Synergistic targeting of senolytic and senomorphic action with dual-engineered biomimetic macrophage nanovesicles for mitigating osteoarthritis.

Osteoarthritis (OA) is a common joint disease characterized by articular cartilage degeneration, with subchondral bone sclerosis being a key pathological change. However, the mechanism underlying subchondral bone sclerosis remains unclear. Senescent stem cells have been found in OA cartilage, synovium, and subchondral bone. This study aimed to explore the role of senescent bone marrow mesenchymal stem cells (BMSCs) in subchondral bone sclerosis and OA progression. This study utilized two mouse OA models: closed anterior cruciate ligament transection and spontaneous OA. Immunostaining was performed to assess the proportion of senescent cells especially BMSCs in subchondral bone during OA progression. A senescence model of BMSCs was constructed using H₂O₂, and transcriptomic sequencing was conducted to analyze secretory phenotypic changes in senescent BMSCs. In vitro experiments were performed to evaluate the effects of senescent BMSC-derived senescence-associated secretory phenotype (SASP) on osteogenic differentiation and osteogenic marker expression in normal BMSCs. We examined the mRNA expression of core molecules in osteogenesis-related pathways, which were activated by SASP. Additionally, OA mice were administered the senolytic combination of dasatinib and quercetin (D + Q), and its effects on senescent BMSCs, osteocalcin expression, subchondral bone sclerosis, and OA progression were assessed. Immunostaining results showed a significant increase in the proportion of senescent BMSCs in subchondral bone during OA progression. Transcriptomic sequencing revealed that senescent BMSCs acquired the SASP. In vitro experiments demonstrated that conditioned medium from senescent BMSCs (containing SASP) significantly promoted osteogenic differentiation and upregulated the expression of osteogenic markers in normal BMSCs. The SASP significantly upregulated Mapk8 and Mapk14 mRNAs in osteogenesis-related pathways. Administration of D + Q to OA mice effectively cleared senescent BMSCs, reduced osteocalcin expression in subchondral bone, inhibited subchondral bone sclerosis, and alleviated OA progression. This study indicates that senescent BMSCs promote subchondral bone sclerosis and OA progression through activating Mapk8 and Mapk14 via SASP in subchondral bone. The senolytic combination D + Q holds potential for OA treatment by eliminating senescent BMSCs in subchondral bone.

Infrapatellar fat pad-derived MSC-extracellular vesicles isolated by anion exchange chromatography can suppress OA progression in a mouse model. Further studies with large-animal models, larger animal groups, and subsequent clinical trials are necessary to confirm the feasibility of this technique for clinical OA treatment.

ObjectivesChondrocyte ferroptosis contributes to osteoarthritis (OA) progression, and D‐mannose shows therapeutic value in many inflammatory conditions. Here, we investigated whether D‐mannose interferes in chondrocyte ferroptotic cell death during osteoarthritic cartilage degeneration.Materials and methodsIn vivo anterior cruciate ligament transection (ACLT)‐induced OA mouse model and an in vitro study of chondrocytes in an OA microenvironment induced by interleukin‐1β (IL‐1β) exposure were employed. Combined with Epas1 gene gain‐ and loss‐of‐function, histology, immunofluorescence, quantitative RT‐PCR, Western blot, cell viability and flow cytometry experiments were performed to evaluate the chondroprotective effects of D‐mannose in OA progression and the role of hypoxia‐inducible factor 2 alpha (HIF‐2 α) in D‐mannose‐induced ferroptosis resistance of chondrocytes.ResultsD‐mannose exerted a chondroprotective effect by attenuating the sensitivity of chondrocytes to ferroptosis and alleviated OA progression. HIF‐2α was identified as a central mediator in D‐mannose‐induced ferroptosis resistance of chondrocytes. Furthermore, overexpression of HIF‐2α in chondrocytes by Ad‐Epas1 intra‐articular injection abolished the chondroprotective effect of D‐mannose during OA progression and eliminated the role of D‐mannose as a ferroptosis suppressor.ConclusionsD‐mannose alleviates osteoarthritis progression by suppressing HIF‐2α‐mediated chondrocyte sensitivity to ferroptosis, indicating D‐mannose to be a potential therapeutic strategy for ferroptosis‐related diseases.

Impact of controlling abnormal joint movement on the effectiveness of subsequent exercise intervention in mouse model of early knee osteoarthritis

Increasing evidence indicates that osteoarthritis (OA) is a musculoskeletal disease affecting the whole joint, including both cartilage and subchondral bone. Reactive oxygen species (ROS) have been demonstrated to be one of the important destructive factors during early‐stage OA development. The objective of this study was to investigate isorhamnetin (Iso) treatment on osteoclast formation and chondrocyte protection to attenuate OA by modulating ROS. Receptor activator of nuclear factor‐kappa B ligand (RANKL) was used to establish the osteoclast differentiation model in bone marrow macrophages (BMMs) in vivo. H2O2 was used to induce ROS, which could further cause chondrocyte apoptosis. We demonstrated that Iso suppressed RANKL‐induced ROS generation, which could mediate osteoclastogenesis. Moreover, we found that Iso inhibited osteoclast formation and function by suppressing the expression of osteoclastogenesis‐related genes and proteins. We proved that Iso inhibited RANKL‐induced activation of mitogen‐activated protein kinase activation of mitogen‐activated protein kinase (MAPK), nuclear factor‐kappa B (NF‐κB) and AKT signalling pathways in BMMs. In addition, Iso inhibited ROS‐induced chondrocyte apoptosis by regulating apoptosis‐related proteins. Moreover, Iso was administered to an anterior cruciate ligament transection (ACLT)‐induced OA mouse model. The results indicated that Iso exerted beneficial effects on inhibiting excessive osteoclast activity and chondrocyte apoptosis, which further remedied cartilage damage. Overall, our data showed that Iso is an effective candidate for treating OA.

This study aims to investigate the molecular mechanisms underlying the therapeutic effects of fire needling acupuncture (FNA) in alleviating knee osteoarthritis (KOA) symptoms, focusing on the SDF-1/CXCR4 signaling pathway and macrophage polarization. A KOA mouse model was established using anterior cruciate ligament transection and destabilization of the medial meniscus (ACLT + DMM) surgery. After four weeks, FNA was administered twice weekly for another four weeks. Pain behavior was assessed via weight-bearing and mechanical withdrawal thresholds. Micro-CT, histological staining, immunohistochemistry, and immunofluorescence were utilized to evaluate cartilage and subchondral bone changes, along with relevant markers. Synovial inflammation and macrophage polarization were analyzed, specifically targeting the SDF-1/CXCR4 pathway. The effects of CXCR4 modulation were assessed using AMD3100 (antagonist) and ATI-2341 (agonist). FNA treatment improved joint function, reduced pain, alleviated synovial inflammation, and mitigated subchondral bone changes. It decreased cartilage damage while upregulating COL2 and SOX9, and downregulating MMP13. The SDF-1/CXCR4 pathway was activated, leading to a shift in macrophage polarization characterized by reduced pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and increased anti-inflammatory cytokine IL-10. M0 macrophage marker F4/80 and M1 marker CD86 were downregulated, whereas M2 marker CD206 was upregulated. The ATI-2341 group exhibited therapeutic effects similar to FNA, while the AMD3100 and AMD3100 + FNA groups showed reduced efficacy. FNA alleviates KOA by activating the SDF-1/CXCR4 pathway, modulating macrophage polarization, and reducing cartilage damage and inflammation. These findings highlight the therapeutic potential of FNA in the management of KOA. Key Points • Fire needling acupuncture (FNA) differentially regulates SDF-1/CXCR4 signaling in knee osteoarthritis (KOA), where synovial/cartilaginous upregulation enhances mesenchymal stem cell (MSC) homing to potentiate tissue repair, while subchondral suppression mitigates pathological bone remodeling. • FNA activates the JAK1/STAT6/PPAR-γ axis to drive macrophage phenotypic switching from pro-inflammatory M1 to anti-inflammatory M2 states, thereby preserving cartilage matrix integrity through reduced collagenase activity. • Combinatorial CXCR4 inhibition/agonist studies demonstrate FNA's targetable effects across tissue compartments, providing a mechanistic framework to refine acupuncture-based regenerative strategies. • This work establishes a precision medicine framework for degenerative joint diseases by bridging traditional acupuncture with immunomodulatory biologics to target immune-microenvironment crosstalk.

Senescent cells (SnCs) accumulate in many vertebrate tissues with age and contribute to age-related pathologies, presumably through their secretion of factors contributing to the senescence-associated secretory phenotype (SASP). Removal of SnCs delays several pathologies and increases healthy lifespan. Aging and trauma are risk factors for the development of osteoarthritis (OA), a chronic disease characterized by degeneration of articular cartilage leading to pain and physical disability. Senescent chondrocytes are found in cartilage tissue isolated from patients undergoing joint replacement surgery, yet their role in disease pathogenesis is unknown. To test the idea that SnCs might play a causative role in OA, we used the p16-3MR transgenic mouse, which harbors a p16INK4a (Cdkn2a) promoter driving the expression of a fusion protein containing synthetic Renilla luciferase and monomeric red fluorescent protein domains, as well as a truncated form of herpes simplex virus 1 thymidine kinase (HSV-TK). This mouse strain allowed us to selectively follow and remove SnCs after anterior cruciate ligament transection (ACLT). We found that SnCs accumulated in the articular cartilage and synovium after ACLT, and selective elimination of these cells attenuated the development of post-traumatic OA, reduced pain and increased cartilage development. Intra-articular injection of a senolytic molecule that selectively killed SnCs validated these results in transgenic, non-transgenic and aged mice. Selective removal of the SnCs from in vitro cultures of chondrocytes isolated from patients with OA undergoing total knee replacement decreased expression of senescent and inflammatory markers while also increasing expression of cartilage tissue extracellular matrix proteins. Collectively, these findings support the use of SnCs as a therapeutic target for treating degenerative joint disease.

The present study aimed to investigate whether dihydroartemisinin (DHA) alleviates osteoarthritis (OA) in a mouse model of OA. Ten-week-old female C57BL/6j mice were used to establish OA models by anterior cruciate ligament transection (ACLT) and ovariectomized (OVX). DHA was then used to treat the OA in the ACLT and OVX mice. Safranin O-fast green staining and Osteoarthritis Research Society International (OARSI)-modified Mankin scores were used to grade articular cartilage degeneration. Expression of metalloproteinase-13 (MMP-13) and vascular endothelial growth factor (VEGF) in the articular cartilage and leukemia inhibitory factor (LIF), sclerostin, and β-catenin in the subchondral bone were analyzed by immunohistochemistry. Expression of RANKL and CD31 were detected by immunofluorescence. Micro-computed tomography was used to ascertain alterations in the microarchitecture of the subchondral bone. The results demonstrated that DHA decreased MMP-13 and VEGF expression in the articular cartilage. DHA decreased OARSI scores and reduced articular cartilage degeneration. In addition, DHA reduced abnormal subchondral bone remodeling, as demonstrated by a reduction in trabecular separation (Tb.Sp), increased bone volume fractions (BV/TV), as well as bone mineral densities (BMD) compared with the ACLT+vehicle group and the OVX+vehicle group. Furthermore, DHA decreased the inhibition of sclerostin through reduction of LIF secretion by osteoclasts and, hence, attenuated aberrant bone remodeling and inhibited angiogenesis in subchondral bone, further reducing the progression of OA. The present study demonstrated that DHA attenuated OA by inhibiting abnormal bone remodeling and angiogenesis in subchondral bone, which may be a potential therapeutic target for this disease.

Suramin ameliorates osteoarthritis by acting on the Nrf2/HO-1 and NF-κB signaling pathways in chondrocytes and promoting M2 polarization in macrophages

BackgroundThe aim of this study was to determine the role of icariin, a Chinese traditional herbal medicine extracted from Epimedium, in osteoarthritis (OA), using the murine anterior cruciate ligament transection (ACLT)-induced model of OA and micromass culture of murine chondrocytes.Material/MethodsTwenty-four three-month-old C57/6J mice were randomly divided into three groups: the sham group (no surgery and joint injection with normal saline) (N=8); the ACLT + ICA group (ACLT surgery and icariin treatment) (N=8); and the ACLT group (ACLT surgery and joint injection with normal saline) (N=8). At 12 weeks after ACLT surgery, murine articular cartilage was harvested from all mice for histological evaluation of any differences in cartilage degeneration. In vitro micromass culture of mouse chondrocytes was used to study the effects of icariin on chondrocyte differentiation and growth from the three mouse groups.ResultsIcariin treatment (mice in the ACLT + ICA group) significantly reduced degeneration of cartilage in OA with increased cartilage thickness, associated with increased expression of collagen type II alpha 1 (COL2A1), decreased chondrocyte hypertrophy, and decreased expression of collagen type X (ColX) and matrix metalloproteinase 13 (MMP13). In vitro, icariin promoted chondrocyte differentiation by upregulating the expression of agrrecan, Sox9 and parathyroid hormone-related protein (PHrP) and down-regulation of Indian hedgehog (Ihh) and genes regulated by Ihh.ConclusionsIn a mouse model of OA icariin treatment reduced destruction of cartilage, promoted chondrocyte differentiation, upregulated expression of PHrP and down-regulated the expression of Ihh.

The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse

The natural initiation and progression of osteoarthritis in the anterior cruciate ligament deficient feline knee

Background Osteoarthritis (OA) is a multifactorial disease with various risk factors, resulting in the degeneration of articular cartilage and whole joints. However, to date, no effective disease-modifying therapy for OA has been developed. Oxymatrine (OMT) is associated with many pharmacological effects, including anti-inflammatory, antiapoptotic, and antioxidative properties. However, the role of OMT in OA remains unclear. Materials and Methods An IL-1β-induced chondrocyte model and anterior cruciate ligament transection (ACLT)-induced murine model of OA were constructed. The effect of OMT on chondrocyte viability was assessed using the CCK-8 assay. The protein level was assessed by Western blot analysis, and the apoptosis rate was assessed by flow cytometry in vitro and TUNEL staining in OA model mice. The effect of OMT on the degradation of articular cartilage in ACLT-induced OA mice was assessed by histological analysis. Results OMT at 0–2 mg/mL showed no conspicuous cytotoxicity on chondrocytes after 24 hours of incubation. OMT at 0.5, 1, and 2 mg/mL inhibited IL-1β-triggered apoptosis, upregulated MMP13, MMP9, and Col X, and upregulated Col II in chondrocytes in vitro. OMT represses the NF-κB signaling cascade in IL-1β-triggered chondrocytes in vitro. In an in vivo study, OMT decreased the apoptosis rate of chondrocytes and exerted a protective effect against the degradation of articular cartilage in ACLT-triggered OA mice. Conclusion OMT plays a protective role against chondrocyte injury induced by IL-1β in vitro or ACLT in vivo. OMT may play a role in chondrocytes during OA by inhibiting NF-κB signaling by decreasing the phosphorylation of p65 and IκB. OMT treatment may be a promising chondroprotective approach to delay OA cartilage progression.

Defining the factors that alter OA risk following ACL injury using pre-clinical animal models

ObjectiveTo investigate the combined effect of bilateral ovariectomy (OVX) and anterior cruciate ligament transection (ACLT) with medial meniscectomy (MM) on the development of osteoarthritis (OA).MethodsTwenty female 15-week-old Sprague-Dawley rats were used. Five rats in each group underwent bilateral OVX (OVX group), bilateral ACLT with MM (ACLT with MM group), bilateral OVX plus ACLT with MM (OVX plus ACLT with MM group), and sham surgery (SHAM group). All the rats were subjected to treadmill running for 4 weeks. The behavioral evaluation for induction of OA used the number of rears method, and this was conducted at 1, 2, and 4 weeks post-surgery. Bone mineral density (BMD) was calculated with micro-computerized tomography images and the modified Mankin's scoring was used for the histological changes.ResultsThe number of rears in the OVX plus ACLT with MM group decreased gradually and more rapidly in the ACLT with MM group. Histologically, the OVX plus ACLT with MM group had a significantly higher modified Mankin's score than the OVX group (p=0.008) and the SHAM group (p=0.008). BMDs of the OVX plus ACLT with MM group were significantly lower than the SHAM group (p=0.002), and the ACLT with MM group (p=0.003).ConclusionWe found that bilateral OVX plus ACLT with MM induced definite OA change in terms of histology and BMD compared to bilateral OVX and ACLT with MM alone. Therefore, OVX and ACLT with MM was an appropriate degenerative OA rat model.