Osteoarthritic Articular Cartilage Research Articles

Green synthesis, chemical characterization, anti-osteoarthritis properties of Cu nanoparticles

In our latest study, we assessed the effectiveness of a green copper nanoparticle formulation containing curcumin on the chick’s knee joint injury. The NPs were characterized using FE-SEM, TEM, XRD, and EDX techniques. The primary cultured chondrocytes were utilized to check the nanoparticles effectiveness on chondrogenesis in the orthopedic experiments. Subsequently, we assessed the functional restoration post copper nanoparticles transplantation using an osteoarthritis articular model in the knee. Histological, PCR, and western blot analyses were conducted to investigate the underlying mechanisms. The XRD results exhibited the CuO formula for CuNPs and a spherical shape with a size <60 nm was obtained for the NPs from the TEM and FE-SEM images. The presence signals in EDX diagram including signals at 0.22 keV for C Lα, 0.51 keV for O Lα, and 0.93 keV for Cu Lα approve the formation of Cu NPs. The use of nanoparticles effectively inhibited abnormal fibrosis and angiogenesis at the injury site. The nanoparticles presence in the histological test resulted in rised synthesis of cartilage matrix and proliferation of chondrocytes, while also reducing the formation of abnormal vasculature. The study unveiled that the use of nanoparticles accelerated the healing process following an injury after three weeks. Furthermore, we highlighted the beneficial effects of copper nanoparticles in promoting cartilage formation in a laboratory setting. In the immunological domain, the copper nanoparticles demonstrated a decrease in the quantities of IL-1β, TNF-α, p-p65, and MMPs expression. This phenomenon could potentially be attributed to the cellular redox homeostasis modulation associated with the Nrf2 pathway. In conclusion, the results of this research confirmed the positive efficacy of copper nanoparticles containing curcumin in accelerating the osteoarthritis articular cartilage repair.

The Hounsfield Unit Values of Talar Subchondral Bone Predict Articular Cartilage Degeneration in Patients With Ankle Osteoarthritis.

Therapeutic strategies for ankle osteoarthritis (OA) are determined based on OA staging, alignment, and articular cartilage conditions. However, it is difficult to evaluate the degeneration of the remaining cartilage using imaging modalities. Subchondral bone plays a crucial role in maintaining cartilage homeostasis. Measurement of local Hounsfield unit (HU) values allows for the quantitative assessment of small changes in the subchondral bone. This study aimed to evaluate a relationship between the HU values of the subchondral bone and the histologic findings of articular cartilage in ankle OA. The talar articular surface was harvested from 14 ankles during arthroplasty. The talus was divided into anterior, middle, and posterior parts, and histologic specimens were prepared. Safranin O staining was performed and histologic findings were evaluated using the modified Mankin score. The regions of interest (ROIs) were set in the medial, central, and lateral regions of the specimens and computed tomography (CT) images, and the relationship between the HU values and histologic findings was analyzed. As OA progressed, cartilage defects increased. In conjunction with cartilage degeneration, the subchondral bone plate thickened, and the HU values increased. The HU value significantly and positively correlated with the modified Mankin score (r = 0.756), subchondral bone thickness (r = 0.674, P < .01), and trabecular bone area (r = 0.637). The cutoff HU values were 594 (sensitivity, 0.813; specificity, 0.944) for 3 points and 727 (sensitivity, 0.929; specificity, 0.782) for 11 points on the modified Mankin score. Significant correlations between HU values and cartilage degeneration in ankle OA were noted. Measuring HU values on CT images can be useful for evaluating the joint surface condition, including histologic findings of the remaining cartilage.

Therapeutic framework nucleic acid complexes targeting oxidative stress and pyroptosis for the treatment of osteoarthritis

Osteoarthritis (OA) is one of the most prevalent joint diseases and severely affects the quality of life in the elderly population. However, there are currently no effective prevention or treatment options for OA. Oxidative stress and pyroptosis play significant roles in the development and progression of OA. To address this issue, we have developed a novel therapeutic approach for OA that targets oxidative stress and pyroptosis. We synthesized tetrahedral framework nucleic acid (tFNAs) to form framework nucleic acid complexes (TNCs), which facilitate the delivery of the naturally occurring polymethoxyflavonoid nobiletin (Nob) to chondrocytes. TNC has demonstrated favorable bioavailability, stability, and biosafety for delivering Nob. Both in vitro and in vivo experiments have shown that TNC can alleviate OA and protect articular cartilage from damage by eliminating oxidative stress, inhibiting pyroptosis, and restoring the extracellular matrix anabolic metabolism of chondrocytes. These findings suggest that TNC has significant potential in the treatment of OA and cartilage injury.

CircRELL1 promotes osteoarthritis progression by regulating miR-200c-3p

BackgroundThere is a growing body of evidence indicating a potential association between circular RNA and the pathogenesis of human osteoarthritis (OA). Nevertheless, the precise extent of their involvement in OA remains largely unexplored. Hence, the objective of this investigation is to elucidate the function of Circular (Circ) RELL1 in the context of OA. Methods24 OA tissue samples and 11 normal tissue samples were collected. The inflammatory OA-like conditions were established by Destabilized Medial Meniscus (DMM) operation in mice and LPS-induced C28/I2 cells. OA severity and articular cartilage degradation were assessed by Safranin-O staining, hematoxylin-eosin (H&E) staining, and International Society for Osteoarthritis Research (OARSI) criteria. CircRELL1, miR-200c-3p, and TCF4 were measured by RT-qPCR and Immunoblot. The cell viability and apoptosis rate were measured by MTT and flow cytometry, respectively. The levels of cytokines interleukin (IL)-1β, IL-6, and TNF-α were determined by ELISA. Apoptosis-associated proteins (cleaved caspase-3, Bax, and Bcl-2) and extracellular matrix (ECM) degradation-associated proteins (MMP13, collagen II, and Aggrecan) were detected by Immunoblot. The interaction between miR-200c-3p and circRELL1 or TCF4 was verified by dual luciferase reporter assay and RIP assay. ResultsCircRELL1 expression was upregulated in OA patients, and the results were consistent in DMM mice and LPS-treated C28/I2 cells. Silencing circRELL1 improved cartilage injury caused by DMM and contributed to a lower OARSI score. Silencing CircRELL1 increased the activity of OA chondrocytes in vivo and in vitro and inhibited cellular inflammatory responses and ECM degradation. In terms of mechanism, circRELL1 functioned by targeting miR-200c-3p, leading to the suppression of inflammatory factor production, cell apoptosis, and ECM degradation, thus inhibiting the progression of OA. ConclusionCircRELL1 may promote the progression of OA by regulating the miR-200c-3p.

Injectable dECM-enhanced hyaluronic microgels with spatiotemporal release of cartilage-specific molecules to improve osteoarthritic chondrocyte’s function

The interior environment of articular cartilage in osteoarthritis (OA) presents substantial hurdles, leading to the malfunction of chondrocytes and the breakdown of collagen II-enriched hyaline cartilage matrix. Despite this, most clinical treatments primarily provide temporary relief from OA discomfort without arresting OA progression. This study aimed to alleviate OA by developing intra-articular injectable dECM-enhanced hyaluronic (HE) microgels. The HE hydrogel was engineered and shaped into uniformly sized microgels using microfluidics and photopolymerization techniques. These microgels provided a spatiotemporal cascade effect, facilitating the rapid release of growth factors and a slower release of ECM macromolecules and proteins. This process assisted in the recovery of OA chondrocytes’ function, promoting cell proliferation, matrix synthesis, and cartilage-specific gene expression in vitro. It also effectively aided repair of the collagen II-enriched hyaline cartilage and significantly reduced the severity of OA, as demonstrated by radiological observation, gross appearance, histological/immunohistochemical staining, and analysis in an OA rat model in vivo. Collectively, the HE injectable microgels with spatiotemporal release of cartilage-specific molecules have shown promise as a potential candidate for a cell-free OA therapy approach.Graphical

Screening of MMP-13 Inhibitors Using a GelMA-Alginate Interpenetrating Network Hydrogel-Based Model Mimicking Cytokine-Induced Key Features of Osteoarthritis In Vitro.

Osteoarthritis (OA) is a chronic joint disease characterized by irreversible cartilage degradation. Current clinical treatment options lack effective pharmaceutical interventions targeting the disease's root causes. MMP (matrix metalloproteinase) inhibitors represent a new approach to slowing OA progression by addressing cartilage degradation mechanisms. However, very few drugs within this class are in preclinical or clinical trial phases. Hydrogel-based 3D in vitro models have shown promise as preclinical testing platforms due to their resemblance to native extracellular matrix (ECM), abundant availability, and ease of use. Metalloproteinase-13 (MMP-13) is thought to be a major contributor to the degradation of articular cartilage in OA by aggressively breaking down type II collagen. This study focused on testing MMP-13 inhibitors using a GelMA-alginate hydrogel-based OA model induced by cytokines interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α). The results demonstrate a significant inhibition of type II collagen breakdown by measuring C2C concentration using ELISA after treatment with MMP-13 inhibitors. However, inconsistencies in human cartilage explant samples led to inconclusive results. Nonetheless, the study highlights the GelMA-alginate hydrogel-based OA model as an alternative to human-sourced cartilage explants for in vitro drug screening.

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.

Матриксные металлопротеиназы — перспективная терапевтическая мишень для лечения остеоартрита

Osteoarthritis (osteoarthrosis) is one of the most common pathologies of the musculoskeletal system, primarily among the elderly, who present typical clinical symptoms such as joint pain, swelling, stiffness and motion limitation. This can lead to a decrease in physical activity and quality of life, in addition to increasing the socio-economic burden on patients and society as a whole. According to data from 2017, more than 303 million people worldwide suffer from osteoarthritis, which makes this disease socially significant. Consonant to modern concepts, osteoarthritis is a joint disease characterized by cellular stress and degradation of the extracellular matrix resulting from macro/micro-injuries that activate pathological adaptive restorative responses, including pro-inflammatory pathways of the immune system. A long-term research period into the pathogenesis of this disease did not allow the development of treatment and prevention techniques that would completely solve the problem of osteoarthritis high incidence. Currently, a pertinent research area is the investigation of this disease phenotypes with particular emphasis on phenotypes associated with inflammation and connective tissue metabolism. An urgent task is to identify the factors regulating these conditions, namely pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukins 1, 6, 18 and catabolism enzymes — matrix metalloproteinases (MMPs), since they can become potential new therapy targets. Most MMPs, including MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13 and MMP-14, are involved in the metabolism of the extracellular matrix and the associated destruction of articular cartilage in osteoarthritis. This article is devoted to determining the role of inflammatory mediators, MMPs in the osteoarthritis pathogenesis and reviewing new pathogenetically justified treatment options for this disease. KEYWORDS: osteoarthritis, inflammation, matrix metalloproteinases, therapy targets, connective tissue. FOR CITATION: Tyurin A.V., Gaydukova I.Z., Akhiyarova K.E., Galina I.I., Khusainova R.I. Matrix metalloproteinases are a promising therapeutic target for the osteoarthritis treatment. Russian Medical Inquiry. 2024;8(7):400–405 (in Russ.). DOI: 10.32364/2587-6821-2024-8-7-5.

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.