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Background: Moderate mechanical stress generated by normal joint loading and movements helps maintain the health of articular cartilage. Despite growing interest in the pathogenesis of cartilage degeneration caused by reduced mechanical stress, its reversibility by mechanical reloading is less understood. This study aimed to investigate the response of articular cartilage exposed to mechanical reloading after unloading in vivo and in vitro., Methods and Results: Disuse atrophy was induced in the knee joint cartilage of adult mice through hindlimb unloading by tail suspension. For in vivo experiments, mice were subjected to reloading with or without daily exercise intervention or surgical destabilization of the knee joint. Microcomputed tomography and histomorphometric analyses were performed on the harvested knee joints. Matrix loss and thinning of articular cartilage due to unloading were fully or partially restored by reloading, and exercise intervention enhanced the restoration. Subchondral bone density decreased by unloading and increased to above-normal levels by reloading. The severity of cartilage damage caused by joint instability was not different even with prior non-weight bearing. For in vitro experiments, articular chondrocytes isolated from the healthy or unloaded joints of the mice were embedded in agarose gel. After dynamic compression loading, the expression levels of anabolic (Sox9, Col2a1, and Acan) and catabolic (Mmp13 and Adamts5) factors of cartilage were analyzed. In chondrocytes isolated from the unloaded joints, similar to those from healthy joints, dynamic compression increased the expression of anabolic factors but suppressed the expression of catabolic factors., Conclusion: The results of this study indicate that the morphological changes in articular cartilage exposed to mechanical unloading may be restored in response to mechanical reloading by shifting extracellular matrix metabolism in chondrocytes to anabolism., (© 2024. The Author(s).)

Background: Understanding how healthy articular cartilage responds to mechanical loading is critical. Moderate mechanical loading has positive effects on the cartilage, such as maintaining cartilage homeostasis. The degree of mechanical loading is determined by a combination of intensity, frequency, and duration; however, the best combination of these parameters for knee cartilage remains unclear. This study aimed to determine which combination of intensity, frequency, and duration provides the best mechanical loading on healthy knee articular cartilage in vitro and in vivo., Methods and Results: In this study, 33 male mice were used. Chondrocytes isolated from mouse knee joints were subjected to different cyclic tensile strains (CTSs) and assessed by measuring the expression of cartilage matrix-related genes. Furthermore, the histological characteristics of mouse tibial cartilages were quantified using different treadmill exercises. Chondrocytes and mice were divided into the control group and eight intervention groups: high-intensity, high-frequency, and long-duration; high-intensity, high-frequency, and short-duration; high-intensity, low-frequency, and long-duration; high-intensity, low-frequency, and short-duration; low-intensity, high-frequency, and long-duration; low-intensity, high-frequency, and short-duration; low-intensity, low-frequency, and long-duration; low-intensity, low-frequency, and short-duration. In low-intensity CTSs, chondrocytes showed anabolic responses by altering the mRNA expression of COL2A1 in short durations and SOX9 in long durations. Furthermore, low-intensity, low-frequency, and long-duration treadmill exercises minimized chondrocyte hypertrophy and enhanced aggrecan synthesis in tibial cartilages., Conclusion: Low-intensity, low-frequency, and long-duration mechanical loading is the best combination for healthy knee cartilage to maintain homeostasis and activate anabolic responses. Our findings provide a significant scientific basis for exercise and lifestyle instructions., (© 2024. The Author(s).)

Background: Traumatic cartilage injury is an important cause of osteoarthritis (OA) and limb disability, and toll-like receptors (TLRs) mediated innate immune response has been confirmed to play a crucial role in cartilage injury. In the previous study, we found that the activation of TLR8 molecules in injured articular cartilage was more obvious than other TLRs by establishing an animal model of knee impact injury in rabbits, and the changes of TLR8 molecules could significantly affect the process of articular cartilage injury and repair., Objective: To verify how mir-99a-5p regulates TLR8 receptor mediated innate immune response to treat traumatic cartilage injury., Methods: The impact of a heavy object on the medial condyle of the rabbit's knee joint caused damage to the medial condylar cartilage. Through pathological and imaging analysis, it was demonstrated whether the establishment of an animal model of traumatic cartilage injury was successful. Establishing a cell model by virus transfection of chondrocytes to demonstrate the role of TLR8 in the innate immune response to impact cartilage injury. Through transcriptome sequencing, potential targets of TLR8, mir-99a-5p, were predicted, and basic experiments were conducted to demonstrate how they interact with innate immune responses to impact cartilage damage., Results: TLR8 is a receptor protein of the immune system, which is widely expressed in immune cells. In our study, we found that TLR8 expression is localized in lysosomes and endosomes. Mir-99a-5p can negatively regulate TLR8 to activate PI3K-AKT molecular pathway and aggravate cartilage damage. Inhibiting TLR8 expression can effectively reduce the incidence of articular cartilage damage., Conclusion: Based on the results from this study, mir-99a-5p may be an effective molecular marker for predicting traumatic cartilage injury and targeting TLR8 is a novel and promising approach for the prevention or early treatment of cartilage damage., (© 2024 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

The aim of this study was to construct rat models of environmental risk factors for Kashin-Beck disease (KBD) with low selenium and T-2 toxin levels and to screen the differentially expressed genes (DEGs) between the rat models exposed to environmental risk factors. The Se-deficient (SD) group and T-2 toxin exposure (T-2) group were constructed. Knee joint samples were stained with hematoxylin-eosin, and cartilage tissue damage was observed. Illumina high-throughput sequencing technology was used to detect the gene expression profiles of the rat models in each group. Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis were performed and five differential gene expression results were verified by quantitative real-time polymerase chain reaction (qRT‒PCR). A total of 124 DEGs were identified from the SD group, including 56 upregulated genes and 68 downregulated genes. A total of 135 DEGs were identified in the T-2 group, including 68 upregulated genes and 67 downregulated genes. The DEGs were significantly enriched in 4 KEGG pathways in the SD group and 9 KEGG pathways in the T-2 group. The expression levels of Dbp, Pc, Selenow, Rpl30, and Mt2A were consistent with the results of transcriptome sequencing by qRT‒PCR. The results of this study confirmed that there were some differences in DEGs between the SD group and the T-2 group and provided new evidence for further exploration of the etiology and pathogenesis of KBD., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Infrapatellar fat pad (IPFP) is closely associated with the development and progression of knee osteoarthritis (OA), but the underlying mechanism remains unclear. Here, it is find that IPFP from OA patients can secret small extracellular vesicles (sEVs) and deliver them into articular chondrocytes. Inhibition the release of endogenous osteoarthritic IPFP-sEVs by GW4869 significantly alleviated IPFP-sEVs-induced cartilage destruction. Functional assays in vitro demonstrated that IPFP-sEVs significantly promoted chondrocyte extracellular matrix (ECM) catabolism and induced cellular senescence. It is further demonstrated that IPFP-sEVs induced ECM degradation in human and mice cartilage explants and aggravated the progression of experimental OA in mice. Mechanistically, highly enriched let-7b-5p and let-7c-5p in IPFP-sEVs are essential to mediate detrimental effects by directly decreasing senescence negative regulator, lamin B receptor (LBR). Notably, intra-articular injection of antagomirs inhibiting let-7b-5p and let-7c-5p in mice increased LBR expression, suppressed chondrocyte senescence and ameliorated the progression of experimental OA model. This study uncovers the function and mechanism of the IPFP-sEVs in the progression of OA. Targeting IPFP-sEVs cargoes of let-7b-5p and let-7c-5p can provide a potential strategy for OA therapy., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Osteoarthritis (OA) is a worldwide joint disease. However, the precise mechanism causing OA remains unclear. Our primary aim was to identify vital biomarkers associated with the mechano-inflammatory aspect of OA, providing potential diagnostic and therapeutic targets for OA. Thirty OA patients who underwent total knee arthroplasty were recruited, and cartilage samples were obtained from both the lateral tibial plateau (LTP) and medial tibial plateau (MTP). GO and KEGG enrichment analyses were performed, and the protein-protein interaction (PPI) assessment was conducted for hub genes. The effect of PSD95 inhibition on cartilage degeneration was also conducted and analyzed. A total of 1247 upregulated and 244 downregulated DEGs were identified. Significant differences were observed between MTP and LTP in mechanical stress-related genes and activated sensory neurons based on a self-contrast model of human knee OA. Cluster analysis identified DLG4 as the hub gene. Cyclic loading stress increased PSD95 (encoded by DLG4 ) expression in LTP cartilage, and PSD95 inhibitors could alleviate OA progression. This study suggests that inhibiting PSD95 could be a potential therapeutic strategy for preventing articular cartilage degradation.

Osteoarthritis (OA) is the most prevalent rheumatic pathology. However, OA is not simply a process of wear and tear affecting articular cartilage but rather a disease of the entire joint. One of the most common locations of OA is the knee. Knee tissues have been studied using molecular strategies, generating a large amount of complex data. As one of the goals of the Rheumatic and Autoimmune Diseases initiative of the Human Proteome Project, we applied a text-mining strategy to publicly available literature to collect relevant information and generate a systematically organized overview of the proteins most closely related to the different knee components. To this end, the PubPular literature-mining software was employed to identify protein-topic relationships and extract the most frequently cited proteins associated with the different knee joint components and OA. The text-mining approach searched over eight million articles in PubMed up to November 2022. Proteins associated with the six most representative knee components (articular cartilage, subchondral bone, synovial membrane, synovial fluid, meniscus, and cruciate ligament) were retrieved and ranked by their relevance to the tissue and OA. Gene ontology analyses showed the biological functions of these proteins. This study provided a systematic and prioritized description of knee-component proteins most frequently cited as associated with OA. The study also explored the relationship of these proteins to OA and identified the processes most relevant to proper knee function and OA pathophysiology., Competing Interests: Conflict of interest We certify that there is no conflict of interest to disclose regarding the materials and data discussed in this manuscript. The contents of this manuscript have not been copyrighted or published previously., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The onset and progression of human inflammatory joint diseases are strongly associated with the activation of resident synovium/infrapatellar fat pad (IFP) pro-inflammatory and pain-transmitting signaling. We recently reported that intra-articularly injected IFP-derived mesenchymal stem/stromal cells (IFP-MSC) acquire a potent immunomodulatory phenotype and actively degrade substance P (SP) via neutral endopeptidase CD10 (neprilysin). Our hypothesis is that IFP-MSC robust immunomodulatory therapeutic effects are largely exerted via their CD10-bound small extracellular vesicles (IFP-MSC sEVs) by attenuating synoviocyte pro-inflammatory activation and articular cartilage degradation. Herein, IFP-MSC sEVs were isolated from CD10High- and CD10Low-expressing IFP-MSC cultures and their sEV miRNA cargo was assessed using multiplex methods. Functionally, we interrogated the effect of CD10High and CD10Low sEVs on stimulated by inflammatory/fibrotic cues synoviocyte monocultures and cocultures with IFP-MSC-derived chondropellets. Finally, CD10High sEVs were tested in vivo for their therapeutic capacity in an animal model of acute synovitis/fat pad fibrosis. Our results showed that CD10High and CD10Low sEVs possess distinct miRNA profiles. Reactome analysis of miRNAs highly present in sEVs showed their involvement in the regulation of six gene groups, particularly those involving the immune system. Stimulated synoviocytes exposed to IFP-MSC sEVs demonstrated significantly reduced proliferation and altered inflammation-related molecular profiles compared to control stimulated synoviocytes. Importantly, CD10High sEV treatment of stimulated chondropellets/synoviocyte cocultures indicated significant chondroprotective effects. Therapeutically, CD10High sEV treatment resulted in robust chondroprotective effects by retaining articular cartilage structure/composition and PRG4 (lubricin)-expressing cartilage cells in the animal model of acute synovitis/IFP fibrosis. Our study suggests that CD10High sEVs possess immunomodulatory miRNA attributes with strong chondroprotective/anabolic effects for articular cartilage in vivo. The results could serve as a foundation for sEV-based therapeutics for the resolution of detrimental aspects of immune-mediated inflammatory joint changes associated with conditions such as osteoarthritis (OA).

Objective: NF-κB signaling is an important modulator in osteoarthritis (OA), and IκB kinase ε (IKKε) regulates the NF-κB pathway. This study was undertaken to identify the functional involvement of IKKε in the pathogenesis of OA and the effectiveness of IKKε inhibition as a modulatory treatment., Methods: IKKε expression in normal and OA human knee joints was analyzed immunohistochemically. Gain- or loss-of-function experiments were performed using human chondrocytes. Furthermore, OA was surgically induced in mice, followed by intraarticular injection of BAY-985, an IKKε/TANK-binding kinase 1 inhibitor, into the left knee joint every 5 days for 8 weeks. Mice were subsequently examined for histologic features of cartilage damage and inflammation., Results: IKKε protein expression was increased in human OA cartilage. In vitro, expression levels of OA-related factors were down-regulated following knockdown of IKKε with the use of small interfering RNA in human OA chondrocytes or following treatment with BAY-985. Conversely, IKKε overexpression significantly increased the expression of OA-related catabolic mediators. In Western blot analysis of human chondrocytes, IKKε overexpression increased the phosphorylation of IκBα and p65. In vivo, intraarticular injection of BAY-985 into the knee joints of mice attenuated OA-related cartilage degradation and hyperalgesia via NF-κB signaling., Conclusion: These results suggest that IKKε regulates cartilage degradation through a catabolic response mediated by NF-κB signaling, and this could represent a potential target for OA treatment. Furthermore, BAY-985 may serve as a major disease-modifying compound among the drugs developed for OA., (© 2022 American College of Rheumatology.)

Objective and Design: The purpose of this study was to explore pathological processes during the first 4 weeks after anterior cruciate ligament reconstruction (ACLR)., Subjects: Sixteen ACL-injured patients (8 females/8 males, mean age = 19.1, mean BMI = 28.6)., Methods: Arthrocentesis was performed 1 and 4 weeks after ACLR. Proteins in the synovial fluid were identified using nanoLC-ESI-MS/MS. Differentially up- or down-regulated proteins were identified and quantified, and a pathway analysis was performed. All identified proteins were mapped into a protein-protein interaction (PPI) network, and networks of PPIs with a combined score > 0.9 were then visualized., Results: Seven pathways were upregulated after ACLR: PI3K-AKT signaling pathway, extracellular matrix (ECM)-receptor interaction, focal adhesion, protein digestion and absorption, ameobiasis, and platelet activation. Network analyses identified 8 proteins that were differentially upregulated with strong PPI interactions (periostin and 7 collagen-related proteins). Increases in periostin moderately correlated with increases in a synovial fluid biomarker of type II cartilage degradation (ρ = 0.51, p = 0.06)., Conclusion: Pro-inflammatory pathways and periostin were upregulated after ACLR. Periostin demonstrated strong network connections with markers of collagen breakdown, and future work is needed to determine whether periostin may offer a biomarker of early cartilage degradation after ACLR and/or play an active role in early post-traumatic osteoarthritis (PTOA) progression., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Objective: Iron accumulation is emerging as a player in aging-related disorders due to its propensity for generating reactive oxygen species (ROS). Studies investigating the role of iron in the pathogenesis of primary osteoarthritis (OA) are limited. We designed a proof-of-principle study to determine the effect of systemic iron deficiency, via an iron deficient diet, on knee OA in an animal model., Methods: Twelve-week-old male Hartley guinea pigs received the standard diet (n = 6) or a diet devoid of iron (n = 6) for 19-weeks. Iron levels were determined in the serum, liver, and articular cartilage. Knees were collected to assess structural changes related to OA (microcomputed tomography, histopathology). Immunohistochemistry was performed to evaluate the presence and distribution of a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) and ROS-driven 4-hydroxynonenal (4-HNE)-induced protein adducts. Transcript expression was also assessed., Results: Relative to control animals, an iron deficient diet reduced the concentration of this mineral in serum, liver, and articular cartilage. Iron deficient animals had lower histologic OA scores; decreased subchondral bone mineral density was also noted. This reduction in knee joint pathology was accompanied by a decrease in: ADAMTS4 in synovium; and 4-HNE protein adducts from lipid peroxidation in both the menisci and articular cartilage of iron deficient animals. Expression of iron-related genes in these tissues was also altered in treated animals., Conclusions: Results from this study suggest that systemic iron levels may play a role in knee OA pathogenesis, with a short-term deficit in dietary iron reducing the severity of knee cartilage lesions., (Copyright © 2022 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: To gain insight in the expression profile of long non-coding RNAs (lncRNAs) in OA subchondral bone., Methods: RNA sequencing data of macroscopically preserved and lesioned OA subchondral bone of patients that underwent joint replacement surgery due to OA (N = 22 pairs; 5 hips, 17 knees, Research osteoArthrits Articular Tissue (RAAK study) was run through an in-house pipeline to detect expression of lncRNAs. Differential expression analysis between preserved and lesioned bone was performed. Spearman correlations were calculated between differentially expressed lncRNAs and differentially expressed mRNAs identified previously in the same samples. Primary osteogenic cells were transfected with locked nucleic acid (LNA) GapmeRs targeting AC005165.1 lncRNA, to functionally investigate its potential mRNA targets., Results: In total, 2816 lncRNAs were well-expressed in subchondral bone and we identified 233 lncRNAs exclusively expressed in knee and 307 lncRNAs exclusively in hip. Differential expression analysis, using all samples (N = 22 pairs; 5 hips, 17 knees), resulted in 21 differentially expressed lncRNAs [false discovery rate (FDR) < 0.05, fold change (FC) range 1.19-7.39], including long intergenic non-protein coding RNA (LINC) 1411 (LINC01411, FC = 7.39, FDR = 2.20 × 10-8), AC005165.1 (FC = 0.44, FDR = 2.37 × 10-6) and empty spiracles homeobox 2 opposite strand RNA (EMX2OS, FC = 0.41, FDR = 7.64 × 10-3). Among the differentially expressed lncRNAs, five were also differentially expressed in articular cartilage, including AC005165.1, showing similar direction of effect. Downregulation of AC005165.1 in primary osteogenic cells resulted in consistent downregulation of highly correlated frizzled related protein (FRZB)., Conclusion: The current study identified a novel lncRNA, AC005165.1, being dysregulated in OA articular cartilage and subchondral bone. Downregulation of AC005165.1 caused a decreased expression of OA risk gene FRZB, an important member of the wnt pathway, suggesting that AC005165.1 could be an attractive potential therapeutic target with effects in articular cartilage and subchondral bone., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Objective: Cartilage collagen has very limited repair potential, though some turnover and incorporation has not been fully excluded. We aim to determine the regional turnover of human osteoarthritis cartilage., Design: Patients scheduled for knee joint replacement surgery due to osteoarthritis were recruited in this prospective study of four weeks duration. Deuterium oxide (D 2 O) was administered orally by weekly boluses at 70% D 2 O, initially 150 ml followed by three boluses of 50 ml. Cartilage from the medial tibia plateau was sampled centrally, under the meniscus, and from osteophytes and treated enzymatically with hyaluronidase and trypsin. Samples were analysed for deuterium incorporation in alanine using mass spectrometry and for gene expression by real-time reverse transcriptase polymerase chain reaction., Results: Twenty participants completed the study: mean (SD) age 64 ± 9.1 years, 45% female, BMI 29.5 ± 4.8 kg/m 2 . Enzymatically treated cartilage from central and submeniscal regions showed similar enrichments at 0.063% APE, while osteophytes showed significantly greater enrichment at 0.072% APE (95% confidence interval of difference) [0.004-0.015]). Fractional synthesis rates were similar for central 0.027%/day and submeniscal cartilage 0.022%/day but 10-fold higher in osteophytes 0.22%/day [0.098-0.363]. When compared to central cartilage, submeniscal cartilage had increased gene expression of MMP-3 and decreased lubricin expression. Untreated cartilage had higher turnover (enrichments at 0.073% APE) than enzymatically treated cartilage (0.063% APE)., Conclusions: In OA, despite regional differences in gene expression, the turnover of the articular cartilage matrix across the entire joint surface is very limited, but higher turnover was observed in osteophyte cartilage., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Nanofat (NF) is a fine emulsion that has been used to treat a variety of diseases given its abundance of bioactive components. However, the biological functions of NF have been limited due to its inability to localize during implantation. In this study, NF was immobilized in microfluidic-generated aldehyde-modified polylactic glycolic acid (PLGA) porous microspheres (PMs) via Schiff base condensation and non-covalent binding in a three-dimensional (3D) porous network (PMs@NF). The PMs effectively enhanced the cartilage-targeted retention efficiency of NF, which also resulted in remarkable lubrication performance, with the friction coefficient being reduced by ∼80%, which was maintained over time. Meanwhile, the 3D penetrating structure of the microspheres stimulated cytokine secretion by the NF-derived stem cells, upregulating the expression of anabolism-related genes and downregulating catabolism, and the expression of inflammation-related and pain-related genes. Injecting PMs@NF into the knee joint cavity of a rat model with destabilization of the medial meniscus (DMM) reduced osteophyte formation and protected the cartilage from degeneration, thereby inhibiting the progression of osteoarthritis and improving animal behavior. In summary, this study developed a multifunctional platform with NF immobilization and super-lubrication, which showed great potential for the minimally invasive treatment of osteoarthritis., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Osteoarthritis (OA) is an aging-related degenerative joint disease with a poorly defined mechanism. Here we report that kindlin-2 is highly expressed in articular chondrocytes and downregulated in the degenerated cartilage of aged mice and patients with OA. Kindlin-2 deletion in articular chondrocytes leads to spontaneous OA and exacerbates instability-induced OA lesions in adult mice. Kindlin-2 deficiency promotes mitochondrial oxidative stress and activates Stat3, leading to Runx2-mediated chondrocyte catabolism. Pharmacological inhibition of Stat3 activation or genetic ablation of Stat3 in chondrocytes reverses aberrant accumulation of Runx2 and extracellular-matrix-degrading enzymes and limits OA deteriorations caused by kindlin-2 deficiency. Deleting Runx2 in chondrocytes reverses structural changes and OA lesions caused by kindlin-2 deletion without downregulating p-Stat3. Intra-articular injection of AAV5-kindlin-2 decelerates progression of aging- and instability-induced knee joint OA in mice. Collectively, we identify a pathway consisting of kindlin-2, Stat3 and Runx2 in articular chondrocytes that is responsible for maintaining articular cartilage integrity and define a potential therapeutic target for OA., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Osteoarthritis (OA) is condition which poses a main concern to the aging population and its severity is expected to increase with the increasing life expectancy. In the future, several possible targets for OA treatment need to be defined. Dickkopf‑related protein 3 (DKK3) is an atypical member of the Wnt‑antagonistic dickkopf‑related protein (DKK) family. The availability of research into the role of DKK3 in the abnormal remodeling of subchondral bone in human knee joints is currently limited. Thus, the aim of the present study was the evaluation of DKK3 expression in the abnormal bone remodeling of subchondral bone in human knee OA in order to clarify the role of DKK3 in subchondral bone remodeling and to acknowledge its potential relevance to β‑catenin. In total, 38 specimens were collected from osteotomies of the medial tibial plateau of the human knee. The patient samples were then divided into the normal, mild, moderate and severe symptom groups, according to the Osteoarthritis Research Society International (OARSI) score. Following hematoxylin and eosin (H&E) and Safranin O‑fast green staining for alkaline phosphatase (AZO method), changes in the distribution and number of osteocytes in the subchondral bone and the degree of sclerosis of the subchondral bone were observed. Immunohistochemical staining, immunofluorescence, western blot analysis and reverse‑transcription quantitative PCR (RT‑qPCR) were used for the detection of DKK3 and β‑catenin expression level changes in osteoblasts in the subchondral bone of the medial tibial plateau. H&E and alkaline phosphatase staining revealed that the total number of osteocytes in the subchondral bone increased with the severity of the disease. The samples were also evaluated using Safranin O‑Fast Green staining and were attributed a score according to the OARSI scoring system: The scoring number and cartilage damage increased along with OA severity. Immunohistochemistry and immunofluorescence assays demonstrated that β‑catenin expression in osteocytes increased from mild to moderate, whereas DKK3 expression decreased with the development of arthritis from normal, mild to moderate. According to the results of western blot analysis, β‑catenin expression was higher in moderate OA and then decreased in severe OA. On the other hand, the DKK3 levels decreased along with the progression from normal, mild to moderate OA. The results of RT‑qPCR demonstrated that β‑catenin and DKK3 gene expression differed with the degree of OA. On the whole, the present study demonstrates that DKK3 and β‑catenin may play opposite roles in OA subchondral bone remodeling.

The calcified cartilage zone (CCZ) is a thin interlayer between the hyaline articular cartilage and the subchondral bone and plays an important role in maintaining the joint homeostasis by providing biological and mechanical support from unmineralized cartilage to the underlying mineralized subchondral bone. The hallmark of CCZ characteristics in osteoarthritis (OA) is less well known. The aim of our study is to evaluate the structural, molecular, and biochemical composition of CCZ in tissues affected by primary knee OA and its relationship with disease severity. We collected osteochondral tissue samples stratified according to disease severity, from 16 knee OA patients who underwent knee replacement surgery. We also used meniscectomy-induced rat samples to confirm the pathophysiologic changes of human samples. We defined the characteristics of the calcified cartilage layer using a combination of morphological, biochemical, proteomic analyses on laser micro-dissected tissue. Our results demonstrated that the Calcium/Phosphate ratio is unchanged during the OA progression, but the calcium-binding protein and cadherin binding protein, as well as carbohydrate metabolism-related proteins, undergo significant changes. These changes were further accompanied by thinning of the CCZ, loss of collagen and proteoglycan content, the occurrence of the endochondral ossification, neovasculature, loss of the elastic module, loss of the collagen direction, and increase of the tortuosity indicating an altered structural and mechanical properties of the CCZ in OA. In conclusion, our results suggest that the calcified cartilage changes can reflect the disease progression., (© 2022 Federation of American Societies for Experimental Biology.)

Current clinical applications of mesenchymal stem cell therapy for osteoarthritis lack consistency because there are no established criteria for clinical processes. We aimed to systematically organize stem cell treatment methods by reviewing the literature. The treatment methods used in 27 clinical trials were examined and reviewed. The clinical processes were separated into seven categories: cell donor, cell source, cell preparation, delivery methods, lesion preparation, concomitant procedures, and evaluation. Stem cell donors were sub-classified as autologous and allogeneic, and stem cell sources included bone marrow, adipose tissue, peripheral blood, synovium, placenta, and umbilical cord. Mesenchymal stem cells can be prepared by the expansion or isolation process and attached directly to cartilage defects using matrices or injected into joints under arthroscopic observation. The lesion preparation category can be divided into three subcategories: chondroplasty, microfracture, and subchondral drilling. The concomitant procedure category describes adjuvant surgery, such as high tibial osteotomy. Classification codes were assigned for each subcategory to provide a useful and convenient method for organizing documents associated with stem cell treatment. This classification system will help researchers choose more unified treatment methods, which will facilitate the efficient comparison and verification of future clinical outcomes of stem cell therapy for osteoarthritis.

Objective: Recent evidence delineates an emerging role of periostin in osteoarthritis (OA), since its expression after knee injury is detrimental to the articular cartilage. We undertook this study to examine whether intraarticular (IA) knockdown of periostin would ameliorate posttraumatic OA in a murine model., Methods: Posttraumatic OA was induced in 10-week-old male C57BL/6J mice (n = 24) by destabilization of the medial meniscus (DMM), and mice were analyzed 8 weeks after surgery. Periostin expression was inhibited by small interfering RNA (siRNA) delivered IA using a novel peptide-nucleotide polyplex. Following histologic assessment of the mouse knee cartilage, the extent of cartilage degeneration was determined using Osteoarthritis Research Society International (OARSI) cartilage damage score, and severity of synovitis was also assessed. Bone changes were measured using micro-computed tomography. The effect and mechanism of periostin silencing were investigated in human chondrocytes that had been stimulated with interleukin-1β (IL-1β) with or without the IκB kinase 2 inhibitor SC-514., Results: Periostin expression in mice with posttraumatic OA was significantly abolished using IA delivery of a peptide-siRNA nanoplatform. OARSI cartilage damage scores were significantly lower in mice receiving periostin siRNA (mean ± SEM 10.94 ± 0.66) compared to untreated mice (22.38 ± 1.30) and mice treated with scrambled siRNA (22.69 ± 0.87) (each P = 0.002). No differences in the severity of synovitis were observed. Subchondral bone sclerosis, bone volume/total volume, volumetric bone mineral density, and heterotopic ossification were significantly lower in mice that had received periostin siRNA treatment. Immunostaining of cartilage revealed that periostin knockdown reduced the intensity of DMM-induced matrix metalloproteinase 13 (MMP-13) expression and also diminished the phosphorylation of p65 and immunoreactivity of the aggrecan neoepitope DIPEN. Periostin knockdown also suppressed IL-1β-induced MMP-13 and ADAMTS-4 expression in chondrocytes. Mechanistically, periostin-induced MMP-13 expression was abrogated by SC-514, demonstrating a link between periostin and NF-κB., Conclusion: IA delivery of the periostin-siRNA nanocomplex represents a promising clinical approach to mitigate the severity of joint degeneration in OA. Our findings may thus provide an unequivocal scientific rationale for longitudinal studies of this approach. Utilizing a cartilage-specific gene-knockout strategy will further illuminate the functional role of periostin in OA., (© 2021, American College of Rheumatology.)

Using the rat model of posttraumatic osteoarthrosis of the knee joint induced by surgical transection of their anterior cruciate ligaments, we showed that irreversible loss of hyaluronan by the extracellular matrix of the joint cartilage tissue against the background of oxidative stress accompanied by accumulation of intermediate LPO products in blood serum and formation of thiol system incompetence was one of the key patterns of dystrophic degeneration of the cartilage tissue. Considerable metabolic shifts were associated with structural modification of the articular hyaline cartilage: its thinning and a decrease of chondrocyte density and their abnormal spatial distribution in the matrix with predominance of solitary isolated cells with signs of karyopyknosis and karyolysis., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: To evaluate differences in pro-inflammatory and degradative mediator production from osteoarthritic knee articular cartilage explants treated with a hyperosmolar saline solution supplemented with anti-inflammatory components (l-glutamine, ascorbic acid, sodium pyruvate, epigallocatechin gallate [EGCG], and dexamethasone) or normal saline using an in vitro model for knee arthroscopy., Design: Full-thickness 6 mm articular cartilage explants ( n = 12/patient) were created from femoral condyle and tibial plateau samples collected from patients who received knee arthroplasty. One explant half was treated for 3 hours with hyperosmolar saline (600 mOsm/L) supplemented with anti-inflammatory components and the corresponding half with normal saline (308 mOsm/L). Explants were cultured for 3 days and then collected for biomarker analyses. Media biomarker concentrations were normalized to the wet weight of the tissue (mg) and were analyzed by a paired t test with significance set at P < 0.05., Results: Cartilage was collected from 9 females and 2 males (mean age = 68 years). Concentrations of MCP-1 ( P < 0.001), IL-8 ( P = 0.03), GRO-α ( P = 0.02), MMP-1 ( P < 0.001), MMP-2 ( P < 0.001), and MMP-3 ( P < 0.001) were significantly lower in explant halves treated with the enhanced hyperosmolar solution. When considering only those cartilage explants in the top tercile of tissue metabolism, IL-6 ( P = 0.005), IL-8 ( P = 0.0001), MCP-1 ( P < 0.001), GRO-α ( P = 0.0003), MMP-1 ( P < 0.001), MMP-2 ( P < 0.001), MMP-3 ( P < 0.001), and GAG expression ( P = 0.0001) was significantly lower in cartilage explant halves treated with the enhanced hyperosmolar solution., Conclusions: Treatment of cartilage explants with a hyperosmolar saline arthroscopic irrigation solution supplemented with anti-inflammatory components was associated with significant decreases in inflammatory and degradative mediator production and mitigation of proteoglycan loss.

Knee cartilage is in an aqueous environment filled with synovial fluid consisting of water, various nutrients, and ions to maintain chondrocyte homeostasis. Aquaporins (AQPs) are water channel proteins that play an important role in water exchange in cells, and AQP1, -3, and -4 are known to be expressed predominantly in cartilage. We evaluated the changes in AQP expression in chondrocytes from human knee articular cartilage in patients of different ages and identified the key factor(s) that mediate age-induced alteration in AQP expression. The mRNA and protein expression of AQP1, -3 and -4 were significantly decreased in fibrocartilage compared to hyaline cartilage and in articular cartilage from older osteoarthritis patients compared to that from young patients. Gene and protein expression of AQP1, -3 and -4 were altered during the chondrogenic differentiation of C3H10T1/2 cells. The causative factors for age-associated decrease in AQP included H 2 O 2 , TNFα, and HMGB1 for AQP1, -3, and -4, respectively. In particular, the protective effect of AQP4 reduction following HMGB1 neutralization was noteworthy. The identification of other potent molecules that regulate AQP expression represents a promising therapeutic approach to suppress cartilage degeneration during aging., (© 2021. The Author(s).)

Chondrocyte-based cartilage repair strategies, such as articular chondrocyte implantation, are widely used, but few studies addressed the communication between native subchondral bone cells and the transplanted chondrocytes. An indirect co-culture model was developed, representing a chondrocyte/scaffold-construct repair of a cartilage defect adjoining bone, where the bone could have varying degrees of degeneration. Human BM-MSCs were isolated from two areas of subchondral bone in each of five osteochondral tissue specimens from five patients undergoing knee arthroplasty. These two areas underlaid the macroscopically and histologically best and worst cartilage, representing early and late-stage OA, respectively. BM-MSCs were co-cultured with normal chondrocytes suspended in agarose, with the two cell types separated by a porous membrane. After 0, 7, 14 and 21 days, chondrocyte-agarose scaffolds were assessed by gene expression and biochemical analyses, and the abundance of selected proteins in conditioned media was assessed by ELISA. Co-culture with late-OA BM-MSCs resulted in a reduction in GAG deposition and a decreased expression of genes encoding matrix-specific proteins ( COL2A1 and ACAN ), compared to culturing with early OA BM-MSCs. The concentration of TGF-β1 was significantly higher in the early OA conditioned media. The results of this study have clinical implications for cartilage repair, suggesting that the health of the subchondral bone may influence the outcomes of chondrocyte-based repair strategies.

Articular cartilage is a connective tissue lining the surfaces of synovial joints. When the cartilage severely wears down, it leads to osteoarthritis (OA), a debilitating disease that affects millions of people globally. The articular cartilage is composed of a dense extracellular matrix (ECM) with a sparse distribution of chondrocytes with varying morphology and potentially different functions. Elucidating the molecular and functional profiles of various chondrocyte subtypes and understanding the interplay between these chondrocyte subtypes and other cell types in the joint will greatly expand our understanding of joint biology and OA pathology. Although recent advances in high-throughput OMICS technologies have enabled molecular-level characterization of tissues and organs at an unprecedented resolution, thorough molecular profiling of articular chondrocytes has not yet been undertaken, which may be in part due to the technical difficulties in isolating chondrocytes from dense cartilage ECM. In this study, we profiled articular cartilage from healthy and injured mouse knee joints at a single-cell resolution and identified nine chondrocyte subtypes with distinct molecular profiles and injury-induced early molecular changes in these chondrocytes. We also compared mouse chondrocyte subpopulations to human chondrocytes and evaluated the extent of molecular similarity between mice and humans. This work expands our view of chondrocyte heterogeneity and rapid molecular changes in chondrocyte populations in response to joint trauma and highlights potential mechanisms that trigger cartilage degeneration.

Developmental engineering strategies aim to recapitulate aspects of development in vitro as a means of forming functional engineered tissues, including cartilage and bone, for tissue repair and regeneration. Biophysical stimuli arising from fetal movements are critical for guiding skeletogenesis, but there have been few investigations of the biomechanical parameters which optimally promote cartilage and bone development events in in vitro explants. The effect of applied flexion-extension movement frequencies (0.33 and 0.67 Hz) and durations (2 h periods, 1, 2 or 3 × per day) on knee (stifle) joint cartilage shape, chondrogenesis and diaphyseal mineralisation of fetal chick hindlimbs, cultured in a mechanostimulation bioreactor, were assessed both quantitatively and qualitatively. It was hypothesised that increasing frequency and duration of movements would synergistically promote cartilage and bone formation in a dose-dependent manner. Increasing loading duration promoted cartilage growth, shape development and mineralisation of the femoral condyles and tibiotarsus. While increasing frequency had a significant positive effect on mineralisation, hyaline cartilage growth and joint shape were unaffected by frequency change within the ranges assessed, and there were limited statistical interactions between the effects of movement frequency and duration on cartilage or bone formation. Increased glycosaminoglycan deposition and cell proliferation may have contributed to the accelerated cartilage growth and shape change under increasing loading duration. The results demonstrated that frequencies and durations of applied biomechanical stimulation differentially promoted cartilage and bone formation, with implications for developmentally inspired tissue engineering strategies aiming to modulate tissue construct properties.

Osteoarthritis (OA) is a complex disease with biologic, biomechanical, and clinical heterogeneity among patients. Relationships among OA tissue metabolism, histopathology, and extracellular matrix (ECM) composition have not been well characterized. It was hypothesized that moderate (r = .4-.69) to strong (r > .7) correlations exist among these different measures of disease severity in osteochondral tissues from OA knees. Joint surfaces were obtained from patients (n = 6) undergoing total knee arthroplasty. Osteochondral explants (n = 136) were created and cultured for 3 days. Culture media were collected for biomarker analyses, and tissue was assessed for viability, histological scoring, and ECM composition. Correlations among media biomarker concentrations, histological scoring, ECM composition, and viability were determined using a Spearman correlation. GRO-α, IL-6, IL-8, and MCP-1 showed strong positive correlations to each other, and moderate positive correlations to NO, PGE2, and MMP-2. Total MMP activity, MMP-9, and MMP-13 had strong positive correlations to each other, and moderate positive correlations to MMP-1. MMP-2 had a moderate to strong positive correlations to histological scores (total and cartilage structure) and collagen content. MMP-2, IL-6, IL-8, and MCP-1 had moderate negative correlations, and MMP-9 had a moderate positive correlation, to viability. GRO-α, IL-6, IL-8, and MCP-1 had moderate positive correlations to collagen content. MMP-9, MMP-13, and total MMP activity had moderate negative correlations to tissue GAG. The data suggest links among proinflammatory and degradative pathways are present in OA osteochondral tissues. Further characterization of these links have the potential to delineate mechanisms of disease and diagnostic and therapeutic targets for knee OA., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Osteoarthritis (OA) is a multifactorial disease which is characterized by a change in the homeostasis of the extracellular matrix (ECM). The ECM is essential for the function of the articular cartilage and plays an important role in cartilage mechanotransduction. To provide a better understanding of the interaction between the ECM and the actin cytoskeleton, we investigated the localization and expression of the Ca 2+ -dependent proteins cartilage oligomeric matrix protein (COMP), thrombospondin-1 (TSP-1), plastin 3 (PLS3) and stromal interaction molecule 1 (STIM1). We investigated 16 patients who suffered from varus knee OA and performed a topographical analysis of the cartilage from the medial and lateral compartment of the proximal tibial plateau. In a varus knee, OA is more pronounced in the medial compared to the lateral compartment as a result of an overloading due to the malalignment. We detected a location-dependent staining of PLS3 and STIM1 in the articular cartilage tissue. The staining intensity for both proteins correlated with the degree of cartilage degeneration. The staining intensity of TSP-1 was clearly reduced in the cartilage of the more affected medial compartment, an observation that was confirmed in cartilage extracts by immunoblotting. The total amount of COMP was unchanged; however, slight changes were detected in the localization of the protein. Our results provide novel information on alterations in OA cartilage suggesting that Ca 2+ -dependent mechanotransduction between the ECM and the actin cytoskeleton might play an essential role in the pathomechanism of OA.

The expression of some chemokines and chemokine receptors is induced during the development of post-traumatic osteoarthritis (PTOA), but their involvement in the pathogenesis of the disease is unclear. The goal of this study was to test whether CCL21 and CXCL13 play a role in PTOA development. For this purpose, we evaluated the expression profiles of the chemokines Ccl21 and Cxcl13, matrix metalloproteinase enzymes Mmp3 and Mmp13, and inflammatory cell markers in response to partial medial meniscectomy and destabilization (MMD). We then assessed the effect of local administration of CCL21 neutralizing antibody on PTOA development and post-knee injury inflammation. The mRNA expression of both Ccl21 and Cxcl13 was induced early post-surgery, but only Ccl21 mRNA levels remained elevated 4 weeks post-surgery in rat MMD-operated knees compared to controls. This suggests that while both CXCL13 and CCL21 are involved in post-surgery inflammation, CCL21 is necessary for development of PTOA. A significant increase in the mRNA levels of Cd4, Cd8 and Cd20 was observed during the first 3 days post-surgery. Significantly, treatment with CCL21 antibody reduced post-surgical inflammation that was accompanied by a reduction in the expression of Mmp3 and Mmp13 and post-MMD cartilage degradation. Our findings are consistent with a role for CCL21 in mediating changes in early inflammation and subsequent cartilage degeneration in response to knee injury. Our results suggest that targeting CCL21 signaling pathways may yield new therapeutic approaches effective in delaying or preventing PTOA development following injury., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Increased intake of dietary saturated fatty acids has been linked to obesity and the development of Osteoarthritis (OA). However, the mechanism by which these fats promote cartilage degradation and the development of OA is not clearly understood. Here, we report the effects of consumption of common dietary saturated and unsaturated fatty acids, palmitate and oleate, respectively, on body weight, metabolic factors, and knee articular cartilage in a mouse model of diet-induced obesity. Mice fed on a diet rich in saturated or unsaturated fatty acid gained an equal amount of weight; however, mice fed a palmitate diet, but not a control or oleate diet, exhibited more cartilage lesions and increased expression of 1) unfolded protein response (UPR)/endoplasmic reticulum (ER) stress markers including BIP, P-IRE1α, XBP1, ATF4, and CHOP; 2) apoptosis markers CC3 and C-PARP; and 3) negative cell survival regulators Nupr1 and TRB3, in knee articular cartilage. Palmitate-induced apoptosis was confirmed by TUNEL staining. Likewise, dietary palmitate was also increased the circulatory levels of classic proinflammatory cytokines, including IL-6 and TNF-α. Taken together, our results demonstrate that increased weight gain is not sufficient for the development of obesity-linked OA and suggest that dietary palmitate promotes UPR/ER stress and cartilage lesions in mouse knee joints. This study validates our previous in vitro findings and suggests that ER stress could be the critical metabolic factor contributing to the development of diet/obesity induced OA., Competing Interests: The authors declare no competing interests.

Objective: TGFβ1 plays an important role in the metabolism of articular cartilage and bone; however, the pathological mechanism and targets of TGFβ1 in cartilage degradation and uncoupling of subchondral bone remodeling remain unclear. Therefore, in this study, we investigated the relationship between TGFβ1 and major protein-degrading enzymes, and evaluated the role of high levels of active TGFβ1 in the thickening of subchondral bone and calcification of articular cartilage., Materials and Methods: The expression of TGFβ1 and protein-degrading enzymes in clinical samples of articular cartilage and subchondral bone obtained from the knee joint of patients with osteoarthritis was detected by immunohistochemistry. The expression levels of TGFβ1, MMP-3, MMP-13 and IL-1β in cartilage and subchondral bone tissues were detected by absolute real-time quantitative RT-PCR. The expression of TGFβ1, nestin and osterix in subchondral bone was detected by Western blot analysis and immunohistochemistry. The degree of subchondral bone thickening was determined by micro-computed tomography (CT) imaging., Results: Expression of TGFβ1 and cartilage-degrading enzymes was higher in the cartilage-disrupted group than that in the intact group. Furthermore, expression of TGFβ1, nestin and osterix was significantly higher in the OA group than that in the control group. Micro-CT imaging showed that in the OA group, the subchondral bone plate is thickened and the density is increased. The trabecular bone structure is thick plate-like structure, the thickness of the trabecular bone is increased and the gap is small., Conclusions: The data suggest that highly active TGFβ1 activates the expression of cartilage-degrading enzymes. Abnormally activated TGFβ1 may induce formation of the subchondral bone and expansion of the calcified cartilage area, eventually leading to degradation of the cartilage tissue., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Objective and Design: This study aimed at evaluating the effect of methylprednisolone (MPA) on messenger ribonucleic acid (mRNA) expression levels in immature ovine knee joint tissue explants following interleukin (IL)1β induction and to assess responsiveness of the explants., Material or Subjects: Explants were harvested from the articular cartilage, synovium, and infrapatellar fat pad (IPFP) from immature female sheep., Treatment: Methylprednisolone., Methods: The samples were allocated into six groups: (1) control, (2) MPA (10 -3  M), (3) MPA (10 -4  M), (4) IL1β, (5) IL1β + 10 -3  M MPA, or (6) IL1β + 10 -4  M MPA. mRNA expression levels for molecules relevant to inflammation, cartilage degradation/anabolism, activation of innate immunity, and adipose tissue/hormones were quantified. Fold changes with MPA treatment were compared via the comparative C T method., Results: Methylprednisolone treatment significantly suppressed MMPs consistently across the cartilage (MMP1, MMP3, and MMP13), synovium (MMP1 and MMP3), and IPFP (MMP13) (all p < 0.05). Other genes that were less consistently suppressed include endogenous IL1β (cartilage) and IL6 (IPFP) (all p < 0.05), and others not affected either by IL-1 exposure or subsequent MPA include TGFβ1, TLR4, and adipose-related molecules., Conclusions: Methylprednisolone significantly mitigated IL1β induced mRNA expression for MMPs in the immature cartilage, synovium, and IPFP, but the extent of the responsiveness was tissue-, location-, and gene-specific.

MicroRNA (miRNA) in situ hybridization (ISH) is a highly sensitive method that allows for the detection of expression and distribution of miRNAs in fixed paraffin-embedded tissues. MiRNA ISH requires time-consuming optimization based on the tissue type analyzed, method of tissue fixation, and miRNA detection probe. Here, we provide the optimized miRNA ISH protocol for human cartilage and mouse whole knee joints that also entails the necessary steps for sample collection, processing, and preparation for high-quality ISH staining.

Objective: For many proteins from osteoarthritic synovial fluid, their intra-articular tissue of origin remains unknown. In this study we performed comparative proteomics to identify osteoarthritis-specific and joint tissue-dependent secreted proteins that may serve as candidates for osteoarthritis biomarker development on a tissue-specific basis., Design: Protein secretomes of cartilage, synovium, Hoffa's fat pad and meniscus from knee osteoarthritis patients were determined using liquid chromatography tandem mass spectrometry, followed by label-free quantification. Validation of tissue-dependent protein species was conducted by ELISA on independent samples. Differential proteomes of osteoarthritic and non-osteoarthritic knee synovial fluids were obtained via similar proteomics approach, followed by ELISA validation., Results: Proteomics revealed 64 proteins highly secreted from cartilage, 94 from synovium, 37 from Hoffa's fat pad and 21 from meniscus. Proteomic analyses of osteoarthritic vs non-osteoarthritic knee synovial fluid revealed 70 proteins with a relatively higher abundance and 264 proteins with a relatively lower abundance in osteoarthritic synovial fluid. Of the 70 higher abundance proteins, 23 were amongst the most highly expressed in the secretomes of a specific intra-articular tissue measured. Tissue-dependent release was validated for SLPI, C8, CLU, FN1, RARRES2, MATN3, MMP3 and TNC. Abundance in synovial fluid of tissue-dependent proteins was validated for IGF2, AHSG, FN1, CFB, KNG and C8., Conclusions: We identified proteins with a tissue-dependent release from intra-articular human knee OA tissues. A number of these proteins also had an osteoarthritis-specific abundance in knee synovial fluid. These proteins may serve as novel candidates for osteoarthritis biomarker development on a tissue-specific basis., Competing Interests: Conflict of Interests TJM Welting is listed as inventor on patents: WO2017178251, WO2017178253 and US 20130123314. PJ Emans and LW van Rhijn are listed as inventors on patent US 20130123314. LW van Rhijn, PJ Emans and TJM Welting have shares in Chondropeptix and are CDO, CMO and CSO of Chondropeptix, respectively., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: As a common joint disease, osteoarthritis (OA) is the main cause of limited joint mobility and disability. The role of lncRNAs in the regulation of OA is increasingly discovered. Therefore, further exploring the function of SNHG7 in OA is of great significance for understanding its occurrence and development., Methods: We used interleukin-1β (IL-1β) to treat to establish an OA model primary on chondrocytes in vitro, and gain- and loss of function assays of SNHG7 and miR-214-5p were conducted. The cell viability and apoptosis of chondrocytes were detected by CCK8 assay, BrdU assay and flow cytometry. The inflammatory cytokines (IL-1β, IL-6 and TNF-α), NLRP3 inflammasome, protein level of PPARGC1B, PPARγ, P38 and NF-κB were determined by RT-PCR and/or western blot., Results: The results showed that SNHG7 was distinctly downregulated, while miR-214-5p was significantly upregulated in OA patients and primary chondrocytes treated with IL-1β. In addition, SNHG7 enhanced cell viability, inhibited apoptosis and inflammation of IL-1β-mediated chondrocytes. In contrast, miR-214-5p upregulation reduced viability, promoted apoptosis and inflammation of chondrocytes. Mechanistically, SNHG7 served as a competitive endogenous RNA by sponging miR-214-5p, which targeted PPARGC1B. Besides, the results of the compensation experiment affirmed that miR-214-5p attenuates SNHG7-mediated protective effects on IL-1β-mediated chondrocytes against apoptosis and inflammation, and activating PPARγ pathway markedly dampened the cytotoxic effects of miR-214-5p., Conclusions: Collectively, The above results confirmed that SNHG7 prevents IL-1β induced OA by inhibiting NLRP3 inflammasome and apoptosis through miR-214-5p/PPARGC1B axis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Osteoarthritis (OA) is the most common arthropathy, characterized by progressive degeneration of the articular cartilage. Currently, there are no disease-modifying approaches for OA treatment. Adeno-associated virus (AAV)-mediated gene therapy has recently become a potential treatment for OA due to its exceptional characteristics; however, the tropism and transduction efficiency of different AAV serotypes to articular joints and the safety profile of AAV applications are still unknown. The present study aims to screen an ideal AAV serotype to efficiently transfer genes to arthritic cartilage. AAV vectors of different serotypes expressing eGFP protein were injected into the knee joint cavities of mice, with all joint tissues collected 30 days after AAV injection. The transduction efficiency of AAVs was quantified by assessing the fluorescent intensities of eGFP in the cartilage of knee joints. Structural and morphological changes were analyzed by toluidine blue staining. Changes to ECM metabolism and pyroptosis of chondrocytes were determined by immunohistochemical staining. Fluorescence analysis of eGFP showed that eGFP was expressed in the cartilage of knee joints injected with each AAV vector. Quantification of eGFP intensity indicated that AAV2, 7 and 8 had the highest transduction efficiencies. Both toluidine blue staining and Mankin score showed that AAV6 aggravated cartilage degeneration. The analysis of key molecules in ECM metabolism suggested that AAV5 and 7 significantly reduced collagen type II, while AAV9 increased ADAMTS-4 but decreased MMP-19. In addition, transduction with AAV2, 5, 7 and 8 had no obvious effect on pyroptosis of chondrocytes. Comprehensive score analysis also showed that AAV2 had the highest score in intra-articular gene transfer. Collectively, our findings point to AAV2 as the best AAV serotype candidate for gene transfer on arthritic cartilage, resulting in minimal impact to ECM metabolism and pyroptosis of chondrocytes., Competing Interests: The authors have declared that no competing interests exist.

Objective: The purpose of this study was to compare the differences of degenerative characteristics of medial, lateral regions, femoral, patellar, and tibia parts of the knee joint in the early stage of knee osteoarthritis (OA)., Patients and Methods: A total of 66 patients with early-stage knee OA and 22 healthy volunteers who have no knee-related clinical symptoms were enrolled in this cross-sectional study. T2 mapping and 3D dual-echo images were acquired with a 3.0T MR scanner. The degenerative changes of the articular cartilage were quantified by a T2 mapping and cartilage thicknesses analysis. Any structural changes were conducted using the Whole Organ Magnetic Resonance Imaging Score (WORMS)., Results: In patients with knee OA, the thicknesses of medial cartilages were significantly thinner than lateral ones (2.13 mm vs. 2.34 mm, p<0.0001), but with higher T2 values (40.38 ms vs. 38.4 ms, p<0.0001) and WORMS scores (12.12 vs. 0.47, p=0.004). No significant differences were observed between medial and lateral cartilage in the healthy volunteers. The T2 values of the femoral (p<0.001) and patellar (p=0.012) cartilages of OA patients were higher than that of the healthy controls. Within OA group, the T2 values of femoral (p<0.0001) and patellar (p<0.0001) cartilages were higher than tibial ones. Moreover, the WORMS scores of femoral (p=0.001) and patellar (p<0.0001) cartilages were higher than that of the tibial ones., Conclusions: This study demonstrates that the medial compartment and patellofemoral knee joint degenerate more severely in patients with early-stage knee OA.

Objective: To determine the role of JNK signaling in the development of osteoarthritis (OA) induced by joint injury or aging in mice., Methods: In the joint injury model, 12-week-old wild-type control, JNK1 -/- , JNK2 -/- , and JNK1 fl/fl JNK2 -/- aggecan-Cre ERT 2 double-knockout mice were subjected to destabilization of the medial meniscus (DMM) (n = 15 mice per group) or sham surgery (n = 9-10 mice per group), and OA was evaluated 8 weeks later. In the aging experiment, wild-type control, JNK1 -/- , and JNK2 -/- mice (n = 15 per group) were evaluated at 18 months of age. Mouse knee joints were evaluated by scoring articular cartilage structure, toluidine blue staining, osteophytes, and synovial hyperplasia, by histomorphometric analysis, and by immunostaining for the senescence marker p16 INK 4a . Production of matrix metalloproteinase 13 (MMP-13) in cartilage explants in response to fibronectin fragments was measured by enzyme-linked immunosorbent assay., Results: There were no differences after DMM surgery between the wild-type and the JNK-knockout mouse groups in articular cartilage structure, toluidine blue, or osteophyte scores or in MMP-13 production in explants. All 3 knockout mouse groups had increased subchondral bone thickness and area of cartilage necrosis compared to wild-type mice. Aged JNK-knockout mice had significantly worse articular cartilage structure scores compared to the aged wild-type control mice (mean ± SD 52 ± 24 in JNK1 -/- mice and 60 ± 25 in JNK2 -/- mice versus 32 ± 18 in controls; P = 0.02 and P = 0.004, respectively). JNK1 -/- mice also had higher osteophyte scores. Deletion of JNK resulted in increased expression of p16 INK 4a in the synovium and cartilage in older mice., Conclusion: JNK1 and JNK2 are not required for the development of OA in the mouse DMM model. Deletion of JNK1 or JNK2 is associated with more severe age-related OA and increased cell senescence, suggesting that JNK may act as a negative regulator of senescence in the joint., (© 2020, American College of Rheumatology.)

Water, collagen, and proteoglycans determine articular cartilage functionality. If altered, susceptibility to premature degeneration is increased. This study investigated the effects of enzymatic proteoglycan depletion on cartilage functionality as assessed by advanced Magnetic Resonance Imaging (MRI) techniques under standardized loading. Lateral femoral condylar cartilage-bone samples from patients undergoing knee replacement (n = 29) were serially imaged by Proton Density-weighted and T1, T1ρ, T2, and T2* mapping sequences on a clinical 3.0 T MRI scanner (Achieva, Philips). Using pressure-controlled indentation loading, samples were imaged unloaded and quasi-statically loaded to 15.1 N and 28.6 N, and both before and after exposure to low-concentrated (LT, 0.1 mg/mL, n = 10) or high-concentrated trypsin (HT, 1.0 mg/mL, n = 10). Controls were not treated (n = 9). Responses to loading were assessed for the entire sample and regionally, i.e. sub- and peri-pistonally, and zonally, i.e. upper and lower sample halves. Trypsin effects were quantified as relative changes (Δ), analysed using appropriate statistical tests, and referenced histologically. Histological proteoglycan depletion was reflected by significant sub-pistonal decreases in T1 (p = 0.003) and T2 (p = 0.008) after HT exposure. Loading-induced changes in T1ρ and T2* were not related. In conclusion, proteoglycan depletion alters cartilage functionality and may be assessed using serial T1 and T2 mapping under loading.

Aim: A limited healing response to focal cartilage lesions is frequently encountered in the clinical cartilage pathology. This study compares the gene expression patterns of damaged and undamaged regions of cartilage obtained from the same patient with focal cartilage lesions. The aim of this study is to provide new genes and proteins, which may be a potential future target of research., Methods: During the autologous chondrocyte implantation (MACI) surgery, cartilage tissues (healthy non-weight bearing and Damaged-lesion side) were obtained from 10 patients with knee focal cartilage lesions. The degeneration status of the cartilage was characterized according to ICRS criteria. Whole genome microarray gene expression profiling was performed and some of the differentially regulated genes were validated with RT-PCR., Results: Damaged and undamaged non-weight bearing cartilage showed distinct gene expression profiles. Genes involved in cell signaling, matrix degradation, hypoxia, and the inflammatory response showed significant up- or down-regulation. In the focal lesions, expression of genes such as HIF1α, TIMP-2, EID1, EID2, NCOA3, NBR1, SP100, and HSP90AA1 was significantly higher compared to healthy non-weight bearing cartilage from the same joint, whereas TIMP-4 was lower., Conclusion: The genes examined in this study differ distinctly between focal cartilage (ICRS 3-4) lesions and undamaged sites of the same joint. We believe that the data set forth in this study may be used for clinical purposes and be a guide in the development of new biological approaches for therapy.

Objective: Iron is emerging as a key player in aging-associated diseases due to its propensity for driving free radical formation. Studies examining the role of iron in the pathogenesis of primary osteoarthritis (OA) are limited. Our objective was to establish a direct relationship between excess iron and OA by administering iron dextran to a guinea pig strain with decreased propensity for developing this disease., Design: Twenty, 12-week-old Strain 13 guinea pigs received either iron dextran or dextran control intraperitoneally once weekly for 4 weeks; termination occurred at 16 weeks of age. Iron levels were determined systemically (serum and liver) and within diarthrodial joints [femoral head articular cartilage and infrapatellar fat pads (IFPs) of knee joints]. One knee was collected to score structural changes associated with OA via microcomputed tomography (microCT) and histology using published grading schemes. Articular cartilage and IFPs were harvested from contralateral knees for gene expression analyses., Results: Iron overload was confirmed systemically via increased serum iron and liver iron concentration. Articular cartilage and IFPs in the iron dextran group also had higher levels of iron. Excess iron worsened knee OA using both microCT and histologic scoring systems. Gene analyses revealed that exogenous iron altered the expression of iron trafficking proteins, select cytokines, and structural components of cartilage., Conclusion: These results demonstrate that systemic iron overload caused cellular iron accumulation in the knee joint. This excess iron is associated with increased expression of local inflammatory mediators and early onset and progression of knee joint OA in Strain 13 animals., (Copyright © 2020 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Purpose: To determine whether (1) human leukocyte-platelet-rich plasma (L-PRP) or (2) leukocyte-platelet-rich fibrin (L-PRF) delivered on a hyaluronic acid (HA) scaffold at a bovine chondral defect, a simulated cartilage tear interface, in vitro would improve tissue formation based on biomechanical, histologic, and biochemical measures., Methods: L-PRF and L-PRP were prepared from 3 healthy volunteer donors and delivered in conjunction with HA scaffolds to defects created in full-thickness bovine cartilage plugs harvested from bovine femoral condyle and trochlea. Specimens were cultured in vitro for up to 42 days. Treatment groups included an HA scaffold alone and scaffolds containing L-PRF or L-PRP. Cartilage repair was assessed using biomechanical testing, histology, DNA quantification, and measurement of sulfated glycosaminoglycan and collagen content at 28 and 42 days., Results: L-PRF elicited the greatest degree of defect filling and improvement in other histologic measures. L-PRF-treated specimens also had the greatest cellularity when compared with L-PRP and control at day 28 (560.4 μg vs 191.4 μg vs 124.2 μg, P = .15); at day 48, there remained a difference, although not significant, between L-PRF versus L-PRP (761.1 μg vs 589.3 μg, P = .219) . L-PRF had greater collagen deposition when compared with L-PRP at day 42 (40.1 μg vs 16.3 μg, P < .0001). L-PRF had significantly greater maximum interfacial strength compared with the control at day 42 (10.92 N vs 0.66 N, P = .015) but had no significant difference compared with L-PRP (10.92 N vs 6.58 N, P = .536). L-PRP facilitated a greater amount of sulfated glycosaminoglycan production at day 42 when compared with L-PRF (15.9 μg vs 4.3 μg, P = .009)., Conclusions: Delivery of leukocyte-rich platelet concentrates in conjunction with a HA scaffold may allow for improvements in cartilage healing through different pathways. L-PRF was not superior to L-PRP in its biomechanical strength, suggesting that both treatments may be effective in improving biomechanical strength of healing cartilage through different pathways., Clinical Relevance: The delivery of platelet-rich concentrates in conjunction HA scaffolds may augment healing cartilaginous injuries., (Copyright © 2019 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Objective: The present study aimed to investigate whether experimentally induced lumbar facet-joint OA lead to degenerative changes and enhanced SP expression within the ipsilateral neurosegmentally linked tibiofemoral cartilage., Methods: Adult male Sprague-Dawley rats were assigned to left side L5-L6 facet mechanical compression injury (surgery) ( n = 6), L5-L6 facet exposure with no compression (sham) ( n = 5), or naïve (no surgery) ( n = 4) groups. The morphology of the tibiofemoral articular cartilage was assessed using a modified Mankin scoring system. Immunohistochemistry was used to examine the density of chondrocytes stained positive for SP (cells/cm 2 ) in the ipsilateral tibiofemoral cartilage at 28 days postintervention., Results: Tibiofemoral cartilage in the surgery group showed consistent loss of superficial zone chondrocytes, mild roughening of the articular surface and occasional chondrocyte clusters as well as a greater density of SP mainly in the superficial cartilage zone compared with sham and naïve groups, although they also had a basic SP-expression., Conclusion: Our results support the hypothesis that neurogenic mechanisms may mediate the spread of SP to neurosegmentally linked heterologous joints affecting the distal cartilage homeostasis. These findings contribute additional insight into the potential role of neurogenic inflammation with implications in the pathophysiology of chronic inflammatory joint disease and OA.

Objective: NF-κB-dependent signaling is an important modulator in osteoarthritis (OA), and G protein-coupled receptor kinase 5 (GRK5) regulates the NF-κB pathway. This study was undertaken to investigate the functional involvement of GRK5 in OA pathogenesis., Methods: GRK5 expression in normal and OA human knee joints was analyzed immunohistochemically. Gain- or loss-of-function experiments were performed using human and mouse chondrocytes. OA was induced in GRK5-knockout mice by destabilization of the medial meniscus, and histologic examination was performed. OA was also induced in wild-type mice, which were then treated with an intraarticular injection of amlexanox, a selective GRK5 inhibitor, every 5 days for 8 weeks., Results: GRK5 protein expression was increased in human OA cartilage. In vitro, expression levels of OA-related factors and NF-κB transcriptional activation were down-regulated by suppression of the GRK5 gene in human OA chondrocytes (3.49-fold decrease in IL6 [P < 0.01], 2.43-fold decrease in MMP13 [P < 0.01], and 2.66-fold decrease in ADAMTS4 [P < 0.01]). Conversely, GRK5 overexpression significantly increased the expression of OA-related catabolic mediators and NF-κB transcriptional activation. On Western blot analysis, GRK5 deletion reduced IκBα phosphorylation (up to 4.4-fold decrease [P < 0.05]) and decreased p65 nuclear translocation (up to 6.4-fold decrease [P < 0.01]) in mouse chondrocytes. In vivo, both GRK5 deletion and intraarticular amlexanox protected mouse cartilage against OA., Conclusion: Our results suggest that GRK5 regulates cartilage degradation through a catabolic response mediated by NF-κB signaling, and is a potential target for OA treatment. Furthermore, amlexanox may be a major compound in relevant drugs., (© 2019, American College of Rheumatology.)

This study was designed to evaluate the anti-inflammatory effects of a curcumin treatment on the knee of rats with induced osteoarthritis. Fifteen adult rats were used and divided in three groups: the osteoarthritis group (OAG), control group (CG-without induction of osteoarthritis), and curcumin-treated osteoarthritis group (COAG). Osteoarthritis was induced in the right knee of rats in the OAG and COAG by administering an intra-articular injection of 1 mg of zymosan. Fourteen days after induction, 50 mg/kg curcumin was administered by gavage daily for 60 days to the COAG. After the treatment period, rats from all groups were euthanized. Medial femoral condyles were collected for light microscopy and immunohistochemical staining. The expression of SOX-5, IHH, MMP-8, MMP-13, and collagen 2 (Col2) was analyzed. The COAG exhibited an increase in the number of chondrocytes in the surface and middle layers compared with that of the OAG and CG, respectively. The COAG also showed a decrease in the thicknesses of the middle and deep layers compared with those of the OAG, and an increase in Col2 expression was observed in all articular layers (surface, middle, and deep) in the COAG compared with that in the OAG. SOX-5 expression was increased in the surface and deep layers of the COAG compared with those in the OAG and CG. Based on the results of this study, the curcumin treatment appeared to exert a protective effect on cartilage, as it did not result in an increase in cartilage thickness or in MMP-8 and MMP-13 expression but led to increased IHH, Col2, and SOX-5 expression and the number of chondrocytes., Competing Interests: The authors have declared that no competing interests exist.

Roughly 20% of Americans run annually, yet how this exercise influences knee cartilage health is poorly understood. To address this question, quantitative magnetic resonance imaging (MRI) can be used to infer the biochemical state of cartilage. Specifically, T1rho relaxation times are inversely related to the proteoglycan concentration in cartilage. In this study, T1rho MRI was performed on the dominant knee of eight asymptomatic, male runners before, immediately after, and 24 hours after running 3 and 10 miles. Overall, (mean ± SEM) patellar, tibial, and femoral cartilage T1rho relaxation times significantly decreased immediately after running 3 (65 ± 3 ms to 62 ± 3 ms; p = 0.04) and 10 (69 ± 4 ms to 62 ± 3 ms; p < 0.001) miles. No significant differences between pre-exercise and recovery T1rho values were observed for either distance (3 mile: p = 0.8; 10 mile: p = 0.08). Percent decreases in T1rho relaxation times were significantly larger following 10 mile runs as compared to 3 mile runs (11 ± 1% vs. 4 ± 1%; p = 0.02). This data suggests that alterations to the relative proteoglycan concentration of knee cartilage due to water flow are mitigated within 24 hours of running up to 10 miles. This information may inform safe exercise and recovery protocols in asymptomatic male runners by characterizing running-induced changes in knee cartilage composition.

Growth and Differentiation Factor 5 (GDF5) is a key risk locus for osteoarthritis (OA). However, little is known regarding regulation of Gdf5 expression following joint tissue damage. Here, we employed Gdf5-LacZ reporter mouse lines to assess the spatiotemporal activity of Gdf5 regulatory sequences in experimental OA following destabilisation of the medial meniscus (DMM) and after acute cartilage injury and repair. Gdf5 expression was upregulated in articular cartilage post-DMM, and was increased in human OA cartilage as determined by immunohistochemistry and microarray analysis. Gdf5 expression was also upregulated during cartilage repair in mice and was switched on in injured synovium in prospective areas of cartilage formation, where it inversely correlated with expression of the transcriptional co-factor Yes-associated protein (Yap). Indeed, overexpression of Yap suppressed Gdf5 expression in chondroprogenitors in vitro. Gdf5 expression in both mouse injury models required regulatory sequence downstream of Gdf5 coding exons. Our findings suggest that Gdf5 upregulation in articular cartilage and synovium is a generic response to knee injury that is dependent on downstream regulatory sequence and in progenitors is associated with chondrogenic specification. We propose a role for Gdf5 in tissue remodelling and repair after injury, which may partly underpin its association with OA risk.

Purpose: Osteoarthritis (OA) of the knee is a multifactorial degenerative disease typically defined as the 'wear and tear' of articular joint cartilage. However, recent studies suggest that OA is a disease arising from chronic low-grade inflammation. We conducted a study to investigate the relationship between chronic inflammatory mediators present in both the systemic peripheral blood system and localised inflammation in synovial fluid (SF) of OA and non-OA knees; and subsequently made direct comparative analyses to understand the mechanisms that may underpin the processes involved in OA., Methods: 20-Plex proteins were quantified using Human Magnetic Luminex® assay (R&D Systems, USA) from plasma and SF of OA (n = 14) and non-OA (n = 14) patients. Ingenuity Pathway Analysis (IPA) software was used to predict the relationship and possible interaction of molecules pertaining to OA., Results: There were significant differences in plasma level for matrix metalloproteinase (MMP)-3, interleukin (IL)-27, IL-8, IL-4, tumour necrosis factor-alpha, MMP-1, IL-15, IL-21, IL-10, and IL-1 beta between the groups, as well as significant differences in SF level for IL-15, IL-8, vascular endothelial growth factor (VEGF), MMP-1, and IL-18. Our predictive OA model demonstrated that toll-like receptor (TLR) 2, macrophage migration inhibitory factor (MIF), TLR4 and IL-1 were the main regulators of IL-1B, IL-4, IL-8, IL-10, IL-15, IL-21, IL-27, MMP-1 and MMP-3 in the plasma system; whilst IL-1B, TLR4, IL-1, and basigin (BSG) were the regulators of IL-4, IL-8, IL-10, IL-15, IL-18, IL-21, IL-27, MMP-1, and MMP-3 in the SF system., Conclusion: The elevated plasma IL-8 and SF IL-18 may be associated with the pathogenesis of OA via the activation of MMP-3., (Copyright © 2019. Published by Elsevier B.V.)

Objective: To investigate the impact of exercise therapy on molecular biomarkers related to cartilage and inflammation in individuals at risk of, or with established, knee osteoarthritis by conducting a systematic review of randomized controlled trials (RCTs)., Methods: We conducted a literature search up to September 2017 in 5 major databases with no restriction on publication year or language. Data were extracted from the first available follow-up time point, and we performed a narrative synthesis for the effect of exercise therapy on molecular biomarkers related to cartilage and inflammation. A subset of studies reporting sufficient data was combined in a meta-analysis, using an adjusted random-effects model., Results: Twelve RCTs involving 57 study comparisons at 4 to 24 weeks following an exercise-therapy intervention were included. Exercise therapy decreased molecular biomarkers in 17 study comparisons (30%), had no effect in 36 (63%), and increased molecular biomarkers in 4 study comparisons (7%). Meta-analyses of 9 biomarkers showed that exercise therapy was associated with nonsignificant reductions of the C-reactive protein level, C-terminal crosslinking telopeptide of type II collagen, tumor necrosis factor (TNF), soluble TNF receptors 1 and 2, C2C neoepitope of type II collagen, and cartilage oligomeric matrix protein, compared to nonexercising control groups, and exercise therapy had no effect on interleukin-6 and soluble interleukin-6 receptor., Conclusion: Exercise therapy is not harmful, because it does not increase the concentration of molecular biomarkers related to cartilage turnover and inflammation, implicated in osteoarthritis progression. The overall quality of evidence was downgraded to low because of the limited number of RCTs available., (© 2018, American College of Rheumatology.)

Objective: WNT signaling is of key importance in chondrogenesis and defective WNT signaling may contribute to the pathogenesis of osteoarthritis and other cartilage diseases. Biochemical composition of articular cartilage in patients with aberrant WNT signaling has not been studied. Our objective was to assess the knee articular cartilage in WNT1 mutation-positive individuals using a 3.0T MRI unit to measure cartilage thickness, relaxation times, and texture features., Design: Cohort comprised mutation-positive (N = 13; age 17-76 years) and mutation-negative (N = 13; 16-77 years) subjects from two Finnish families with autosomal dominant WNT1 osteoporosis due to a heterozygous missense mutation c.652T>G (p.C218G) in WNT1. All subjects were imaged with a 3.0T MRI unit and assessed for cartilage thickness, T2 and T1ρ relaxation times, and T2 texture features contrast, dissimilarity and homogeneity of T2 relaxation time maps in six regions of interest (ROIs) in the tibiofemoral cartilage., Results: All three texture features showed opposing trends with age between the groups in the medial tibiofemoral cartilage (P = 0.020-0.085 for the difference of the regression coefficients), the mutation-positive individuals showing signs of cartilage preservation. No significant differences were observed in the lateral tibiofemoral cartilage. Cartilage thickness and means of T2 relaxation time did not differ between groups. Means of T1ρ relaxation time were significantly different in one ROI but the regression analysis displayed no differences., Conclusions: Our results show less age-related cartilage deterioration in the WNT1 mutation-positive than the mutation-negative subjects. This suggests, that the WNT1 mutation may alter cartilage turnover and even have a potential cartilage-preserving effect., (Copyright © 2019 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

The articular cartilage is an avascular and aneural tissue and its injuries result mostly in osteoarthritic changes and formation of fibrous tissue. Efforts of scientists worldwide are focused on restoration of cartilage with increase in life quality of patients. Novel polymeric polyhydroxybutyrate/chitosan (PCH) porous 3D scaffolds were developed and characterized. The rat mesenchymal stem cells (MSCs) were seeded in vitro on PCH scaffolds by a simple filtration of MSCs suspension over scaffolds using syringe. The chondrogenesis of cell-scaffold constructs was carried out in supplemented chondrogenic cultivation medium. After 2 and 4 weeks of in vitro culturing cell-scaffold constructs in chondrogenic differentiation medium, the cartilage extracellular matrix components like glycosaminoglycans and collagens were identified in scaffolds by biochemical assays and histological and immunohistochemical staining. Preliminary in vivo experiments with acellular scaffolds, which filled the artificially created cartilage defect in sheep knee were done and evaluated. Cells released from the bone marrow cavity have penetrated into acellular PCH scaffold in cartilage defect and induced tissue formation similar to hyaline cartilage. The results demonstrated that PCH scaffolds supported chondrogenic differentiation of MSCs in vitro. Acellular PCH scaffolds were successfully utilized in vivo for reparation of artificially created knee cartilage defects in sheep and supported wound healing and formation of hyaline cartilage-like tissue.