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

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

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

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice