Post-traumatic Osteoarthritis Model Research Articles

Plasma and synovial fluid extracellular vesicles display altered microRNA profiles in horses with naturally occurring post-traumatic osteoarthritis: an exploratory study.

The objective of this study was to characterize extracellular vesicles (EVs) in plasma and synovial fluid obtained from horses with and without naturally occurring post-traumatic osteoarthritis (PTOA). EVs were isolated from plasma and synovial fluid from horses with (n = 6) and without (n = 6) PTOA. Plasma and synovial fluid EVs were characterized with respect to quantity, size, and surface markers. Small RNA sequencing was performed, and differentially expressed microRNAs (miRNAs) underwent bioinformatic analysis to identify putative targets and to explore potential associations with specific biological processes. Plasma and synovial fluid samples from horses with PTOA had a significantly higher proportion of exosomes and a lower proportion of microvesicles compared to horses without PTOA. Small RNA sequencing revealed several differentially expressed miRNAs, including miR-144, miR-219-3p, and miR-199a-3l in plasma and miR-199a-3p, miR-214, and miR-9094 in synovial fluid EVs. Bioinformatics analysis of the differentially expressed miRNAs highlighted their potential role in fibrosis, differentiation of chondrocytes, apoptosis, and inflammation pathways in PTOA. We have identified dynamic molecular changes in the small noncoding signatures of plasma and synovial fluid EVs in horses with naturally occurring PTOA. These findings could serve to identify promising biomarkers in the pathogenesis of PTOA, to facilitate the development of targeted therapies, and to aid in establishing appropriate translational models of PTOA.

The BMP7-Derived Peptide p[63-82] Reduces Cartilage Degeneration in the Rat ACLT-pMMx Model for Posttraumatic Osteoarthritis.

Osteoarthritis (OA) is characterized by articular cartilage erosion, pathological subchondral bone changes, and signs of synovial inflammation and pain. We previously identified p[63-82], a bone morphogenetic protein 7 (BMP7)-derived bioactive peptide that attenuates structural cartilage degeneration in the rat medial meniscal tear-model for posttraumatic OA. This study aimed to evaluate the cartilage erosion-attenuating activity of p[63-82] in a different preclinical model for OA (anterior cruciate ligament transection-partial medial meniscectomy [anterior cruciate ligament transection (ACLT)-pMMx]). The disease-modifying action of the p[63-82] was followed-up in this model for 5 and 10 weeks. Skeletally mature male Lewis rats underwent ACLT-pMMx surgery. Rats received weekly intra-articular injections with either saline or 500 ng p[63-82]. Five and 10 weeks postsurgery, rats were sacrificed, and subchondral bone characteristics were determined using microcomputed tomography (µCT). Histopathological evaluation of cartilage degradation and Osteoarthritis Research Society International (OARSI)-scoring was performed following Safranin-O/Fast Green staining. Pain-related behavior was measured by incapacitance testing and footprint analysis. Histopathological evaluation at 5 and 10 weeks postsurgery showed reduced cartilage degeneration and a significantly reduced OARSI score, whereas no significant changes in subchondral bone characteristics were found in the p[63-82]-treated rats compared to the saline-treated rats. ACLT-pMMx-induced imbalance of static weightbearing capacity in the p[63-82] group was significantly improved compared to the saline-treated rats at weeks 5 postsurgery. Footprint analysis scores in the p[63-82]-treated rats demonstrated improvement at week 10 postsurgery. Weekly intra-articular injections of p[63-82] in the rat ACLT-pMMx posttraumatic OA model resulted in reduced degenerative cartilage changes and induced functional improvement in static weightbearing capacity during follow-up.

Low-intensity pulsed ultrasound delays the progression of osteoarthritis by regulating the YAP-RIPK1-NF-κB axis and influencing autophagy.

Osteoarthritis (OA) is a degenerative disease characterized by chronic inflammation of the joint. As the disease progresses, patients will gradually develop symptoms such as pain, physical limitations and even disability. The risk factors for OA include genetics, gender, trauma, obesity, and age. Unfortunately, due to limited understanding of its pathological mechanism, there are currently no effective drugs or treatments to suspend the progression of osteoarthritis. In recent years, some studies found that low-intensity pulsed ultrasound (LIPUS) may have a positive effect on osteoarthritis. Nonetheless, the exact mechanism by which LIPUS affects osteoarthritis remains unknown. It is valuable to explore the specific mechanism of LIPUS in the treatment of OA. In this study, we validated the potential therapeutic effect of LIPUS on osteoarthritis by regulating the YAP-RIPK1-NF-κB axis at both cellular and animal levels. To verify the effect of YAP on OA, the expression of YAP was knocked down or overexpressed by siRNA and plasmid in chondrocytes and adeno-associated virus was injected into the knee joint of rats. The effect of LIPUS was investigated in inflammation chondrocytes induced by IL-1β and in the post-traumatic OA model. In this study, we observed that YAP plays an important role in the development of osteoarthritis and knocking down of YAP significantly inhibited the inflammation and alleviated cartilage degeneration. We also demonstrated that the expression of YAP was increased in osteoarthritis chondrocytes and YAP could interact with RIPK1, thereby regulating the NF-κB signal pathway and influencing inflammation. Moreover, we also discovered that LIPUS decreased the expression of YAP by restoring the impaired autophagy capacity and inhibiting the binding between YAP and RIPK1, thereby delaying the progression of osteoarthritis. Animal experiment showed that LIPUS could inhibit cartilage degeneration and alleviate the progression of OA. These results showed that LIPUS is effective in inhibiting inflammation and cartilage degeneration and alleviate the progression of OA. As a result, our results provide new insight of mechanism by which LIPUS delays the development of osteoarthritis, offering a novel therapeutic regimen for osteoarthritis.

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.

Inhibition of Phlpp1 preserves the mechanical integrity of articular cartilage in a murine model of post-traumatic osteoarthritis

ObjectivePhlpp1 inhibition is a potential therapeutic strategy for cartilage regeneration and prevention of post-traumatic osteoarthritis (PTOA). To understand how Phlpp1 loss affects cartilage structure, cartilage elastic modulus was measured with atomic force microscopy (AFM) in male and female mice after injury. MethodsOsteoarthritis was induced in male and female Wildtype (WT) and Phlpp1-/- mice by destabilization of the medial meniscus (DMM). At various timepoints post-injury, activity was measured, and knee joints examined with AFM and histology. In another cohort of WT mice, the PHLPP inhibitor NSC117079 was intraarticularly injected four weeks after injury. ResultsMale WT mice showed decreased activity and histological signs of cartilage damage at 12 but not 6 weeks post-DMM. Female mice showed a less severe response to DMM by comparison, with no histological changes seen at any timepoint. In both sexes the elastic modulus of medial condylar cartilage was decreased in WT mice but not Phlpp1-/- mice after DMM as measured by AFM. By 6 weeks, cartilage modulus had decreased from 2MPa to 1MPa in WT mice. Phlpp1-/- mice showed no change in modulus at 6 weeks and only a 25% decrease at 12 weeks. The PHLPP inhibitor NSC117079 protected cartilage structure and prevented signs of OA 6 weeks post-injury. ConclusionsAFM is a sensitive method for detecting early changes in articular cartilage post-injury. Phlpp1 suppression, either through genetic deletion or pharmacological inhibition, protects cartilage degradation in a model of PTOA, validating Phlpp1 as a therapeutic target for PTOA.

Sex-specific effects of injury and beta-adrenergic activation on metabolic and inflammatory mediators in a murine model of post-traumatic osteoarthritis

ObjectiveMetabolic processes are intricately linked to the resolution of innate inflammation and tissue repair, two critical steps for treating post-traumatic osteoarthritis (PTOA). Based on lipolytic and immunoregulatory actions of norepinephrine, we hypothesized that intra-articular β-adrenergic receptor (βAR) stimulation would suppress PTOA-associated inflammation in the infrapatellar fat pad (IFP) and synovium. DesignWe used the βAR agonist isoproterenol to perturb intra-articular metabolism 3.5 weeks after applying a non-invasive single-load compression injury to knees of 12-week-old male and female mice. We examined the acute effects of intra-articular isoproterenol treatment relative to saline on IFP histology, multiplex gene expression of synovium-IFP tissue, synovial fluid metabolomics, and mechanical allodynia. ResultsInjured knees developed PTOA pathology characterized by heterotopic ossification, articular cartilage loss, and IFP atrophy and fibrosis. Isoproterenol suppressed the upregulation of pro-fibrotic genes and downregulated the expression of adipose genes and pro-inflammatory genes (Adam17, Cd14, Icam1, Csf1r, and Casp1) in injured joints of female (but not male) mice. Analysis of published single-cell RNA-seq data identified elevated catecholamine-associated gene expression in resident-like synovial-IFP macrophages after injury. Injury substantially altered synovial fluid metabolites by increasing amino acids, peptides, sphingolipids, phospholipids, bile acids, and dicarboxylic acids, but these changes were not appreciably altered by isoproterenol. Intra-articular injection of either isoproterenol or saline increased mechanical allodynia in female mice, whereas neither substance affected male mice. ConclusionsAcute βAR activation altered synovial-IFP transcription in a sex and injury-dependent manner, suggesting that women with PTOA may be more sensitive than men to treatments targeting sympathetic neural signaling pathways.

Transcript-dependent effects of the CALCA gene on the progression of post-traumatic osteoarthritis in mice

Osteoarthritis represents a chronic degenerative joint disease with exceptional clinical relevance. Polymorphisms of the CALCA gene, giving rise to either a procalcitonin/calcitonin (PCT/CT) or a calcitonin gene-related peptide alpha (αCGRP) transcript by alternative splicing, were reported to be associated with the development of osteoarthritis. The objective of this study was to investigate the role of both PCT/CT and αCGRP transcripts in a mouse model of post-traumatic osteoarthritis (ptOA). WT, αCGRP−/− and CALCA−/− mice were subjected to anterior cruciate ligament transection (ACLT) to induce ptOA of the knee. Mice were sacrificed 4 and 8 weeks post-surgery, followed by micro-CT and histological evaluation. Here we show that the expression of both PCT/CT and αCGRP transcripts is induced in ptOA knees. CALCA−/− mice show increased cartilage degeneration and subchondral bone loss with elevated osteoclast numbers compared to αCGRP−/− and WT mice. Osteophyte formation is reduced to the same extent in CALCA−/− and αCGRP−/− mice compared to WT controls, while a reduced synovitis score is noticed exclusively in mice lacking CALCA. Our data show that expression of the PCT/CT transcript protects from the progression of ptOA, while αCGRP promotes osteophyte formation, suggesting that CALCA-encoded peptides may represent novel targets for the treatment of ptOA.

Collagen type II solution extracted from supercritical carbon dioxide decellularized porcine cartilage: regenerative efficacy on post-traumatic osteoarthritis model

Osteoarthritis (OA) of the knee is a common degenerative articular disorder and is one of the main causes of pain and functional disability. Cartilage damage is frequently linked to elevated osteoarthritis incidence. Supercritical carbon dioxide (scCO2) decellularized cartilage graft produced from the porcine cartilage is an ideal candidate for cartilage tissue engineering. In the present study, we derived collagen type II (Col II) solution from the scCO2 decellularized porcine cartilage graft (dPCG) and compared its efficacy with hyaluronic acid (HA) in the surgical medial meniscectomy (MNX) induced post-traumatic osteoarthritis (PTOA) model. Dose-dependent attenuation of the OA (12.3 ± 0.8) progression was observed in the intra‐articular administration of Col II solution (7.3 ± 1.2) which significantly decreased the MNX-induced OA symptoms similar to HA. The pain of the OA group (37.4 ± 2.7) was attenuated dose-dependently by Col II solution (45.9 ± 4.1) similar to HA (43.1 ± 3.5) as evaluated by a capacitance meter. Micro‐CT depicted a dose-dependent attenuation of articular cartilage damage by the Col II solution similar to HA treatment. A significant (p < 0.001) dose-dependent elevation in the bone volume was also observed in Col II solution-treated OA animals. The protective competence of Col II solution on articular cartilage damage is due to its significant (p < 0.001) increase in the expression of type II collagen, aggrecan and SOX‐9 similar to HA. To conclude, intra‐articular administration of type II collagen solution and HA reestablished the injured cartilage and decreased osteoarthritis progression in the experimental PTOA model.Graphical

Exercise-induced modulation of myokine irisin on muscle-bone unit in the rat model of post-traumatic osteoarthritis

Background and aimPost-traumatic osteoarthritis (PTOA) is a subtype of osteoarthritis (OA). Exercise may produce and release the myokine irisin through muscle fiber contraction. However, the effect of exercise-promoted irisin production on the internal interactions of the muscle–bone unit in PTOA studies remains unclear.MethodsEighteen 8-week-old Sprague–Dawley (SD) rats were randomly divided into three groups: Sham/sedentary (Sham/Sed), PTOA/sedentary (PTOA/Sed), and PTOA/treadmill-walking (PTOA/TW). The PTOA model was established by transection of anterior cruciate ligament (ACLT) and destabilization of medial meniscus (DMM). After 4 weeks of modeling, the PTOA/TW group underwent treadmill exercise (15 m/min, 30 min/d, 5 d/ week, 8 weeks), and the other two groups were free to move in the cage. Evaluation and correlation analysis of muscle, cartilage, subchondral bone and serological indexes were performed after euthanasia.ResultsEight weeks of treadmill exercise effectively alleviated the trauma-induced OA phenotype, thereby maintaining cartilage and subchondral bone integrity in PTOA, and reducing quadriceps atrophy and myofibril degradation. Exercise reversed the down-regulated expression of peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) and fibronectin type III structural domain protein 5 (FNDC5) in muscle tissue of PTOA rats, and increased the blood irisin level, and the irisin level was positively correlated with the expression of PGC-1α and FNDC5. In addition, correlation analysis showed that irisin metabolism level was strongly negatively correlated with Osteoarthritis Research Society International (OARSI) and subchondral bone loss, indicating that irisin may be involved in cartilage biology and PTOA-related changes in cartilage and subchondral bone. Moreover, the metabolic level of irisin was strongly negatively correlated with muscle fiber cross-sectional area (CSA), Atrogin-1 and muscle ring-finger protein-1(MuRF-1) expression, suggesting that irisin may alleviate muscle atrophy through autocrine action.ConclusionTreadmill exercise can alleviate the atrophy and degeneration of muscle fibers in PTOA rats, reduce the degradation of muscle fibrin, promote the expression of serum irisin, and alleviate the degeneration of articular cartilage and subchondral bone loss in PTOA rats. These results indicate that treadmill exercise can affect the process of PTOA by promoting the expression of myokine irisin in rat muscle–bone unit.

Diverse Response to Local Pharmacological Blockade of Sirt1 Cleavage in Age-Induced versus Trauma-Induced Osteoarthritis Female Mice.

Objective: Previous studies have shown that the cleavage of Sirt1 contributes to the development of osteoarthritis (OA). In fact, OA was effectively abrogated by the intra-articular (IA) administration of two compounds, one blocking Sirt1 cleavage (CA074me) and the other activating Sirt1 (SRT1720), using a post-traumatically induced model (PTOA) in young female mice. In this study, we attempted to understand if this local treatment is effective in preventing age-associated OA (AOA) progression and symptoms. Design: A group of 17-month-old female C57BL/6J mice were IA administered with CA074me and/or SRT1720 or their combination. Joint histopathological analysis and bone histomorphometry were carried out, with an assessment of knee mechanical hyperalgesia. A serum analysis for NT/CT Sirt1 was carried out along with immunohistochemistry for articular cartilage to detect p16INK4A or γH2A.X. Similarly, meniscal cartilage was monitored for Lef1 and Col1a1 deposition. The data were compared for young female mice subjected to post-traumatic OA (PTOA). Results: Similar to PTOA, combination-treated AOA exhibited improved knee hyperalgesia, yet structural improvements were undetected, corresponding to unchanged NT/CT Sirt1 serum levels. Both AOA and PTOA exhibited unchanged staining for nuclear p16INK4A or γH2A.X and lacked a correlation with OA severity. Contrarily to PTOA, the combination treatment with AOA did not exhibit a local reduction in the Lef1 and Col1 targets. Conclusions: When targeting Sirt1 cleavage, the PTOA and AOA models exhibited a similar pain response to the combination treatment; however, they displayed diverse structural outcomes for joint-related damage, related to Lef1-dependent signaling. Interestingly, nuclear p16INK4A was unaffected in both models, regardless of the treatment's effectiveness. Finally, these findings highlight the variations in the responses between two highly researched OA preclinical models, reflecting OA pathophysiology heterogeneity and variations in gender-related drug-response mechanisms.

Wnt5a deficiency in osteocalcin-expressing cells could not alleviate the osteoarthritic phenotype in a mouse model of post-traumatic osteoarthritis.

Wnt5a, which regulates the activities of osteoblasts and osteoclasts, is reportedly overexpressed in osteoarthritis (OA) tissues. The purpose of this study was to elucidate its role in the development of OA by deleting Wnt5a in osteocalcin (OCN)-expressing cells. Knee OA was induced by anterior cruciate ligament transection (ACLT) in OCN-Cre;Wnt5afl/fl knockout (Wnt5a-cKO) mice and control littermates. Eight weeks after surgery, histological changes, cell apoptosis, and matrix metabolism of cartilage were evaluated by toluidine blue, TUNEL staining, and im-immunohistochemistry analyses, respectively. In addition, the subchondral bone microarchitecture of mice was examined by micro-computed tomography (micro-CT). Histological scores show substantial cartilage degeneration occurred in ACLT knees, coupled with decreased collagen type II expression and enhanced matrix metalloproteinase 13 expression, as well as higher proportions of apoptotic cells. Micro-CT results show that ACLT resulted in decreased bone mineral density, bone volume/trabecular volume, trabecular number, and structure model index of subchondral bones in both Wnt5a-cKO and control littermates; although Wnt5a-cKO mice display lower BMD and BV/TV values, no significant difference was observed between Wnt5a-cKO and control mice for any of these values. Our findings indicate that Wnt5a deficiency in OCN-expressing cells could not prevent an osteoarthritic phenotype in a mouse model of post-traumatic OA.

GDF8 inhibition enhances musculoskeletal recovery and mitigates posttraumatic osteoarthritis following joint injury.

Musculoskeletal disorders contribute substantially to worldwide disability. Anterior cruciate ligament (ACL) tears result in unresolved muscle weakness and posttraumatic osteoarthritis (PTOA). Growth differentiation factor 8 (GDF8) has been implicated in the pathogenesis of musculoskeletal degeneration following ACL injury. We investigated GDF8 levels in ACL-injured human skeletal muscle and serum and tested a humanized monoclonal GDF8 antibody against a placebo in a mouse model of PTOA (surgically induced ACL tear). In patients, muscle GDF8 was predictive of atrophy, weakness, and periarticular bone loss 6 months following surgical ACL reconstruction. In mice, GDF8 antibody administration substantially mitigated muscle atrophy, weakness, and fibrosis. GDF8 antibody treatment rescued the skeletal muscle and articular cartilage transcriptomic response to ACL injury and attenuated PTOA severity and deficits in periarticular bone microarchitecture. Furthermore, GDF8 genetic deletion neutralized musculoskeletal deficits in response to ACL injury. Our findings support an opportunity for rapid targeting of GDF8 to enhance functional musculoskeletal recovery and mitigate the severity of PTOA after injury.

A Reproducible Cartilage Impact Model to Generate Post-Traumatic Osteoarthritis in the Rabbit.

Post-traumatic osteoarthritis (PTOA) is responsible for 12% of all osteoarthritis cases in the United States. PTOA can be initiated by a single traumatic event, such as a high-impact load acting on articular cartilage, or by joint instability, as occurs with anterior cruciate ligament rupture. There are no effective therapeutics to prevent PTOA currently. Developing a reliable animal model of PTOA is necessary to better understand the mechanisms by which cartilage damage proceeds and to investigate novel treatment strategies to alleviate or prevent the progression of PTOA. This protocol describes an open, drop tower-based rabbit femoral condyle impact model to induce cartilage damage. This model delivered peak loads of 579.1 ± 71.1 N, and peak stresses of 81.9 ± 10.1 MPa with a time-to-peak load of 2.4 ± 0.5 ms. Articular cartilage from impacted medial femoral condyles (MFCs) had higher rates of apoptotic cells (p = 0.0058) and possessed higher Osteoarthritis Research Society International (OARSI) scores of 3.38 ± 1.43 compared to the non-impacted contralateral MFCs (0.56 ± 0.42), and other cartilage surfaces of the impacted knee (p < 0.0001). No differences in OARSI scores were detected among the non-impacted articular surfaces (p > 0.05).

New approach to testing treatments for osteoarthritis: FastOA

Animal models of post traumatic osteoarthritis have shown many promising treatments for disease, but human trials have mostly failed to identify effective treatments. This viewpoint suggests that the frequent failure...

Senolytic treatment reduces oxidative protein stress in an aging male murine model of post-traumatic osteoarthritis.

Senolytic drugs are designed to selectively clear senescent cells (SnCs) that accumulate with injury or aging. In a mouse model of osteoarthritis (OA), senolysis yields a pro-regenerative response, but the therapeutic benefit is reduced in aged mice. Increased oxidative stress is a hallmark of advanced age. Therefore, here we investigate whether senolytic treatment differentially affects joint oxidative load in young and aged animals. We find that senolysis by a p53/MDM2 interaction inhibitor, UBX0101, reduces protein oxidative modification in the aged arthritic knee joint. Mass spectrometry coupled with protein interaction network analysis and biophysical stability prediction of extracted joint proteins revealed divergent responses to senolysis between young and aged animals, broadly suggesting that knee regeneration and cellular stress programs are contrarily poised to respond as a function of age. These opposing responses include differing signatures of protein-by-protein oxidative modification and abundance change, disparate quantitative trends in modified protein network centrality, and contrasting patterns of oxidation-induced folding free energy perturbation between young and old. We develop a composite sensitivity score to identify specific key proteins in the proteomes of aged osteoarthritic joints, thereby nominating prospective therapeutic targets to complement senolytics.

A pilot study to determine the optimal dose of scAAVIL-1ra in a large animal model of post-traumatic osteoarthritis

Gene therapy approaches using adeno-associated viral vectors have been successfully tested in the equine post-traumatic osteoarthritis (PTOA) model. Owing to differences in the levels of transgene expression and adverse tissue reactions observed in published studies, we sought to identify a safe therapeutic dose of scAAVIL-1ra in an inflamed and injured joint that would result in improved functional outcomes without any adverse events. scAAVIL-1ra was delivered intra-articularly over a 100-fold range, and horses were evaluated throughout and at the end of the 10-week study. A dose-related increase in IL-1ra levels with a decrease in PGE2 levels was observed, with the peak IL-1ra concentration being observed 7 days post-treatment in all groups. Perivascular infiltration with mononuclear cells was observed within the synovial membrane of the joint treated with the highest viral dose of 5 × 1012 vg, but this was absent in the lower-dosed joints. The second-highest dose of scAAVeqIL-1ra 5 × 1011 vg demonstrated elevated IL-1ra levels without any cellular response in the synovium. Taken together, the data suggest that the 10-fold lower dose of 5 × 1011vg scAAVIL-1ra would be a safe therapeutic dose in an equine model of PTOA.

Sexual dimorphism of the synovial transcriptome underpins greater PTOA disease severity in male mice following joint injury

Osteoarthritis is a disease with sex-dependent prevalence and severity in both humans and animal models. We sought to elucidate sex differences in synovitis, mechanical sensitization, structural damage, bone remodeling, and the synovial transcriptome in the anterior cruciate ligament rupture (ACLR) mouse model of post-traumatic osteoarthritis (PTOA). Male and female 12-week-old C57/BL6J mice were randomized to Sham or noninvasive ACLR with harvests at 7d or 28d post-ACLR (n=9 per sex in each group - Sham, 7d ACLR, 28d ACLR). Knee hyperalgesia, mechanical allodynia, and intra-articular MMP activity (via intravital imaging) were measured longitudinally. Trabecular and subchondral bone remodeling and osteophyte formation were assessed by µCT. Histological scoring of PTOA and synovitis and anti-MMP13 immunostaining were performed. NaV1.8-Cre;tdTomato mice were used to document localization and sprouting of nociceptors. Bulk RNA-seq of synovium in Sham, 7d, and 28d post-ACLR, and contralateral joints (n=6 per group per sex) assessed injury-induced and sex-dependent gene expression. Male mice exhibited more severe joint damage at 7d and 28d and more severe synovitis at 28d, accompanied by 19% greater MMP activity, 8% lower knee hyperalgesia threshold, and 43% lower hindpaw withdrawal threshold in injured limbs compared to female injured limbs. Females had injury-induced catabolic responses in trabecular and subchondral bone, whereas males exhibited 133% greater normalized osteophyte volume relative to females and sclerotic remodeling of trabecular and subchondral bone. NaV1.8+ nociceptor sprouting in subchondral bone and medial synovium was induced by injury and comparable between sexes. RNA-seq of synovium demonstrated similar injury-induced transcriptomic programs between the sexes at 7d, but only female mice exhibited a transcriptomic signature indicative of synovial inflammatory resolution by 28d, whereas males had persistent pro-inflammatory, pro-fibrotic, pro-neurogenic, and pro-angiogenic gene expression. Male mice exhibited more severe overall joint damage and pain behavior after ACLR, which was associated with persistent activation of synovial inflammatory, fibrotic, and neuroangiogenic processes, implicating persistent synovitis in driving sex differences in murine PTOA.

High-fat diet aggravates the severity of the in vitro posttraumatic osteoarthritis model through macrophagic FBW7.

Osteoarthritis (OA) is a prevalent and distressing chronic degenerative joint disorder characterized by damaged articular cartilage and inflamed joints. Among risk factors, obesity has emerged as the second-leading contributor to OA after age. Obesity is believed to play a key role in the development and progression of OA. This study aimed to investigate the role and underlying mechanisms of high-fat diet (HFD)-induced obesity in the development of OA. Our findings revealed that HFD could aggravate the destabilization of the medial meniscus (DMM)-induced damage in the mouse model of obesity. Similar results were observed when macrophages obtained from HFD-fed mice were cocultured with cartilage and subsequently stimulated with interleukin-1β (IL-1β). Mechanistically, we observed a decrease in the expression of intraarticular macrophagic FBW7, which was implicated in the aggravation of OA in the HFD-fed animal. Furthermore, by modulating the immune status of macrophages, we found that reversing the macrophagic expression of FBW7 in these cells can alleviate the chondrocyte damage. In conclusion, this study provides novel insights into the pathological mechanisms underlying HFD-related OA development by identifying the role of FBW7 in synovial macrophages. These findings open up new avenues for research and therapeutic interventions targeting HFD-related OA.

Poster 150: Immunophenotyping of Synovial Tissue in Adolescents Undergoing ACL Reconstruction Reveals Distinct Inflammatory Responses to Injury

Objectives: Tears of the anterior cruciate ligament (ACL) are common in the pediatric athletic population. In spite of trends towards early surgical reconstruction to minimize the risk of secondary meniscus and cartilage injuries, long term disruption of knee joint homeostasis and progression to post- traumatic osteoarthritis (PTOA) occurs in 50-90% of patients - a particularly challenging problem in this young cohort. The exact pathogenesis of PTOA after ACL injury and reconstruction remains unknown. Evidence from animal models of PTOA suggests a critical role of synovial inflammatory infiltration in both the onset and progression of PTOA. However, there is a scarcity of in vivo human data documenting the synovial inflammatory response to ACL injury. One recent study has looked at immune cell composition in synovial fluid, but there are no comparable publications using synovial tissue. Our primary objective was to use flow cytometry with an advanced immune cell panel to provide a snapshot of the cellular composition of immune infiltrates in the synovium of adolescent patients at the time of ACL reconstruction. Our secondary objective was to look for correlations between demographic and injury parameters and inflammatory cell composition. Methods: Patients between the ages of 12 and 18 years who were undergoing primary ACL reconstruction were enrolled in an IRB-approved prospective study. Exclusion criteria included multiligament knee injuries, revision ACL reconstruction, history of prior knee surgery, and history of inflammatory joint disease. Demographic variables and intraoperative data were recorded. At the time of surgery, an arthroscopic synovial biopsy from the prefemoral synovium was obtained. Synovium tissue was enzymatically digested into a single cell suspension for immune profiling by multicolor flow cytometry. Data was acquired on a BD lsrFortessa flow cytometer and analyzed with FlowJo v10. Statistical analyses were performed with GraphPad Prism including parametric ANOVA (one-way or two- way), simple linear regression, Kruskal Wallis analysis of variance, Mann Whitney T-test, and parallel Principal component analyses (Eigenvalue with Monte Carlo simulation). Heat map variable scaling was calculated relative to the mean and standard deviation of all measurements. Results: Twelve patients were prospectively enrolled in the study (33% male, 67% female, n = 12). Mean age was 15.5 +/- 2.02 years. Median time between injury and surgery was 35.5 days (23.3-67.5). Meniscal tears were present in 58.3% of patients (8.3% medial only, 33.3% lateral only, 16.7% both). Cells isolated from synovium were 36.8% fibroblasts, 9.89% fibrocytes, and 36.0% hematopoietic lineage cells (Fig. 1A). Within the hematopoietic lineage, the dominant cell types were monocytes, macrophages, and T-cells (Fig. 1B). Principle component analysis (PCA) revealed three immunophenotypes. Immunotype 1 had higher mast cell content than other groups and fewer inflammatory cells such as T cells, B cells, and macrophages. Immunotype 2 had strong T/B cell infiltration and lower levels of innate immune cells. These patients also had the highest overall levels of inflammatory cells (Figure 2). Immunotype 3 had a strong innate immune response - in particular higher levels of M2 polarized macrophages - and significantly fewer T/B cells. There were no obvious effects of age, skeletal maturity, or the presence of meniscal tears on immunotype (Table 1). There was a trend towards a correlation between delay in surgery and classification within immunotype 1 (p = 0.09, Table 1). Conclusions: Our study provides the first in vivo analysis of inflammatory cell behavior in the synovium of ACL injured knees. In young patients undergoing primary ACL reconstruction, three immunotypes were discovered. Our pilot data suggest a possible relationship between delay in surgery and a high prevalence of synovial mast cells. This would be of particular interest since mast cells are known to play a role in the development of OA and possibly PTOA. In the more acute setting, two separate immunotypes were encountered, one with heavy inflammation and a predominance of T/B cells, and the other with more moderate inflammation and a higher percentage of innate immune cells / M2 polarized macrophages. These were not clearly tied to patient demographic factors, and could represent intrinsic differences in the response to injury. Future work investigating the correlation between initial immunotype and clinical outcomes – in particular the development of PTOA – may provide insight into the role of synovial inflammation in post-ACL joint health and degeneration, as well as provide strategies for personalized treatments in the prevention of PTOA. [Table: see text]

3D T1 relaxation time measurements in an equine model of subtle post-traumatic osteoarthritis using MB-SWIFT.

The aim of this study is to assess whether articular cartilage changes in an equine model of post-traumatic osteoarthritis (PTOA), induced by surgical creation of standard (blunt) grooves, and very subtle sharp grooves, could be detected with ex vivo T1 relaxation time mapping utilizing three-dimensional (3D) readout sequence with zero echo time. Grooves were made on the articular surfaces of the middle carpal and radiocarpal joints of nine mature Shetland ponies and osteochondral samples were harvested at 39 weeks after being euthanized under respective ethical permissions. T1 relaxation times of the samples (n = 8 + 8 for experimental and n = 12 for contralateral controls) were measured with a variable flip angle 3D multiband-sweep imaging with Fourier transform sequence. Equilibrium and instantaneous Young's moduli and proteoglycan (PG) content from OD of Safranin-O-stained histological sections were measured and utilized as reference parameters for the T1 relaxation times. T1 relaxation time was significantly (p < 0.05) increased in both groove areas, particularly in the blunt grooves, compared with control samples, with the largest changes observed in the superficial half of the cartilage. T1 relaxation times correlated weakly (Rs ≈ 0.33) with equilibrium modulus and PG content (Rs ≈ 0.21). T1 relaxation time in the superficial articular cartilage is sensitive to changes induced by the blunt grooves but not to the much subtler sharp grooves, at the 39-week timepoint post-injury. These findings support that T1 relaxation time has potential in detection of mild PTOA, albeit the most subtle changes could not be detected.