Osteoarthritic Articular Cartilage Research Articles

Cbfβ regulates Wnt/β-catenin, Hippo/Yap, and Tgfβ signaling pathways in articular cartilage homeostasis and protects from ACLT surgery-induced osteoarthritis.

As the most common degenerative joint disease, osteoarthritis (OA) contributes significantly to pain and disability during aging. Several genes of interest involved in articular cartilage damage in OA have been identified. However, the direct causes of OA are poorly understood. Evaluating the public human RNA-seq dataset showed that CBFB (subunit of a heterodimeric Cbfβ/Runx1, Runx2, or Runx3 complex) expression is decreased in the cartilage of patients with OA. Here, we found that the chondrocyte-specific deletion of Cbfb in tamoxifen-induced Cbfbf/f;Col2a1-CreERT mice caused a spontaneous OA phenotype, worn articular cartilage, increased inflammation, and osteophytes. RNA-sequencing analysis showed that Cbfβ deficiency in articular cartilage resulted in reduced cartilage regeneration, increased canonical Wnt signaling and inflammatory response, and decreased Hippo/Yap signaling and Tgfβ signaling. Immunostaining and western blot validated these RNA-seq analysis results. ACLT surgery-induced OA decreased Cbfβ and Yap expression and increased active β-catenin expression in articular cartilage, while local AAV-mediated Cbfb overexpression promoted Yap expression and diminished active β-catenin expression in OA lesions. Remarkably, AAV-mediated Cbfb overexpression in knee joints of mice with OA showed the significant protective effect of Cbfβ on articular cartilage in the ACLT OA mouse model. Overall, this study, using loss-of-function and gain-of-function approaches, uncovered that low expression of Cbfβ may be the cause of OA. Moreover, Local admission of Cbfb may rescue and protect OA through decreasing Wnt/β-catenin signaling, and increasing Hippo/Yap signaling and Tgfβ/Smad2/3 signaling in OA articular cartilage, indicating that local Cbfb overexpression could be an effective strategy for treatment of OA.

Site-specific elastic and viscoelastic biomechanical properties of healthy and osteoarthritic human knee joint articular cartilage

Articular cartilage exhibits site-specific biomechanical properties. However, no study has comprehensively characterized site-specific cartilage properties from the same knee joints at different stages of osteoarthritis (OA).Cylindrical osteochondral explants (n = 381) were harvested from donor-matched lateral and medial tibia, lateral and medial femur, patella, and trochlea of cadaveric knees (N = 17). Indentation test was used to measure the elastic and viscoelastic mechanical properties of the samples, and Osteoarthritis Research Society International (OARSI) grading system was used to categorize the samples into normal (OARSI 0–1), early OA (OARSI 2–3), and advanced OA (OARSI 4–5) groups.OA-related changes in cartilage mechanical properties were site-specific. In the lateral and medial tibia and trochlea sites, equilibrium, instantaneous and dynamic moduli were higher (p < 0.001) in normal tissue than in early and advanced OA tissue. In lateral and medial femur, equilibrium, instantaneous and dynamic moduli were smaller in advanced OA, but not in early OA, than in normal tissue. The phase difference (0.1–0.25 Hz) between stress and strain was significantly smaller (p < 0.05) in advanced OA than in normal tissue across all sites except medial tibia.Our results indicated that in contrast to femoral and patellar cartilage, equilibrium, instantaneous and dynamic moduli of the tibia and trochlear cartilage decreased in early OA. These may suggest that the tibia and trochlear cartilage degrades faster than the femoral and patellar cartilage. The information is relevant for developing site-specific computational models and engineered cartilage constructs.

Glycosphingolipids in Osteoarthritis and Cartilage-Regeneration Therapy: Mechanisms and Therapeutic Prospects Based on a Narrative Review of the Literature.

Glycosphingolipids (GSLs), a subtype of glycolipids containing sphingosine, are critical components of vertebrate plasma membranes, playing a pivotal role in cellular signaling and interactions. In human articular cartilage in osteoarthritis (OA), GSL expression is known notably to decrease. This review focuses on the roles of gangliosides, a specific type of GSL, in cartilage degeneration and regeneration, emphasizing their regulatory function in signal transduction. The expression of gangliosides, whether endogenous or augmented exogenously, is regulated at the enzymatic level, targeting specific glycosyltransferases. This regulation has significant implications for the composition of cell-surface gangliosides and their impact on signal transduction in chondrocytes and progenitor cells. Different levels of ganglioside expression can influence signaling pathways in various ways, potentially affecting cell properties, including malignancy. Moreover, gene manipulations against gangliosides have been shown to regulate cartilage metabolisms and chondrocyte differentiation in vivo and in vitro. This review highlights the potential of targeting gangliosides in the development of therapeutic strategies for osteoarthritis and cartilage injury and addresses promising directions for future research and treatment.

Preparation of an inclusion complex of nickel-based β-cyclodextrin: Characterization and accelerating the osteoarthritis articular cartilage repair

Abstract Osteoarthritis is caused by the cartilage destruction of the bones of the joint surfaces and structures that produce synovium fluid. Osteoarthritis treatment includes the use of surgical methods and non-surgical or maintenance treatments including knee orthoses, medical insoles with external edges, use of physiotherapy techniques, exercise, weight loss in obese people, and teaching the principles of joint care. The main goal of treatment in osteoarthritis of the knee is to ameliorate physical function, decrease pain, and reduce the progression of the disease, through correcting the knee alignment and reducing the varus torque. Previous studies have indicated that medicinal plants and herbal nanoparticles (NPs) have the best anti-inflammatory effects. β-Cyclodextrin is a cyclic carbohydrate molecule that is used as a host to prepare inclusion complexes. In this study, the synthesis of nickel NPs is based on β-cyclodextrin (NiBCD NPs) for accelerating the osteoarthritis articular cartilage repair. The FT-IR and XRD techniques confirmed the formula of NiO for the NiBCD NPs. The FE-SEM imaging shows a non-spherical structure for NiBCD NPs with a size of less than 100 nm. In EDX, the signals at the energy levels of 8.3, 7.5, and 0.87 keV are assigned for the electron migration of Ni Kβ, Ni Kα, and Ni Lα. Furthermore, the signals for the elements of oxygen and carbon of BCD appeared at 0.52 and 0.28 keV. The effectiveness of NiBCD NPs in promoting chondrogenesis was examined in orthopedic experiments using primary cultured chondrocytes. Subsequently, we determined the functional restoration following NiBCD NPs’ transplantation in a knee osteoarthritis articular cartilage injury model. We conducted histological, PCR, and Western blot assays. In the immunological analysis, the levels of MMPs, IL-1β, TNF-α, and p-p65 expression were found to be reduced by NiBCD NPs. This reduction may be attributed to the regulation of cellular redox homeostasis through Nrf2. Furthermore, our findings demonstrated the positive impact of NiBCD NPs on stimulating chondrogenesis in vitro. Notably, the NiBCD NPs’ application accelerated the recovery of injury-induced dysfunction. Additionally, the presence of NiBCD NPs at the injury site suppressed abnormal fibrosis and angiogenesis. The histological assay revealed the chondrocytes’ proliferation and increased cartilage matrix synthesis in the NiBCD NPs’ presence.

Metformin inhibits knee osteoarthritis induced by type 2 diabetes mellitus in rats: S100A8/9 and S100A12 as players and therapeutic targets

Abstract Type 2 diabetes mellitus (T2DM) is a significant risk factor for osteoarthritis (OA), and metformin, as the main therapeutic drug for T2DM, has shown positive effects on OA without a clear mechanism. This study aimed to explore the protective effects and mechanisms of oral metformin in T2DM-induced OA. We identified differentially expressed genes, using the GSE117999 and GSE98918 datasets, and protein–protein interaction networks were analyzed using the MCODE algorithm in cytospace to finalize the OA hub genes (S100A8, S100A9, and S100A12). To validate whether S100A8, S100A9, and S100A12 are potential targets of action for OA, we randomly divided 40 SD rats into a control group (CG, n = 10) and a T2DM group (n = 30). We modeled rats in the T2DM group with streptozotocin (35 mg/kg, i.p.) and a high carbohydrate and fat diet. Finally, 20 were randomly selected and divided into the T2DM group (n = 10) and the treated group (Met + T2DM, n = 10), and the treated group was given Met (180 mg/kg/day) by gavage for 8 weeks. We subsequently used histological assessment to show that oral metformin mitigated the development of T2DM-associated OA as indicated by the OA Research Society International score and articular cartilage thickness, and immunohistochemistry also confirmed that metformin significantly reduced the expression of S100A8, S100A9, and S100A12 in the knee joints of OA rats. In conclusion, metformin demonstrated a protective effect against OA in T2DM-induced rats, slowing knee OA progression by inhibiting S100A8, S100A9, and S100A12 expression. These findings suggest potential biological targets for future OA treatments.

Study on the mechanism of cross-linked hyaluronic acid-dexamethasone hydrogelin post-traumatic osteoarthritis

Objective: To explore the mechanism of cross-linked hyaluronic acid-dexamethasone hydrogel (cHA-Dex) in inhibiting chondrocyte apoptosis and alleviating early post-traumatic osteoarthritis (PTOA). Methods: To generate PTOA model, anterior cruciate ligament transection (ACLT)was performed on SD rats (n=70), and the sham surgery group (n=70) was set as control. The changes in inflammatory indicators such as interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), matrix metalloproteinase-3 (MMP-3), and matrix metalloproteinase-13 (MMP-13) in the joint lavage fluid were measured at different time points (1-14 days, 5 rats at each time point) after surgery. The cHA-Dex (0.5 mg/ml) hydrogel (experimental group, n=70) and ordinary low-molecular-weight hyaluronic acid (HA) hydrogel premixed with Dex, that was, HA-Dex (0.5 mg/ml) hydrogel (control group, n=70) were injected into the joint cavity of PTOA rats, and the release amount and cumulative release amount of Dex in the joint fluid of rats at each time point(1-14 days, 5 rats at each time point) were detected to reveal the release mechanism of cHA-Dex hydrogel. The cartilage of knee joint of patients with osteoarthritis (OA) who underwent knee arthroplasty in the Second Hospital of Shanxi Medical University from January 2020 to December 2022 was taken for in vitro tissue block culture (Outbridge score=1 or 2,n=18). After the cartilage tissue block was treated with cHA-Dex hydrogel premixed with 0.1, 0.2, and 0.5 mg/ml Dex, the mRNA expression levels of IL-1β, IL-6, TNF-α, MMP-3, and MMP-13 in the articular cartilage tissue block were detected. OA chondrocytes were isolated from cartilage samples using enzymatic hydrolysis and cultured in vitro (n=18). Chondrocytes were divided into 4 groups: saline, cHA hydrogel, Dex (0.5 mg/ml), and cHA-Dex (0.5 mg/ml) hydrogel group. The effects of different interventions on chondrocyte proliferation and apoptosis were tested. Results: The Osteoarthritis Research Society International (OARSI) score of safranine O-solid green staining in PTOA group was 3.34±0.35, and it was 1.17±0.21 in Sham group(P=0.010). The Meachim score of knee joint osteophytes in PTOA rats was significantly higher than that in the Sham group (2.66±0.41 vs 0.22±0.17, P=0.010), indicating PTOA model in rat was established successfully. The cHA-Dex hydrogel, which corresponded to the peak changes of inflammatory factors in the joints of PTOA rats in the early stage, was also released in the early stage and sustained-released in the late stage. After the OA articular cartilage tissue block was treated with cHA-Dex hydrogel premixed with 0.1, 0.2, and 0.5 mg/ml Dex, the mRNA expression levels of IL-1 β, IL-6, TNF-α, MMP-3, and MMP-13 in the tissue block were reduced significantly (all P<0.05) and in a dose-dependent manner. Compared with Dex (0.5 mg/ml) alone group, the apoptosis rate of cHA-Dex (0.5 mg/ml) hydrogel group was significantly reduced (0.60±0.07 vs 6.63±0.98, P=0.010).Compared with the normal saline or the cHA hydrogel alone group, the cHA-Dex (0.5 mg/ml) hydrogel group had significant cell proliferation, and the difference at each time point were all significant statistically (all P<0.05). Conclusion: For the early inflammation of PTOA, cHA-Dex hydrogel can not only inhibit cartilage inflammation, but also reverse the increased apoptosis and decreased proliferation rate of chondrocytes caused by Dex, and finally alleviate the progress of PTOA by releasing Dex.

Dual Enzyme Sequential Digestion Protocol for Isolation of Human Primary Chondrocytes From the Articular Cartilage Derived From Knee Osteoarthritis Patients

Introduction: Majority of available protocols for isolation of chondrocytes from articular cartilage tissue rely on the enzymatic digestion of the tissue by collagenase type 2. The yield of chondrocytes in such protocols is low. Herein, we designed a novel indigenous sequential digestion by dual enzyme Pronase and Collagenase Type 1 for isolating human Chondrocytes from articular cartilage. Methods: Articular cartilage of Osteoarthritis (OA) patients undergoing total knee replacement were collected for the isolation of chondrocyte cells and subjected to sequential digestion by Pronase for three hours followed by Collagenase 1 overnight. Pellet of cells collected after digestion was plated on culture flask in 5% CO2 incubator. Results: From day three onwards, round to elongated cells adhered to the flask were visible which developed into elongated cell population of homogenous morphology, expressed Aggrecan (Agg), Collagen 2a (Col2a) and SRY-box transcription factor (Sox9) and had chondrogenic differentiation similar to a commercially available healthy chondrocyte. These cells were negative for Alizarin red stain, thus confirming the purity of chondrocytes. Conclusion: We have successfully established a sequential dual enzyme digestion-based culture technique for isolating the human chondrocytes from the articular cartilage biopsy derived from OA knee joints.

Inhibition of HOXD11 promotes cartilage degradation and induces osteoarthritis development

The 5′-HOXD genes are important for chondrogenesis in vertebrates, but their roles in osteoarthritis (OA) are still ambiguous. In our study, 5′-HOXD genes involvement contributing to cartilage degradation and OA was investigated. In bioinformatics analysis of 5′-HOXD genes, we obtained the GSE169077 data set related to OA in the GEO and analyzed DEGs using the GEO2R tool attached to the GEO. Then, we screened the mRNA levels of 5′-HOXD genes by quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR). We discovered that OA chondrocyte proliferation was inhibited, and apoptosis was increased. Moreover, it was discovered that SOX9 and COL2A1 were downregulated at mRNA and protein levels, while matrix metalloproteinases (MMPs) and a disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTSs) were upregulated. According to the results of differentially expressed genes (DEGs) and qRT-PCR, we evaluated the protein level of HOXD11 and found that the expression of HOXD11 was downregulated, reversed to MMPs and ADAMTSs but consistent with the cartilage-specific factors, SOX9 and COL2A1. In the lentivirus transfection experiments, HOXD11 overexpression reversed the effects in OA chondrocytes. In human OA articular cartilage, aberrant subchondral bone was formed in hematoxylin–eosin (H&E) and Safranin O and fast green (SOFG) staining results. Furthermore, according to immunohistochemistry findings, SOX9 and HOXD11 expression was inhibited. The results of this study established that HOXD11 was downregulated in OA cartilage and that overexpression of HOXD11 could prevent cartilage degradation in OA.

The CREB1 inhibitor 666-15 maintains cartilage homeostasis and mitigates osteoarthritis progression.

cAMP response element binding protein (CREB1) is involved in the progression of osteoarthritis (OA). However, available findings about the role of CREB1 in OA are inconsistent. 666-15 is a potent and selective CREB1 inhibitor, but its role in OA is unclear. This study aimed to investigate the precise role of CREB1 in OA, and whether 666-15 exerts an anti-OA effect. CREB1 activity and expression of a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) in cells and tissues were measured by immunoblotting and immunohistochemical (IHC) staining. The effect of 666-15 on chondrocyte viability and apoptosis was examined by cell counting kit-8 (CCK-8) assay, JC-10, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) staining. The effect of 666-15 on the microstructure of subchondral bone, and the synthesis and catabolism of cartilage, in anterior cruciate ligament transection mice were detected by micro-CT, safranin O and fast green (S/F), immunohistochemical staining, and enzyme-linked immunosorbent assay (ELISA). CREB1 was hyperactive in osteoarthritic articular cartilage, interleukin (IL)-1β-treated cartilage explants, and IL-1β- or carbonyl cyanide 3-chlorophenylhydrazone (CCCP)-treated chondrocytes. 666-15 enhanced cell viability of OA-like chondrocytes and alleviated IL-1β- or CCCP-induced chondrocyte injury through inhibition of mitochondrial dysfunction-associated apoptosis. Moreover, inhibition of CREB1 by 666-15 suppressed expression of ADAMTS4. Additionally, 666-15 alleviated joint degeneration in an ACLT mouse model. Hyperactive CREB1 played a critical role in OA development, and 666-15 exerted anti-IL-1β or anti-CCCP effects in vitro as well as joint-protective effects in vivo. 666-15 may therefore be used as a promising anti-OA drug.

Identification of autophagy-related genes in osteoarthritis articular cartilage and their roles in immune infiltration.

Autophagy plays a critical role in the progression of osteoarthritis (OA), mainly by regulating inflammatory and immune responses. However, the underlying mechanisms remain unclear. This study aimed to investigate the potential relevance of autophagy-related genes (ARGs) associated with infiltrating immune cells in OA. GSE114007, GSE169077, and ARGs were obtained from the Gene Expression Omnibus (GEO) database and the Human Autophagy database. R software was used to identify the differentially expressed autophagy-related genes (DEARGs) in OA. Functional enrichment and protein-protein interaction (PPI) analyses were performed to explore the role of DEARGs in OA cartilage, and then Cytoscape was utilized to screen hub ARGs. Single-sample gene set enrichment analysis (ssGSEA) was used to conduct immune infiltration analysis and evaluate the potential correlation of key ARGs and immune cell infiltration. Then, the expression levels of hub ARGs in OA were further verified by the GSE169077 and qRT-PCR. Finally, Western blotting and immunohistochemistry were used to validate the final hub ARGs. A total of 24 downregulated genes and five upregulated genes were identified, and these genes were enriched in autophagy, mitophagy, and inflammation-related pathways. The intersection results identified nine hub genes, namely, CDKN1A, DDIT3, FOS, VEGFA, RELA, MAP1LC3B, MYC, HSPA5, and HSPA8. GSE169077 and qRT-PCR validation results showed that only four genes, CDKN1A, DDT3, MAP1LC3B, and MYC, were consistent with the bioinformatics analysis results. Western blotting and immunohistochemical (IHC) showed that the expression of these four genes was significantly downregulated in the OA group, which is consistent with the qPCR results. Immune infiltration correlation analysis indicated that DDIT3 was negatively correlated with immature dendritic cells in OA, and FOS was positively correlated with eosinophils. CDKN1A, DDIT3, MAP1LC3B, and MYC were identified as ARGs that were closely associated with immune infiltration in OA cartilage. Among them, DDIT3 showed a strong negative correlation with immature dendritic cells. This study found that the interaction between ARGs and immune cell infiltration may play a crucial role in the pathogenesis of OA; however, the specific interaction mechanism needs further research to be clarified. This study provides new insights to further understand the molecular mechanisms of immunity involved in the process of OA by autophagy.

Human osteoarthritic articular cartilage stem cells suppress osteoclasts and improve subchondral bone remodeling in experimental knee osteoarthritis partially by releasing TNFAIP3

BackgroundThough articular cartilage stem cell (ACSC)-based therapies have been demonstrated to be a promising option in the treatment of diseased joints, the wide variety of cell isolation, the unknown therapeutic targets, and the incomplete understanding of the interactions of ACSCs with diseased microenvironments have limited the applications of ACSCs.MethodsIn this study, the human ACSCs have been isolated from osteoarthritic articular cartilage by advantage of selection of anatomical location, the migratory property of the cells, and the combination of traumatic injury, mechanical stimuli and enzymatic digestion. The protective effects of ACSC infusion into osteoarthritis (OA) rat knees on osteochondral tissues were evaluated using micro-CT and pathological analyses. Moreover, the regulation of ACSCs on osteoarthritic osteoclasts and the underlying mechanisms in vivo and in vitro were explored by RNA-sequencing, pathological analyses and functional gain and loss experiments. The one-way ANOVA was used in multiple group data analysis.ResultsThe ACSCs showed typical stem cell-like characteristics including colony formation and committed osteo-chondrogenic capacity. In addition, intra-articular injection into knee joints yielded significant improvement on the abnormal subchondral bone remodeling of osteoarthritic rats. Bioinformatic and functional analysis showed that ACSCs suppressed osteoarthritic osteoclasts formation, and inflammatory joint microenvironment augmented the inhibitory effects. Further explorations demonstrated that ACSC-derived tumor necrosis factor alpha-induced protein 3 (TNFAIP3) remarkably contributed to the inhibition on osteoarhtritic osteoclasts and the improvement of abnormal subchondral bone remodeling.ConclusionIn summary, we have reported an easy and reproducible human ACSC isolation strategy and revealed their effects on subchondral bone remodeling in OA rats by releasing TNFAIP3 and suppressing osteoclasts in a diseased microenvironment responsive manner.Graphical abstract

MiR-335-5p inhibits endochondral ossification by directly targeting SP1 in TMJ OA.

During the development of temporomandibular joint osteoarthritis, endochondral ossification is compromised which leads to condylar degeneration; miR-335-5p in endochondral ossification in osteoarthritic condylar cartilage tissue remains unclear. Up-regulated microRNA and its target gene were searched for endochondral ossification in osteoarthritis articular cartilage. The effect of increased or decreased miR-335-5p on endochondral ossification was evaluated by transfecting miR-335-5p mimics or miR-335-5p inhibitor in vitro in chondrocytes C28/I2. Finally, we injected the temporomandibular joint of rats intra-articularly with agomiR-335 in a unilateral anterior crossbite rat model to determine the in vivo regulation of miR-335. After the onset of temporomandibular joint osteoarthritis, miR-335-5p levels were gradually up-regulated, whereas endochondral ossification-related genes were down-regulated in condylar cartilage specimens. Our results showed that miR-335 inhibited endochondral ossification after administration of a miR-335 antagonist into the temporomandibular joint articular cavity of a unilateral anterior crossbite rat model. AgomiR-335, a miR-335 agonist, inhibited matrix mineralization in fibrocartilage stem cells in vitro and then miR-335-5p negatively regulated chondrocyte activity by directly targeting SP1 via promoting transforming growth factor-β/Smad signalling. miR-335-5p can significantly inhibit endochondral ossification; therefore, its inhibition may be beneficial for the treatment of temporomandibular joint osteoarthritis.

Impaired autophagy contributes to the aggravated deterioration of osteoarthritis articular cartilage by peroxisome proliferator-activated receptor α deficiency, associated with decreased ERK and Akt activation

BackgroundAlthough the chondroprotection of peroxisome proliferator-activated receptor α (PPARα) activation against osteoarthritis (OA) has been revealed, the regulatory mechanism of PPARα deficiency to aggravate osteoarthritic cartilage deterioration remains unclear. Here, we aimed to investigate whether and how autophagy is involved in OA pathological progression.MethodsModel of experimental OA was established using destabilization of the medial meniscus in PPARα-KO 129S4/SvJae male mice, followed by histopathological detection of articular cartilage and immunohistochemistry detection of extracellular matrix (ECM) or autophagy-related signal molecules. Meanwhile, human OA chondrocytes obtained from total knee replacement surgery patients with OA were cultured with the pretreatment of IL-1β, followed with the treatment of PPARα agonist WY14643 and the detection of related signal molecules.ResultsPPARα deficiency aggravated cartilage damage with decreased LC3B level in combination with an increase in P62 level, accompanied with reduced p-Akt and p-ERK levels in PPARα-KO mouse model of experimental OA. On the contrary, PPARα activation by WY14643 promoted ECM synthesis in IL-1β-treated human OA chondrocytes, accompanied with increased LC3B-II/I ratio and Beclin 1 level and decreased P62 and Bcl2 levels. Meanwhile, it was observed that activated ERK and Akt by PPARα activation contributed to the enhancement of autophagy and ECM synthesis in human OA chondrocytes.ConclusionsImpaired autophagy contributed to the aggravated deterioration of osteoarthritis articular cartilage by PPARα deficiency associated with the suppression of ERK and Akt, with an implication that triggering PPARα activation ought to be a potential promising therapeutic target for OA therapy.

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

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

Role of Syndecan-4 in the Inhibition of Articular Cartilage Degeneration in Osteoarthritis.

Despite its widespread existence, there are relatively few drugs that can inhibit the progression of osteoarthritis (OA). Syndecan-4 (SDC4) is a transmembrane heparan sulfate proteoglycan that modulates cellular interactions with the extracellular matrix. Upregulated SDC4 expression in articular cartilage chondrocytes correlates with OA progression. In the present study, we treated osteoarthritic cartilage with SDC4 to elucidate its role in the disease's pathology. In this in vitro study, we used real-time polymerase chain reaction (PCR) to investigate the effects of SDC4 on anabolic and catabolic factors in cultured chondrocytes. In the in vivo study, we investigated the effect of intra-articular injection of SDC4 into the knee joints of an OA mouse model. In vitro, SDC4 upregulated the expression of tissue inhibitor of metalloproteinase (TIMP)-3 and downregulated the expression of matrix metalloproteinase (MMP)-13 and disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 in chondrocytes. Injection of SDC4 into the knee joints of OA model mice prevented articular cartilage degeneration 6 and 8 weeks postoperatively. Immunohistochemical analysis 8 weeks after SDC4 injection into the knee joint revealed decreased ADAMTS-5 expression and increased TIMP-3 expression. The results of this study suggest that the treatment of osteoarthritic articular cartilage with SDC4 inhibits cartilage degeneration.

FINITE ELEMENT ANALYSIS OF HUMAN AND ARTIFICIAL ARTICULAR CARTILAGE

Joint diseases, such as osteoarthritis, induce pain and loss of mobility to millions of people around the world. Current clinical methods for the diagnosis of osteoarthritis include X-ray, magnetic resonance imaging, and arthroscopy. These methods may be insensitive to the earliest signs of osteoarthritis. This study investigates a new procedure that was developed and validated numerically for use in the evaluation of cartilage quality. This finite element model of the human articular cartilage could be helpful in providing insight into mechanisms of injury, effects of treatment, and the role of mechanical factors in degenerativeconditions, this three-dimensional finite element model is a useful tool for understanding of the stress distributions within articular cartilage in response to external loads and investigating both the prevention of injury and the pathological degeneration of the joints.In this study, 21 models were analysed by using ANSYS workbench v12.1: four normal articular cartilage models (distal femur, patella, medial and lateral tibia). A redesign to the distal femur model was done to get osteoarthritis articular cartilage (simple and deep) seven models by making partial cut without affecting the subchondral bone, and full cut with part of the subchondral bone in different diameters. Finally a treatment done by replacing the defective parts with artificial articular cartilages with different types of treatment. The finite element analysis studied depending on a Von Mises criteria and total deformation in different activities. The results shows that Autologous Chondrocyte Implementation is the best treatment way and it is close by 87.50% to normal cartilage. This procedure can be used as a diagnostic procedure for osteoarthritic patients and to choose the best treatment options.

Osteoarthritis related epigenetic variations in miRNA expression and DNA methylation

Osteoarthritis (OA) is chronic arthritis characterized by articular cartilage degradation. However, a comprehensive regulatory network for OA-related microRNAs and DNA methylation modifications has yet to be established. Thus, we aimed to identify epigenetic changes in microRNAs and DNA methylation and establish the regulatory network between miRNAs and DNA methylation. The mRNA, miRNA, and DNA methylation expression profiles of healthy or osteoarthritis articular cartilage samples were downloaded from Gene Expression Omnibus (GEO) database, including GSE169077, GSE175961, and GSE162484. The differentially expressed genes (DEGs), differentially expressed miRNAs (DEMs), and differentially methylated genes (DMGs) were analyzed by the online tool GEO2R. DAVID and STRING databases were applied for functional enrichment analysis and protein-protein interaction (PPI) network. Potential therapeutic compounds for the treatment of OA were identified by Connectivity map (CMap) analysis. A total of 1424 up-regulated DEGs, 1558 down-regulated DEGs, 5 DEMs with high expression, 6 DEMs with low expression, 1436 hypermethylated genes, and 455 hypomethylated genes were selected. A total of 136 up-regulated and 65 downregulated genes were identified by overlapping DEGs and DEMs predicted target genes which were enriched in apoptosis and circadian rhythm. A total of 39 hypomethylated and 117 hypermethylated genes were obtained by overlapping DEGs and DMGs, which were associated with ECM receptor interactions and cellular metabolic processes, cell connectivity, and transcription. Moreover, The PPI network showed COL5A1, COL6A1, LAMA4, T3GAL6A, and TP53 were the most connective proteins. After overlapping of DEGs, DMGs and DEMs predicted targeted genes, 4 up-regulated genes and 11 down-regulated genes were enriched in the Axon guidance pathway. The top ten genes ranked by PPI network connectivity degree in the up-regulated and downregulated overlapping genes of DEGs and DMGs were further analyzed by the CMap database, and nine chemicals were predicted as potential drugs for the treatment of OA. In conclusion, TP53, COL5A1, COL6A1, LAMA4, and ST3GAL6 may play important roles in OA genesis and development.

Cartilaginous glycoprotein‑39 as laboratory marker of dynamics of course and effectiveness of treatment in patients with osteoarthritis

Backgrounds. Osteoarthritis (OA) occupies the first rank among diseases of the musculoskeletal system and accounts for 18.4% of the structure of this pathology. Currently, OA is considered as a disease in which there is a violation of the processes of remodeling (degradation and synthesis) of cartilage tissues, subchondral bone, joint capsule, tendon-ligamentous and muscular apparatus. An increased concentration of cartilage glycoprotein-39 (CGP-39) in the circulation is associated with inflammatory diseases and the processes of active tissue restructuring. To date, CGP-39 is a marker of chondrocyte activation and a sign of progressive OA, which determines its clinical significance. Along with this, the role of this biomarker in evaluating the effectiveness of anti-inflammatory therapy is relevant.The purpose. Comparative assessment of the quantitative content of CGP-39 in patients with knee joint OA, depending on the severity of inflammatory changes in the joints, the dynamics of the disease and the effectiveness of therapy.Methods. The study included 36 patients with OA and 30 practically healthy volunteers, identical in gender and age to the examined patients. The mean age of patients was 64 years, the average duration of the disease was 6 years. The diagnosis was determined according to the diagnostic criteria of the American College of Rheumatology (ACR, 1991). Along with the generally accepted clinical, laboratory and instrumental diagnosis of OA, in all patients and in control persons the levels of circulating CGP-39 were measured by enzyme immunoassay (ELISA). All examined patients received Ambene® Bio for 20 days, followed by repeated determination of the level of CGP-39. For statistical processing of the obtained data, we have used the programs Microsoft Excel 2007 and Statistica 10.0. Using the application program KRelRisk 1.1, the relative risk indicator for the studied factor was determined.Results. In the blood of patients with OA, a significant increase in the levels of CGP-39 has been found in comparison with the group of healthy individuals, which indicates increased degradation of articular cartilage in OA. As the severity of knee OA increased, the concentration of CGP-39 in the blood serum significantly raised, including in comparison with the control group. Reliable direct correlations were found between CGP-39 and clinical indices of knee OA severity. The content of CGP-39 in serum in patients with stage III radiological OA was significantly higher than in patients with stage I–II. The highest level of CGP-39 in patients with OA is associated with the presence of synovitis. TThe level of CGP-39 significantly decreased in patients with knee OA after a course of Ambene® Bio therapy, which confirms the participation of CGP-39 in inflammation on the one hand and the possibility of its use as an indicator of the effectiveness of anti-inflammatory therapy on the other hand.Conclusions. CGP-39 as a marker of inflammation and degradation of articular cartilage reflects the severity of the course of OA. The investigation of this biomarker is useful not only for diagnostic purposes, but also to assess the response to anti-inflammatory treatment in patients with knee OA.

Visible and Near-Infrared Spectroscopy Enables Differentiation of Normal and Early Osteoarthritic Human Knee Joint Articular Cartilage

Osteoarthritis degenerates cartilage and impairs joint function. Early intervention opportunities are missed as current diagnostic methods are insensitive to early tissue degeneration. We investigated the capability of visible light-near-infrared spectroscopy (Vis-NIRS) to differentiate normal human cartilage from early osteoarthritic one. Vis-NIRS spectra, biomechanical properties and the state of osteoarthritis (OARSI grade) were quantified from osteochondral samples harvested from different anatomical sites of human cadaver knees. Two support vector machines (SVM) classifiers were developed based on the Vis-NIRS spectra and OARSI scores. The first classifier was designed to distinguish normal (OARSI: 0–1) from general osteoarthritic cartilage (OARSI: 2–5) to check the general suitability of the approach yielding an average accuracy of 75% (AUC = 0.77). Then, the second classifier was designed to distinguish normal from early osteoarthritic cartilage (OARSI: 2–3) yielding an average accuracy of 71% (AUC = 0.73). Important wavelength regions for differentiating normal from early osteoarthritic cartilage were related to collagen organization (wavelength region: 400–600 nm), collagen content (1000–1300 nm) and proteoglycan content (1600–1850 nm). The findings suggest that Vis-NIRS allows objective differentiation of normal and early osteoarthritic tissue, e.g., during arthroscopic repair surgeries.

Label-free, multi-parametric assessments of cell metabolism and matrix remodeling within human and early-stage murine osteoarthritic articular cartilage

Osteoarthritis (OA) is characterized by the progressive deterioration of articular cartilage, involving complicated cell-matrix interactions. Systematic investigations of dynamic cellular and matrix changes during OA progression are lacking. In this study, we use label-free two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging to assess cellular and extracellular matrix features of murine articular cartilage during several time points at early stages of OA development following destabilization of medial meniscus surgery. We detect significant changes in the organization of collagen fibers and crosslink-associated fluorescence of the superficial zone as early as one week following surgery. Such changes become significant within the deeper transitional and radial zones at later time-points, highlighting the importance of high spatial resolution. Cellular metabolic changes exhibit a highly dynamic behavior, and indicate metabolic reprogramming from enhanced oxidative phosphorylation to enhanced glycolysis or fatty acid oxidation over the ten-week observation period. The optical metabolic and matrix changes detected within this mouse model are consistent with differences identified in excised human cartilage specimens from OA and healthy cartilage specimens. Thus, our studies reveal important cell-matrix interactions at the onset of OA that may enable improved understanding of OA development and identification of new potential treatment targets.