chemotactic agent, and MSC migration was assessed in the context of physical impediment to cell recruitment by testing Fibrin-HA and HA-Tyramine hydrogels of different cross-linking densities. Supplementation of PDGF-BB stimulated progressive migration of MSC through the gels over time. We then investigated in situ cell migration into the hydrogels with and without PDGF-BB, using a cartilage-bone explant model implanted subcutaneously in athymic mice. In vivo studies show that when placed into an osteochondral defect, both hydrogels supported endogenous cell infiltration and provided an amenable microenvironment for cartilage production. These processes were best supported in Fibrin-HA hydrogel in the absence of PDGF-BB. This study used an advanced preclinical testing platform to select an appropriate microenvironment provided by implanted hydrogels, demonstrating that HA-based hydrogels can promote the initial and critical step of endogenous cell recruitment and circumvent some of the clinical challenges in cartilage tissue repair. STATEMENT OF SIGNIFICANCE: The challenge of articular cartilage repair arises from its complex structure and architecture, which confers the unique mechanical behavior of the extracellular matrix. The aim of our research is to identify biomaterials for implants that can support migration of endogenous stem and progenitor cell populations from cartilage and bone tissue, in order to permanently replace damaged cartilage with the original hyaline structure. Here, we present an in vitro 3D spheroid-based migration assay and an osteochondral defect model, which provide the opportunity to assess biomaterials and biomolecules, and to get stronger experimental evidence of the not well-characterized dynamic process of endogenous cells colonization in an osteochondral defect. Furthermore, the delicate step of early cell migration into biomaterials towards functional tissue engineering is reproduced. These tests can be used for pre-clinical testing of newly developed material designs in the field of scaffold engineering.

Cell-surface markers identify tissue resident multipotential stem/stromal cell subsets in synovial intimal and sub-intimal compartments with distinct chondrogenic properties.

OBJECTIVE: Synovium contains multipotent progenitor/stromal cells (MPCs) with potential to participate in cartilage repair. Understanding the identity of these MPCs will allow their therapeutic potential to be fully exploited. Hence this study aimed to identify primary synovial MPCs and characterize them in the context of cartilage regeneration. METHODS: Primary MPC/MPC-subset specific markers in synovium were identified by FACS analysis of uncultured cells. MPC-subsets from human synovium obtained from patients undergoing total knee arthroplasty were FACS sorted, cultured, immunophenotyped and chondrogenically differentiated. The anatomical localization of MPCs in synovium was examined using immunohistochemistry. Finally, the presence of these MPC subsets in healthy synovium obtained from human organ donors was examined. RESULTS: A combination of CD45, CD31, CD73 and CD90 can isolate two distinct MPC-subsets in synovium. These MPC-subsets, freshly isolated from synovium, did not express CD45 or CD31, but expressed CD73. Additionally, a sub-population of CD73+ cells also expressed CD90. CD45-CD31-CD73+CD90- cells were significantly more chondrogenic than CD45-CD31-CD73+CD90+ cells in the presence of TGFß1. Interestingly, reduced chondrogenic ability of CD73+CD90+ cells could be reversed by the addition of BMP2, showing discrete chondrogenic factor requirements by distinct cell-subsets. In addition, these MPCs had distinct anatomical localization; CD73 was expressed both in intimal and sub-intimal region while CD90 was enriched in the sub-intimal region. We further demonstrated that these subsets are also present in healthy synovium. CONCLUSIONS: We provide indications that primary MPCs in synovial intima and sub-intima are phenotypically and functionally distinct with different chondrogenic properties.

pH-responsive poly(lactide-co-glycolide) nanoparticles containing near-infrared dye for visualization and hyaluronic acid for treatment of osteoarthritis.

This study focuses on intra-articular (IA) drug delivery system for the treatment of knee osteoarthritis (OA). In osteoarthritic condition the synovial fluid presents pockets with lower pH environment. To take advantage of these pH differences, poly(lactic-co-glycolic acid (PLGA) nanoparticles (NPs) and pH- responsive PLGA NPs encapsulated with ammonium bicarbonate (NH4HCO3) were generated. The nanoparticles were loaded with hyaluronic acid (HA) as a possible model drug for OA and with near-infrared dye (NIR) that was used to visualize the NPs with molecular imaging techniques. These NPs were characterized by dynamic light scattering, transmission electron microscopy and compared in in vitro, in vivo and ex vivo experiments in the treatment of OA. The results indicate that the NPs were sufficiently small, displayed a uniform size distribution and were non-toxic both in vitro and in vivo. Both NPs treatment seem to induced a reduction in OA progression, with pH- responsive NPs showing the more pronounced effect. This is probably because the pockets of low pH environment in the synovial fluid trigger a burst release of the pH-responsive NPs. This result is corroborated by in vitro experiments since the pH- responsive NPs showed an extracellular burst release behavior and higher chondrocyte vitality than non-responsive NPs. This study demonstrates that PLGA NPs containing HA and NH4HCO3 are candidates for the treatment of knee OA.

Mechanically stimulated osteochondral organ culture for evaluation of biomaterials in cartilage repair studies.

Surgical procedures such as microfracture or autologous chondrocyte implantation have been used to treat articular cartilage lesions; however, repair often fails in terms of matrix organization and mechanical behaviour. Advanced biomaterials and tissue engineered constructs have been developed to improve cartilage repair; nevertheless, their clinical translation has been hampered by the lack of reliable in vitro models suitable for pre-clinical screening of new implants and compounds. In this study, an osteochondral defect model in a bioreactor that mimics the multi-axial motion of an articulating joint, was developed. Osteochondral explants were obtained from bovine stifle joints, and cartilage defects of 4 mm diameter were created. The explants were used as an interface against a ceramic ball applying dynamic compressive and shear loading. Osteochondral defects were filled with chondrocytes-seeded fibrin-polyurethane constructs and subjected to mechanical stimulation. Cartilage viability, proteoglycan accumulation and gene expression of seeded chondrocytes were compared to free swelling controls. Cells within both cartilage and bone remained viable throughout the 10-day culture period. Loading did not wear the cartilage, as indicated by histological evaluation and glycosaminoglycan release. The gene expression of seeded chondrocytes indicated a chondrogenic response to the mechanical stimulation. Proteoglycan 4 and cartilage oligomeric matrix protein were markedly increased, while mRNA ratios of collagen type II to type I and aggrecan to versican were also enhanced. This mechanically stimulated osteochondral defect culture model provides a viable microenvironment and will be a useful pre-clinical tool to screen new biomaterials and biological regenerative therapies under relevant complex mechanical stimuli. STATEMENT OF SIGNIFICANCE: Articular cartilage lesions have a poor healing capacity and reflect one of the most challenging problems in orthopedic clinical practice. The aim of current research is to develop a testing system to assess biomaterials for implants, that can permanently replace damaged cartilage with the original hyaline structure and can withstand the mechanical forces long term. Here, we present an osteochondral ex vivo culture model within a cartilage bioreactor, which mimics the complex motion of an articulating joint in vivo. The implementation of mechanical forces is essential for pre-clinical testing of novel technologies in the field of cartilage repair, biomaterial engineering and regenerative medicine. Our model provides a unique opportunity to investigate healing of articular cartilage defects in a physiological joint-like environment.

Trophic effects of adipose-tissue-derived and bone-marrow-derived mesenchymal stem cells enhance cartilage generation by chondrocytes in co-culture.

AIMS: Combining mesenchymal stem cells (MSCs) and chondrocytes has great potential for cell-based cartilage repair. However, there is much debate regarding the mechanisms behind this concept. We aimed to clarify the mechanisms that lead to chondrogenesis (chondrocyte driven MSC-differentiation versus MSC driven chondroinduction) and whether their effect was dependent on MSC-origin. Therefore, chondrogenesis of human adipose-tissue-derived MSCs (hAMSCs) and bone-marrow-derived MSCs (hBMSCs) combined with bovine articular chondrocytes (bACs) was compared. METHODS: hAMSCs or hBMSCs were combined with bACs in alginate and cultured in vitro or implanted subcutaneously in mice. Cartilage formation was evaluated with biochemical, histological and biomechanical analyses. To further investigate the interactions between bACs and hMSCs, (1) co-culture, (2) pellet, (3) Transwell® and (4) conditioned media studies were conducted. RESULTS: The presence of hMSCs-either hAMSCs or hBMSCs-increased chondrogenesis in culture; deposition of GAG was most evidently enhanced in hBMSC/bACs. This effect was similar when hMSCs and bAC were combined in pellet culture, in alginate culture or when conditioned media of hMSCs were used on bAC. Species-specific gene-expression analyses demonstrated that aggrecan was expressed by bACs only, indicating a predominantly trophic role for hMSCs. Collagen-10-gene expression of bACs was not affected by hBMSCs, but slightly enhanced by hAMSCs. After in-vivo implantation, hAMSC/bACs and hBMSC/bACs had similar cartilage matrix production, both appeared stable and did not calcify. CONCLUSIONS: This study demonstrates that replacing 80% of bACs by either hAMSCs or hBMSCs does not influence cartilage matrix production or stability. The remaining chondrocytes produce more matrix due to trophic factors produced by hMSCs.

Celecoxib-mediated reduction of prostanoid release in Hoffa's fat pad from donors with cartilage pathology results in an attenuated inflammatory phenotype.

OBJECTIVE: The Hoffa's fat pad (HFP) is an intra-articular adipose tissue which is situated under and behind the patella. It contains immune cells next to adipocytes and secretes inflammatory factors during osteoarthritis (OA). In this study, we compared the release profile of prostanoids, which are involved in inflammation, of HFP from OA patients vs patients with a focal cartilage defect (CD) without evidence for OA on MRI and investigated the prostanoid modulatory anti-inflammatory action of celecoxib on HFP. DESIGN: Prostanoid release was analyzed in conditioned medium of HFP explant cultures from 17 osteoarthritic patients and 12 CD patients, in the presence or absence of celecoxib. Furthermore, gene expression of COX enzymes and expression of genes indicative of a pro-inflammatory or anti-inflammatory phenotype of HFP was analyzed. RESULTS: Prostanoid release by HFP from knee OA patients clustered in two subgroups with high and low prostanoid producers. HFP from high prostanoid producers released higher amounts of PGE2, PGF2α and PGD2 compared to HFP from CD patients. PGE2 release by OA HFP was positively associated with expression of genes known to be expressed by M1 macrophages, indicating a role for macrophages. Celecoxib modulated prostanoid release by HFP, and also modulated the inflammation ratio towards a more favorable anti-inflammatory M2 phenotype, most effectively in patients with higher prostanoid release profiles. CONCLUSION: In knee OA patients with inflamed HFP's, celecoxib may exert positive effects in the knee joint via decreasing the release of prostanoids produced by the HFP and by favorably modulating the anti-inflammatory marker expression in HFP.

Cartilage inflammation and degeneration is enhanced by pro-inflammatory (M1) macrophages in vitro, but not inhibited directly by anti-inflammatory (M2) macrophages.

OBJECTIVE: Macrophages play a crucial role in the progression of osteoarthritis (OA). Their phenotype may range from pro-inflammatory to anti-inflammatory. The aim of this study was to evaluate the direct effects of macrophage subtypes on cartilage by culturing macrophage conditioned medium (MCM) on human articular cartilage. DESIGN: Human OA cartilage explants were cultured with MCM of pro-inflammatory M(IFNγ+TNFα), or anti-inflammatory M(IL-4) or M(IL-10) human monocyte-derived macrophages. To assess effects of anti-inflammatory macrophages, the cartilage was cultured with a combination of MCM phenotypes as well as pre-stimulated with IFNγ+TNFα cartilage before culture with MCM. The reactions of the explants were assessed by gene expression, nitric oxide (NO) production and release of glycosaminoglycans (GAGs). RESULTS: M(IFNγ+TNFα) MCM affected OA cartilage by upregulation of IL1B (Interleukin 1ß), IL6, MMP13 (Matrix Metalloproteinase-13) and ADAMTS5 (A Disintegrin And Metalloproteinase with Thrombospondin Motifs-5), while inhibiting ACAN (aggrecan) and COL2A1 (collagen type II). M(IL-10) upregulated IL1B and Suppressor of cytokine signaling 1 (SOCS1). NO production and GAG release by the cartilage was increased when cultured with M(IFNγ+TNFα) MCM. M(IL-4) and M(IL-10) did not inhibit the effects of M(IFNγ+TNFα) MCM of neither phenotype affected IFNγ+TNFα pre-stimulated cartilage, in which an inflammatory gene response was deliberately induced. CONCLUSION: M(IFNγ+TNFα) macrophages have a prominent direct effect on OA cartilage, while M(IL-4) and M(IL-10) do not inhibit the effects of M(IFNγ+TNFα), or IFNγ+TNFα induced inflammation of the cartilage. Therapies aiming at inhibiting cartilage degeneration may take this into account by directing suppression of pro-inflammatory macrophages or stimulation of anti-inflammatory macrophages.

Guiding synovial inflammation by macrophage phenotype modulation: an in vitro study towards a therapy for osteoarthritis.

OBJECTIVE: The aims of this study were to modulate inflammation in synovial explants with the compounds: dexamethasone, rapamycin, bone morphogenetic protein 7 (BMP-7) and pravastatin, and to investigate the modulatory capacity of the compounds on specific macrophage phenotypes. DESIGN: Synovial explants from osteoarthritis (OA) patients were treated with 10(-6) M dexamethasone, 100 ng/mL rapamycin, 500 ng/mL BMP-7 or 50 µM pravastatin. Half of the explants were pre-stimulated with IFNγ + TNFα to simulate acute inflammation. Inflammatory state of the synovium was assessed with gene expression analysis. Primary human monocytes were isolated and stimulated towards macrophage phenotypes M(IFNγ + TNFα), M(IL-4) and M(IL-10) with the respective cytokines, followed by treatment with the compounds. RESULTS: Dexamethasone had an anti-inflammatory effect on IFNγ + TNFα stimulated and osteoarthritic synovium, likely due to suppression of pro-inflammatory M(IFNγ + TNFα) macrophages while enhancing anti-inflammatory M(IL4) and M(IL10) macrophages. Rapamycin and BMP-7 further enhanced inflammation in stimulated synovium, but rapamycin did not have a clear effect on non-stimulated synovium. Rapamycin suppressed M(IL-4) and M(IL-10) macrophages without affecting M(IFNγ + TNFα). BMP-7 suppressed M(IFNγ + TNFα) and enhanced M(IL-10) in the macrophage cultures. Pravastatin did not affect synovium, but enhanced M(IL-10). CONCLUSIONS: These data indicate that macrophage phenotype modulation can be used to guide joint inflammation and thereby contribute to the development of new therapies to delay the progression of OA. The varying effects of the compounds on synovium of different degrees of inflammation, indicate that the modulatory capacity of the compounds depends on OA stage and underlines the importance of identifying this stadium for adequate treatment.

Quantitative in vivo CT arthrography of the human osteoarthritic knee to estimate cartilage sulphated glycosaminoglycan content: correlation with ex-vivo reference standards.

OBJECTIVE: Recently, computed tomography arthrography (CTa) was introduced as quantitative imaging biomarker to estimate cartilage sulphated glycosaminoglycan (sGAG) content in human cadaveric knees. Our aim was to assess the correlation between in vivo CTa in human osteoarthritis (OA) knees and ex vivo reference standards for sGAG and collagen content. DESIGN: In this prospective observational study 11 knee OA patients underwent CTa before total knee replacement (TKR). Cartilage X-ray attenuation was determined in six cartilage regions. Femoral and tibial cartilage specimens harvested during TKR were re-scanned using equilibrium partitioning of an ionic contrast agent with micro-CT (EPIC-µCT), which served as reference standard for sGAG. Next, cartilage sGAG and collagen content were determined using dimethylmethylene blue (DMMB) and hydroxyproline assays. The correlation between CTa X-ray attenuation, EPIC-µCT X-ray attenuation, sGAG content and collagen content was assessed. RESULTS: CTa X-ray attenuation correlated well with EPIC-µCT (r = 0.76, 95% credibility interval (95%CI) 0.64 to 0.85). CTa correlated moderately with the DMMB assay (sGAG content) (r = -0.66, 95%CI -0.87 to -0.49) and to lesser extent with the hydroxyproline assay (collagen content) (r = -0.56, 95%CI -0.70 to -0.36). CONCLUSIONS: Outcomes of in vivo CTa in human OA knees correlate well with sGAG content. Outcomes of CTa also slightly correlate with cartilage collagen content. Since outcomes of CTa are mainly sGAG dependent and despite the fact that further validation using hyaline cartilage of other joints with different biochemical composition should be conducted, CTa may be suitable as quantitative imaging biomarker to estimate cartilage sGAG content in future clinical OA research.

Expression of CD105 on expanded mesenchymal stem cells does not predict their chondrogenic potential.

OBJECTIVE: Total bone marrow-derived mesenchymal stem cell (BMSC) populations differ in their potential to undergo chondrogenesis, with individual BMSCs differing in their chondrogenic capacity. The aim of this study was to explore the use of CD105 as a marker to isolate a chondrogenic subpopulation of BMSCs from the total, heterogeneous population. DESIGN: BMSCs were isolated from patients undergoing total hip replacement and following expansion (Passage 1-Passage 5), CD105 expression was investigated by FACS analysis. FACS was also used to sort BMSCs based on the presence of CD105 (CD105(+)/CD105(-)) or their amount of CD105 expression (CD105(Bright)/CD105(Dim)). After 3 or 5 weeks of differentiation, chondrogenic potential was determined by thionine staining for glycosaminoglycan (GAG) content and by detection of collagen type II using immunohistochemistry. RESULTS: Expanded total BMSC populations were composed almost exclusively of CD105(+) cells, the percentage of which did not correlate to subsequent chondrogenic potential; chondrogenic potential was observed to diminish with culture although CD105 expression remained stable. Similarly, differences in chondrogenic potential were observed between donors despite similar levels of CD105(+) BMSCs. Comparison of CD105(Bright) and CD105(Dim) BMSCs did not reveal a subpopulation with superior chondrogenic potential. CONCLUSIONS: Chondrogenic potential of BMSCs is often linked to CD105 expression. This study demonstrates that CD105 expression on culture expanded BMSC populations does not associate with a chondroprogenitor phenotype and CD105 should not be pursued as a marker to obtain a chondroprogenitor population from BMSCs.

Arthritic and non-arthritic synovial fluids modulate IL10 and IL1RA gene expression in differentially activated primary human monocytes.

OBJECTIVE: Synovitis with an increased presence of macrophages is observed in osteoarthritis (OA) and rheumatoid arthritis (RA). Given the important role of macrophages in arthritis, we investigated the influence of OA and RA synovial fluid (SF) on primary human monocytes (Mo), their lineage precursors. METHOD: Adherent monocytes without any stimulation (Mo(-)) or stimulated with IFN-γ and TNF-α (Mo(IFN-γ/TNF-α)) or IL-4 (Mo(IL-4)) were exposed to SF from 6 donors without any known joint disease (SF-Ctrl), 10 OA donors (SF-OA), and 10 RA donors (SF-RA). The transcriptional expression of IL6, IL1B, TNFA, IL10, CCL18, CD206, and IL1RA was analyzed. RESULTS: Mo(-) exposed to SF-RA had a lower expression of IL10 and a higher expression of IL1RA than when exposed to SF-Ctrl. Mo(IL-4) exposed to SF-RA had a lower expression of IL10 and CCL18 than when exposed to SF-Ctrl and Mo(IFN-γ/TNF-α) were not affected by SF-RA. Mo exposed to SF-OA also expressed less IL10, but only upon stimulation with IL-4, and expressed more IL1RA than when exposed to SF-Ctrl in any condition. CONCLUSION: A lower expression of IL10 may be regarded as a response to less inflammatory conditions since IL10 expression is higher in response to IFN-γ/TNF-α stimulation, probably as a feedback mechanism. Therefore, the lower expression of IL10 and the higher expression of IL1RA in Mo exposed to arthritic than to non-arthritic SF suggest that arthritic SF is mainly reducing the inflammatory responses in Mo. This may mimic the response of monocytes/macrophages recruited to the joint, where feedback mechanisms counteract pro-inflammatory processes.

TruFit Plug for Repair of Osteochondral Defects-Where Is the Evidence? Systematic Review of Literature.

OBJECTIVE: Treatment of osteochondral defects remains a challenge in orthopedic surgery. The TruFit plug has been investigated as a potential treatment method for osteochondral defects. This is a biphasic scaffold designed to stimulate cartilage and subchondral bone formation. The aim of this study is to investigate clinical, radiological, and histological efficacy of the TruFit plug in restoring osteochondral defects in the joint. DESIGN: We performed a systematic search in five databases for clinical trials in which patients were treated with a TruFit plug for osteochondral defects. Studies had to report clinical, radiological, or histological outcome data. Quality of the included studies was assessed. RESULTS: Five studies describe clinical results, all indicating improvement at follow-up of 12 months compared to preoperative status. However, two studies reporting longer follow-up show deterioration of early improvement. Radiological evaluation indicates favorable MRI findings regarding filling of the defect and incorporation with adjacent cartilage at 24 months follow-up, but conflicting evidence exists on the properties of the newly formed overlying cartilage surface. None of the included studies showed evidence for bone ingrowth. The few histological data available confirmed these results. CONCLUSION: There are no data available that support superiority or equality of TruFit compared to conservative treatment or mosaicplasty/microfracture. Further investigation is needed to improve synthetic biphasic implants as therapy for osteochondral lesions. Randomized controlled clinical trials comparing TruFit plugs with an established treatment method are needed before further clinical use can be supported.

Association of urinary biomarker COLL2-1NO2 with incident clinical and radiographic knee OA in overweight and obese women.

OBJECTIVE: To investigate the association between urinary biomarker Coll2-1NO2 (uColl2-1NO2) and incident knee OA after 2.5 years follow-up in middle-aged overweight and obese women at high risk for knee osteoarthritis (OA). DESIGN: Data were used from PROOF, a randomized controlled trial with 2.5 years follow-up evaluating the preventive effects of a diet and exercise program and oral glucosamine sulphate (double blind and placebo controlled), on development of incident knee OA in women with body mass index ≥ 27 kg/m(2) without signs of knee OA at baseline. Baseline and 2.5 years uColl2-1NO2 concentrations were assessed with enzyme-linked immunosorbent assay (ELISA). Primary outcome measure was incidence of knee OA in one or both knees, defined as incidence of either Kellgren & Lawrence grade ≥2, joint space narrowing of ≥1.0 mm or knee OA according to the combined clinical and radiographic ACR-criteria. We used binary logistic regression for the association analyses. RESULTS: 254 women were available for analyses. At 2.5 years follow-up, incident knee OA was present in 72 of 254 women (28.3%). An inversed association was found between baseline uColl2-1NO2 and incident knee OA at 2.5 years (OR 0.74, 95% CI 0.55-0.99). The concentration at 2.5 years and the change in concentration over 2.5 years did not show significant associations with the outcome. CONCLUSIONS: In overweight and obese middle-aged women, not higher but lower baseline uColl2-1NO2 concentration was significantly associated with an increased risk for incident knee OA. This interesting but counterintuitive outcome makes further validation of this biomarker warranted.

Association of pain in knee osteoarthritis with distinct patterns of synovitis.

OBJECTIVE: To determine possible patterns of synovitis on contrast-enhanced magnetic resonance imaging (CE-MRI) and its relation to pain and severity in patients with radiographic knee osteoarthritis (OA). METHODS: In total, 86 patients (mean age 62 years, 66% women, median body mass index 29 kg/m(2) ) with symptomatic knee OA (Kellgren/Lawrence radiographic score 3) were included. T1-weighted, gadolinium-chelate-enhanced MRI with fat suppression was used to semiquantitatively score the extent of synovitis at 11 knee sites (total score range 0-22). Self-reported pain was assessed with 3 standardized questionnaires. Principal components analysis (PCA) was used to investigate patterns (the location and severity) of synovitis. Subsequently, these patterns were assessed for associations with pain measures and radiographic severity in adjusted logistic regression models. RESULTS: Synovitis was observed in 86 patients and was found to be generally mild on CE-MRI (median total synovitis score 7, range 0-16). The median pain scores were 53 (range 0-96) on the visual analog scale for pain, 51.4 (range 2.8-97.2) on the Knee Injury and Osteoarthritis Outcome Score (KOOS) for pain, 35 (range 0-75) on the Intermittent and Constant Osteoarthritis Pain (ICOAP) score for constant pain, and 40.6 (range 0-87.5) on the ICOAP score for intermittent pain. PCA resulted in extraction of 3 components, explaining 53.4% of the variance. Component 1 was characterized by synovitis at 7 sites (mainly medial parapatellar involvement) and was associated with scores on the KOOS pain subscale and the ICOAP constant pain subscale. Component 2 was characterized by synovitis at 4 sites (mainly the site adjacent to the anterior cruciate ligament), but was not associated with pain measures or with radiographic severity. Component 3, characterized by synovitis at 3 sites (mainly at the loose body site), was associated with radiographic severity. CONCLUSION: Different patterns of synovitis in knee OA were observed. The pattern that included several patellar sites was associated with pain, whereas other patterns showed no association, suggesting that pain perception in patients with knee OA is a localized response.