Disrupted intestinal epithelial barrier function has been proposed to be integral to rheumatoid arthritis (RA) progression and pathogenesis. To further define the molecular pathways in synovial inflammation and the response of the intestinal tissues, we have used a rat model of mono-joint inflammatory arthritis, induced by intra-articular injection of Complete Freund's adjuvant (CFA). The predominant inflammatory response of a single injection of the adjuvant into the knee joint resulted in rapid and reproducible formation of a fibrotic myeloid-infiltrated synovial pannus. Our aim was to determine how intestinal tissues, including the proximal and distal ileum and distal colon, responded to inflammatory changes in the synovium in a temporally coordinated manner by comparing their transcriptomic landscapes using RNASeq analyses. We confirmed the timeline of joint inflammation by knee joint swelling measurement, increased synovial fluid levels of bikunin (a component of both the acute phase protein pre-alpha-inhibitor and inter-alpha-inhibitor) and demonstrated a self-correcting response of trabecular and cortical bone to the CFA challenge. Intestine-specific responses were monitored by 16S microbiome amplicon sequencing, histopathology for mucus layer integrity, and immune cell immunohistochemistry. We present data that shows the intestinal tissue displays an allostatic response to the acute joint inflammation and was region specific. The ileum primarily responded with increased mucus secretion and silencing of T-cell specific pathways, whereas the colon showed a transient upregulation of macrophages, with a broader suppression of immune related and metabolic pathway related transcripts. Interestingly, many neuropathways were activated early but then suppressed later in both the ileum and colon. There were only insignificant changes in the fecal microbiome composition in ileum or colon post-CFA administration. In summary, our data show for the first time a suppression of intestinal inflammatory and immune responses following the induction of joint inflammation and only minimal and transient changes in the microbiome. The results help clarify the molecular responses of intestinal tissues to inflammatory stresses that accompany the pathogenesis of inflammatory joint diseases.

BACKGROUND:Multiple fixation techniques are available for securing the graft on the patellar side during medial patellofemoral ligament reconstruction. The double curved semi-patellar tunnels (DCST) method is a novel fixation technology, but its biomechanical properties have not yet been investigated. METHODS:72 porcine patellae and bovine tendons were used to establish MPFL reconstruction models employing the three fixation methods: suture anchor (SA), single semi-patellar tunnel (SST), DCST. Load to failure and cyclic loading testing were performed to compare biomechanical performance among the groups. FINDINGS:In the Load to failure testing, the DCST group demonstrated a higher ultimate failure load than both SST and SA (P = 0.006, P = 0.016). The DCST group showed lower maximum load elongation compared to SST group (P = 0.048), but not significantly different from SA (P = 0.147). Stiffness was greater with the DCST group than with SST and SA (P = 0.002, P = 0.003). Under cyclic loading, both SA and SST exhibited higher elongation compared to the DCST group (P = 0.007, P = 0.019). Failures of the DCST group were predominantly due to graft tear. INTERPRETATION:All three fixation techniques provided stable graft fixation under the tested experimental conditions. The DCST technique provides higher initial fixation strength and stability in vitro. These results appear worthy of further clinical investigation.

OBJECTIVE:Nerve growth factor (NGF), a key mediator of pain, is increased in osteoarthritic (OA) joints. Antibodies against NGF show analgesic effects in painful knee OA, but clinical development was stopped because of side effects in the joints. Knowledge about the biologic effects of NGF on joint tissues is limited. Therefore, we explored the effects of repeated intra-articular (IA) injections of NGF into naïve murine knee joints on sensitization, joint innervation, and histopathology. METHODS:Naïve 10- to 15-week-old male wildtype C57BL/6 mice were injected with NGF (50 or 500 ng) or vehicle IA twice a week for four weeks, and effects on knee swelling, knee hyperalgesia, joint histopathology, and bone were assessed. Single cell RNA sequencing (scRNAseq) of the synovium was performed. NaV1.8-tdTomato reporter mice were used to assess joint innervation. Dorsal root ganglia (DRGs) of mice underwent bulk RNAseq after three IA injections of NGF or vehicle. RESULTS:Compared with vehicle, repeated IA injections of NGF caused dose-dependent increases in knee swelling, knee hyperalgesia, synovial pathology, bone mineral density in the medial subchondral bone, and medial preosteophytes, but no overt cartilage damage. NGF caused increased sprouting of nociceptors in the medial synovium that was preceded by up-regulation of axonal growth pathways in the DRGs. ScRNAseq of the synovium revealed up-regulated genes related to neuronal sprouting, synovial fibrosis, and ossification, with a key role for lining fibroblasts. CONCLUSION:In naïve mouse knees, NGF induced many pathologic changes observed in OA, including nociceptor sprouting, suggesting a critical role for NGF in OA pathogenesis.

Heat stroke (HS) causes high mortality via multiple organ dysfunction syndrome, with intestinal barrier dysfunction as an early trigger of systemic inflammation. However, the roles of annexin A1 (ANXA1) in HS-induced intestinal barrier dysfunction remain unclear. Therefore, this study investigated the roles of ANXA1 in HS-induced intestinal barrier dysfunction through integrating single-cell transcriptomics and in vivo experiments. The single-cell RNA sequencing (scRNA-seq) data were used to analyze the cell subpopulation changes between healthy controls and HS patients, as well as between the inflammatory sites and non-inflammatory sites in inflammatory bowel disease. In vivo, the H&E staining, flow cytometry, ELISA, and Western blot were used to determine the pathological injuries, the proportion of neutrophils and monocytes, inflammatory cytokine levels, and related protein expression levels in the colon tissue. Immunofluorescence co-localization analysis was performed to detect the interaction between ANXA1 and immune cells. The scRNA-seq data showed that the proportion of neutrophil and CD14+Mono subpopulations was increased in HS patients. Concurrently, the ANXA1 expression was significantly upregulated in multiple immune cell subpopulations, including the CD14+Mono, CD16+Mono, mDC, MDSC, and neutrophil subpopulations. Cell-cell communication analysis further demonstrated that the ANXA1-formyl peptide receptor 2 (FPR2) ligand-receptor pairs were the dominant mode of interaction during HS. Importantly, there was partial overlap between cell populations expressing ANXA1 and barrier genes. In addition, ANXA1 was positively associated with M2 monocyte phenotype in both inflammatory and non-inflammatory sites. After establishing the HS model, there were some alterations in the colon tissue, including the exacerbated pathological injuries, upregulated ANXA1 and FPR2 protein expression levels, downregulated ZO-1 and occludin protein expression levels, increased inflammatory cytokine levels, and activated TLR4/ERK/NF-κB pathway. Immunofluorescence co-localization analysis revealed that the mean density of ANXA1, CD14+, and Ly6G in the colon tissue of HS mice was significantly elevated compared with the control group, and the co-localization of ANXA1 with CD14+ and ANXA1 with Ly6G was enhanced. The treatment of Boc1 led to a dramatic reduction in ANXA1 mean density, a further increase in CD14+ and Ly6G mean density, and a reduction in the co-localization of ANXA1 with CD14+ and Ly6G. Apart from reversing the ANXA1 and FPR2 protein expression levels, inhibiting ANXA1 aggravated the damaging effects of HS on the colon tissue. In conclusion, ANXA1 protected against HS-induced intestinal barrier dysfunction by regulating neutrophil-monocyte interaction and inhibiting TLR4/ERK/NF-κB, with ANXA1-FPR2 as a key axis, which offers a novel target and strategy for the clinical treatment of HS-induced intestinal barrier dysfunction.

Osteoarthritis (OA) is a leading cause of chronic pain, affecting millions worldwide. Conventional pharmacological treatments provide only short-term relief and pose risks with long-term use, highlighting the need for safer, sustained analgesic strategies. Voltage-gated sodium channels (NaV), particularly NaV1.7, are key mediators of OA pain and represent a promising therapeutic target. Neosaxitoxin (NSTX), a potent site-1 sodium channel blocker (S1SCB), exhibits high affinity for NaV1.7. Clinical studies demonstrate that subcutaneous administration of NSTX can safely block sensory pain in healthy individuals, but its anesthetic effect is short-lived, limiting its clinical utility for OA. Here, we report the first use of NSTX for OA pain relief with a local intra-articular (IA) delivery approach. A low dose (10 pg) produced effective pain relief in a post-traumatic OA mouse model, as assessed by knee hyperalgesia testing. However, the effect persisted for only a few hours even with a 100-fold higher dose, highlighting the need for sustained delivery. To prolong this short-lived pain relief and overcome the challenges of encapsulating small, hydrophilic S1SCBs like NSTX, we developed a sustained-release platform using alginate-poly(lactic-co-glycolic acid) microparticles (AlgPLGA-MPs). Incorporation of a negatively charged alginate core in the inner aqueous phase enhanced NSTX encapsulation through electrostatic interactions and minimized burst release. IA injection of NSTX encapsulated AlgPLGA-MP in a murine OA model produced significant pain relief for up to one week. To further prolong joint residence time, the microparticle surface was functionalized with cationic avidin (AvAlgPLGA-MP) that reversed the net surface charge from anionic to cationic. This modification promoted electrostatic binding to negatively charged synovial matrix components and prolonged intra-joint retention time, while maintaining biocompatibility. Together, these results establish NSTX as a highly potent analgesic for chronic OA pain and present alginate-core PLGA microparticles as a safe and effective sustained-release platform. This delivery system holds broad translational potential for other small, hydrophilic analgesics and therapeutic agents.

With the rising prevalence of osteoarthritis (OA), this disease places a substantial economic burden on society. Nevertheless, there is still a lack of effective therapeutic agents to completely cure OA. This study thus aims to identify potential genetic targets that might influence the progression of OA. Using Mendelian randomization (MR), the correlation between the levels of macrophage colony-stimulating factor (CSF-1) and its associated genetic loci with OA at different anatomical sites was investigated. Data were primarily obtained from the arcOGEN Consortium, the UK Biobank, and other relevant sources. Additionally, Summary Data-based Mendelian Randomization (SMR), Mendelian Randomization, and colocalization analysis were used to examine the relation of 16,989 cis-eQTL genes from the eQTLGen database with OA at various locations, to facilitate the identification of potential genetic loci that might affect the progression of OA. Through MR analysis of two databases, we found a direct correlation between CSF-1 levels and knee OA (OR = 1.08, 95

BACKGROUND:Nerve growth factor (NGF), a key mediator of pain and inflammation, is increased in joints with osteoarthritis (OA). Neutralizing NGF with monoclonal antibodies has shown analgesic effects in painful knee OA, but clinical development was stopped due to side effects in the joints. Knowledge about the biological effects of long-term exposure of joint tissues to NGF is limited. Therefore, we aimed to explore the effects of repeated intra-articular (IA) injections of NGF into the knee joints of healthy mice on pain and sensitization, as well as joint innervation and structure. METHODS:We conducted five experiments in male C57BL/6 mice. In Experiment 1, NGF (50ng or 500ng) or vehicle was injected IA into the knee of naive wildtype (WT) mice, twice a week for 4 weeks. We assessed knee swelling, knee hyperalgesia and histopathology. In Experiment 2, mice were injected with 500ng NGF or vehicle, twice a week for 4 weeks and microCT of the knee was performed. In Experiment 3, NaV1.8-tdTomato reporter mice were injected with 500ng NGF or vehicle, twice a week for 4 weeks, and joint innervation was assessed. In Experiment 4, WT mice received 500ng NGF or vehicle twice a week for 4 weeks and were used for single cell RNA sequencing (scRNAseq) of the synovium. In Experiment 5, L3-L5 DRGs of mice that received 3 IA injections of 500ng NGF or vehicle twice a week were used for bulk RNA sequencing. RESULTS:Repeated bi-weekly IA injections of NGF caused knee hyperalgesia in naïve mice. NGF caused dose-dependent knee swelling, synovial pathology, increased bone mineral density and trabecular bone thickness in the medial subchondral bone, growth of pre-osteophytes in the medial compartment, but no cartilage degeneration. NGF injection caused sprouting of NaV1.8+ neurons in the medial but not the lateral synovium. ScRNAseq of the synovium revealed upregulated genes related to neuronal sprouting, synovial fibrosis and ossification, confirming histopathological findings. Bulk RNA seq of DRG showed upregulated pathways related to axonal growth. CONCLUSIONS:In healthy mouse knees, NGF induced mechanical sensitization, synovitis, neoinnervation in the medial synovium, subchondral bone changes and pre-osteophyte growth in the medial compartment, thus capturing many pathological changes observed in OA, except cartilage damage.

Background:Load-bearing structural degradation is crucial in knee osteoarthritis (KOA) progression, yet limited prediction models use load-bearing tissue radiomics for radiographic (structural) KOA incident. Purpose:We aim to develop and test a Load-Bearing Tissue plus Clinical variable Radiomic Model (LBTC-RM) to predict radiographic KOA incidents. Study design:Risk prediction study. Methods:The 700 knees without radiographic KOA at baseline were included from Osteoarthritis Initiative cohort. We selected 2164 knee MRIs during 4-year follow-up. LBTC-RM, which integrated MRI features of meniscus, femur, tibia, femorotibial cartilage, and clinical variables, was developed in total development cohort (n = 1082, 542 cases vs. 540 controls) using neural network algorithm. Final predictive model was tested in total test cohort (n = 1082, 534 cases vs. 548 controls), which integrated data from five visits: baseline (n = 353, 191 cases vs. 162 controls), 3 years prior KOA (n = 46, 19 cases vs. 27 controls), 2 years prior KOA (n = 143, 77 cases vs. 66 controls), 1 year prior KOA (n = 220, 105 cases vs. 115 controls), and at KOA incident (n = 320, 156 cases vs. 164 controls). Results:In total test cohort, LBTC-RM predicted KOA incident with AUC (95 % CI) of 0.85 (0.82-0.87); with LBTC-RM aid, performance of resident physicians for KOA prediction were improved, with specificity, sensitivity, and accuracy increasing from 50 %, 60 %, and 55 %-72 %, 73 %, and 72 %, respectively. The LBTC-RM output indicated an increased KOA risk (OR: 20.6, 95 % CI: 13.8-30.6, p < .001). Radiomic scores of load-bearing tissue raised KOA risk (ORs: 1.02-1.9) from 4-year prior KOA whereas 3-dimensional feature score of medial meniscus decreased the OR (0.99) of KOA incident at KOA confirmed. The 2-dimensional feature score of medial meniscus increased the ORs (1.1-1.2) of KOA symptom score from 2-year prior KOA. Conclusions:We provided radiomic features of load-bearing tissue to improved KOA risk level assessment and incident prediction. The model has potential clinical applicability in predicting KOA incidents early, enabling physicians to identify high-risk patients before significant radiographic evidence appears. This can facilitate timely interventions and personalized management strategies, improving patient outcomes. The Translational Potential of this Article:This study presents a novel approach integrating longitudinal MRI-based radiomics and clinical variables to predict knee osteoarthritis (KOA) incidence using machine learning. By leveraging deep learning for auto-segmentation and machine learning for predictive modeling, this research provides a more interpretable and clinically applicable method for early KOA detection. The introduction of a Radiomics Score System enhances the potential for radiomics as a virtual image-based biopsy tool, facilitating non-invasive, personalized risk assessment for KOA patients. The findings support the translation of advanced imaging and AI-driven predictive models into clinical practice, aiding early diagnosis, personalized treatment planning, and risk stratification for KOA progression. This model has the potential to be integrated into routine musculoskeletal imaging workflows, optimizing early intervention strategies and resource allocation for high-risk populations. Future validation across diverse cohorts will further enhance its clinical utility and generalizability.

ObjectiveKnee 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.MethodsSham 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.ResultsProgressive 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.ConclusionThese 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.

ObjectiveSynovial pathology has been linked to osteoarthritis (OA) pain in patients. Microscopic grading systems for synovial changes in human OA have been described, but a standardized approach for murine models of OA is needed. We sought to develop a reproducible approach and set of minimum recommendations for reporting of synovial histopathology in mouse models of OA.MethodsCoronal and sagittal sections from male mouse knee joints subjected to destabilization of medial meniscus (DMM) or partial meniscectomy (PMX) were collected as part of other studies. Stains included Hematoxylin and Eosin (H&E), Toluidine Blue (T-Blue) and Safranin O/Fast Green (Saf-O). Four blinded readers graded pathological features (hyperplasia, cellularity, and fibrosis) at specific anatomic locations. Inter-reader agreement of each feature score was determined.ResultsThere was acceptable to very good agreement when using 3-4 individual readers. After DMM and PMX, expected medial predominant changes in hyperplasia and cellularity were observed, with fibrosis noted at 12 weeks post-PMX. Synovial changes were consistent from section to section in the mid-joint area. When comparing stains, H&E and T-blue resulted in better agreement compared to Saf-O stain.ConclusionsTo account for the pathologic and anatomic variability in synovial pathology and allow for a more standardized evaluation that can be compared across studies, we recommend evaluating a minimum set of 3 pathological features at standardized anatomic areas. Further, we suggest reporting individual feature scores separately before relying on a single summed “synovitis” score. H&E or T-blue are preferred, inter-reader agreement for each feature should be considered.

Articular cartilage defects caused by trauma or osteoarthritis remain a significant challenge in clinical practice due to their poor self-healing capacity. Current clinical approaches are generally limited by their complex procedures or certain postoperation complications. Therefore, it is crucial to develop an effective strategy for one-step cartilage repair with long-term functional cartilage regeneration. This study reports the creation of an injectable hydrogel composed of cartilage decellularized extracellular matrix (CdECM) and hyaluronic acid methacrylate (HAMA). In the presence of photoinitiator ruthenium (Ru)/sodium persulfate (SPS), visible light (450 nm) not only triggered the polymerization of CdECM and HAMA to form stable hydrogel in 1 min but also facilitated the tyrosine cross-links between biotissue and CdECM without any further moiety grafting, which cannot be achieved by routinely used photoinitiators and provides the hybrid hydrogel with bioadhesive properties. This dual-network hybrid hydrogel demonstrated robust adhesion strength to cartilage and significantly enhanced mechanical performance in comparison to that of CdECM or HAMA alone. Further in vitro culture demonstrated the promotive effect of hybrid hydrogel on encapsulated porcine bone marrow mesenchymal stem cells toward chondrogenic differentiation after 21 days of incubation compared with HAMA. Through in vivo subcutaneous implantation in a mouse model for two and 4 weeks, we further illustrated that the hybrid hydrogel has improved biocompatibility in comparison to HAMA alone. Thus, the presented hydrogel is a promising biomaterial for cartilage regeneration through minimally invasive arthroscopic injection in clinical practice.