Implementation of the 3R principle in musculoskeletal research – Refinement measures and in vitro replacement methods

Musculoskeletal disorders are a challenging clinical problem. Each year, millions of patients worldwide experience bone fractures and 10–15% of these fractures suffer from impaired healing. The global prevalence for osteoarthritis is higher than ever before due to an increased life expectancy and rise in associated risk factors such as physical inactivity and obesity. Sophisticated complex treatment plans with novels biologics allowed to effectively achieve remission in patients with rheumatoid arthritis, however, about 25% of the patients still suffers from moderate or even high disease activity. Thus, further fundamental, and translational preclinical research is imperative to tackle the unmet medical needs for musculoskeletal conditions and ensure health throughout the life course. The current goldstandard in preclinical research is the use of animal models, i.e. mainly rodents (mouse, rat). However, during recent years, we have witnessed the failure of promising therapeutics in clinical testing albeit being based on strong evidence from animal experiments. Therefore, it can be speculated that trans-species differences might be responsible for the limited transferability of findings to the human patient. The 3R principle (replace, reduce, refine) published by Russell and Burch in 1959 can be used as a framework for the humane use of animals in research. Moreover, it can enhance and ensure scientific quality and integrity in studies using animals, thereby accelerating the translational process. To enhance the current knowledge on refinement measures in fundamental research studies and to provide evidence-based data on pain management protocols in laboratory animals, we evaluated two analgesics, tramadol and buprenorphine in the drinking water, for their efficiency and side effects on experimental readout in the mouse-osteotomy model. Furthermore, we developed novel in vitro approaches to evade cross-species differences and to replace lab animal usage with a specific focus on fracture healing and joint pathologies. In detail, to recapitulate the initial phase of fracture healing, we specifically focused on integrating the interaction between immune cells and mesenchymal stromal cells/bone-related cells, exemplified by artificial fracture hematoma models containing mesenchymal stromal cells and the combination with three-dimensional scaffold-free bone-like constructs (fracture gap model). This tissue-engineered macroscale approach was used in parallel to mimic cartilage degradation during the onset of osteoarthritis in vitro, which was later extended towards an osteochondral unit model by integrating a tricalcium phosphate-based bone equivalent to recapitulate key features of rheumatoid arthritis. Together, within this thesis, I provide an overview of the variety of approaches towards the active implementation of the 3R principle in musculoskeletal-related preclinical research. Thereby, I specifically underline the importance of equivalently prioritizing all 3Rs to effectively rethink traditional research approaches in biomedicine for continuous improvement in animal welfare and successful human patient-driven translation.Erkrankungen des muskuloskelettalen Systems sind ein herausforderndes klinisches Problem. Jedes Jahr erleiden Millionen von Patienten weltweit Knochenbrüche und bei 10-15 % dieser Frakturen kommt es zu Heilungsstörungen. Die weltweite Prävalenz von Osteoarthrose ist aufgrund der gestiegenen Lebenserwartung und der Zunahme der damit verbundenen Risikofaktoren, wie Bewegungsmangel und Übergewicht, höher als je zuvor. Dank ausgeklügelter komplexer Behandlungspläne mit neuartigen Biologika konnte bei Patienten mit rheumatoider Arthritis eine wirksame Remission erreicht werden, allerdings leiden etwa 25 % der Patienten immer noch unter einer mäßigen oder sogar hohen Krankheitsaktivität. Daher ist weiterführende Forschung unerlässlich, um den verbleibenden medizinischen Bedarf im Bereich der muskuloskelettalen Erkrankungen zu decken. Der derzeitige Goldstandard in der präklinischen Forschung ist die Verwendung von Tiermodellen, insbesondere Nagetieren (Maus, Ratte). Dennoch sind in den letzten Jahren immer wieder neue Therapeutika in der klinischen Testung gescheitert, trotz vielversprechender Daten aus dem Tierversuch. Speziesübergreifende Unterschiede werden für die begrenzte Übertragbarkeit der Ergebnisse auf den menschlichen Patienten verantwortlich gemacht. Das von Russell und Burch 1959 veröffentlichte 3R-Prinzip (Replace, Reduce, Refine) kann als Rahmen für den humanen Einsatz von Tieren in der Forschung dienen sowie die Qualität und Integrität von Tierversuchen sicherstellen und so den Translationsprozess beschleunigen. Um das derzeitige Wissen über Refinement-Maßnahmen zu erweitern und evidenzbasierte Daten zu Schmerzbehandlungsprotokollen bei Labortieren bereitzustellen, haben wir zwei Analgetika, Tramadol und Buprenorphin im Trinkwasser, auf ihre Wirksamkeit und ihre Nebenwirkungen im Maus-Osteotomie-Modell untersucht. Darüber hinaus haben wir neue in vitro Ansätze entwickelt mit speziellem Fokus auf die Frakturheilung und Gelenkpathologien. Um die Anfangsphase der Frakturheilung zu rekapitulieren, konzentrierten wir uns insbe-sondere auf die Interaktion zwischen Immunzellen und mesenchymalen Stromazellen/Knochenzellen, z. B. durch die Kombination von Frakturhämatom-Modellen mit dreidimensionalen trägerfreien knochenähnlichen Konstrukten (Frakturspaltmodell). Ein vergleichbarer Ansatz wurde verwendet, um den Knorpelabbau während der beginnenden Osteoarthrose in vitro zu imitieren. Später wurde dieser Ansatz auf ein Modell der osteochondralen Einheit ausgeweitet, um die Hauptmerkmale der rheumatoiden Arthritis zu rekapitulieren. In dieser Arbeit gebe ich einen Überblick über die Vielfalt der Ansätze zur aktiven Implementierung des 3R-Prinzips in der präklinischen muskuloskelettalen Forschung. Dabei unterstreiche ich insbesondere die gleichwertige Priorisierung aller 3R, um eine kontinuierliche Verbesserung des Tierschutzes und eine erfolgreiche, auf den menschlichen Patienten ausgerichtete Translation zu gewährleisten

Working in Regional Agro-food Networks – Strengthening Rural Development through Cooperation

Regional agro-food networks have an impact on the development of rural regions. Networks give small and medium sized enterprises the opportunity to gain access to further markets (e.g. through offering a wider common product range), to conduct more effective marketing or to synergize the variety of skills and know-how of the network partners. Networks of the agricultural and food economy are also seen as a chance for rural regions because they can positively influence social and cultural lives as well as the natural and economic areas in regions.We analysed regional networks of the agricultural and food economy, investigated the strengths and weaknesses in the structure of agro-food networks and developed options for action to strengthen the collaboration within the networks and their regional marketing.In our paper we present the results of one case study in Eastern Germany. We show our findings of a strengths and weaknesses analysis and a constellation analysis. Therefore, success factors were identified and used to evaluate the networks qualitatively. In addition, we discuss how regional networks can support regional marketing and sustainable regional development

Administration of Tramadol or Buprenorphine via the drinking water for post-operative analgesia in a mouse-osteotomy model

Adequate analgesia is essential whenever pain might occur in animal experiments. Unfortunately, the selection of suitable analgesics for mice in bone-linked models is limited. Here, we evaluated two analgesics - Tramadol [0.1 mg/ml (Tlow) vs. 1 mg/ml (Thigh)] and Buprenorphine (Bup; 0.009 mg/ml) - after a pre-surgical injection of Buprenorphine, in a mouse-osteotomy model. The aim of this study was to verify the efficacy of these opioids in alleviating pain-related behaviors, to provide evidence for adequate dosages and to examine potential side effects. High concentrations of Tramadol affected water intake, drinking frequency, food intake and body weight negatively in the first 2-3 days post-osteotomy, while home cage activity was comparable between all groups. General wellbeing parameters were strongly influenced by anesthesia and analgesics. Model-specific pain parameters did not indicate more effective pain relief at high concentrations of Tramadol. In addition, ex vivo high-resolution micro computed tomography (µCT) analysis and histology analyzing bone healing outcomes showed no differences between analgesic groups with respect to newly formed mineralized bone, cartilage and vessels. Our results show that high concentrations of Tramadol do not improve pain relief compared to low dosage Tramadol and Buprenorphine, but rather negatively affect animal wellbeing

The In Vitro Human Fracture Hematoma Model - A Tool for Preclinical Drug Testing

The aim of the study was to establish an in vitro fracture hematoma (FH) model, which mimics the in vivo situation of the human fracture gap in order to assess drug efficacy and effectiveness for the treatment of fracture healing disorders. Therefore, human peripheral blood and mesenchymal stromal cells (MSCs) were coagulated to produce in vitro FH models, incubated in osteogenic medium under normoxia/hypoxia, and analyzed for cell composition, gene expression and cytokine/chemokine secretion. To evaluate the model, we studied the impact of dexamethasone (impairing fracture healing) and deferoxamine (promoting fracture healing). Under hypoxic conditions, MSCs represented the predominant cell population, while the frequencies of leukocytes decreased. Marker gene expression of osteogenesis, angiogenesis, inflammation, migration and hypoxic adaptation increased significantly over time and compared to normoxia while cytokine/chemokine secretion remained unchanged. Finally, dexamethasone favored the frequency of immune cells compared to MSCs, suppressed osteogenic and pro-angiogenic gene expression and enhanced the secretion of inflammatory cytokines. Conversely, deferoxamine favored the frequency of MSCs over that of immune cells and enhanced the expression of the osteogenic marker RUNX2 and markers of the hypoxic adaptation. In summary, we demonstrate that hypoxia is an important factor for in vitro modeling the initial phase of fracture healing, that both fracture-healing disrupting and promoting substances can influence the in vitro model comparable to the in vivo situation. Therefore, we conclude that our model is able to mimic in part the human FH and to reduce the number of animal experiments in early preclinical studies

Impact of Janus Kinase Inhibition with Tofacitinib on Fundamental Processes of Bone Healing

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

Spatial Distribution of Macrophages During Callus Formation and Maturation Reveals Close Crosstalk Between Macrophages and Newly Forming Vessels

Macrophages are essential players in the process of fracture healing, acting by remodeling of the extracellular matrix and enabling vascularization. Whilst activated macrophages of M1-like phenotype are present in the initial pro-inflammatory phase of hours to days of fracture healing, an anti-inflammatory M2-like macrophage phenotype is supposed to be crucial for the induction of downstream cascades of healing, especially the initiation of vascularization. In a mouse-osteotomy model, we provide a comprehensive characterization of vessel (CD31+, Emcn+) and macrophage phenotypes (F4/80, CD206, CD80, Mac-2) during the process of fracture healing. To this end, we phenotype the phases of vascular regeneration-the expansion phase (d1-d7 after injury) and the remodeling phase of the endothelial network, until tissue integrity is restored (d14-d21 after injury). Vessels which appear during the bone formation process resemble type H endothelium (CD31hiEmcnhi), and are closely connected to osteoprogenitors (Runx2+, Osx+) and F4/80+ macrophages. M1-like macrophages are present in the initial phase of vascularization until day 3 post osteotomy, but they are rare during later regeneration phases. M2-like macrophages localize mainly extramedullary, and CD206+ macrophages are found to express Mac-2+ during the expansion phase. VEGFA expression is initiated by CD80+ cells, including F4/80+ macrophages, until day 3, while subsequently osteoblasts and chondrocytes are main contributors to VEGFA production at the fracture site. Using Longitudinal Intravital Microendoscopy of the Bone (LIMB) we observe changes in the motility and organization of CX3CR1+ cells, which infiltrate the injury site after an osteotomy. A transient accumulation, resulting in spatial polarization of both, endothelial cells and macrophages, in regions distal to the fracture site, is evident. Immunofluorescence histology followed by histocytometric analysis reveals that F4/80+CX3CR1+ myeloid cells precede vascularization

Macroscale mesenchymal condensation to study cytokine-driven cellular and matrix-related changes during cartilage degradation

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.publishedVersio

Functional Scaffold‐Free Bone Equivalents Induce Osteogenic and Angiogenic Processes in a Human In Vitro Fracture Hematoma Model

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)

CTLA-4 mediates inhibitory function of mesenchymal stem/stromal cells

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