Cartilage extracellular matrix-derived matrikines in osteoarthritis.

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.

Generation of Matrix Degradation Products Using an In Vitro MMP Cleavage Assay.

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.

Reduced Terminal Complement Complex Formation in Mice Manifests in Low Bone Mass and Impaired Fracture Healing.

The terminal complement complex (TCC) is formed on activation of the complement system, a crucial arm of innate immunity. TCC formation on cell membranes results in a transmembrane pore leading to cell lysis. In addition, sublytic TCC concentrations can modulate various cellular functions. TCC-induced effects may play a role in the pathomechanisms of inflammatory disorders of the bone, including rheumatoid arthritis and osteoarthritis. In this study, we investigated the effect of the TCC on bone turnover and repair. Mice deficient for complement component 6 (C6), an essential component for TCC assembly, and mice with a knockout of CD59, which is a negative regulator of TCC formation, were used in this study. The bone phenotype was analyzed in vivo, and bone cell behavior was analyzed ex vivo. In addition, the mice were subjected to a femur osteotomy. Under homeostatic conditions, C6-deficient mice displayed a reduced bone mass, mainly because of increased osteoclast activity. After femur fracture, the inflammatory response was altered and bone formation was disturbed, which negatively affected the healing outcome. By contrast, CD59-knockout mice only displayed minor skeletal alterations and uneventful bone healing, although the early inflammatory reaction to femur fracture was marginally enhanced. These results demonstrate that TCC-mediated effects regulate bone turnover and promote an adequate response to fracture, contributing to an uneventful healing outcome.

Distinct Effects of IL-6 Classic and Trans-Signaling in Bone Fracture Healing.

Bone healing is a complex process with closely linked phases of inflammation, regeneration, and remodeling. IL-6 may crucially regulate this process; however, the underlying mechanisms are unclear. IL-6 signals are transmitted via the transmembrane glycoprotein 130 by two distinct mechanisms: classic signaling using the membrane-anchored IL-6 receptor and trans-signaling using its soluble form. Herein, we investigated the hypothesis that IL-6 classic and trans-signaling have different functions during bone healing. To investigate fracture healing, 12-week-old C57BL/6J mice underwent a femur osteotomy. To study the function of IL-6 during the inflammatory phase, either an anti-IL-6 antibody, which inhibits IL-6 classic and trans-signaling, or soluble glycoprotein 130 fusion protein, which selectively blocks trans-signaling, was injected after 30 minutes and 48 hours. To analyze IL-6 effects in the repair phase, compounds were injected from day 7 onwards. Global IL-6 inhibition in the early phase after fracture reduced systemic inflammation, the recruitment of immune cells, and bone regeneration, resulting in delayed fracture healing. Global IL-6 inhibition during the repair phase disturbed bone formation and remodeling. In contrast, inhibition of IL-6 trans-signaling exerted minor effects on the immune response and did not influence bone repair, suggesting that the classic pathway accounts for most of the effects observed after global IL-6 inhibition. Our results reveal that IL-6 classic signaling, but not IL-6 trans-signaling, is essential for bone repair.

Induced global deletion of glucocorticoid receptor impairs fracture healing.

Although endogenous glucocorticoids (GCs) are important regulators of bone integrity and the immune system, their role in bone repair after fracture-a process highly dependent on inflammation and bone formation-is unclear. Because most effects of GCs are mediated by the glucocorticoid receptor (GR), we used an inducible global GR knockout (GRgtROSACreERT2) mouse model to eliminate endogenous GC action in all cells contributing to bone repair. The healing process was analyzed by cytokine/chemokine multiplex analysis, flow cytometry, histology, gene-expression analysis, microcomputed tomography, and biomechanical analysis. We observed increased early systemic and local inflammatory responses, as well as a significantly higher number of T cells infiltrating the fracture callus. Later in the healing process, we found impaired endochondral ossification in the absence of the GR, leading to persistent cartilage in the calli of the GRgtROSACreERT2 mice, decreased bending stiffness, and a significantly lower proportion of healed bones. Collectively, our data show that the absence of the GR significantly impairs fracture healing associated with a defective cartilage-to-bone transition, underscoring an important role of GCs during fracture healing.-Rapp, A. E., Hachemi, Y., Kemmler, J., Koenen, M., Tuckermann, J., Ignatius, A. Induced global deletion of glucocorticoid receptor impairs fracture healing.

Autologous Mesenchymal Stroma Cells Are Superior to Allogeneic Ones in Bone Defect Regeneration.

The application of autologous mesenchymal stem cells (MSC) for the treatment of bone defects requires two invasive procedures and several weeks of ex vivo cell expansion. To overcome these limitations, the administration of allogeneic MSC may be attractive, because they are anticipated to be immunoprivileged. Because preclinical studies using various animal models are conflicting with respect to the efficacy of allogeneic MSC, we investigated whether autologous and allogeneic human MSC (hMSC) are equally effective in regenerating bone in a humanized mouse model resembling the human immune system. Applying autologous and allogeneic hMSC in critically sized femoral defects, we found that allogeneic hMSC elicited a mild immune response early after implantation, whereas early angiogenic processes were similar in both treatments. At later healing time points, the transplantation of allogeneic hMSC resulted in less bone formation than autologous hMSC, associated with a reduced expression of the osteogenic factor Runx2 and impaired angiogenesis. We found by species-specific staining for collagen-type-1α2 that MSCs of either source did not synthesize new bone matrix, indicating an indirect contribution of transplanted hMSC to bone regeneration. In conclusion, our data suggest that the application of autologous hMSC is superior to that of allogeneic cells for bone defect treatment.

Evaluating rearing behaviour as a model-specific pain indicator in mouse osteotomy models.

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.

A Lightweight Browser-Based Tool for Collaborative and Blinded Image Analysis.

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.

A buprenorphine depot formulation provides effective sustained post-surgical analgesia for 72 h in mouse femoral fracture models.

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.

The Corpus Adiposum Infrapatellare (Hoffa's Fat Pad)-The Role of the Infrapatellar Fat Pad in Osteoarthritis Pathogenesis.

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.

Animal Models of Osteoarthritis Part 1-Preclinical Small Animal Models: Challenges and Opportunities for Drug Development.

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.

Systematic review on the reporting accuracy of experimental details in publications using mouse femoral fracture models.

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.

Intact Glucocorticoid Receptor Dimerization Is Deleterious in Trauma-Induced Impaired Fracture Healing.

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 (GRdim). The healing process was analyzed by cytokine/chemokine multiplex analysis, flow cytometry, gene-expression analysis, histomorphometry, micro-computed tomography, and biomechanical testing. GRdim mice did not display a systemic or local hyper-inflammation upon combined fracture and thorax trauma. Strikingly, we discovered that GRdim 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.

Molecular mechanisms of glucocorticoids on skeleton and bone regeneration after fracture.

Glucocorticoid hormones (GCs) have profound effects on bone metabolism. Via their nuclear hormone receptor - the GR - they act locally within bone cells and modulate their proliferation, differentiation, and cell death. Consequently, high glucocorticoid levels - as present during steroid therapy or stress - impair bone growth and integrity, leading to retarded growth and glucocorticoid-induced osteoporosis, respectively. Because of their profound impact on the immune system and bone cell differentiation, GCs also affect bone regeneration and fracture healing. The use of conditional-mutant mouse strains in recent research provided insights into the cell-type-specific actions of the GR. However, despite recent advances in system biology approaches addressing GR genomics in general, little is still known about the molecular mechanisms of GCs and GR in bone cells. Here, we review the most recent findings on the molecular mechanisms of the GR in general and the known cell-type-specific actions of the GR in mesenchymal cells and their derivatives as well as in osteoclasts during bone homeostasis, GC excess, bone regeneration and fracture healing.

Inhibition of Midkine Augments Osteoporotic Fracture Healing.

The heparin-binding growth and differentiation factor midkine (Mdk) is proposed to negatively regulate osteoblast activity and bone formation in the adult skeleton. As Mdk-deficient mice were protected from ovariectomy (OVX)-induced bone loss, this factor may also play a role in the pathogenesis of postmenopausal osteoporosis. We have previously demonstrated that Mdk negatively influences bone regeneration during fracture healing. Here, we investigated whether the inhibition of Mdk using an Mdk-antibody (Mdk-Ab) improves compromised bone healing in osteoporotic OVX-mice. Using a standardized femur osteotomy model, we demonstrated that Mdk serum levels were significantly enhanced after fracture in both non-OVX and OVX-mice, however, the increase was considerably greater in osteoporotic mice. Systemic treatment with the Mdk-Ab significantly improved bone healing in osteoporotic mice by increasing bone formation in the fracture callus. On the molecular level, we demonstrated that the OVX-induced reduction of the osteoanabolic beta-catenin signaling in the bony callus was abolished by Mdk-Ab treatment. Furthermore, the injection of the Mdk-Ab increased trabecular bone mass in the skeleton of the osteoporotic mice. These results implicate that antagonizing Mdk may be useful for the therapy of osteoporosis and osteoporotic fracture-healing complications.

Fracture Healing Is Delayed in Immunodeficient NOD/scid­ï»¿IL2Rγcnull Mice.

Following bone fracture, the repair process starts with an inflammatory reaction at the fracture site. Fracture healing is disturbed when the initial inflammation is increased or prolonged, whereby, a balanced inflammatory response is anticipated to be crucial for fracture healing, because it may induce down-stream responses leading to tissue repair. However, the impact of the immune response on fracture healing remains poorly understood. Here, we investigated bone healing in NOD/scid-IL2Rγcnull mice, which exhibit severe defects in innate and adaptive immunity, by biomechanical testing, histomorphometry and micro-computed tomography. We demonstrated that NOD/scid-IL2Rγcnull mice exhibited normal skeletal anatomy and a mild bone phenotype with a slightly reduced bone mass in the trabecular compartment in comparison to immunocompetent Balb/c mice. Fracture healing was impaired in immunodeficient NOD/scid-IL2Rγcnull mice. Callus bone content was unaffected during the early healing stage, whereas it was significantly reduced during the later healing period. Concomitantly, the amount of cartilage was significantly increased, indicating delayed endochondral ossification, most likely due to the decreased osteoclast activity observed in cells isolated from NOD/scid-IL2Rγcnull mice. Our results suggest that--under aseptic, uncomplicated conditions--the immediate immune response after fracture is non-essential for the initiation of bone formation. However, an intact immune system in general is important for successful bone healing, because endochondral ossification is delayed in immunodeficient NOD/scid-IL2Rγcnull mice.

Analgesia via blockade of NGF/TrkA signaling does not influence fracture healing in mice.

Abatement of fracture-related pain is important in patient welfare. However, the frequently used non-steroidal anti-inflammatory drugs are considered to impair fracture healing through blockade of cyclooxygenase-2. An alternative for fracture-related pain treatment may be blockade of nerve growth factor (NGF)/neurotrophic tyrosine kinase receptor type 1 (TrkA) signaling. Because the effect of blocking this signal-pathway on bone healing has not been extensively investigated, we addressed this issue by applying neutralizing antibodies that target NGF and TrkA, respectively, in a mouse fracture model. Mice with a knock-in for human TrkA underwent femur osteotomy and were randomly allocated to phosphate-buffered-saline, anti-NGF-antibody, or anti-TrkA-antibody treatment. The analgesic effect of the antibodies was determined from the activity and the ground reaction force of the operated limb. The effect of antibody administration on fracture healing was assessed by histomorphometry, micro-computed tomography, and biomechanics. NGF/TrkA-signaling blockade had no negative effect on fracture healing as callus formation and maturation were not altered. Mice treated with anti-TrkA antibody displayed significantly greater activity on post-operative day 2 compared to PBS treatment indicating effective analgesia. Our data indicate, that blockade of NGF/TrkA signaling via specific neutralizing antibodies for pain reduction during fracture healing does not influence fracture healing.