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Objectives: Adequate pain management is essential for ethical and scientific reasons in animal experiments. Choice of analgesics in fracture models is limited due to potential interference of anti-inflammatory drugs during the initial phase of healing. Depot formulations of Buprenorphine are only available [for full text, please go to the a.m. URL]

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.

Supplemental material, sj-pdf-1-lan-10.1177_00236772211012841 for Do you want to join our Editorial Board? by Paulin Jirkof, Annemarie Lang, J-P Mocho, José Sanchez-Morgado and Jordi L. Tremoleda in Laboratory Animals

The bone vasculature is essential for skeletal development, homeostasis, and regeneration. In long bones, specific capillary subtypes couple angiogenesis to osteogenesis, especially at the growth plate (metaphysis) and endosteum. These metaphyseal and endosteal vessels, termed “type H,” express high levels of CD31 and endomucin (CD31hiEmcnhi), show a columnar structure and are closely related to Osterix (Osx)- and Runt-related transcription factor 2 (Runx2)-expressing osteoblasts and their progenitors [1]. Sinusoidal vessels with low expression of CD31 and Emcn (CD31lowEmcnlow, type L vessels) are mainly found in the diaphysis/bone marrow cavity [1]. The crosstalk between endothelial cells (ECs) of type H vessels and bone-related cells is characterized by osteogenesis-promoting factors provided by ECs and angiogenic mediators secreted by osteoblast-lineage cells. Bone morphogenetic proteins (BMP) are major regulators of vessel formation, however their role within angiogenic and osteogenic coupling and in particular, the involvement of BMP-related intracellular effectors SMAD1 and SMAD5 (SMAD1/5) is unclear. Previously, we demonstrated that during mouse embryonic development, the synergy of Notch and SMAD1/5 is responsible for the balanced selection of tip and stalk cells in vascular sprouting [2]. Furthermore, we reported that endothelium-specific deletion of SMAD1/5 during early postnatal retinal angiogenesis resulted in arteriovenous malformations, a reduced number of tip cells and a hyperdensity in the vascular plexus [3]. Therefore, we hypothesized that functional EC-specific SMAD1/5 signaling is required for (i) formation and maturation of long bone vasculature and (ii) coupling of osteogenesis and angiogenesis, specifically of type H vessel formation in the metaphysis.

Fracture healing disorders occur in approximately 10% of human patients and cause severe pain and reduced quality of life. To develop and test new therapeutic strategies, the mouse is a frequently used laboratory animal in bone healing research. But the assessment of pain in laboratory mice is often difficult as these animals tend to hide symptoms of reduced wellbeing and pain. In addition, the selection of analgesics in bone healing models is restricted due to potential interfering properties of anti-inflammatory drugs during the initial and inflammatory phase of bone healing. Therefore, the opioids and opioid analogs buprenorphine and tramadol are commonly used. The main administration route of buprenorphine is the application via s.c. or i.p. injection. However, recent studies on buprenorphine have shown a short pharmacokinetic half-life (, Since refinement studies are often performed by lab animal scientists and separated from basic research approaches, the translation of these results back to basic research is challenging due to the unawareness of refinement approaches by the basic research-oriented experimenter. In order to enhance the knowledge on refinement options in basic research studies, and to effectively reduce lab animal usage, we conceive a refinement study embedded in a basic research study to therefore show the possibility to combine both studies.Within our study, we evaluate and compare two analgesic protocols, Tramadol (0.1 mg/ml) administered via the drinking water from one day pre-operatively until 3 days post-operatively and a sustained-release depot Buprenorphine (1,2 mg/kg) administered via an intra-operative s.c. injection for their efficiency and possible side effects on experimental readouts in a mouse osteotomy model. A sequential study design is employed in order to re-use the same animals that underwent an isoflurane anaesthesia for baseline measurements to reduce inter-animal variations which would appear when using a parallel control group and to reduce the animal number used in this study. For the study, male and female ‎C57BL/6 are used, as it is well known that sex can influence pain perception and behaviour. Therefore, we use both sexes to provide evidence for a wider field of application.The impact on the wellbeing and the effectiveness of pain relief is evaluated by the assessment of body weight , food and water intake, and the usage of different scoring systems such as the Mouse-Grimace-Scale (MGS) combined with a clinical scoring, a model-specific pain scoring, the nest complexity score and an explorative test as well as a gait analyses. Finally, to evaluate the influence of pain management on bone healing and experimental readout and to answer our explorative research question on the role of macrophages, including subpopulations and vessels, in fracture healing under different biomechanical loading conditions, we are going to perform µCT analysis and histological staining of the osteotomized bone. The mice will be randomly allocated to a cage (two mice per cage) using simple randomization. An optimal cage setup must either consist of 2 female or 2 male mice with the same fixation method as well as the same pain management protocol. The analgesic protocols comprise either tramadol application via the drinking water from 1 day pre- to 3 days post-operative, or a single injection of a sustained-release buprenorphine formula. The assigned protocol is the same for both procedures (anesthesia only and anesthesia + osteotomy two weeks later). After acclimatization, training phase and baseline measurements, all animals are subjected to anesthesia (15 min isoflurane anesthesia) without surgery but with the assigned analgesia (tramadol via drinking water vs. buprenorphine retard preparation). Then, all behavioral and model-specific examinations are performed 12, 24, 48 and 72 h post-anesthesia and are then examined every 2 days. This process is followed by a wash-out phase of 10-11 days. Afterwards, animals are undergoing an osteotomy. Measurements are then conducted 12, 24, 48, and 72 h post-osteotomy and subsequently in intervals of 2 days. Two weeks post-osteotomy, mice are euthanized by cervical dislocation. The osteotomized left femur is collected for µCT and histology. The contralateral intact femur as well as both tibiae are also collected for possible further analyses., Since animal studies are commonly enrolled in research to test hypotheses and to study mechanism in a physiological organism, researchers start to realize the importance of animal welfare during experiments. Animal welfare includes appropriate housing, handling, pain management and more. It is known that pain and stress can possibly lead to wound healing and blood flow disorders as well as immunosuppression and increased infection risk [1-3]. Therefore, adequate pain management in animal experiments is essential for ethical and scientific reasons. Unfortunately, there is less known on the pain pathology and subsequent treatment in commonly used preclinical animal models and evidence-based data is only scarcely available [4]. Common published guidelines are mainly based on assumptions or experience. An optimal pain management includes i) perioperative or pre-emptive analgesia to avoid pain development and establishment of a pain memory and to reduce the intensity of post-operative pain, ii) intra-operative analgesia to e.g. reduce the dosage of the anaesthetics resulting in a decreased convalescence time and ii) postoperative analgesia that sustains the pain relief and prevents pain-induced symptoms [5]. Besides the good intentions, it is crucial to closely assess and monitor the pain management during the experiments. Buprenorphine is a commonly used, fast acting and potent opioid that is mainly administered s.c. or i.p. in dosages of 0.05–0.75 mg/kg every 12 h although recent studies indicate the short half-life and recommend the administration at every 6 h [6]. With the frequently used application intervals of 6-12 h, the animals could suffer periods without or only insufficient pain relief. Moreover, laboratory rodents can easily be stressed by handling for medical procedures such as injections and this distress is magnified if an aversive procedure must be repeated multiple times per day. Tramadol is an opioid analogue and its application via injection is, due to its short half-life of approx. 2 hours, not recommended, but still carried out nonetheless [7-9]. However, the application of Tramadol via the drinking water in bone healing research is more common, with concentrations ranging from 0.025 mg/ml up to 0.1 mg/ml. During the last years, the application of pain medications via the drinking water has therefore become more of interest, as additional handling is avoided, resulting in reduced stress and strain. Nevertheless, the guaranteed administration via drinking water is controversially discussed as the water intake can be reduced due to the reduced wellbeing, day-night rhythm and different taste due to the analgesic. An orientation towards application methods such as depot formulation that require less frequent manipulation and simultaneously achieve an adequately high effective level over a long period of time and require less frequent manipulations is therefore urgently advised. In the USA, such formulations are already used successfully in mouse models, but an import into the EU is not possible and a corresponding product is currently not available on the European market. A sustained-release formulation of Buprenorphine being available in Europe would lead to an improvement in the field of pain management in laboratory animals and improved the experimental conditions for many miceThe team of Prof. Jörg Huwyler (Department of Pharmaceutical Sciences, Pharmaceutical Technology, University of Basel) has recently been able to develop a Buprenorphine retard preparation and has already successfully tested the new preparation in vivo with regard to pharmacokinetics and effectiveness in initial studies. The efficacy of the buprenorphine retard preparation provided by AG Huwyler will be tested in a procedure of moderate severity, in this case in two mouse osteotomy model (rigid and flexible). It will be evaluated to what extent the minimization of model-unspecific, clinical and non-specific behavioural abnormalities indicating pain, changes in well-being or side effects, are observed by the application of the buprenorphine retard preparation. This study can provide the basis for the further use of the preparation in other (bone-related) mouse models. A commercialisation of the preparation on the European market could effectively close a treatment gap especially in the field of laboratory animal science. The potential and the benefit in the field of pain management, especially for rodents, is therefore immense.1. Carbone, L. and J. Austin, Pain and Laboratory Animals: Publication Practices for Better Data Reproducibility and Better Animal Welfare. PLoS One, 2016. 11(5): p. e0155001.2. Jirkof, P., Side effects of pain and analgesia in animal experimentation. Lab Anim (NY), 2017. 46(4): p. 123-128.3. Peterson, N.C., E.A. Nunamaker, and P.V. Turner, To Treat or Not to Treat: The Effects of Pain on Experimental Parameters. Comp Med, 2017. 67(6): p. 469-482.4. Flecknell, P., Rodent analgesia: Assessment and therapeutics. Vet J, 2018. 232: p. 70-77.5. Lang, A., et al., Osteotomy models - the current status on pain scoring and management in small rodents. Lab Anim, 2016. 50(6): p. 433-441.6. Jirkof, P., et al., Buprenorphine for pain relief in mice: repeated injections vs sustained-release depot formulation. Lab Anim, 2015. 49(3): p. 177-87.7. Aydin, O.N., et al., The antinociceptive effects of systemic administration of tramadol, gabapentin and their combination on mice model of acute pain. Agri, 2012. 24(2): p. 49-55.8. Matthiesen, T., et al., The experimental toxicology of tramadol: an overview. Toxicol Lett, 1998. 95(1): p. 63-71.9. Mouedden, M.E. and T.F. Meert, Pharmacological evaluation of opioid and non-opioid analgesics in a murine bone cancer model of pain. Pharmacol Biochem Behav, 2007. 86(3): p. 458-67.

The hypoxic microenvironment during the initial phase of fracture healing is essential for initiating immunological process that further the regeneration and restoration of the bone. The hypoxia-inducible factor (HIF)-1�� closely regulates the cellular adaption under hypoxic conditions. HIF-1�� can be chemically stabilized by different factors which either inhibit the O2-sensing prolyl hydroxylase e.g. deferoxamine (DFO) or directly interfere with the downstream effects after nuclear translocation e.g. the macrophage-migration inhibitory factor (MIF). As a transcription factor, HIF-1�� initiates the consecutive processes of bone regeneration and human mesenchymal stromal cell (hMSC) differentiation. Hence, the aim was to promote the cellular adaptation towards hypoxia in order to specifically accelerate fracture healing under compromised conditions (inhibited mineralization and vascularization). First, in vitro studies were performed to i) evaluate the potential of DFO and MIF in combination to counteract a glucocorticoid-induced inhibition of hMSC calcification and ii) determine an effective concentration of both substances for further testing in a mouseosteotomy-model. Concurrently, two absorbable bovine Col I scaffolds (ACSs) were tested for their suitability to be used as a delivery system of these substances into the osteotomy gap. Finally, both HIF-stabilizers were evaluated for their potential to accelerate fracture healing in a mouse-osteotomy-model. In vitro, a concentration-dependent inhibitory effect of the glucocorticoid dexamethasone was observed on osteogenic differentiation and calcification of hMSCs via a quantitative Alizarin red assay. This inhibition was counteracted by applying different concentrations DFO and MIF in combination. As suitable scaffolds for the in vivo application Lyostypt�� (ACS-L) and Helistat�� (ACS-H) were investigated in vitro for their structural components and impact on hMSC osteogenesis, cytotoxicity and immunogenicity. Proteomics analysis of both scaffolds yielded several proteins beside collagens that might be advantageous or disadvantageous regarding the fracture healing outcomes. Moreover, ACS-H induced a strong tumor necrosis factor (TNF��) release when applied to whole human blood and inhibited the calcification during the osteogenic differentiation of hMSCs. Consequently, ACS-L was examined in more detail in a mouse-osteotomy-model and revealed an inhibitory effect on mineralized callus formation, cellular recruitment to the osteotomy gap and vessel formation. The application of DFO and MIF in combination and DFO alone during the initial healing phase accelerated the vessel formation, the ACS degradation and the callus mineralization. The results support the fact that stabilization of HIF-1�� enhances osteogenic differentiation in vitro and is capable to counteract e.g. glucocorticoid-induced inhibition. Both ACSs negatively influenced either the hMSC differentiation in vitro or the bone healing process in vivo, although being routinely used in research and clinic routine. These results display a delayed healing process that parallels observed compromised conditions in Rheumatoid arthritis patients or smokers ��� reduced vessel and bone formation. The combination of MIF and DFO was evaluated in this model of compromised condition to test their counteracting ability in this clinically relevant model. This study provides evidence for a promising therapeutic strategy to accelerate fracture healing capacities and to prevent disorders by applying potent HIF-stabilizers in a specific patient cohort with a higher risk of a bone healing delay., Das hypoxische Mikromilieu w��hrend der initialen Phase der Frakturheilung ist essenziell f��r die Knochenregeneration und -wiederherstellung. Dadurch werden wichtige immunologische Prozesse f��r den weiteren Verlauf der Heilung angeschoben. Der Hypoxie-induzierte Faktor (HIF)-1�� reguliert ma��geblich die zellul��re Adaptation unter hypoxischen Bedingungen. HIF-1 �� kann durch verschiedene Faktoren chemisch stabilisiert werden, wobei diese entweder die O2-sensitive Prolyl-Hydroxylase hemmen, wie z.B. Deferoxamin (DFO) oder direkt mit dem Signalweg nach HIF-Translokation in den Nukleus interagieren, wie z.B. der Macrophage-migration Inhibitory Factor (MIF). Als Transkriptionsfaktor aktiviert HIF-1�� wichtige Schritte der Knochenregeneration, wie z.B. die Differenzierung von humanen Mesenchymalen Stromazellen (hMSC) oder die Gef����bildung. Dementsprechend ist die F��rderung der zellul��ren Adaptation gegen��ber Hypoxie ein vielversprechender Ansatz und Inhalt dieser Arbeit, um die Frakturheilung vor allem unter kompromittierten Bedingungen (Hemmung der Mineralisierung und Vaskularisierung) zu verbessern. In einem ersten Schritt wurden In-vitro-Studien durchgef��hrt um (i) das Potential von MIF und DFO in Kombination zu testen, einer Glukokortikoid-induzierten Hemmung der Kalzifizierung von hMSCs entgegenzuwirken und (ii) die optimale Dosierung beider Substanzen f��r weitere Studien in vivo zu bestimmen. Parallel dazu wurden zwei absorbierbare bovine Kollagen-I Scaffolds (ACS) auf ihre Eignung, als Tr��germaterialien f��r die Substanzkombination f��r die weiteren In-vivo-Testung zu dienen, evaluiert. Im letzten Schritt wurde der Effekt von MIF und DFO nach lokaler Applikation in einem Maus-Osteotomie-Modell untersucht. In vitro wurde ein konzentrationsabh��ngiger hemmender Effekt des Glukokortikoids Dexamethason auf die osteogene Differenzierung und Kalzifizierung von hMSC mittels quantitativem Alizarin-Rot-Assay beobachtet. Diese Hemmung konnte durch Applikation von verschiedenen Konzentrationen von MIF und DFO in Kombination aufgehoben werden. Die ausgew��hlten Scaffolds f��r die In-vivo-Studien, Lyostypt�� (ACS-L) und Helistat�� (ACS-H), wurden einer genaueren Analyse ihrer strukturelle Beschaffenheit und ihres potentiellen Einfluss auf die osteogene Differenzierung von hMSC, Zytotoxizit��t und Immunogenit��t unterzogen. Mittels Proteomics-Analysen konnten neben den zu erwartenden Kollagenen weitere Proteine, die potenziell die Knochenregeneration beeinflussen k��nnen, nachgewiesen werden. Weiterhin induzierte ACS-H eine signifikante Freisetzung des Tumor-Nekrosefaktors (TNF)-�� in humanem Vollblut und hemmte die Kalzifizierung von hMSC w��hrend der Osteogenese. Folglich wurde nur ACS-L f��r weitere In-vivo-Studien eingesetzt. Dieser zeigte nach Applikation in einem Maus-Osteotomie-Modell eine hemmende Wirkung auf die mineralisierte Kallusformation, die Zellmigration in den Osteotomiespalt und die Gef����bildung. Die Applikation von DFO und MIF oder nur DFO allein konnte diesem hemmenden Effekt entgegenwirken. Die Ergebnisse der Studie unterst��tzen den Fakt, dass die Stabilisierung von HIF-1��, die osteogene Differenzierung in vitro f��rdert und z.B. Glukokortikoid-induzierten negativen Effekten entgegenwirken kann. Interessanterweise zeigten beide Scaffolds eine hemmendende Wirkung auf hMSC in vitro oder den Knochenregenerationsprozess in vivo, obwohl sie beide routinem����ig in der Klinik oder Forschung zur Behandlung von Knochenbr��chen eingesetzt werden. Die Nutzung des ACS-L, als Tr��germaterial, f��r die In-vivo-Studien ist entsprechend den hier gewonnen Daten eher vergleichbar mit kompromittierenden Bedingungen, wie sie z.B. w��hrend der Knochenheilung in Rheuma-Patienten oder Rauchern beobachtet werden (reduzierte Knochen- und Gef����bildung). Die Kombination von MIF und DFO wurde in diesem Modell untersucht und konnte in einem klinisch-relevanten Modell ihr Potential zur Verbesserung der Knochenregeneration zeigen. Die Ergebnisse dieser Arbeit sind ��u��erst vielversprechend im Hinblick auf die m��gliche Therapie von spezifischen Patientenkohorten, die zu Frakturheilungsst��rungen neigen, mit HIF-Stabilisatoren, um die Knochenregeneration nebenwirkungsarm zu f��rdern.