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The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA’s 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU ‘on the ground’ in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

With increasing age, the risk of bone fractures increases while regenerative capacity decreases. This variation in healing potential appears to be linked to adaptive immunity, but the underlying mechanism is still unknown. This study sheds light on immunoaging/inflammaging, which impacts regenerative processes in aging individuals. In an aged preclinical model system, different levels of immunoaging were analyzed to identify key factors that connect immunoaged/inflammaged conditions with bone formation after long bone fracture. Immunological facets, progenitor cells, the microbiome, and confounders were monitored locally at the injury site and systemically in relation to healing outcomes in 12-month-old mice with distinct individual levels of immunoaging. Bone tissue formation during healing was delayed in the immunoaged group and could be associated with significant changes in cytokine levels. A prolonged and amplified pro-inflammatory reaction was caused by upregulated immune cell activation markers, increased chemokine receptor availability and a lack of inhibitory signaling. In immunoaged mice, interleukin-22 was identified as a core cell signaling protein that played a central role in delayed healing. Therapeutic neutralization of IL-22 reversed this specific immunoaging-related disturbed healing. Immunoaging was found to be an influencing factor of decreased regenerative capacity in aged individuals. Furthermore, a novel therapeutic strategy of neutralizing IL-22 may successfully rejuvenate healing in individuals with advanced immune experiences.

Endochondral ossification requires coordinated mobilization of osteoblast precursors with blood vessels. During adult bone homeostasis, vessel adjacent osteoblast precursors respond to and are maintained by mechanical stimuli; however, the mechanisms by which these cells mobilize and respond to mechanical cues during embryonic development are unknown. Previously, we found that deletion of the mechanoresponsive transcriptional regulators, YAP and TAZ, from Osterix-expressing osteoblast precursors and their progeny caused perinatal lethality. Here, we show that embryonic YAP/TAZ signaling couples vessel-associated osteoblast precursor mobilization to angiogenesis in developing long bones. Osterix-conditional YAP/TAZ deletion impaired endochondral ossification in the primary ossification center but not intramembranous osteogenesis in the bone collar. Single-cell RNA sequencing revealed YAP/TAZ regulation of the angiogenic chemokine, Cxcl12, which was expressed uniquely in vessel-associated osteoblast precursors. YAP/TAZ signaling spatially coupled osteoblast precursors to blood vessels and regulated vascular morphogenesis and vessel barrier function. Further, YAP/TAZ signaling regulated vascular loop morphogenesis at the chondro-osseous junction to control hypertrophic growth plate remodeling. In human cells, mesenchymal stromal cell co-culture promoted 3D vascular network formation, which was impaired by stromal cell YAP/TAZ depletion, but rescued by recombinant CXCL12 treatment. Lastly, YAP and TAZ mediated mechanotransduction for load-induced osteogenesis in embryonic bone.

Skeletal development depends on coordinated angiogenesis and osteogenesis. Bone morphogenetic proteins direct bone development by activating SMAD1/5 signaling in osteoblasts. However, the role of SMAD1/5 in skeletal endothelium is unknown. Here, we found that endothelial cell-conditional SMAD1/5 depletion in juvenile mice caused metaphyseal and diaphyseal hypervascularity, resulting in altered cancellous and cortical bone formation. SMAD1/5 depletion induced excessive sprouting, disrupting the columnar structure of the metaphyseal vessels and impaired anastomotic loop morphogenesis at the chondro-osseous junction. Endothelial SMAD1/5 depletion impaired growth plate resorption and, upon long term depletion, abrogated osteoprogenitor recruitment to the primary spongiosa. Finally, in the diaphysis, endothelial SMAD1/5 activity was necessary to maintain the sinusoidal phenotype, with SMAD1/5 depletion inducing formation of large vascular loops, featuring elevated endomucin expression, ectopic tip cell formation, and hyperpermeability. Together, endothelial SMAD1/5 activity sustains skeletal vascular morphogenesis and function and coordinates growth plate remodeling and osteoprogenitor recruitment dynamics during bone growth. ispartof: bioRxiv ispartof: location:United States status: Published online

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.

ObjectiveDue to the small size of the murine knee joint, extracting the chondrocyte transcriptome from articular cartilage (AC) is a major technical challenge. In this study, we demonstrate a new and pragmatic approach of combining bulk RNA-sequencing (RNA-seq) and single cell (sc)RNA-seq to address this problem.DesignWe propose a new cutting strategy of the murine femur which produces three segments with a predictable mixed cell populations, where one segment contains AC and growth plate (GP) chondrocytes, another contains GP chondrocytes, and the last segment contains only bone and bone marrow. We analysed the bulk RNA-seq of the different segments to find common and distinct genes between the segments. Then, the segment containing AC chondrocytes was digested and analysed via scRNA-seq.ResultsDifferential expression analysis using bulk RNA-seq identified 350 candidate chondrocyte gene in the AC segment. Gene set enrichment analysis of these genes revealed biological processes related- and non-related to chondrocytes, including, cartilage development (adj. p-value: 3.45E-17) and endochondral bone growth (adj. p-value 1.22E-4), respectively. ScRNA-seq of the AC segment found a cluster of 131 cells containing mainly chondrocytes. This cluster had 759 differentially expressed genes which enriched for extracellular matrix organisation (adj. p-value 7.76E-40) and other joint development processes. The intersection of the gene sets of bulk- and scRNA-seq contained 75 genes, where all but ten genes were previously implicated in cartilage homeostasis or osteoarthritis (OA) progression.ConclusionsOur approach has the potential to detect the scarce disease phenotypes of chondrocytes in murine OA models.

After trauma, the formed fracture hematoma within the fracture gap contains all the important components (immune/stem cells, mediators) to initiate bone regeneration immediately. Thus, it is of great importance but also the most susceptible to negative influences. To study the interaction between bone and immune cells within the fracture gap, up-to-date in vitro systems should be capable of recapitulating cellular and humoral interactions and the physicochemical microenvironment (eg, hypoxia). Here, we first developed and characterized scaffold-free bone-like constructs (SFBCs), which were produced from bone marrow-derived mesenchymal stromal cells (MSCs) using a macroscale mesenchymal condensation approach. SFBCs revealed permeating mineralization characterized by increased bone volume (mu CT, histology) and expression of osteogenic markers (RUNX2, SPP1, RANKL). Fracture hematoma (FH) models, consisting of human peripheral blood (immune cells) mixed with MSCs, were co-cultivated with SFBCs under hypoxic conditions. As a result, FH models revealed an increased expression of osteogenic (RUNX2, SPP1), angiogenic (MMP2, VEGF), HIF-related (LDHA, PGK1), and inflammatory (IL6, IL8) markers after 12 and 48 hours co-cultivation. Osteogenic and angiogenic gene expression of the FH indicate the osteoinductive potential and, thus, the biological functionality of the SFBCs. IL-6, IL-8, GM-CSF, and MIP-1 beta were detectable within the supernatant after 24 and 48 hours of co-cultivation. To confirm the responsiveness of our model to modifying substances (eg, therapeutics), we used deferoxamine (DFO), which is well known to induce a cellular hypoxic adaptation response. Indeed, DFO particularly increased hypoxia-adaptive, osteogenic, and angiogenic processes within the FH models but had little effect on the SFBCs, indicating different response dynamics within the co-cultivation system. Therefore, based on our data, we have successfully modeled processes within the initial fracture healing phase in vitro and concluded that the cross-talk between bone and immune cells in the initial fracture healing phase is of particular importance for preclinical studies. (c) 2021 American Society for Bone and Mineral Research (ASBMR).

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term ‘the Three Rs’, which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years — not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA’s 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as ‘on the ground’ facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

The initial phase of fracture healing is crucial for the success of bone regeneration and is characterized by an inflammatory milieu and low oxygen tension (hypoxia). Negative interference with or prolongation of this fine-tuned initiation phase will ultimately lead to a delayed or incomplete healing such as non-unions which then requires an effective and gentle therapeutic intervention. Common reasons include a dysregulated immune response, immunosuppression or a failure in cellular adaptation to the inflammatory hypoxic milieu of the fracture gap and a reduction in vascularizing capacity by environmental noxious agents (e.g. rheumatoid arthritis or smoking). The hypoxia-inducible factor (HIF)-1α is responsible for the cellular adaptation to hypoxia, activating angiogenesis and supporting cell attraction and migration to the fracture gap. Here, we hypothesized that stabilizing HIF-1α could be a cost-effective and low-risk prevention strategy for fracture healing disorders. Therefore, we combined a well-known HIF-stabilizer – deferoxamine (DFO) – and a less known HIF-enhancer – macrophage migration inhibitory factor (MIF) – to synergistically induce improved fracture healing. Stabilization of HIF-1α enhanced calcification and osteogenic differentiation of MSCs in vitro. In vivo, only the application of DFO without MIF during the initial healing phase increased callus mineralization and vessel formation in a preclinical mouse-osteotomy-model modified to display a compromised healing. Although we did not find a synergistically effect of MIF when added to DFO, our findings provide additional support for a preventive strategy towards bone healing disorders in patients with a higher risk by accelerating fracture healing using DFO to stabilize HIF-1α.

25 years after the first Berlin Workshop on Developmental Toxicity this 10th Berlin Workshop aimed to bring together international experts from authorities, academia and industry to consider scientific, methodologic and regulatory aspects in risk assessment of developmental toxicity and to debate alternative strategies in testing developmental effects in the future. Proposals for improvement of the categorization of developmental effects were discussed as well as the update of the DevTox database as valuable tool for harmonization. The development of adverse outcome pathways relevant to developmental neurotoxicity (DNT) was debated as a fundamental improvement to guide the screening and testing for DNT using alternatives to animal methods. A further focus was the implementation of an in vitro mechanism-based battery, which can support various regulatory applications associated with the assessment of chemicals and mixtures. More interdisciplinary and translation research should be initiated to accelerate the development of new technologies to test developmental toxicity. Technologies in the pipeline are (i) high throughput imaging techniques, (ii) models for DNT screening tests, (iii) use of computer tomography for assessment of thoracolumbar supernumerary ribs in animal models, and (iv) 3D biofabrication of bone development and regeneration tissue models. In addition, increased collaboration with the medical community was suggested to improve the relevance of test results to humans and identify more clinically relevant endpoints. Finally, the participants agreed that this conference facilitated better understanding innovative approaches that can be useful for the identification of developmental health risks due to exposure to chemical substances.

Osteoarthritis (OA) is the most common form of musculoskeletal disease with significant healthcare costs and unmet needs in terms of early diagnosis and treatment. Many of the drugs that have been developed to treat OA failed in phase 2 and phase 3 clinical trials or produced inconclusive and ambiguous results. High throughput omics technologies are a powerful tool to better understand the mechanisms of the development of OA and other arthritic diseases. In this paper we outline the strategic reasons for increasingly applying deep phenotyping in OA for the benefit of gaining a better understanding of disease mechanisms and developing targeted treatments. This editorial is intended to launch a special themed issue of Osteoarthritis and Cartilage Open addressing the timely topic of “Advances in omics technologies for deep phenotyping in osteoarthritis”. High throughput omics technologies are increasingly being applied in mechanistic studies of OA and other arthritic diseases. Applying multi-omics approaches in OA is a high priority and will allow us to gather new information on disease pathogenesis at the cellular level, and integrate data from diverse omics technology platforms to enable deep phenotyping. We anticipate that new knowledge in this area will allow us to harness the power of Big Data Analytics and resolve the extremely complex and overlapping clinical phenotypes into molecular endotypes, revealing new information about the cellular taxonomy of OA and “druggable pathways”, thus facilitating future drug development.

The initial phase of fracture healing decides on success of bone regeneration and is characterized by an inflammatory milieu and low oxygen tension (hypoxia). Negative interference with or prolongation of this fine-tuned initiation phase will ultimately lead to a delayed or incomplete healing such as non-unions which then requires an effective and gentle therapeutic intervention. Common reasons include a dysregulated immune response, immunosuppression or a failure in cellular adaptation to the inflammatory hypoxic milieu of the fracture gap and a reduction in vascularizing capacity by environmental noxious agents (e.g. rheumatoid arthritis, smoking). The hypoxia-inducible factor (HIF)-1α is responsible for the cellular adaptation to hypoxia, activating angiogenesis and supporting cell attraction and migration to the fracture gap. Here, we hypothesized that stabilizing HIF-1α could be a cost-effective and low-risk prevention strategy of fracture healing disorders. Therefore, we combined a well-known HIF-stabilizer – deferoxamine (DFO) – and a less known HIF-enhancer – macrophage migration inhibitory factor (MIF) – to synergistically induce improved fracture healing. Stabilization of HIF-1α enhanced calcification and osteogenic differentiation of MSCs in vitro. In vivo, the application of DFO with or without MIF during the initial healing phase accelerated callus mineralization and vessel formation in a clinically relevant mouse-osteotomy-model in a compromised healing setting. Our findings provide support for a promising preventive strategy towards bone healing disorders in patients with a higher risk due to e.g. delayed neovascularization by accelerating fracture healing using DFO and MIF to stabilize HIF-1α.

The interaction between the bone and immune cells plays a crucial role in bone pathologies such as disturbed fracture healing. After a trauma, the initially formed fracture hematoma in the fracture gap contains all important components (immune/stem cells, mediators) to directly induce bone regeneration and is therefore of great importance but most susceptible to negative influences. Thus, reliable in vitro models are needed to study the underlying mechanisms and to predict the efficiency of novel therapeutic approaches. Since common bioengineering approaches exclude the immune component, we introduce an in vitro 3D fracture gap model which combines scaffold-free bone-like constructs with a fracture hematoma model consisting of human peripheral blood (immune cells) and bone marrow-derived mesenchymal stromal cells. Our in vitro 3D fracture gap model provides all osteogenic cues to induce the initial bone healing processes, which were further promoted by applying the osteoinductive deferoxamine (DFO). Thus, we were able to distinctly mimic processes of the initial fracture phase and demonstrated the importance of including the crosstalk between bone and immune cells.

Adequate tissue engineered models are required to further understand the (patho)physiological mechanism involved in the destructive processes of cartilage and subchondral bone during rheumatoid arthritis (RA). Therefore, we developed a human in vitro 3D osteochondral tissue model (OTM), mimicking cytokine-induced cellular and matrix-related changes leading to cartilage degradation and bone destruction in order to ultimately provide a preclinical drug screening tool. To this end, the OTM was engineered by co-cultivation of mesenchymal stromal cell (MSC)-derived bone and cartilage components in a 3D environment. It was comprehensively characterized on cell, protein, and mRNA level. Stimulating the OTM with proinflammatory cytokines, relevant in RA (tumor necrosis factor &alpha, interleukin-6, macrophage migration inhibitory factor ), caused cell- and matrix-related changes, resulting in a significantly induced the gene expression of lactate dehydrogenase A, interleukin-8 and tumor necrosis factor &alpha, in both, cartilage, and bone, while the matrix metalloproteases (MMPs) MMP1 and MMP3 expression were only induced in cartilage. Finally, application of target-specific drugs prevented the induction of inflammation and matrix-degradation. Thus, we here provide evidence that our human in vitro 3D OTM mimics cytokine-induced cell- and matrix-related changes&mdash, key features of RA&mdash, and may serve as a preclinical tool for the evaluation of both new targets and potential drugs in a more translational setup.

Both inflammatory diseases like rheumatoid arthritis (RA) and anti-inflammatory treatment of RA with glucocorticoids (GCs) or non-steroidal anti-inflammatory drugs (NSAIDs) negatively influence bone metabolism and fracture healing. Janus kinase (JAK) inhibition with tofacitinib has been demonstrated to act as a potent anti-inflammatory therapeutic agent in the treatment of RA, but its impact on the fundamental processes of bone regeneration is currently controversially discussed and at least in part elusive. Therefore, in this study, we aimed to examine the effects of tofacitinib on processes of bone healing focusing on recruitment of human mesenchymal stromal cells (hMSCs) into the inflammatory microenvironment of the fracture gap, chondrogenesis, osteogenesis and osteoclastogenesis. We performed our analyses under conditions of reduced oxygen availability in order to mimic the in vivo situation of the fracture gap most optimal. We demonstrate that tofacitinib dose-dependently promotes the recruitment of hMSCs under hypoxia but inhibits recruitment of hMSCs under normoxia. With regard to the chondrogenic differentiation of hMSCs, we demonstrate that tofacitinib does not inhibit survival at therapeutically relevant doses of 10−100 nM. Moreover, tofacitinib dose-dependently enhances osteogenic differentiation of hMSCs and reduces osteoclast differentiation and activity. We conclude from our data that tofacitinib may influence bone healing by promotion of hMSC recruitment into the hypoxic microenvironment of the fracture gap but does not interfere with the cartilaginous phase of the soft callus phase of fracture healing process. We assume that tofacitinib may promote bone formation and reduce bone resorption, which could in part explain the positive impact of tofacitinib on bone erosions in RA. Thus, we hypothesize that it will be unnecessary to stop this medication in case of fracture and suggest that positive effects on osteoporosis are likely.

Understanding the pathophysiological processes of cartilage degradation requires adequate model systems to develop therapeutic strategies towards osteoarthritis (OA). Although different in vitro or in vivo models have been described, further comprehensive approaches are needed to study specific disease aspects. This study aimed to combine in vitro and in silico modeling based on a tissue-engineering approach using mesenchymal condensation to mimic cytokine-induced cellular and matrix-related changes during cartilage degradation. Thus, scaffold-free cartilage-like constructs (SFCCs) were produced based on self-organization of mesenchymal stromal cells (mesenchymal condensation) and (i) characterized regarding their cellular and matrix composition or secondly (ii) treated with interleukin-1β (IL–1β) and tumor necrosis factor α (TNFα) for 3 weeks to simulate OA-related matrix degradation. In addition, an existing mathematical model based on partial differential equations was optimized and transferred to the underlying settings to simulate the distribution of IL–1β, type II collagen degradation and cell number reduction. By combining in vitro and in silico methods, we aimed to develop a valid, efficient alternative approach to examine and predict disease progression and effects of new therapeutics. publishedVersion

ObjectiveUnderstanding the pathophysiological processes of osteoarthritis (OA) require adequate model systems. Although different in vitro or in vivo models have been described, further comprehensive approaches are needed to study specific parts of the disease. This study aimed to combine in vitro and in silico modeling to describe cellular and matrix-related changes during the early phase of OA. We developed an in vitro OA model based on scaffold-free cartilage-like constructs (SFCCs), which was mathematically modeled using a partial differential equation (PDE) system to resemble the processes during the onset of OA.DesignSFCCs were produced from mesenchymal stromal cells and analyzed weekly by histology and qPCR to characterize the cellular and matrix-related composition. To simulate the early phase of OA, SFCCs were treated with interleukin-1β (IL-1β), tumor necrosis factor α (TNFα) and examined after 3 weeks or cultivated another 3 weeks without inflammatory cytokines to validate the regeneration potential. Mathematical modeling was performed in parallel to the in vitro experiments.ResultsSFCCs expressed cartilage-specific markers, and after stimulation an increased expression of inflammatory markers, matrix degrading enzymes, a loss of collagen II (Col-2) and a reduced cell density was observed which could be partially reversed by retraction of stimulation. Based on the PDEs, the distribution processes within the SFCCs, including those of IL-1β, Col-2 degradation and cell number reduction was simulated.ConclusionsBy combining in vitro and in silico methods, we aimed to develop a valid, efficient alternative approach to examine and predict disease progression and new therapeutic strategies.

Background:While osteoarthritis (OA) is the most common joint disease worldwide, rheumatoid arthritis (RA) represents the most common type of autoimmune arthritis. In both diseases, fibroblast-like synoviocytes (FLS), which maintain the structural and dynamic integrity of the joint, have been identified as key drivers of cartilage degradation. FLS can be divided into two major populations. The destructive phenotype which is restricted to the THY1- FLS of the synovial lining promotes bone erosion, while THY1+ FLS of the sublining layer drives synovitis. The FLS phenotype is shaped by glucose metabolism, which promotes disease progression in patients with synovitis. However, profound knowledge about the contribution of FLS to pathogenic mechanisms in cartilage degradation is limited.Objectives:Here, we present the phenotypic features of FLS obtained from patients with OA (OA-FLS) compared to bone marrow-derived mesenchymal stromal cells (MSC) on transcriptomic, proteomic and metabolic levels with the aims (i) to identify novel targets for the development of disease-modifying osteoarthritis drugs and (ii) to distinguish both cell types.Methods:To this end, we comprehensively compared human bone marrow-derived MSC with OA-FLS isolated from human knee joint sections. MSC and OA-FLS were characterized in detail according to their multipotency, surface marker pattern, cell viability, proliferation rate, morphology and expression of fibroblast- and metabolic-related markers using flow cytometry, immunofluorescence and SeahorseTM. More in-depth, selected gene and protein expression patterns were analyzed using qPCR and mass spectrometry.Results:We observed a similar phenotype of OA-FLS and MSC with regard to the minimal criteria that define a MSC phenotype. In-depth comparison of OA-FLS and MSC on proteome level revealed 598 differentially expressed proteins. We observed no differences in the expression of classical fibroblast markers such as vimentin, tenascin C and decorin as confirmed on RNA level. Remarkably, fibronectin, which is mainly produced by fibroblasts, is significantly lower expressed at both protein and RNA levels in OA-FLS together with collagen type 1 and CD106. Conversely, CD9, CD54 and fibroblast-specific protein-1 were expressed significantly higher in FLS at both levels, while hyaluronan synthase 1-3 remained unchanged. Of note, in terms of mitochondrial function, human OA-FLS show a significantly lower basal respiration and ATP production than MSC, but a comparable spare respiratory capacity and cellular mitochondrial dehydrogenase activity (NADH amount) per cell. Additionally, we identified the pyruvate dehydrogenase kinase (PDK) 3 to be highly expressed in OA-FLS, while the expression of mitochondrial ATP synthase subunits, electron transport chain complexes and glycolytic enzymes was comparable with MSC. Finally, inhibition of PDK by using DCA resulted in a significant increase in oxygen consumption rate and ATP production in OA-FLS. Thus, our data newly suggest, that PDKs may play a crucial role in the pathogenesis of OA and possibly RA.Conclusion:Our data provide evidence that, although the classical fibroblast markers do not discriminate between MSC and FLS, the latter demonstrate a significantly higher expression of PDKs, known to inhibit the pyruvate entry into the TCA cycle which finally limits the mitochondrial ATP production. Therefore, shifting the metabolism of FLS from glycolysis to mitochondrial respiration via inhibition of PDKs might be a novel approach in OA for the development of disease-modifying osteoarthritis drugs in order to unleash the metabolic flow.Disclosure of Interests:None declared

The initial inflammatory phase of fracture healing is of great importance for the clinical outcome. We aimed to develop a detailed time-dependent analysis of the initial fracture hematoma. We analyzed the composition of immune cell subpopulations by flow cytometry and the concentration of cytokines and chemokines by bioplex in 42 samples from human fractures of long bones

Background:Our ultimate goal is to study potential drug candidates in an experimental setting of arthritis. Therefore, we aim to develop a valid humanin vitro3D joint model mimicking features of joint inflammation by applying inflammatory conditions namely immune cells and pro-inflammatory cytokines. Our in vitro3D joint model consists of different components including an osteogenic and chondrogenic part, the joint space filled with synovial fluid, and the synovial membrane. Developed as an alternative experimental setup to animal experiments, our 3D joint model will enable us to study efficiently the effects of potential drug candidates in a human-basedin vitromodel.Objectives:Here, we aimed to demonstrate the suitability of our human-basedin vitro3D osteochondral model by analyzing the influence of the main cytokines involved in the pathogenesis of RA as well as the impact of a specific therapeutic intervention.Methods:Based on human bone marrow-derived mesenchymal stromal cells (hMSCs), we developed 3D bone and cartilage tissue components that were characterized in detail (e.g. cell vitality, morphology, structural integrity) using histological, biochemical and molecular biological methods as well as µCT and scanning electron microscope (SEM). In brief, to establish the osteogenic component, we populated β-tricalcium phosphate (TCP) – mimicking the mineral bony part – with hMSCs, while the scaffold-free cartilage component was generated by cellular self-assembly and intermittent mechanical stimulation (fzmb GmbH). Subsequently, we co-cultivated both tissue components for three weeks to generate an interconnected 3D osteochondral model. To test the suitability, we applied a cocktail of TNFα, IL-6 and MIF using concentrations reported from RA synovial fluid alone or in combination with specific therapeutic drugs and analyzed their impact by qPCR.Results:We verified the osteogenic phenotype of our 3D bone tissue component by demonstrating an increase in mineralized bone volume and the induction of bone-related gene expression (RUNX2,SPP1andCOL1A1) as compared to the corresponding control. Secondly, we verified the chondrogenic phenotype of our cartilage tissue component by HE and Alcian Blue staining as well as by the reduced expression ofCOL1A1and an abundant expression ofCOL2A1. Interestingly, co-cultivation of both components for up to 3 weeks demonstrated colonization, connectivity and initial calcification implying a transitional bridging area. Cytokine stimulation with a cocktail of TNF, IL-6 and MIF leads to an upregulation of the metabolic markerLDHAand the angiogenic markerVEGFin both bone and cartilage. The inflammation markersIL8andTNFare also upregulated in both components, whileIL6is downregulated in bone compared to the unstimulated control. In addition, a cytokine-induced upregulation of matrix-metalloproteases was observed especially in the cartilage component. All these cytokine-related effects could be antagonized with a cocktail of therapeutics (milatuzumab, adalimumab and tocilizumab).Conclusion:The results of our study showed cytokine related effects of both tissue components, which can be therapeutically antagonized. By combining the components in a 96 well format, we aim to provide a mid-throughput system for preclinical drug testing.Acknowledgments:This project is funded by the Federal Ministery of Education and Research (BMBF)Disclosure of Interests:Alexandra Damerau: None declared, Moritz Pfeiffenberger: None declared, Annemarie Lang: None declared, Timo Gaber: None declared, Frank Buttgereit Grant/research support from: Amgen, BMS, Celgene, Generic Assays, GSK, Hexal, Horizon, Lilly, medac, Mundipharma, Novartis, Pfizer, Roche, and Sanofi.

Background: Tanezumab, a monoclonal antibody against nerve growth factor, is in development for the treatment of signs and symptoms of osteoarthritis (OA). Objectives: To assess the safety and tolerability of subcutaneous (SC) tanezumab in patients with OA. Methods: Data were derived from 3 randomized placebo-controlled OA trials. SC treatment (every 8 weeks for 16–24 weeks with 8–24 week follow-up) included placebo, tanezumab 2.5 mg, tanezumab 2.5/5 mg (2.5 mg at day 1 and 5 mg at week 8), and tanezumab 5 mg. Overall treatment-emergent adverse events (TEAEs) and TEAEs of abnormal peripheral sensation were pooled from all 3 trials (placebo N = 586; tanezumab: 2.5 mg N = 602, 2.5/5 mg N = 219, 5 mg N = 347). Pre-specified TEAEs potentially associated with sympathetic neuropathy (anhidrosis, bradycardia, hypohidrosis, orthostatic hypotension, or syncope) and pre-specified joint events (primary osteonecrosis, rapidly progressive OA [RPOA] type 1 or type 2, subchondral insufficiency fracture, or pathological fracture; adjudicated by an independent committee of experts) were pooled from the 2 trials that included prospective evaluation of sympathetic and joint safety (placebo N = 514; tanezumab: 2.5 mg N = 528, 2.5/5 mg N = 219, 5 mg N = 284). TEAEs are presented for the treatment period; joint safety is presented for the full study (treatment plus follow up) period. Results: Patient demographics (80.7% white, 66.8% female, mean age ≈ 63 years) and clinical characteristics were similar across groups. TEAE rates were: placebo = 51.7%, tanezumab 2.5 mg = 52.3%, tanezumab 2.5/5 mg = 47.0%, and tanezumab 5 mg = 54.8%. Of TEAEs occurring in ≥2% of patients in any group, only oedema peripheral, joint stiffness, and paraesthesia had a higher incidence (95% confidence interval excluded 0) in any tanezumab group relative to placebo. Serious TEAE rates were: placebo = 1.5%, tanezumab 2.5 mg = 2.2%, tanezumab 2.5/5 mg = 1.4%, and tanezumab 5 mg = 2.6%. Rates of treatment and/or study discontinuation due to TEAEs were: placebo = 2.2%, tanezumab 2.5 mg = 1.8%, tanezumab 2.5/5 mg = 0.5%, and tanezumab 5 mg = 1.4%. Only arthralgia and OA led to discontinuation in >1 patient in any group. TEAEs of abnormal peripheral sensation rates were: placebo = 2.2%, tanezumab 2.5 mg = 5.1%, tanezumab 2.5/5 mg = 3.2%, and tanezumab 5 mg = 6.1%. Paraesthesia and hypoaesthesia were the most common events. Potential sympathetic neuropathy TEAE rates were: placebo = 0.8%, tanezumab 2.5 mg = 1.5%, tanezumab 2.5/5 mg = 0.5%, and tanezumab 5 mg = 2.8%; exposure-adjusted rates were not statistically different between any tanezumab group and placebo. Bradycardia and orthostatic hypotension were the most common events. No patient was considered to have a sympathetic neuropathy. TEAEs of abnormal peripheral sensation and potential sympathetic neuropathy were mostly mild and resolved. Joint safety event rates were statistically different for tanezumab 5mg (3.2%), but not 2.5mg (1.9%) or 2.5/5mg (0.5%), compared to placebo (0%). RPOA type-1 was the most common event. Total joint replacement rates were: placebo = 4.5%, tanezumab 2.5 mg = 5.9%, tanezumab 2.5/5 mg = 6.8%, and tanezumab 5 mg = 7.0%; rates were not statistically different between any tanezumab group and placebo. Conclusion: Tanezumab was generally safe and well tolerated in most patients, with rates of overall TEAEs and treatment/study discontinuations similar to placebo and no evidence of a sympathetic safety signal. TEAEs of abnormal peripheral sensation and joint safety events were infrequent but more common with tanezumab than placebo. Disclosure of Interests: Francis Berenbaum Grant/research support from: TRB Chemedica (through institution), MSD (through institution), Pfizer (through institution), Consultant of: Novartis, MSD, Pfizer, Lilly, UCB, Abbvie, Roche, Servier, Sanofi-Aventis, Flexion Therapeutics, Expanscience, GSK, Biogen, Nordic, Sandoz, Regeneron, Gilead, Bone Therapeutics, Regulaxis, Peptinov, 4P Pharma, Paid instructor for: Sandoz, Speakers bureau: Novartis, MSD, Pfizer, Lilly, UCB, Abbvie, Roche, Servier, Sanofi-Aventis, Flexion Therapeutics, Expanscience, GSK, Biogen, Nordic, Sandoz, Regeneron, Gilead, Sandoz, Thomas Schnitzer Consultant of: Pfizer, Lilly, AstraZeneca, GSK, Alan Kivitz Shareholder of: AbbVie, Amgen, Gilead, GSK, Pfizer Inc, Sanofi, Consultant of: AbbVie, Boehringer Ingelheim,,Flexion, Genzyme, Gilead, Janssen, Novartis, Pfizer Inc, Regeneron, Sanofi, SUN Pharma Advanced Research, UCB, Paid instructor for: Celgene, Genzyme, Horizon, Merck, Novartis, Pfizer, Regeneron, Sanofi, Speakers bureau: AbbVie, Celgene, Flexion, Genzyme, Horizon, Merck, Novartis, Pfizer Inc, Regeneron, Sanofi, Lars Viktrup Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Anne Hickman Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, Glenn Pixton Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, Mark Brown Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, Isabelle Davignon Shareholder of: Pfizer, Employee of: Pfizer, Christine West Shareholder of: Pfizer Inc, Employee of: Pfizer Inc

Background:Approximately 10% of fractures lead to significant fracture healing disorders, with a tendency to further increase due to the aging population. Of note, especially immunosuppressed patients with ongoing inflammation show difficulties in the correct course of fracture healing leading to fracture healing disorders. Most notably, invading immune cells and secreted cytokines are considered to provide an inflammatory microenvironment within the fracture gap, primarily during the initial phase of fracture healing. Current research has the focus on small animal models, facing the problem of translation towards the human system. In order to improve the therapy of fracture healing disorders, we have developed a human cell-basedin vitromodel to mimic the initial phase of fracture healing adequately. This model will be used for the development of new therapeutic strategies.Objectives:Our aim is to develop anin vitro3D fracture gap model (FG model) which mimics thein vivosituation in order to provide a reliable preclinical test system for fracture healing disorders.Methods:To assemble our FG model, we co-cultivated coagulated peripheral blood and primary human mesenchymal stromal cells (MSCs) mimicking the fracture hematoma (FH model) together with a scaffold-free bone-like construct mimicking the bony part of the fracture gap for 48 h under hypoxic conditions (n=3), in order to reflect thein vivosituation after fracture most adequately. To analyze the impact of the bone-like construct on thein vitroFH model with regard to its osteogenic induction capacity, we cultivated the fracture gap models in either medium with or without osteogenic supplements. To analyze the impact of Deferoxamine (DFO, known to foster fracture healing) on the FG model, we further treated our FG models with either 250 µmol DFO or left them untreated. After incubation and subsequent preparation of the fracture hematomas, we evaluated gene expression of osteogenic (RUNX2,SPP1), angiogenic (VEGF,IL8), inflammatory markers (IL6,IL8) and markers for the adaptation towards hypoxia (LDHA,PGK1) as well as secretion of cytokines/chemokines using quantitative PCR and multiplex suspension assay, respectively.Results:We found via histology that both the fracture hematoma model and the bone-like construct had close contact during the incubation, allowing the cells to interact with each other through direct cell-cell contact, signal molecules or metabolites. Additionally, we could show that the bone-like constructs induced the upregulation of osteogenic markers (RUNX2, SPP1) within the FH models irrespective of the supplementation of osteogenic supplements. Furthermore, we observed an upregulation of hypoxia-related, angiogenic and osteogenic markers (RUNX2,SPP1) under the influence of DFO, and the downregulation of inflammatory markers (IL6,IL8) as compared to the untreated control. The latter was also confirmed on protein level (e.g. IL-6 and IL-8). Within the bone-like constructs, we observed an upregulation of angiogenic markers (RNA-expression ofVEGF,IL8), even more pronounced under the treatment of DFO.Conclusion:In summary, our findings demonstrate that our establishedin vitroFG model provides all osteogenic cues to induce the initial bone healing process, which could be enhanced by the fracture-healing promoting substance DFO. Therefore, we conclude that our model is indeed able to mimic correctly the human fracture gap situation and is therefore suitable to study the influence and efficacy of potential therapeutics for the treatment of bone healing disorders in immunosuppressed patients with ongoing inflammation.Disclosure of Interests:Moritz Pfeiffenberger: None declared, Alexandra Damerau: None declared, Paula Hoff: None declared, Annemarie Lang: None declared, Frank Buttgereit Grant/research support from: Amgen, BMS, Celgene, Generic Assays, GSK, Hexal, Horizon, Lilly, medac, Mundipharma, Novartis, Pfizer, Roche, and Sanofi., Timo Gaber: None declared

Although several biomaterials for bone regeneration have been developed in the last decades, clinical application of bone morphogenetic protein 2 is clinically only approved when applied on an absorbable bovine collagen I scaffold (ACS) (Helistat; ACS-H). In research, another ACS, namely Lyostypt (ACS-L) is frequently used as a scaffold in bone-linked studies. Nevertheless, until today, the influence of ACS alone on bone healing remains unknown. Unexpectedly, in vitro studies using ASC-H revealed a suppression of osteogenic differentiation and a significant reduction of cell vitality when compared to ASC-L. In mice, we observed a significant delay in bone healing when applying ACS-L in the fracture gap during femoral osteotomy. The results of our study show for the first time a negative influence of both ACS-H and ACS-L on bone formation demonstrating a substantial need for more sophisticated delivery systems for local stimulation of bone healing in both clinical application and research. STATEMENT OF SIGNIFICANCE: Our study provides evidence-based justification to promote the development and approval of more suitable and sophisticated delivery systems in bone healing research. Additionally, we stimulate researchers of the field to consider that the application of those scaffolds as a delivery system for new substances represents a delayed healing approach rather than a normal bone healing which could greatly impact the outcome of those studies and play a pivotal role in the translation to the clinics. Moreover, we provide impulses on underlying mechanism involving the roles of small-leucine rich proteoglycans (SLRP) for further detailed investigations.

Mesenchymal stem/stromal cells (MSCs) are stem cells of the connective tissue, possess a plastic phenotype, and are able to differentiate into various tissues. Besides their role in tissue regeneration, MSCs perform additional functions as a modulator or inhibitor of immune responses. Due to their pleiotropic function, MSCs have also gained therapeutic importance for the treatment of autoimmune diseases and for improving fracture healing and cartilage regeneration. However, the therapeutic/immunomodulatory mode of action of MSCs is largely unknown. Here, we describe that MSCs express the inhibitory receptor CTLA-4 (cytotoxic T lymphocyte antigen 4). We show that depending on the environmental conditions, MSCs express different isoforms of CTLA-4 with the secreted isoform (sCTLA-4) being the most abundant under hypoxic conditions. Furthermore, we demonstrate that the immunosuppressive function of MSCs is mediated mainly by the secretion of CTLA-4. These findings open new ways for treatment when tissue regeneration/fracture healing is difficult.

Background Our ultimate goal is to develop a valid human in vitro 3D joint model to simulate the pathogenesis of arthritis. The in vitro 3D joint model consists of different components including an1 osteogenic and2 chondrogenic part,3 the synovial fluid and4 the synovial membrane and contains all involved cell types and thus, to allow interactions between cells by cell contacts and signalling molecules. As an alternative experimental setup for animal models, our in vitro 3D joint model will enable us to study the influence and efficacy of drug treatment. Currently, there is no valid 3D model which is able to mimic an arthritic joint. Objectives Here, we aim to mimic the1 osteogenic and2 chondrogenic part,3 the joint space with synovial fluid and4 the synovial membrane. Methods For the osteogenic component of the 3D joint model, we populated β-tricalcium phosphate (TCP) – mimicking the mineral bony part – with osteogenic pre-differentiated human bone marrow-derived mesenchymal stromal cells (hMSC) and coated the particles with an hMSC monolayer cell-sheet to get a compact bony component. Survival, adhesion and structural integrity of the cells were evaluated by Scanning Electron Microscopy (SEM), LIVE/DEAD staining and cellular release of LDH. Osteogenic differentiation was analysed by µCT for mineralization and on gene expression level using qRT-PCR. To mimic the chondrogenic part, a scaffold-free 3D cartilage construct was generated by chondrogenic differentiation of hMSC under hypoxia with intermittent mechanical stimulation. Constructs were analysed by histology and qRT-PCR. Simulating the synovial fluid, hyaluronic acid was applied to the osteochondral model. To model the synovial membrane, a confluent monolayer of hMSC was formed on a polycarbonate membrane and visualised by hemacolor staining. Results We developed an in vitro 3D bone model by successfully seeding pre-differentiated hMSC on a β-TCP scaffold. Cells consistently adhere onto the scaffold for up to 3 weeks as observed by SEM. The analysis of cell viability via LDH detection and LIVE/DEAD staining showed no toxic effects on the cells even after 3 weeks of incubation as compared to the corresponding control. mRNA expression of bone-related genes such as RUNX2, SPP1 and COL1A1 as well as µCT analysis confirmed the osteogenic phenotypic of hMSC grown in 3D. Mimicking the articular cartilage component, we verified its chondrogenic phenotype by HE and Alcian Blue staining as well as by the reduced mRNA expression of COL1A1 and an abundant expression of COL2A1. Interestingly, co-cultivation of the osteogenic and chondrogenic part for up to 3 weeks demonstrated successful colonisation, connectivity and initial calcification implying a functional transitional bridging area. Modelling the synovial membrane, we successfully and reproducibly created a confluent monolayer of hMSC, which is easily transferable to the model. Conclusions First steps towards the in vitro simulation of an arthritic joint based on a multi-component model confirm good cell vitality and phenotypic stability which indicates successful progression. To finalise the development of healthy joint model, we will combine the established parts to provide a suitable 3D multi-component joint model which enables us to study the efficacy of drug treatment in vitro. Disclosure of Interest None declared

Bone is a complex tissue with a variety of functions, such as providing mechanical stability for locomotion, protection of the inner organs, mineral homeostasis and haematopoiesis. To fulfil these diverse roles in the human body, bone consists of a multitude of different cells and an extracellular matrix that is mechanically stable, yet flexible at the same time. Unlike most tissues, bone is under constant renewal facilitated by a coordinated interaction of bone-forming and bone-resorbing cells. It is thus challenging to recreate bone in its complexity in vitro and most current models rather focus on certain aspects of bone biology that are of relevance for the research question addressed. In addition, animal models are still regarded as the gold-standard in the context of bone biology and pathology, especially for the development of novel treatment strategies. However, species-specific differences impede the translation of findings from animal models to humans. The current review summarizes and discusses the latest developments in bone tissue engineering and organoid culture including suitable cell sources, extracellular matrices and microfluidic bioreactor systems. With available technology in mind, a best possible bone model will be hypothesized. Furthermore, the future need and application of such a complex model will be discussed.

A seminar and interactive workshop on “In silico Methods – Computational Alternatives to Animal Testing” was held in Berlin, Germany, organized by Annemarie Lang, Frank Butt- gereit and Andrea Volkamer at the Charite-Universitatsmedizin Berlin, on August 17-18, 2017. During the half-day seminar, the variety and applications of in silico methods as alternatives to animal testing were presented with room for scientific discus- sions with experts from academia, industry and the German fed- eral ministry (Fig. 1). Talks on computational systems biology were followed by detailed information on predictive toxicology in order to display the diversity of in silico methods and the potential to embrace them in current approaches (Hartung and Hoffmann, 2009; Luechtefeld and Hartung, 2017). The follow- ing interactive one-day Design Thinking Workshop was aimed at experts, interested researchers and PhD-students interested in the use of in silico as alternative methods to promote the 3Rs (Fig. 2). Forty participants took part in the seminar while the workshop was restricted to sixteen participants.

Background Immunosuppressed patients with ongoing inflammation experience more often difficulties in the process of fracture healing. Hereby amongst others immunomodulated activation of osteoclasts leads to augmented osteolysis. Most notably numerous cytokines and invading lymphocytes provide an inflammatory environment within the fracture gap, utmost during the first phase of fracture healing. Nowadays researchers, whilst investigating fracture healing, use small rodent models, facing the problem of translation towards the human system. Hence, there is a lack of valid in vitro models to examine the first phase of fracture healing. To test new therapeutic strategies, we develop a valid 3D-model using human cells to mimic the first phase of fracture healing in vitro which is characterized by the formation of a fracture hematoma, a hypoxic microenvironment as well as inflammation that initiate the healing cascade and the process of endochondral ossification. Objectives To develop 3D bone-like models displaying endochondral ossification. Methods As a first step, we focus on establishing 3D bone-like, matrix-free models consisting of human mesenchymal stromal cells (hMSC). MSC were isolated from bone-marrow samples of patients undergoing total hip replacement and characterized with regard to their typical surface markers as well as their differentiation potential towards osteogenic, adipogenic and chondrogenic lineage. 3D bone-like models were generated from micro-mass culture of hMSC (Research Center of Medical Technology and Biotechnology, Bad Langensalza). After initial maturation, 3D bone-like models were cultured under hypoxic conditions (37°C, 1% O2) in osteogenic medium for up to 3 months. In vitro μCT analyses were performed at day 0, after one and two months focusing on the total volume (TV) and bone volume (BV). Additionally osteogenic-relevant genes/factors (Runx2, SPP1, Dlx5, ALP, RANKL, SPI1) as well as exclusion markers (SOX9, PPARg2) were investigated after 3 months of cultivation using qRT-PCR. Finally, we implemented histological/immunohistochemical methods (van Kossa, ALP, Col1 staining). Results The 3D bone-like models achieved diameters between 0.5 and 0.7 and a thickness of 0.3 cm. In vitro μCt analysis revealed a high amount of mineralized tissue at day 0 and substantial increase in the bone volume (BV/TV) after cultivation. At day 0 in vitro μ Ct analysis implied mineralization mostly at the margin but penetrated the tissue within further cultivation. These results are also supported by positive van Kossa staining. Additionally, qRT-PCR results yielded higher expressions of RUNX2, SPP1, SPI1, RANKL and DLx5. Furthermore, immunohistochemistry showed high ALP-activity and ColI-expression. Conclusions Preliminary results of our study focusing at developing a 3D bone-like model displayed a promising trend towards modelling endochondral ossification in vitro by increased mineralization (in vitro μCt analysis and van Kossa staining), and upregulation of osteogenic-relevant RUNX2 and SPP1 expression as well as ALP-activity and ColI-expression. High expression of SPI1 and RANKL could refer to osteoclast-like activities, which will be in the focus of further investigations. Finally, the complete 3D model will leave us the opportunity for studying the first phase of fracture healing under in vitro conditions. Disclosure of Interest None declared

Immunologically restricted patients such as those with autoimmune diseases or malignancies often suffer from delayed or insufficient fracture healing. In human fracture hematomas and the surrounding bone marrow obtained from immunologically restricted patients, we analyzed the initial inflammatory phase on cellular and humoral level via flow cytometry and multiplex suspension array. Compared with controls, we demonstrated higher numbers of immune cells like monocytes/macrophages, natural killer T (NKT) cells, and activated T helper cells within the fracture hematomas and/or the surrounding bone marrow. Also, several pro-inflammatory cytokines such as Interleukin (IL)-6 and Tumor necrosis factor α (TNFα), chemokines (e.g., Eotaxin and RANTES), pro-angiogenic factors (e.g., IL-8 and Macrophage migration inhibitory factor: MIF), and regulatory cytokines (e.g., IL-10) were found at higher levels within the fracture hematomas and/or the surrounding bone marrow of immunologically restricted patients when compared to controls. We conclude here that the inflammatory activity on cellular and humoral levels at fracture sites of immunologically restricted patients considerably exceeds that of control patients. The initial inflammatory phase profoundly differs between these patient groups and is probably one of the reasons for prolonged or insufficient fracture healing often occurring within immunologically restricted patients. View Full-Text

Background Gene therapy appears to have the potential for achieving a long-term remedy for osteoarthritis (OA). However, there is a risk of adverse reactions, especially when using cytomegalovirus-controlled expression. To provide a safe application, we focused on the expression of therapeutic cytokines [e.g. interleukin (IL)-4] in a disease-responsive manner by use of the previously cloned Cox-2 promoter as ‘genetic switch’. In the present study, we report the functionality of a controlled gene therapeutic system in an equine osteoarthritic cell model. Methods Different nonviral transfection reagents were tested for their efficiency on equine chondrocytes stimulated with equine IL-1β or lipopolysaccharide to create an inflammatory environment. To optimize the transfection, we successfully redesigned the vector by excluding the internal ribosomal entry site (IRES). The functionality of our Cox-2 promoter construct with respect to expressing IL-4 was proven at the mRNA and protein levels and the anti-inflammatory potential of IL-4 was confirmed by analyzing the expression of IL-1β, IL-6, IL-8, matrix metalloproteinase (MMP)-1, MMP-3 and tumor necrosis factor (TNF)-α using a quantitative polymerase chain reaction. Results Nonviral transfection reagents yielded transfection rates from 21% to 44% with control vectors with and without IRES, respectively. Stimulation of equine chondrocytes resulted in a 20-fold increase of mRNA expression of IL-1β. Such exogenous stimulation of chondrocytes transfected with pNCox2-IL4 led to an increase of IL-4 mRNA expression, whereas expression of inflammatory mediators decreased. The timely link between these events confirms the anti-inflammatory potential of synthesized IL-4. Conclusions We consider that this approach has significant potential for translation into a useful anti-inflammation therapy. Molecular tools such as the described therapeutic plasmid pave the way for a local-controlled, self-limiting gene therapy. Copyright © 2014 John Wiley & Sons, Ltd.