Inflammation is a primary driver of osteoarthritis (OA), and no therapies exist to halt or delay disease progression or substantially ameliorate the chronic pain, inflammation and disability that are characteristic of disease. Soluble CD14 (sCD14), a co-factor that enhances inflammatory toll-like receptor signaling, is present in synovial fluid in patients with OA and positively associates with joint space narrowing, synovial macrophage content, and pain. In this study, we show that increased sCD14 within human synovial fluid correlates with joint effusion volume and increased knee hyperalgesia. Next, we evaluated CD14 as a potential therapeutic target in three pre-clinical models of post traumatic OA (PTOA), using both genetic deficiency and pharmacologic blockade to modulate its activity. We demonstrate that deficiency or blockade of CD14 results in significant protection from increased evoked pain behaviors and from OA driven mobility impairments (i.e. decreased cage activity) across models that differ in severity, and across male and female cohorts. Using flow cytometry, single cell transcriptomics, and spatial proteomics, we further show that CD14-deficiency drastically influences the local synovial inflammatory landscape post-injury, reducing monocyte and macrophage populations and modulating local fibroblast populations. Targeting CD14 via genetic deficiency or therapeutic blockade revealed no substantial protection, but no worsening, of cartilage degeneration. Ultimately, our results provide strong support that targeting synovial inflammation through blockade of CD14 can safely ameliorate OA pain and disability after a pre-disposing injury. One Sentence Summary:The study demonstrates the key role of CD14 in pain, mobility loss, and inflammation in PTOA, and demonstrates the therapeutic potential of CD14 blockade for OA pain relief.

During osteoarthritis (OA) pathogenesis, the infrapatellar fat pad (IFP) undergoes fibrotic changes that might contribute to pain development. Recent studies have demonstrated that thrombospondin-4 (TSP-4), first detected in the extracellular matrix of cartilage and released during its degradation, has been implicated in the pathogenesis of pain. Therefore, we analyzed TSP-4 levels in the IFP and synovial fluid and correlated this data with IFP fibrosis and knee joint pain. IFP and synovial fluid were collected from patients undergoing total knee replacement surgery. Total WOMAC total and pain scores were determined preoperatively. IFP sections were stained using standard Masson trichrome and hematoxylin/eosin dyes to assess fibrotic changes, number of vessels and lymphocytic infiltration. TSP-4 expression in the IFP was detected immunohistochemically. TSP-4 in synovial fluid samples was quantified using ELISA. TSP-4 was detectable in human IFP tissue at the protein level and its expression levels showed a positive correlation with the degree of tissue fibrosis. Regarding the degree of fibrosis and TSP-4-stained areas, four patient subgroups could be distinguished. Notably, moderate levels of TSP-4 expression were already detectable in samples exhibiting a low degree of fibrosis. There was no significant correlation between TSP-4 staining intensity in IFP and pain. There was no correlation between TSP-4 staining intensity and synovial fluid TSP-4 concentrations. A significant relationship between synovial fluid TSP-4 concentration and pain intensity was only found in female OA patients. TSP-4 has been detected in the IFP for the first time. The correlation between TSP-4 expression and fibrotic severity indicates a possible involvement of TSP-4 in the development of fibrosis. Although TSP-4 within the IFP may not directly mediate pain, its presence in synovial fluid could be of functional relevance in pain-related mechanisms. Further analysis of synovial fluid and even serum samples from larger patient populations will determine whether TSP-4 could serve as a biomarker for pain or potentially represent a novel target for analgesic therapies.

Osteoarthritis (OA) is a chronic degenerative disease, characterized by cartilage degradation, synovial inflammation, and subchondral bone changes, leading to joint pain and stiffness. Recent research implicates the sympathetic nervous system (SNS), particularly the neurotransmitter noradrenaline (NE), in OA pathogenesis. Chronic stress, a prevalent health issue, sustains SNS activation and stimulates the hypothalamic-pituitary-adrenal (HPA) axis, elevating stress hormones such as NE and cortisol, both of which exert catabolic effects on joint cells in vitro . This study investigates in vivo the impact of chronic stress on OA progression. OA was induced in 12-week old male C57BL/6J mice using the destabilization of the medial meniscus (DMM), with sham-operated mice as controls. Half of the mice underwent 12 weeks of chronic unpredictable mild stress (CUMS). Stress was validated by monitoring behavioral changes, body weight gain, and stress hormone levels including splenic NE and serum corticosterone (CORT). OA progression was assessed by histological evaluation of cartilage and synovial tissue, micro-computed tomography (μCT), pain behavior using the Dynamic weight-bearing system, and immune cell profiling via flow cytometry in the synovium and dorsal root ganglia (DRGs). CUMS significantly elevated stress parameters, with increased splenic NE ( p <0.01) and serum corticosterone (p<0.01). Interestingly, OA alone increases NE levels in DMM mice. Stress led to reduced activity and impaired body weight gain (p<0.01). Chronic stress exacerbated OA progression, with increased cartilage damage (p<0.01), synovial inflammation (p<0.01), and calcified cartilage thickness (p<0.05). Pain behavior worsened in stressed DMM mice, as shown by reduced weight-bearing on the affected paw (p<0.01) and diminished rearing time (p<0.01). In the synovium, CUMS elevated proinflammatory CD3 + T-cells (p<0.01) and CD11b + F4/80 + Ly6C high proinflammatory monocytes (p<0.01), while reducing anti-inflammatory CD301 + CD150 - M2a macrophages (p<0.05). These immune changes appeared early after 4 weeks but partially regressed by week 12. In the DRGs, CUMS increased proinflammatory CD11b + Ly6G + neutrophils (p<0.05) and CD3 + T-cells (p<0.01). Our results demonstrate that chronic stress exacerbates OA progression, indicated by increased cartilage degeneration, calcification, synovial inflammation, and pain. The observed elevation in synovial inflammation and pain can be attributed to the stress-induced mobilization and migration of pro-inflammatory immune cells to the synovium and DRGs. Interestingly, OA itself induced an increased release of NE and CORT. Our findings highlight the bidirectional relationship between OA and chronic stress, suggesting that stress can both contribute to OA development and be a consequence of the disease. Targeting stress pathways may represent a promising therapeutic strategy for managing OA symptoms.

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.

Purpose (the aim of the study): Osteoarthritis (OA) affects more than 590 million people worldwide, and the economic and personal burden of the disease continues to increase, because there is still no causal treatment. OA is a chronic degenerative disease of the whole joint characterized by articular cartilage degradation, synovial inflammation, subchondral bone (SB) sclerosis and osteophyte formation, resulting in the main symptoms of joint stiffness and chronic pain. Our recent study demonstrated that dampening of sympathetic nervous activity by peripheral sympathectomy resulted in exacerbated SB sclerosis in a murine DMM model.

To assess pain in mouse models of bone fractures, currently applied assessment batteries use combinations of clinical signs with spontaneous behaviours and model-specific behaviours, including walking and weight-bearing behaviour. Rearing behaviour - an upright position on the hindlimbs - has a motivational and an ambulatory component. Thus, rearing behaviour might have the potential to be an indicator for model-specific pain in mouse fracture models. To date, the assessment of rearing behaviour in bone fracture models using mice is only scarcely described. In this study, we aimed to determine whether the duration of rearing behaviour is affected by osteotomy of the femur in male and female C57BL/6N mice with external fixation (rigid vs. flexible) and could be an additional sign for model-specific pain, such as the presence of limping. Rearing duration was significantly decreased after osteotomy in male and female mice at 24 h, 48 h and 72 h, but was not affected by anaesthesia/analgesia alone. In male mice, the relative rearing duration increased over 72 h (both fixations) and at 10 days in the rigid fixation group but remained significantly lower in the flexible fixation group. In contrast, in female mice, no increase in rearing duration was observed within 72 h and at 10 days post-osteotomy, independent of the fixation. We did not identify any association between relative rearing time and presence or absence of limping. In summary, our results do not provide sufficient evidence that altered rearing behaviour might be an indicative sign for pain in this model.

IntroductionOsteoarthritis (OA) is a chronic degenerative disease of the entire joint leading to joint stiffness and pain (PMID:33571663). Recent evidence suggests that the sympathetic nervous system (SNS) plays a role in the pathogenesis of OA (PMID:34864169). A typical cause for long-term hyperactivity of the SNS is chronic stress. To study the contribution of increased sympathetic activity, we analyzed the progression of OA in chronically stressed mice.MethodWe induced OA in male C57BL/6J mice by destabilizing the medial meniscus (DMM)(PMID:17470400) and exposed half of these mice to chronic unpredictable mild stress (CUMS)(PMID:28808696). Control groups consisted of sham-operated mice with and without CUMS exposure. After 12 weeks, CUMS efficacy was determined by assessing changes in body weight gain and activity of mice, measuring splenic norepinephrine and serum corticosterone levels. OA progression was studied by histological analysis of cartilage degeneration and synovitis, and by μCT to evaluate changes in calcified cartilage and subchondral bone microarchitecture. A dynamic weight-bearing system was used to assess OA-related pain.ResultCUMS resulted in significantly decreased body weight gain and activity, as well as increased splenic norepinephrine and serum corticosterone concentrations compared to the respective controls. Surprisingly, already DMM alone resulted in elevated stress hormone levels. CUMS significantly exacerbated cartilage degeneration and synovial inflammation and increased OA pain in DMM mice. The underlying cellular and molecular mechanisms are currently being analyzed using FACS, single cell RNAseq, and spatial proteomics.ConclusionOverall, chronic stress exacerbates OA severity and pain. Moreover, increased levels of stress hormones were observed in OA mice without CUMS induction, suggesting a complex bi-directional interaction between the SNS and OA. Targeting the autonomic nervous system, such as attenuating the SNS but also stimulating the activity of the parasympathetic nervous system, as a counterpart of the SNS, may therefore be promising for novel preventive or causal treatments of OA.

Purpose: Osteoarthritis (OA) is a multifactorial disease classically associated to cartilage destruction. However, the effect of this disease in other tissues of the articular organ has been scarcely studied. In the present work, we used the dislocation of the medial meniscus model (DMM) in the mouse to induce post-traumatic OA (PTOA). We assessed the cellular immune response occurring locally in the synovial membrane and capsule, regionally, by studying the popliteal and inguinal lymph nodes and, systemically, in peripheral blood during early (3 days, 4 weeks) and late (12 weeks) development of PTOA.

Substance P (SP) and a calcitonin‐related gene alpha (αCGRP−/−) are implicated in musculoskeletal pain perception and were shown to have different effects on the pathogenesis of osteoarthritis (OA). However, it has not been investigated, whether deficiency for SP or αCGRP impacts pain‐related behavior and well‐being as well as gait during development of experimental OA. We induced OA in the right knee of wild‐type (WT) mice and mice either deficient for SP (tachykinin 1, Tac‐1) or αCGRP (male, n = 8 per genotype) by destabilizing the medial meniscus (DMM). We monitored body weight and food and water intake as indicators of wellbeing, determined nest building and composite pain score, and performed CatWalk gait analysis over 12 weeks. Cartilage degeneration was determined by OARSI scoring. The 12‐week post‐DMM, cartilage degradation in the medial compartment was significantly reduced in Tac1−/− mice compared to the WT and to αCGRP−/− mice, coinciding with highest unloading of the operated limb in Tac1−/−. Behavioral and gait analysis revealed only minor differences between the genotypes. Paw print area was most prominently reduced in Tac1−/− over the observation period; at 12 weeks, we found a significant reduction in normalized print area in Tac1−/− compared to presurgery and to the WT at the same time‐point. Calculated weight bearing was significantly reduced only in Tac1−/−. Overall, we observed minor impact of DMM on gait and behavior in the present study. The reduced cartilage damage in the absence of SP might be in part due to reduced loading, however, the mechanism is not clear yet.

Collaborative manual image analysis by multiple experts in different locations is an essential workflow in biomedical science. However, sharing the images and writing down results by hand or merging results from separate spreadsheets can be error-prone. Moreover, blinding and anonymization are essential to address subjectivity and bias. Here, we propose a new workflow for collaborative image analysis using a lightweight online tool named Tyche. The new workflow allows experts to access images via temporarily valid URLs and analyze them blind in a random order inside a web browser with the means to store the results in the same window. The results are then immediately computed and visible to the project master. The new workflow could be used for multi-center studies, inter- and intraobserver studies, and score validations.

Osteoarthritis (OA) is among the most frequent diseases of the musculoskeletal system. Degradation of cartilage extracellular matrix (ECM) is a hallmark of OA. During the degradation process, intact/full-length proteins and proteolytic fragments are released which then might induce different downstream responses via diverse receptors, therefore leading to different biological consequences. Collagen type II and the proteoglycan aggrecan are the most abundant components of the cartilage ECM. However, over the last decades, a large number of minor components have been identified and for some of those, a role in the manifold processes associated with OA has already been demonstrated. To date, there is still no therapy able to halt or cure OA. A better understanding of the matrikine landscape occurring with or even preceding obvious degenerative changes in joint tissues is needed and might help to identify molecules that could serve as biomarkers, druggable targets, or even be blueprints for disease modifying drug OA drugs. For this narrative review, we screened PubMed for relevant literature in the English language and summarized the current knowledge regarding the function of selected ECM molecules and the derived matrikines in the context of cartilage and OA.

Adequate pain management is essential for ethical and scientific reasons in animal experiments and should completely cover the period of expected pain without the need for frequent re-application. However, current depot formulations of Buprenorphine are only available in the USA and have limited duration of action. Recently, a new microparticulate Buprenorphine formulation (BUP-Depot) for sustained release has been developed as a potential future alternative to standard formulations available in Europe. Pharmacokinetics indicate a possible effectiveness for about 72 h. Here, we investigated whether the administration of the BUP-Depot ensures continuous and sufficient analgesia in two mouse fracture models (femoral osteotomy) and could, therefore, serve as a potent alternative to the application of Tramadol via the drinking water. Both protocols were examined for analgesic effectiveness, side effects on experimental readout, and effects on fracture healing outcomes in male and female C57BL/6N mice. The BUP-Depot provided effective analgesia for 72 h, comparable to the effectiveness of Tramadol in the drinking water. Fracture healing outcome was not different between analgesic regimes. The availability of a Buprenorphine depot formulation for rodents in Europe would be a beneficial addition for extended pain relief in mice, thereby increasing animal welfare.

In recent years, the infrapatellar fat pad (IFP) has gained increasing research interest. The contribution of the IFP to the development and progression of knee osteoarthritis (OA) through extensive interactions with the synovium, articular cartilage, and subchondral bone is being considered. As part of the initiation process of OA, IFP secretes abundant pro-inflammatory mediators among many other factors. Today, the IFP is (partially) resected in most total knee arthroplasties (TKA) allowing better visualization during surgical procedures. Currently, there is no clear guideline providing evidence in favor of or against IFP resection. With increasing numbers of TKAs, there is a focus on preventing adverse postoperative outcomes. Therefore, anatomic features, role in the development of knee OA, and consequences of resecting versus preserving the IFP during TKA are reviewed in the following article.

Purpose: Cartilage extracellular matrix (ECM) degradation by matrix metalloproteinase (MMPs) is a hallmark of osteoarthritis (OA). Although main MMPs in cartilage degradation were identified, their individual role during degradation of specific components is unclear. Besides collagens and proteoglycans, a large number of gylcoproteins such as the thrombospondin family members thrombospondin-4 (TSP-4) and -5 (TSP-5, also known as cartilage oligomeric matrix protein COMP) play important roles in cartilage homeostasis.

Abstract Background Scientific image analysis is crucial in many research fields but it can be hampered by subjectivity and bias. Thus, image analysis should be performed anonymized by multiple observers and images displayed in a random order. Yet in radiology for example most PACS-viewers display patient metadata which could influence the analysis. Although desirable, including multiple observers can be logistically difficult as files have to be provided including spreadsheets to document the analyzed parameters. Methods To address these challenges we created a browser-based program displaying scientific images anonymized and in a random order. Observers are invited by the project master via a project-specific URL that is only temporarily valid. The project master can design an unlimited number of project-specific questions which will be displayed next to each image. As the analysis is performed online, results from all observers are summarized in real-time and the data are immediately visible to the project master. In this study, we present the process of analysis using histological and radiological images and compare Tyche with the traditional “offline” desktop bound way. Results Tyche allows users to upload anonymized images and create single choice and numeric questions specifically for these images. Via temporarily active URLs, access to the data can be provided to observers who can analyze images within any web browser using the integrated tools (zoom, length, etc.). Next to each image, the defined questions and answers are displayed and results are immediately visible. Aspects of traditional image analysis like laborious file sharing via clouds or e-mail with observers and data gathering via potentially error-prone spreadsheets become obsolete. Performing a histologic image analysis, no difference in results comparing Tyche with the offline analysis were found. Conclusion Tyche is a lightweight, web-based tool that facilitates anonymized image analysis by multiple observers and reduces subjectivity and bias.

Osteoarthritis (OA) is the most common form of arthritis and a major source of pain and disability in the adult population. There is a significant unmet medical need for the development of effective pharmacological therapies for the treatment of OA. In addition to spontaneously occurring animal models of OA, many experimental animal models have been developed to provide insights into mechanisms of pathogenesis and progression. Many of these animal models are also being used in the drug development pipeline. Here, we provide an overview of commonly used and emerging preclinical small animal models of OA and highlight the strengths and limitations of small animal models in the context of translational drug development. There is limited information in the published literature regarding the technical reliability of these small animal models and their ability to accurately predict clinical drug development outcomes. The cost and complexity of the available models however is an important consideration for pharmaceutical companies, biotechnology startups, and contract research organizations wishing to incorporate preclinical models in target validation, discovery, and development pipelines. Further considerations relevant to industry include timelines, methods of induction, the key issue of reproducibility, and appropriate outcome measures needed to objectively assess outcomes of experimental therapeutics. Preclinical small animal models are indispensable tools that will shine some light on the pathogenesis of OA and its molecular endotypes in the context of drug development. This paper will focus on small animal models used in preclinical OA research. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.

Matrix metalloproteinases (MMPs) play crucial roles in tissue homeostasis and pathologies by remodeling the extracellular matrix. Previous studies have demonstrated the biological activities of MMP-derived cleavage products. Furthermore, specific fragments can serve as biomarkers. Therefore, an in vitro cleavage assay to identify substrates and characterize cleavage patterns could provide important insight in disease-relevant mechanisms and the identification of novel biomarkers. In the pathogenesis of osteoarthritis (OA), MMP-2, -8, -9 and -13 are of vital importance. However, it is unclear which protease can cleave which matrix component. To address this question, we established an in vitro cleavage assay using recombinantly expressed MMPs and the two cartilage matrix components, COMP and thrombospondin-4. We found a time- and concentration-dependent degradation and an MMP-specific cleavage pattern for both proteins. Cleavage products can now be enriched and purified to investigate their biological activity. To verify the in vivo relevance, we compared the in vitro cleavage patterns with serum and synovial fluid from OA patients and could indeed detect fragments of similar size in the human samples. The cleavage assay can be adapted to other MMPs and substrates, making it a valuable tool for many research fields.

The outcomes of animal experiments can be influenced by a variety of factors. Thus, precise reporting is necessary to provide reliable and reproducible data. Initiatives such as the ARRIVE guidelines have been enrolled during the last decade to provide a road map for sufficient reporting. To understand the sophisticated process of bone regeneration and to develop new therapeutic strategies, small rodents, especially mice, are frequently used in bone healing research. Since many factors might influence the results from those studies, we performed a systematic literature search from 2010 to 2019 to identify studies involving mouse femoral fracture models (stable fixation) and evaluated the reporting of general and model-specific experimental details. 254 pre-selected publications were systematically analyzed, showing a high reporting accuracy for the used mouse strain, the age or developmental stage and sex of mice as well as model-specific information on fixation methods and fracturing procedures. However, reporting was more often insufficient in terms of mouse substrains and genetic backgrounds of genetically modified mice, body weight, hygiene monitoring/immune status of the animal, anesthesia, and analgesia. Consistent and reliable reporting of experimental variables in mouse fracture surgeries will improve scientific quality, enhance animal welfare, and foster translation into the clinic.

Following severe trauma, fracture healing is impaired because of overwhelming systemic and local inflammation. Glucocorticoids (GCs), acting via the glucocorticoid receptor (GR), influence fracture healing by modulating the trauma-induced immune response. GR dimerization-dependent gene regulation is essential for the anti-inflammatory effects of GCs. Therefore, we investigated in a murine trauma model of combined femur fracture and thoracic trauma, whether effective GR dimerization influences the pathomechanisms of trauma-induced compromised fracture healing. To this end, we used mice with decreased GR dimerization ability (GR dim ). The healing process was analyzed by cytokine/chemokine multiplex analysis, flow cytometry, gene-expression analysis, histomorphometry, micro-computed tomography, and biomechanical testing. GR dim mice did not display a systemic or local hyper-inflammation upon combined fracture and thorax trauma. Strikingly, we discovered that GR dim mice were protected from fracture healing impairment induced by the additional thorax trauma. Collectively and in contrast to previous studies describing the beneficial effects of intact GR dimerization in inflammatory models, we report here an adverse role of intact GR dimerization in trauma-induced compromised fracture healing.