Thyroid hormone induces ossification and terminal maturation in a preserved OA cartilage biomimetic model.

OBJECTIVE: To characterize aspects of triiodothyronine (T3) induced chondrocyte terminal maturation within the molecular osteoarthritis pathophysiology using the previously established T3 human ex vivo osteochondral explant model. DESIGNS: RNA-sequencing was performed on explant cartilage obtained from OA patients (n = 8), that was cultured ex vivo with or without T3 (10 ng/ml), and main findings were validated using RT-qPCR in an independent sample set (n = 22). Enrichment analysis was used for functional clustering and comparisons with available OA patient RNA-sequencing and GWAS datasets were used to establish relevance for OA pathophysiology by linking to OA patient genomic profiles. RESULTS: Besides the upregulation of known hypertrophic genes EPAS1 and ANKH, T3 treatment resulted in differential expression of 247 genes with main pathways linked to extracellular matrix and ossification. CCDC80, CDON, ANKH and ATOH8 were among the genes found to consistently mark early, ongoing and terminal maturational OA processes in patients. Furthermore, among the 37 OA risk genes that were significantly affected in cartilage by T3 were COL12A1, TNC, SPARC and PAPPA. CONCLUSIONS: RNA-sequencing results show that metabolic activation and recuperation of growth plate morphology are induced by T3 in OA chondrocytes, indicating terminal maturation is accelerated. The molecular mechanisms involved in hypertrophy were linked to all stages of OA pathophysiology and will be used to validate disease models for drug testing.

Effects of different obesogenic diets on joint integrity, inflammation and intermediate monocyte levels in a rat groove model of osteoarthritis.

Introduction: Obesogenic diets aggravate osteoarthritis (OA) by inducing low-grade systemic inflammation, and diet composition may affect OA severity. Here, we investigated the effect of diet on joint damage and inflammation in an OA rat model. Methods: Wistar-Han rats (n = 24) were fed a chow, a high-fat (HF) diet, or a high-fat/high-sucrose (HFS) for 24 weeks. OA was induced unilaterally 12 weeks after the diet onset by groove surgery, and compared to sham surgery or no surgical intervention (contralateral limb). Knee OA severity was determined by OARSI histopathology scoring system. At several timepoints monocyte populations were measured using flow cytometry, and joint macrophage response was determined via CD68 immunohistochemistry staining. Results: Groove surgery combined with HF or HFS diet resulted in higher OARSI scores, and both HF and HFS diet showed increased circulating intermediate monocytes compared to chow fed rats. Additionally, in the HFS group, minimal damage by sham surgery resulted in an increased OARSI score. HFS diet resulted in the largest metabolic dysregulation, synovial inflammation and increased CD68 staining in tibia epiphysis bone marrow. Conclusion: Obesogenic diets resulted in aggravated OA development, even with very minimal joint damage when combined with the sucrose/fat-rich diet. We hypothesize that diet-induced low-grade inflammation primes monocytes and macrophages in the blood, bone marrow, and synovium, resulting in joint damage when triggered by groove OA inducing surgery. When the metabolic dysregulation is larger, as observed here for the HFS diet, the surgical trigger required to induce joint damage may be smaller, or even redundant.

The lipopolysaccharide model for the experimental induction of transient lameness and synovitis in Standardbred horses.

An established lipopolysaccharide (LPS) model previously described in Warmbloods, was inconsistent in Standardbred horses, where lameness was not detected despite the presence of synovitis. The present study aimed to determine the dose of LPS from E. coli O55:B5 required to induce mild to moderate lameness following middle carpal joint injection in Standardbred horses and to quantitate the induced lameness over time, with and without anti-inflammatory pre-treatment. In a baseline trial, eight healthy, clinically sound Standardbred horses were used in a rule-based dose-escalation design trial, starting at a dose of 10 endotoxin units (EU). Lameness at trot was evaluated visually and quantitatively (using an inertial-sensor system and pressure plate analysis). Synovial fluid aspirates were analysed for total nucleated cell counts, total protein and prostaglandin E2 (PGE2). Following 2 months wash-out, the effective LPS-dose determined in the baseline trial was used to evaluate the effect of anti-inflammatory treatment. A mixed model for repeated measures with horse as random effect was used for analysis. After injection of 10 EU LPS, the desired degree of lameness was observed in the baseline trial, with maximal lameness at post-injection hour (PIH) 4, followed by a rapid decline and return to baseline by PIH 48. No lameness was observed following pre-treatment with meloxicam. In synovial fluid, PGE2 was significantly higher at PIH 8 and PIH 24 in the baseline trial compared with following meloxicam pre-treatment. In conclusion, injection of the middle carpal joint with 10 EU LPS consistently induces a transient lameness and synovitis in Standardbred horses.

High-fat feeding primes the mouse knee joint to develop osteoarthritis and pathologic infrapatellar fat pad changes after surgically induced injury.

OBJECTIVE: Obesity is one of the greatest risk factors for osteoarthritis (OA) and evidence is accumulating that inflammatory mediators and innate immunity play an important role. The infrapatellar fat pad (IPFP) could be a potential local source of inflammatory mediators in the knee. Here, we combine surgical joint damage with high-fat feeding in mice to investigate inflammatory responses in the IPFP during OA development. DESIGN: Mice (n = 30) received either a low-fat diet (LFD), high-fat diet (HFD) for 18 weeks or switched diets (LFD > HFD) after 10 weeks. OA was induced by surgical destabilization of the medial meniscus (DMM), contralateral knees served as sham controls. An additional HFD-only group (n = 15) received no DMM. RESULTS: The most pronounced inflammation, characterized by macrophage crown-like structures (CLS), was found in HFD + DMM mice, CLS increased compared to HFD only (mean difference = 7.26, 95%CI [1.52-13.0]) and LFD + DMM (mean difference = 6.35, 95%CI [0.53-12.18). The M1 macrophage marker iNOS increased by DMM (ratio = 2.48, 95%CI [1.37-4.50]), while no change in M2 macrophage marker CD206 was observed. Fibrosis was minimal by HFD alone, but in combination with DMM it increased with 23.45% (95%CI [13.67-33.24]). CONCLUSIONS: These findings indicate that a high-fat diet alone does not trigger inflammation or fibrosis in the infrapatellar fat pad, but in combination with an extra damage trigger, like DMM, induces inflammation and fibrosis in the infrapatellar fat pad. These data suggest that HFD provides a priming effect on the infrapatellar fat pad and that combined actions bring the joint in a metabolic state of progressive OA.

A short-term evaluation of a thermoplastic polyurethane implant for osteochondral defect repair in an equine model.

Cartilage repair remains a major challenge and treatment of (osteo)chondral defects generally results in poor quality fibrous repair tissue. Our approach aims to address some of the major biomechanical issues encountered in scaffold-based cartilage repair, such as insufficient stiffness of the scaffolds, step formation at the interface with the native tissue and inadequate integration with the original tissue. Two osteochondral defects were created on the medial femoral trochlear ridge in each stifle of six Shetland ponies. The defects were filled with a bi-layered implant consisting of a polyetherketoneketone (PEKK) bone anchor and a polyurethane elastomer. The defects in the contralateral joint served as unfilled controls. After 12 weeks, the ponies were euthanased and tissues were evaluated macroscopically and using micro-computed tomography, histology and immunohistochemistry. Post-operative recovery was good in all ponies and minimal lameness was observed. After 12 weeks, the proximally located plug was partially covered (mean±standard deviation [SD] percentage surface area covered 72.5±19.7%) and the distal plug was nearly completely covered (mean±SD percentage surface area covered 98.5±6.1%) with stiff and smooth repair tissue. Histology and immunohistochemistry confirmed that the repair tissue was well connected to the native cartilage but contained negligible amounts of collagen type II and glycosaminoglycans (GAGs). The repair tissue was stiff and fibrous in nature and presented a nearly flush surface with the surrounding native cartilage distally. This approach therefore resolves a number of issues related to scaffold-based cartilage repair and compares favourably with results of several other studies in large animal models. However, long-term follow-up is needed to evaluate the true potential of this type of implant.

Human C-reactive protein aggravates osteoarthritis development in mice on a high-fat diet.

OBJECTIVE: C-reactive protein (CRP) levels can be elevated in osteoarthritis (OA) patients. In addition to indicating systemic inflammation, it is suggested that CRP itself can play a role in OA development. Obesity and metabolic syndrome are important risk factors for OA and also induce elevated CRP levels. Here we evaluated in a human CRP (hCRP)-transgenic mouse model whether CRP itself contributes to the development of 'metabolic' OA. DESIGN: Metabolic OA was induced by feeding 12-week-old hCRP-transgenic males (hCRP-tg, n = 30) and wild-type littermates (n = 15) a 45 kcal% high-fat diet (HFD) for 38 weeks. Cartilage degradation, osteophytes and synovitis were graded on Safranin O-stained histological knee joint sections. Inflammatory status was assessed by plasma lipid profiling, flow cytometric analyses of blood immune cell populations and immunohistochemical staining of synovial macrophage subsets. RESULTS: Male hCRP-tg mice showed aggravated OA severity and increased osteophytosis compared with their wild-type littermates. Both classical and non-classical monocytes showed increased expression of CCR2 and CD86 in hCRP-tg males. HFD-induced effects were evident for nearly all lipids measured and indicated a similar low-grade systemic inflammation for both genotypes. Synovitis scores and synovial macrophage subsets were similar in the two groups. CONCLUSIONS: Human CRP expression in a background of HFD-induced metabolic dysfunction resulted in the aggravation of OA through increased cartilage degeneration and osteophytosis. Increased recruitment of classical and non-classical monocytes might be a mechanism of action through which CRP is involved in aggravating this process. These findings suggest interventions selectively directed against CRP activity could ameliorate metabolic OA development.

The preventive effects of two nutraceuticals on experimentally induced acute synovitis.

BACKGROUND: Nutraceuticals are often used in the management of equine osteoarthritis, but scientific evidence of their efficacy is lacking. OBJECTIVES: To study the preventive effects of two new nutraceuticals after the experimental induction of synovitis in comparison with positive and negative control treatments. STUDY DESIGN: Blinded, controlled, randomised experiment. METHODS: Twenty-four healthy Standardbred horses were randomly allocated to supplement AT (multi-ingredient, 28 days), supplement HP (collagen hydrolysate, 60 days), meloxicam (4 days) or placebo (60 days). Synovitis was induced in the right intercarpal joint by intra-articular injection of 0.5 ng lipopolysaccharide (LPS) of Escherichia coli while treatments were continued. Blood and synovial fluid were sampled before treatment, immediately prior to LPS injection, and at 8, 24 and 48 h post-injection. Synovial fluid samples were analysed for total nucleated cell count (TNCC), total protein (TP) and selected biomarkers (prostaglandin E2 [PGE2 ], interleukin-6 [IL-6], glycosaminoglycans [GAGs], type II collagen synthesis [CPII], matrix metalloproteinase [MMP]). Lameness was scored by visual examination and pressure plate analysis immediately prior to LPS injection, and at 8, 24 and 48 h post-injection. Clinical examinations were performed before treatment, immediately prior to LPS injection, at 2, 4 and 6 h post-injection, and then twice per day during the test period. RESULTS: Before treatment and intra-articular challenge, there were no statistically significant differences among the treatment groups for any of the parameters. After intra-articular challenge, the placebo group showed significantly higher synovial fluid TP, TNCC and PGE2 compared with the meloxicam group, although the model did not induce a relevant amount of lameness. Both nutraceuticals resulted in significantly lower synovial fluid TP, TNCC and PGE2 compared with placebo. No statistical differences in IL-6, GAGs, CPII or MMPs were observed among treatment groups. No adverse effects were observed. MAIN LIMITATIONS: Despite evidence of synovitis, lameness was too mild to detect. CONCLUSIONS: The preventive administration of these nutraceuticals showed anti-inflammatory effects in this validated synovitis model. Therefore, further studies of their clinical applicability are warranted.

Micro- and nano-mechanics of osteoarthritic cartilage: The effects of tonicity and disease severity.

The present study aims to discover the contribution of glycosaminoglycans (GAGs) and collagen fibers to the mechanical properties of the osteoarthritic (OA) cartilage tissue. We used nanoindentation experiments to understand the mechanical behavior of mild and severe osteoarthritic cartilage at micro- and nano-scale at different swelling conditions. Contrast enhanced micro-computed tomography (EPIC-µCT) was used to confirm that mild OA specimens had significantly higher GAGs content compared to severe OA specimens. In micro-scale, the semi-equilibrium modulus of mild OA specimens significantly dropped after immersion in a hypertonic solution and at nano-scale, the histograms of the measured elastic modulus revealed three to four components. Comparing the peaks with those observed for healthy cartilage in a previous study indicated that the first and third peaks represent the mechanical properties of GAGs and the collagen network. The third peak shows considerably stiffer elastic modulus for mild OA samples as compared to the severe OA samples in isotonic conditions. Furthermore, this peak clearly dropped when the tonicity increased, indicating the loss of collagen (pre-) stress in the shrunk specimen. Our observations support the association of the third peak with the collagen network. However, our results did not provide any direct evidence to support the association of the first peak with GAGs. For severe OA specimens, the peak associated with the collagen network did not drop when the tonicity increased, indicating a change in the response of OA cartilage to hypertonicity, likely collagen damage, as the disease progresses to its latest stages.

YKL-40 and matrix metalloproteinases as potential biomarkers of inflammation and fibrosis in the development of bronchiolitis obliterans syndrome.

BACKGROUND AND OBJECTIVE: The development of bronchiolitis obliterans syndrome (BOS) after lung transplantation is characterized by inflammation, remodeling and fibrosis. Both YKL-40 and matrix metalloproteinase (MMP)-9 have shown to be involved in these processes. We measured serial YKL-40 and MMP-9 serum levels in lung transplant recipients and assessed their usefulness as biomarker for BOS. Furthermore, we investigate the relationship between these two potential biomarkers of BOS and MMP-7. DESIGN: Ten patients with BOS (BOS(pos)) and 10 matched patients without BOS (BOS(neg)) were included. Serial serum samples were collected after lung transplantation and prior to BOS. YKL-40, MMP-9 and MMP-7 serum levels were determined by ELISA. RESULTS: The median concentrations of YKL-40 did not differ between BOS(pos) and BOS(neg) patients (p > 0.05). The median concentration of MMP-9 in BOS(pos) patients was significantly higher than in BOS(neg) patients (p < 0.0001). For MMP-9 as possible risk factor for BOS, a cut off value of 145 ng/ml has a sensitivity of 90% and a negative predictive value of 83%. Longitudinal analysis of YKL-40 and MMP-9 serum levels from the early post-transplant period onwards did not reveal a significant trend in time in both serum levels preceding BOS. In BOS(neg) patients MMP-9 showed an inverse relationship with MMP-7, that was absent in BOS(pos) patients. CONCLUSIONS: From the moment of transplantation onwards, patients who eventually developed BOS had significantly increased MMP-9 serum levels in comparison with patients who did not develop BOS. Therefore, increased MMP-9 serum levels might be useful as risk factor for BOS.

Genetic variability in the IL1RN gene and the balance between interleukin (IL)-1 receptor agonist and IL-1ß in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a rapidly progressive interstitial lung disease of unknown aetiology. Interleukin (IL)-1ß plays an important role in inflammation and has been associated with fibrotic remodelling. We investigated the balance between IL-1ß and IL-1 receptor antagonist (IL-1Ra) in bronchoalveolar lavage fluid (BALF) and serum as well as the influence of genetic variability in the IL1B and IL1RN gene on disease susceptibility and cytokine levels. In 77 IPF patients and 349 healthy controls, single nucleotide polymorphisms (SNPs) in the IL1RN and IL1B genes were determined. Serum and BALF IL-1Ra and IL-1ß levels were measured using a multiplex suspension bead array system and were correlated with genotypes. Both in serum and BALF a significantly decreased IL-1Ra/IL-1ß ratio was found in IPF patients compared to healthy controls. In the IL1RN gene, one SNP was associated with both the susceptibility to IPF and reduced IL-1Ra/IL-1ß ratios in BALF. Our results show that genetic variability in the IL1RN gene may play a role in the pathogenesis of IPF and that this role may be more important than thought until recently. The imbalance between IL-1Ra and IL-1ß might contribute to a proinflammatory and pro-fibrotic environment in their lungs.

MRP14 is elevated in the bronchoalveolar lavage fluid of fibrosing interstitial lung diseases.

Pulmonary fibrosis is defined by an overgrowth of fibroblasts and extracellular matrix deposition, and results in respiratory dysfunction that is often fatal. It is the end stage in many chronic inflammatory interstitial lung diseases (ILD) such as sarcoidosis and idiopathic pulmonary fibrosis (IPF). The myeloid-related proteins (MRPs) belong to the S100 family of calcium-binding proteins and are highly expressed by neutrophils, macrophages and epithelial cells during chronic inflammation. MRP14 stimulates fibroblast proliferation in vitro and is expressed in granulomas from sarcoidosis patients. We hypothesized that MRP14 may be a biomarker for fibrotic interstitial lung diseases. The objective of this study was to investigate whether levels of MRP14 in the bronchoalveolar lavage fluid (BALF) of patients with sarcoidosis and IPF correlate with clinical parameters. We used an enzyme-linked immunosorbent assay (ELISA) to measure MRP14 in BALF of 74 sarcoidosis patients, 54 IPF patients and 19 controls. Mean BALF levels of MRP14 were elevated significantly in IPF (P < 0.001) and sarcoidosis (P < 0.05) patients compared to controls. MRP14 levels were associated linearly with sarcoidosis disease severity based on chest radiographic stage. Moreover, BALF MRP14 levels were correlated inversely with diffusion capacity and forced vital capacity in sarcoidosis patients. In IPF patients, a correlation with BALF neutrophil percentage was found. In conclusion, BALF MRP14 levels are elevated in IPF and sarcoidosis and are associated with disease severity in sarcoidosis. The results support the need for further studies into the role of MRP14 in the pathogenesis of lung fibrosis.