Destabilization Of The Medial Meniscus Surgery Research Articles

Acute systemic macrophage depletion in osteoarthritic mice alleviates pain-related behaviors and does not affect joint damage.

Osteoarthritis (OA) is a painful degenerative joint disease and a leading source of years lived with disability globally due to inadequate treatment options. Neuroimmune interactions reportedly contribute to OA pain pathogenesis. Notably, in rodents, macrophages in the DRG are associated with onset of persistent OA pain. Our objective was to determine the effects of acute systemic macrophage depletion on pain-related behaviors and joint damage using surgical mouse models in both sexes. We depleted CSF1R+ macrophages by treating male macrophage Fas-induced apoptosis (MaFIA) transgenic mice 8- or 16-weeks post destabilization of the medial meniscus (DMM) with AP20187 or vehicle control (10 mg/kg i.p., 1x/day for 5 days), or treating female MaFIA mice 12 weeks post partial meniscectomy (PMX) with AP20187 or vehicle control. We measured pain-related behaviors 1-3 days before and after depletion, and, 3-4 days after the last injection we examined joint histopathology and performed flow cytometry of the dorsal root ganglia (DRGs). In a separate cohort of male 8-week DMM mice or age-matched naïve vehicle controls, we conducted DRG bulk RNA-sequencing analyses after the 5-day vehicle or AP20187 treatment. Eight- and 16-weeks post DMM in male mice, AP20187-induced macrophage depletion resulted in attenuated mechanical allodynia and knee hyperalgesia. Female mice showed alleviation of mechanical allodynia, knee hyperalgesia, and weight bearing deficits after macrophage depletion at 12 weeks post PMX. Macrophage depletion did not affect the degree of cartilage degeneration, osteophyte width, or synovitis in either sex. Flow cytometry of the DRG revealed that macrophages and neutrophils were reduced after AP20187 treatment. In addition, in the DRG, only MHCII+ M1-like macrophages were significantly decreased, while CD163+MHCII- M2-like macrophages were not affected in both sexes. DRG bulk RNA-seq revealed that Cxcl10 and Il1b were upregulated with DMM surgery compared to naïve mice, and downregulated in DMM after acute macrophage depletion. Acute systemic macrophage depletion reduced the levels of pro-inflammatory macrophages in the DRG and alleviated pain-related behaviors in established surgically induced OA in mice of both sexes, without affecting joint damage. Overall, these studies provide insight into immune cell regulation in the DRG during OA.

Intra-articular sprouting of nociceptors accompanies progressive osteoarthritis: comparative evidence in four murine models.

Knee joints are densely innervated by nociceptors. In human knees and rodent models, sprouting of nociceptors has been reported in late-stage osteoarthritis (OA). Here, we sought to describe progressive nociceptor remodeling in early and late-stage OA, using four distinct experimental mouse models. Sham surgery, destabilization of the medial meniscus (DMM), partial meniscectomy (PMX), or non-invasive anterior cruciate ligament rupture (ACLR) was performed in the right knee of 10-12-week old male C57BL/6 NaV1.8-tdTomato mice. Mice were euthanized (1) 4, 8 or 16 weeks after DMM or sham surgery; (2) 4 or 12 weeks after PMX or sham; (3) 1 or 4 weeks after ACLR injury or sham. Additionally, a cohort of naïve male wildtype mice was evaluated at age 6 and 24 months. Mid-joint cryosections were assessed qualitatively and quantitatively for NaV1.8+ or PGP9.5+ innervation. Cartilage damage, synovitis, and osteophytes were assessed. Progressive OA developed in the medial compartment after DMM, PMX, and ACLR. Synovitis and associated neo-innervation of the synovium by nociceptors peaked in early-stage OA. In the subchondral bone, channels containing sprouting nociceptors appeared early, and progressed with worsening joint damage. Two-year old mice developed primary OA in the medial and the lateral compartment, accompanied by nociceptor sprouting in the synovium and the subchondral bone. All four models showed increased nerve signal in osteophytes. These findings suggest that anatomical neuroplasticity of nociceptors is intrinsic to OA pathology. The detailed description of innervation of the OA joint and its relationship to joint damage might help in understanding OA pain.

TBK1 pharmacological inhibition mitigates osteoarthritis through attenuating inflammation and cellular senescence in chondrocytes

ObjectivesTANK-binding kinase 1 (TBK1) is pivotal in autoimmune and inflammatory diseases, yet its role in osteoarthritis (OA) remains elusive. This study sought to elucidate the effect of the TBK1 inhibitor BX795 on OA and to delineate the underlying mechanism by which it mitigates OA. MethodsInterleukin-1 Beta (IL-1β) was utilized to simulate inflammatory responses and extracellular matrix degradation in vitro. In vivo, OA was induced in 8-week-old mice through destabilization of the medial meniscus surgery. The impact of BX795 on OA was evaluated using histological analysis, X-ray, micro-CT, and the von Frey test. Additionally, Western blot, RT-qPCR, and immunofluorescence assays were conducted to investigate the underlying mechanisms of BX795. ResultsPhosphorylated TBK1 (P-TBK1) levels were found to be elevated in OA knee cartilage of both human and mice. Furthermore, intra-articular injection of BX795 ameliorated cartilage degeneration and alleviated OA-associated pain. BX795 also counteracted the suppression of anabolic processes and the augmentation of catabolic activity, inflammation, and senescence observed in the OA mice. In vitro studies revealed that BX795 reduced P-TBK1 levels and reversed the effects of anabolism inhibition, catabolism promotion, and senescence induction triggered by IL-1β. Mechanistically, BX795 inhibited the IL-1β-induced activation of the cGAS–STING and TLR3–TRIF signaling pathways in chondrocytes. ConclusionsPharmacological inhibition of TBK1 with BX795 protects articular cartilage by inhibiting the activation of the cGAS–STING and TLR3–TRIF signaling pathways. This action attenuates inflammatory responses and cellular senescence, positioning BX795 as a promising therapeutic candidate for OA treatment. The translational potential of this articleThis study furnishes experimental evidence and offers a potential mechanistic explanation supporting the efficacy of BX795 as a promising candidate for OA treatment.

CHONDROCYTE-SPECIFIC KNOCKOUT OF PIEZO ION CHANNELS PROTECTS AGAINST POST-TRAUMATIC OSTEOARTHRITIS

Osteoarthritis (OA) is the leading cause of pain and disability worldwide and is characterized by the degenerative changes of articular cartilage. Joint loading is required for cartilage maintenance; however, hyper-physiologic loading is a risk factor for OA. Mechanosensitive ion channels Piezo1 and Piezo2 synergistically transduce hyper-physiologic compression of chondrocytes, leading to chondrocyte death and onset of OA. This injury response is inhibited by Piezo channel loss of function, however the mechanistic role of Piezo channels in vivo is unknown. We examined the hypothesis that deletion of Piezo in chondrocytes will protect mice from joint damage and pain-related behaviors following a surgical destabilization of the medial meniscus (DMM), investigating a key mechanistic and mechanobiological role of these channels in the pathogenesis of OA.Aggrecan-Cre Piezo1 and Piezo1/2 knockout mice ((Agc)1-CREERT2;Piezo1fl/flPiezo2fl/fl) were generated and given a 5-day Tamoxifen regimen at 12-weeks of age (n=6–12/group/sex). Cre-negative mice served as controls. At 16-weeks, mice received DMM surgery on the left knee. 12-weeks following DMM prior to sacrifice, activity and hyperalgesia were measured using spontaneous running wheels and a small animal algometer. Structural changes in bone, cartilage, and synovium were characterized using microCT, histology, and Modified Mankin Score criteria.Knockout of Piezo1/2 channels was chondroprotective in both sexes following DMM surgery as demonstrated by reduced Modified Mankin Score compared to control animals. Piezo1 KO was chondroprotective in only female mice, indicating a sexually dimorphic response. Piezo1 and Piezo1/2 KO was protective against pain in male mice, while females displayed no differences compared to controls. No changes were observed in bone morphology.Chondrocyte-specific Piezo1/2 knockout protects the knee joint from structural damage, hyperalgesia and functional deficits in a surgical model of PTOA in male and female mice, illustrating the importance of Piezo channels in response to injury in vivo. Future work aims to interrogate potential sexually dimorphic responses to cartilage damage and investigating Piezo2 KO mice.

CHRONIC STRESS RESULTS IN INCREASED PAIN PERCEPTION IN OSTEOARTHRITIS IN VIVO

Osteoarthritis (OA) affects the whole joint and leads to chronic pain. The sympathetic nervous system (SNS) seems to be involved in OA pathogenesis, as indicated by in vitro studies as well as by our latest work demonstrating that sympathectomy in mice results in increased subchondral bone volume in the OA knee joint. We assume that chronic stress may lead to opposite effects, such as an increased bone loss in OA due to an elevated sympathetic tone. Therefore, we analyzed experimental OA progression in mice exposed to chronic stress. OA was induced in male C57BL/6J mice by surgical destabilization of the medial meniscus (DMM) and Sham as well as non-operated mice served as controls. Half of these groups were exposed to chronic unpredictable mild stress (CUMS). After 12 weeks, chronic stress efficiency was assessed using behavioral tests. In addition to measuring body weight and length, changes in subchondral bone were analyzed by μCT. Dynamic Weight Bearing system was used to monitor OA-related pain. Histological scoring will be conducted to investigate the severity cartilage degeneration and synovial inflammation. CUMS resulted in increased anxiety and significant decrease in body weight gain in all CUMS groups compared to non-CUMS groups. CUMS also increased serum corticosterone in healthy mice, with even higher levels in CUMS mice after DMM surgery. CUMS had no significant effect on subchondral bone, but subarticular bone mineral density and trabecular thickness were increased. Moreover, CUMS resulted in significant potentiation of DMM-associated pain. Our results suggest that the autonomic imbalance with increased sympathetic nervous activity induced by chronic stress exacerbates the severity of OA pain perception. We expect significantly increased cartilage degeneration as well as more severe synovial inflammation in CUMS DMM mice compared to DMM mice.

Picroside II suppresses chondrocyte pyroptosis through MAPK/NF-κB/NLRP3 signaling pathway alleviates osteoarthritis.

Picroside II (P-II) is the main bioactive constituent of Picrorhiza Kurroa, a traditional Chinese herb of interest for its proven anti-inflammatory properties. Its beneficial effects have been noted across several physiological systems, including the nervous, circulatory, and digestive, capable of treating a wide range of diseases. Nevertheless, the potential of Picroside II to treat osteoarthritis (OA) and the mechanisms behind its efficacy remain largely unexplored. This study aims to evaluate the efficacy of Picroside II in the treatment of osteoarthritis and its potential molecular mechanisms. In vitro, we induced cellular inflammation in chondrocytes with lipopolysaccharide (LPS) and subsequently treated with Picroside II to assess protective effect on chondrocyte. We employed the Cell Counting Kit-8 (CCK-8) assay to assess the impact of Picroside II on cell viability and select the optimal Picroside II concentration for subsequent experiments. We explored the effect of Picroside II on chondrocyte pyroptosis and its underlying molecular mechanisms by qRT-PCR, Western blot (WB) and immunofluorescence. In vivo, we established the destabilization of the medial meniscus surgery to create an OA mouse model. The therapeutic effects of Picroside II were then assessed through Micro-CT scanning, Hematoxylin-eosin (H&E) staining, Safranin O-Fast Green (S&F) staining, immunohistochemistry and immunofluorescence. In in vitro studies, toluidine blue and CCK-8 results showed that a certain concentration of Picroside II had a restorative effect on the viability of chondrocytes inhibited by LPS. Picroside II notably suppressed the expression levels of caspase-1, IL-18, and IL-1β, which consequently led to the reduction of pyroptosis. Moreover, Picroside II was shown to decrease NLRP3 inflammasome activation, via the MAPK/NF-κB signaling pathway. In vivo studies have shown that Picroside II can effectively reduce subchondral bone destruction and osteophyte formation in the knee joint of mice after DMM surgery. Our research suggests that Picroside II can inhibit chondrocyte pyroptosis and ameliorate osteoarthritis progression by modulating the MAPK/NF-κB signaling pathway.

Curculigoside inhibits osteoarthritis via the regulation of NLRP3 pathway.

Osteoarthritis (OA) is characterized by degenerative articular cartilage. Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) plays an important role in inflammation. This studyaims to investigate whether protective effects of curculigoside on OA are medicated by the regulation of NLRP3 pathway. Destabilization of the medial meniscus (DMM) was performed to build an OA mouse model.After surgery, OA mice were treated with curculigoside. Immunohistochemistry was conducted to evaluate OA cartilage. In addition, human chondrocytes were isolated and treated with curculigoside. The mRNA and protein expression of iNOS, MMP-9, NLRP3 was detected by PCR and Western blot analysis. Curculigoside inhibitedmRNA and protein levels of iNOS and MMP-9 induced by DMM surgery in a dose-dependent manner. Furthermore, the expression of NLRP3, NF-κB and PKR was downregulated after curculigoside administration.Moreover, curculigoside reversed the effects of IL-1β on MMP-9, iNOS and type II collagen expression at mRNA and protein levels in human chondrocytes in a dose-dependent manner. In conclusion, curculigoside exhibits beneficial effect on cartilage via the inhibition of NLRP3 pathway.

The Preventive Effects of Platelet-Rich Plasma Against Knee Osteoarthritis Progression in Rats.

In recent years, the intra-articular administration of platelet-rich plasma (PRP), a novel therapeutic strategy for knee osteoarthritis (KOA), has gained attention. However, the efficacy of PRP in inhibiting degenerative joint changes remains unclear. The current study aimed to evaluate the therapeutic effect of the intra-articular administration of PRP in rats with induced KOA. PRP was prepared from the whole blood of nine-week-old male Wistar rats via centrifugation at 25°C, 200 × g, for seven minutes. KOA was induced in the right knees of the rats via destabilization of the medial meniscus (DMM) surgery. The animals were divided into the control, sham, DMM, and DMM + PRP groups (n = 5 each). The rats in the DMM + PRP group received 50 μL of intra-articular PRP in the right knee joint four weeks after surgery. The rotarod test was conducted to assess locomotive function. Eight weeks after DMM surgery, the degree of medial meniscus extrusion was measured via computed tomography (CT) images on the right knee. Then, a histological analysis of the harvested knees was conducted. KOA progression was assessed using the Osteoarthritis Research Society International (OARSI) score. The number of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in the subchondral bone was counted via histological analysis. The degree of medial meniscus extrusion did not significantly differ between the DMM and DMM + PRP groups. Similarly, there were no significant differences in the walking time based on the rotarod test between the DMM and DMM + PRP groups. However, the DMM group had a significantly higher OARSI score than the DMM + PRP group. The number of TRAP-positive osteoclasts in the subchondral bone of the DMM group increased over time, peaking four weeks after surgery. The DMM + PRP group had a higher number of TRAP-positive osteoclasts in the subchondral bone than the control group. However, there was no significant difference between the number of TRAP-positive osteoclasts between the DMM group and the control and sham groups. The intra-articular administration of PRP may inhibitKOA progression in a rat model, especially in the articular cartilage degradation and osteophyte formation. The results can provide further evidence about the efficacy of PRP against KOA progression and can contribute to the current practice of healthcare professionals based on accurate knowledge.

Comparative Effects of Intra-Articular versus Intravenous Mesenchymal Stromal Cells Therapy in a Rat Model of Osteoarthritis by Destabilization of Medial Meniscus.

Transplanted mesenchymal stromal cells (MSCs) exhibit a robust anti-inflammatory and homing capacity in response to high inflammatory signals, as observed in studies focused on rheumatic diseases that target articular cartilage (AC) health. However, AC degradation in osteoarthritis (OA) does not necessarily coincide with a highly inflammatory joint profile. Often, by the time patients seek medical attention, they already have damaged AC. In this study, we examined the therapeutic potential of a single bone marrow MSC transplant (2 × 106 cells/kgbw) through two different routes: intra-articular (MSCs-IAt) and intravenous (MSCs-IVt) in a preclinical model of low-grade inflammatory OA with an established AC degeneration. OA was induced through the destabilization of the medial meniscus (DMM) in female Wistar Kyoto rats. The animals received MSCs 9 weeks after surgery and were euthanized 4 and 12 weeks post-transplant. In vivo and ex vivo tracking of MSCs were analyzed via bioluminescence and imaging flow cytometry, respectively. Cytokine/chemokine modulation in serum and synovial fluid was measured using a multiplex panel. AC degeneration was quantified through histology, and hindlimb muscle balance was assessed with precision weighing. To our knowledge, we are the first group to show the in vivo (8 h) and ex vivo (12 h) homing of cells to the DMM-OA joint following MSCs-IVt. In the case of MSCs-IAt, the detection of cellular bioluminescence at the knee joint persisted for up to 1 week. Intriguingly, intra-articular saline injection (placebo-IAt) resulted in a worse prognosis of OA when compared to a non-invasive control (placebo-IVt) without joint injection. The systemic cytokines/chemokines profile exhibited a time-dependent variation between transplant routes, displaying a transient anti-inflammatory systemic response for both MSCs-IVt and MSCs-IAt. A single injection of MSCs, whether administered via the intra-articular or intravenous route, performed 9 weeks after DMM surgery, did not effectively inhibit AC degeneration when compared to a non-invasive control.

Acetyl zingerone ameliorates osteoarthritis by inhibiting chondrocyte programmed cell death.

Osteoarthritis (OA) is a degenerative disease that ultimately leads to joint deformity. The pathogenesis of OA is believed to involve abnormal chondrocyte death, with ferroptosis serving a key role in chondrocyte damage. The present study investigated whether acetyl zingerone (AZ), a newly identified antioxidant derived from curcumin, can alleviate the progression of OA. To investigate this, the present study performed various experiments, including crystal violet staining, flow cytometry, immunofluorescence and western blot analysis. In addition, dual validation was performed using invivo and invitro experiments; a mouse OA model was constructed for the invivo experiments, and chondrocytes were used for the invitro experiments. Destabilization of the medial meniscus (DMM) surgery was performed to establish an OA model in mice and IL‑1β was used to induce an OA model invitro. The results indicated that AZ may promote chondrocyte viability and the expression of extracellular matrix components. Furthermore, AZ reduced the occurrence of ferroptosis by promoting the expression of glutathione peroxidase 4, inhibiting cartilage destruction and osteophyte formation, and alleviating damage to articular cartilage caused by DMM surgery. Mechanistically, the activation of nuclear factor erythroid 2‑related factor 2 and heme oxygenase‑1 may be responsible for the anti‑ferroptosis effects of AZ on chondrocytes. These findings indicated that AZ may be considered a promising candidate for OA therapy.

Ginsenoside compound K alleviates osteoarthritis by inhibiting NLRP3‑mediated pyroptosis.

Ginsenoside compound K (GCK) has been previously reported to be a potent antiarthritic and bone-protective agent. Therefore, the present study aimed to explore the potential effects of GCK on osteoarthritis and its regulatory effects on the pyroptosis of chondrocytes. Primary mouse chondrocytes (PMCs) were used for in vitro analysis. ELISA assays revealed that compared with the untreated cells, TNF-α induced a significant increase in IL-6, MMP13, A disintegrin and metalloproteinase with thrombospondin motifs 5 and MMP3 expression but induced a significant decrease in aggrecan and collagen II expression. By contrast, GCK reversed the aforementioned alterations in a dose-dependent manner. Experimental osteoarthritis was subsequently induced in mice through transection of the medial meniscotibial ligament and medial collateral ligament in the right knee [destabilization of the medial meniscus (DMM) mice]. GCK was found to reduce cartilage degradation in vivo in DMM mice, which was assessed using the Osteoarthritis Research Society International (OARSI) score, collagen II and MMP13 expression. Cartilage degradation is associated with higher OARSI score, decreased collagen II and increased MMP13 expression. In PMCs, TNF-α treatment stimulated an increase in the expression of NLR family pyrin domain containing 3 (NLRP3), Gasdermin D-N terminal (GSDMD-NT), cleaved caspase-1 and mature IL-1β, markers that indicate the occurrence of pyroptosis. However, GCK treatment suppressed the increase of the aforementioned proteins in a dose-dependent manner. Immunohistochemistry staining of the knee joint tissue sections from the DMM mice confirmed that GCK attenuated the NLRP3 and GSDMD-NT expression that was induced by DMM surgery. In conclusion, the present study revealed that GCK can reduce cartilage degradation in an osteoarthritis model by inhibiting the NLRP3-inflammasome activation and subsequent pyroptosis.

MATN3 delivered by exosome from synovial mesenchymal stem cells relieves knee osteoarthritis: Evidence from in vitro and in vivo studies

BackgroundSynovial mesenchymal stem cell (SMSC) exerts chondroprotective effects in osteoarthritis (OA) clinical models. However, the regulatory potentials of SMSC-derived exosomes (SMSC-Exo) in OA still need to be discovered, which attracted our attention. MethodsThe destabilization of the medial meniscus surgery was performed on the knee joints of a mouse OA model, followed by injection of SMSC-Exo. In addition, SMSC-Exo was administrated to mouse chondrocytes to observe the functional and molecular alterations. ResultsBoth of SMSC-Exo and overexpression of Matrilin-3 (MATN3) alleviated cartilage destruction and suppressed degradation of extracellular matrix (ECM) in the OA rat model. In addition, assays concerning the in vitro OA model induced by IL-1β showed that SMSC-Exo could promote chondrocyte viability and inhibit autophagy defects. Furthermore, SMSC-Exo achieved the chondroprotective effects through the delivery of MATN3/IL-17A, and MATN3 could suppress the activation of PI3K/AKT/mTOR signaling through IL-17A. ConclusionSMSC-Exo exerts beneficial therapeutic effects on OA by preventing ECM degradation and autophagy defects by delivering MATN3/IL-17A. The Translational Potential of this ArticleThe translational potential of this study is not only limited to the treatment of knee osteoarthritis but also provides new insights for the treatment of other joint diseases by exploring the mechanism of MATN3. In addition, SMSCExo, as a novel drug carrier, has great potential for treating and diagnosing other diseases. With further research, these findings will provide new directions for developing personalized and innovative treatment options.

Notch signaling is activated in knee-innervating dorsal root ganglia in experimental models of osteoarthritis joint pain

BackgroundWe aimed to explore activation of the Notch signaling pathway in knee-innervating lumbar dorsal root ganglia (DRG) in the course of experimental osteoarthritis (OA) in mice, and its role in knee hyperalgesia.MethodsCultured DRG cells were stimulated with the TLR4 agonist, lipopolysaccharide (LPS). Notch signaling in the cells was either inhibited with the γ-secretase inhibitor, DAPT, or with soluble Jagged1, or activated through immobilized Jagged1. CCL2 production was analyzed at mRNA and protein levels. In in vivo experiments, knee hyperalgesia was induced in naïve mice through intra-articular (IA) injection of LPS. The effect of inhibiting Notch signaling was examined by pre-injecting DAPT one hour before LPS. OA was induced through surgical destabilization of the medial meniscus (DMM) in male C57BL/6 mice. Gene expression in DRG was analyzed by qRT-PCR and RNAscope in situ hybridization. Activated Notch protein (NICD) expression in DRG was evaluated by ELISA and immunofluorescence staining. DAPT was injected IA 12 weeks post DMM to inhibit Notch signaling, followed by assessing knee hyperalgesia and CCL2 expression in the DRG.ResultsIn DRG cell cultures, LPS increased NICD in neuronal cells. Inhibition of Notch signaling with either DAPT or soluble Jagged1 attenuated LPS-induced increases of Ccl2 mRNA and CCL2 protein. Conversely, activating Notch signaling with immobilized Jagged1 enhanced these LPS effects. In vivo, IA injection of LPS increased expression of Notch genes and NICD in the DRG. Pre-injection of DAPT prior to LPS alleviated LPS-induced knee hyperalgesia, and decreased LPS-induced CCL2 expression in the DRG. Notch signaling genes were differentially expressed in the DRG from late-stage experimental OA. Notch1, Hes1, and NICD were increased in the neuronal cell bodies in DRG after DMM surgery. IA administration of DAPT alleviated knee hyperalgesia post DMM, and decreased CCL2 expression in the DRG.ConclusionsThese findings suggest a synergistic effect of Notch signaling with TLR4 in promoting CCL2 production and mediating knee hyperalgesia. Notch signaling is activated in knee-innervating lumbar DRG in mice with experimental OA, and is involved in mediating knee hyperalgesia. The pathway may therefore be explored as a target for alleviating OA pain.

Effects of uphill and downhill walking on post-traumatic osteoarthritis development in mice.

Using destabilization of the medial meniscus (DMM) induced models of osteoarthritis (OA), we sought to clarify how flat, uphill, and downhill walking affects OA-related inflammation and articular cartilage degeneration. Thirty-two male C57BL/6J mice of 7 weeks old underwent DMM surgery in their right knee and sham surgery in their left knee, and then were assigned to either the no walking after DMM group or the flat, uphill, or downhill walking after DMM group (n = 8/group). After creating the knee OA model, the mice in the walking groups were subjected to treadmill walking 1 day after surgery, which included walking for 12 m/min, 30 min/day, and 7 days/week, with inclines of 0, 20, or - 20 degrees. Knee joints were harvested at the end of the intervention period. Non-demineralized frozen sections were prepared and samples were examined histologically. Osteoarthritis Research Society International scores were significantly decreased in both the uphill and flat walking groups, compared with the no-walking group. Immunohistochemical staining showed increased levels of aggrecan and Sry-related high mobility group box 9; conversely, decreased levels of Matrix metalloproteinase-13 and A disintegrin and metalloproteinase with thrombospondin motifs 5 in both the uphill and flat walking groups. Micro-CT results showed higher bone volume fraction in the uphill and flat walking groups than that in the no-walking group. Our findings indicate that flat and uphill walking may prevent the progression of OA. KEY POINTS: Flat and uphill treadmill walking can prevent the development of post-traumatic osteoarthritis in mice. Flat and uphill walking increases anabolic proteins and decreases catabolic proteins and inflammatory cytokines in articular cartilage, resulting in protection against cartilage degeneration. Downhill walking increases catabolic proteins and inflammatory cytokines in cartilage, which has negative effects on articular cartilage. Abstract figure legend This study sought to verify how flat, uphill, and downhill walking affects osteoarthritis development. Mice of the post-traumatic osteoarthritis model were subjected to treadmill walking. Flat and uphill walking increased cartilage anabolic proteins (Sry-related high mobility group box 9 [Sox9] and/or Aggrecan) and decreased catabolic proteins (Matrix metalloproteinase-13 [MMP-13] or A disintegrin and metalloproteinase with thrombospondin motifs 5 [ADAMTS5]), resulting in protection against osteoarthritis development. Downhill walking decreased Sox9 protein levels in cartilage compared to uphill walking. In addition, downhill walking increased MMP-13 levels, suggesting negative effects on articular cartilage. This article is protected by copyright. All rights reserved.

PROTEIN PHOSPHATASE PPM1A INHIBITION ATTENUATES OSTEOARTHRITIS VIA REGULATION OF TGF-Β/SMAD2 SIGNALLING IN CHONDROCYTES

TGF-β/Smad2 signaling is considered to be one of the important pathways involved in osteoarthritis (OA) and protein phosphatase magnesium-dependent 1A (PPM1A) functions as an exclusive phosphatase of Smad2 and regulates TGF-β signaling, here, we investigated the functional role of PPM1A in OA pathogenesis.PPM1A expressions in both human OA cartilage and experimental OA mice chondrocytes were analyzed immunohistochemically. Besides, the mRNA and protein expression of PPM1A induced by IL-1β treatment were also detected by q-PCR and immunofluorescence in vitro. OA was induced in PPM1A knockout (KO) mice by destabilization of the medial meniscus (DMM), and histopathological examination was performed. OA was also induced in wild-type (WT) mice, which were then treated with an intra-articular injection of a selective PPM1A inhibitor for 8 weeks.PPM1A protein expressions were increased in both human OA cartilage and experimental OA mice chondrocytes. We also found that treatment with IL-1β in mouse primary chondrocytes significantly increased both mRNA and protein expression of PPM1A in vitro. Importantly, our data showed that PPM1A deletion could substantially protect against surgically induced OA. Concretely, the average OARSI score and quantification of BV/TV of subchondral bone in KO mice were significantly lower than that in WT mice 8 weeks after DMM surgery. Besides, TUNEL staining revealed a significant decrease in apoptotic chondrocytes in PPM1A-KO mice with DMM operation. With OA induction, the rates of chondrocytes positive for Mmp-13 and Adamts-5 in KO mice were also significantly lower than those in WT mice. Moreover, compared with WT mice, the phosphorylation of Smad2 in chondrocytes was increased in KO mice underwent DMM surgery. However, articular-injection with SD-208, a selective inhibitor of TGF-β/Smad2 signaling could significantly abolish the chondroprotective phenotypes in PPM1A-KO mice. Additionally, both cartilage degeneration and subchondral bone subchondral bone sclerosis in DMM model were blunted following intra-articular injection with BC-21, a small-molecule inhibitor for PPM1A.Our study demonstrated that PPM1A inhibition attenuates OA by regulating TGF-β/Smad2 signaling. Furthermore, PPM1A is a potential target for OA treatment and BC-21 may be employed as alternative therapeutic agents for the management of OA.

Synovial mesenchymal stem cell-derived exosomal microRNA-320c facilitates cartilage damage repair by targeting ADAM19-dependent Wnt signalling in osteoarthritis rats.

Our previous study revealed that synovial mesenchymal stem cell (SMSC)-derived exosomal microRNA-302c enhanced chondrogenesis by targeting a disintegrin and metalloproteinase 19 (ADAM19) in vitro. This study aimed to validate the potential of SMSC-derived exosomal microRNA-302c for the treatment of osteoarthritis in vivo. After 4weeks of destabilization of the medial meniscus surgery (DMM) to establish an osteoarthritis model, the rats received weekly articular cavity injection of SMSCs with or without GW4869 treatment (exosome inhibitor) or exosomes from SMSCs with or without microRNA-320c overexpression for another 4weeks. SMSCs and SMSC-derived exosomes reduced the Osteoarthritis Research Society International (OARSI) score, improved cartilage damage repair, suppressed cartilage inflammation, suppressed extracellular matrix (ECM) degradation, and inhibited chondrocyte apoptosis in DMM rats. However, these effects were largely hampered in rats that were injected with GW4869-treated SMSCs. Moreover, exosomes from microRNA-320c-overexpressing SMSCs exerted a better effect than exosomes from negative control SMSCs on decreasing the OARSI score, enhancing cartilage damage repair, suppressing cartilage inflammation, and inhibiting ECM degradation and chondrocyte apoptosis. Mechanistically, exosomes from microRNA-320c-overexpressing SMSCs reduced the levels of ADAM19, as well as β-catenin and MYC, which are two critical proteins in Wnt signalling. SMSC-derived exosomal microRNA-320c suppresses ECM degradation and chondrocyte apoptosis to facilitate cartilage damage repair in osteoarthritis rats by targeting ADAM19-dependent Wnt signalling.

African Spiny Mice (Acomys) Exhibit Mild Osteoarthritis Following Meniscal Injury.

Hyaline cartilage has limited innate healing abilities and hyaline cartilage loss is a hallmark of osteoarthritis (OA). Animal models can provide important insights into cartilage regeneration potential. One such animal model, the African spiny mouse (Acomys), is capable of regenerating skin, skeletal muscle, and elastic cartilage. This study aims to evaluate whether these regenerative abilities protect Acomys with meniscal injury from OA-related joint damage and behaviors indicative of joint pain and dysfunction. Acomys received destabilization of the medial meniscus (DMM) surgery (n = 11) or a skin incision (n = 10). Gait testing occurred at 4, 6, 8, 10, and 12 weeks after surgery. At endpoint, joints were processed for histology to assess cartilage damage. Following joint injury, Acomys with DMM surgery altered their walking patterns by increasing the percent stance time on the contralateral limb relative to the operated limb, thereby reducing the amount of time the injured limb must bear weight on its own throughout the gait cycle. Histological grading indicated evidence of OA-related joint damage in Acomys with DMM surgery; these changes were primarily driven by loss of structural integrity in the hyaline cartilage. Acomys developed gait compensations, and the hyaline cartilage in Acomys is not fully protected from OA-related joint damage following meniscal injury, although this damage was less severe than that historically found in C57BL/6 mice with an identical injury. Thus, Acomys do not appear to be completely protected from OA-related changes, despite the ability to regenerate other wounded tissues.

Limited roles of Piezo mechanosensing channels in articular cartilage development and osteoarthritis progression

To investigate the role of Piezo1 and Piezo2 in surgically induced osteoarthritis (OA) in mice. Male conditional knockout (cKO) mice missing Piezo1 and Piezo2 in the joint using Gdf5-Cre transgenic mice were induced with post-traumatic OAby destabilization of the medial meniscus (DMM) of the right knee joint at 12 weeks of age. The severity of OA was histologically assessed at 24 weeks of age. OA-associated pain was evaluated by static weight bearing analysis. Additionally, articular chondrocytes isolated from cKO mice were exposed to fluid flow shear stress (FFSS) to evaluate the expression of OA-associated genes. Mice with conditional deletion of Piezo1 and Piezo2 showed normal joint development with no overt histological changes in the knee joint at 12 weeks and 24 weeks. DMM surgery induced moderate to severe OA in both control and cKO mice (median OARSI score: control, 4.67; cKO, 4.23, P=0.3082), although a few cKO mice showed milder OA. Pain assessment by static weight-bearing analysis suggested Piezo ablation in the joint has no beneficial effects on pain. FFSS increased the expression of OA-related genes both in control and cKO mice to similar extents. Piezo1 and Piezo2 are not essential for normal joint development. Genetic ablation of Piezo channels did not confer evident protective effects on OA progression in mice. In vitro data suggests that different mechanotransducers other than Piezo channels mediate FFSS in mechanical stress-induced gene expression.

Targeting CD38 to Suppress Osteoarthritis Development and Associated Pain After Joint Injury in Mice.

This study was undertaken to determine the role of CD38, which can function as an enzyme to degrade NAD+ , in osteoarthritis (OA) development. Human knee cartilage from normal donors and OA donors were examined for CD38 expression. "Gain-of-function," through overexpression of CD38 via transient transfection, and "loss-of-function," through pharmacologic inhibition of CD38, approaches were used to assess the effects of CD38 on intracellular NAD+ :NADH ratio and catabolic activity in chondrocytes. We also initiated joint injury-induced OA by surgical destabilization of the medial meniscus (DMM) in CD38 knockout mice and wild-type (WT; C57BL/6) mice and in WT male mice in the presence or absence of apigenin treatment. Cartilage degradation, synovial inflammation, subchondral bone changes, and pain behavior were evaluated after DMM surgery. We also examined expression of CD38 and the neuropeptide calcitonin gene-related peptide (CGRP) in knee sections from these mice. CD38 expression was up-regulated in human knee OA cartilage and in chondrocytes stimulated with the proinflammatory cytokine interleukin-1β (IL-1β). Overexpression of CD38 in chondrocytes resulted in reduced cellular NAD+ :NADH ratio and augmented catabolic responses to IL-1β. These effects were reversed by pharmacologic inhibition of CD38. Cartilage degradation and synovial inflammation, associated with increased CD38 expression in cartilage and synovium, osteophyte formation and subchondral bone sclerosis, and pain-like behavior linked to increased CGRP expression in the synovium were observed in WT mice after joint injury. Such effects were significantly reduced in mice deficient in CD38 through either genetic knockout or pharmacologic inhibition. CD38 deficiency exerts OA disease-modifying effects. Inhibition of CD38 has the potential to be a novel therapeutic approach for OA treatment.

Accuracy of in vivo microCT imaging in assessing the microstructural properties of the mouse tibia subchondral bone.

Osteoarthritis (OA) is one of the most common musculoskeletal diseases. OA is characterized by degeneration of the articular cartilage as well as the underlying subchondral bone. Post-traumatic osteoarthritis (PTOA) is a subset of OA caused by mechanical trauma. Mouse models, such as destabilization of the medial meniscus (DMM), are useful to study PTOA. Ex vivo micro-Computed Tomography (microCT) imaging is the predominant technique used to scan the mouse knee in OA studies. Nevertheless, in vivo microCT enables the longitudinal assessment of bone microstructure, reducing measurement variability and number of animals required. The effect of image resolution in measuring subchondral bone parameters was previously evaluated only for a limited number of parameters. The aim of this study was to evaluate the ability of in vivo microCT imaging in measuring the microstructural properties of the mouse tibia trabecular and cortical subchondral bone, with respect to ex vivo high resolution imaging, in a DMM model of PTOA. Sixteen male C57BL/6J mice received DMM surgery or sham operation at 14 weeks of age (N=8 per group). The right knee of each mouse was microCT scanned in vivo (10.4μm voxel size) and ex vivo (4.35μm voxel size) at the age of 26 weeks. Each image was aligned to a reference image using rigid registration. The subchondral cortical bone plate thickness was measured at the lateral and medial condyles. Standard morphometric parameters were measured in the subchondral trabecular bone. In vivo microCT imaging led to significant underestimation of bone volume fraction (-14%), bone surface density (-3%) and trabecular number (-16%), whereas trabecular thickness (+3%) and separation (+5%) were significantly overestimated. Nevertheless, most trabecular parameters measured in vivo were well correlated with ex vivo measurements (R2 = 0.69-0.81). Degree of anisotropy, structure model index and connectivity density were measured in vivo with lower accuracy. Excellent accuracy was found for cortical thickness measurements. In conclusion, this study identified what bone morphological parameters can be reliably measured by in vivo microCT imaging of the subchondral bone in the mouse tibia. It highlights that this approach can be used to study longitudinal effects of diseases and treatments on the subchondral cortical bone and on most subchondral trabecular bone parameters, but systematic over- or under-estimations should be considered when interpreting the results.