• We conduct research to understand what is going on in the body during a disease.
• We want to find new starting points for advanced diagnostics and therapeutic options and test their feasibility as quickly and efficiently as possible
• We use non-animal methods whenever possible, but according to the current state of science and regulatory requirements, we cannot completely eliminate animal testing.

We do not treat the issue of animal testing in science lightly. The protection of laboratory animals is a very high priority and all scientists, together with those working in animal husbandry, are committed to the consistent implementation of the 3Rs principle. The three R's stand for Replace, Reduce, Refine and describe ethical principles for research with animals. This means that we use non-animal methods whenever possible. If experiments are unavoidable, we keep animal suffering to a minimum.

In Berlin, we are supported by

• Charité 3R
• BB3R
• Bf3R

There are many questions about animal testing, answers to some of them can be found here:

• Tierversuche verstehen, eine Informationsinitiative der Wissenschaft
• Themenportal Tierversuche, Max-Planck-Gesellschaft
• Deutsches Primatenzentrum – häufig gestellte Fragen
• Tierversuche, wie denken Menschen in Europa darüber?
• Bedeutung des Tierversuchs in der Forschung – Deutsches Regerenzzentrum für Ethik in den Biowissenschaften
• Pro-Test Deutschland e.V.: Ihr frag. Wir antworten

Replace:

At present, there are no in vitro models that can realistically represent the complexity of bone healing or the cellular immune system. However, individual phases and situations of bone regeneration can now be studied in vitro. We use these methods to best prepare our pre-clinical in vivo research and are actively involved in the development of further in vitro models. Similarly, immunological experiments are supported by extensive in vitro experiments, in particular to better design necessary animal experiments.

• In vitro model of the early bone healing phase, an artificial bone hematoma (Pfeiffenberger et al., Functional Scaffold-Free Bone Equivalents Induce Osteogenic and Angiogenic Processes in a Human In Vitro Fracture Hematoma Model, J Bone Miner Res. 2021- 36(6):1189-1201. doi: 10.1002/jbmr.4267)
• In vitro model of bone homoeostasis (Schon et al, Metal-Specific Biomaterial Accumulation in Human Peri-Implant Bone and Bone Marrow, Adv Sci (Weinh) . 2020 Aug 3;7(20):2000412.doi: 10.1002/advs.202000412.) include a link to AG Geissler here (BCRT).
• Use of in silico models (Perier-Metz et al., Initial mechanical conditions within an optimised bone scaffold do not ensure bone regeneration - an in silico analysis. Biomech Model Mechanobiol. 2021 Jun 7. doi: 10.1007/s10237-021-01472-2.; Borgiani et la., Bone morphogenetic protein 2-induced cellular chemotaxis drives tissue patterning during critical-sized bone defect healing: an in silico study. Biomech Model Mechanobiol. 2021 May 28. doi: 10.1007/s10237-021-01466-0.; Orassi et al, Biomechanical Assessment of the Validity of Sheep as a Preclinical Model for Testing Mandibular Fracture Fixation Devices. Front Bioeng Biotechnol. 2021 May 6;9:672176. doi: 10.3389/fbioe.2021.672176.; Borgiani et al, Age-Related Changes in the Mechanical Regulation of Bone Healing Are Explained by Altered Cellular Mechanoresponse. J Bone Miner Res. 2019 Oct;34(10):1923-1937. doi: 10.1002/jbmr.3801.) include a link to AG Checa here (JWI).
• In vitro models for understanding cell behaviour in biomaterials (Herrera et al, From macroscopic mechanics to cell-effective stiffness within highly aligned macroporous collagen scaffolds. Mater Sci Eng C Mater Biol Appl. 2019 Oct;103:109760. doi: 10.1016/j.msec.2019.109760.)
• Use of primary cells in collaboration with the Core Unit Cell Harvesting for in vitro assay of, inter alia, osteogenic differentiation.
• In vitro modelling of cellular therapies. (Drzeniek et al, Bio-instructive hydrogel expands the paracrine potency of mesenchymal stem cells; Biofabrication. 2021 Jul 8;13(4). doi: 10.1088/1758-5090/ac0a32.)
• In silico analyses of immunological correlations. (Vollmer et al, The intratumoral CXCR3 chemokine system is predictive of chemotherapy response in human bladder cancer; Sci Transl Med. 2021 Jan 13;13(576):eabb3735. doi: 10.1126/scitranslmed.abb3735.)
• Evaluation of potential in vitro animal replacement methods. (Wendering et al, The Value of a Rapid Test of Human Regulatory T Cell Function Needs to be Revised; J Front Immunol. 2019 Feb 5;10:150. doi: 10.3389/fimmu.2019.00150. eCollection 2019.)

Reduce:

To keep animal numbers as low as possible we use several strategies:

• AniMatch - a platform that makes it possible to use animals as optimally as possible
• Share Animals - optimally several questions can be answered with one animal, however this can only work if the animal experiments are bundled over a few people who have an overview and can network researchers, which is possible in this unit.
• Sharing of control groups - similar questions and thus research projects with related topics can access the same control groups
• We work with group sizes of 6-8 animals - biologically relevant results are achieved by combining different analytical methods. Our publications and stable project acquisitions and translational successes prove that we are able to generate reliable data with these methods.
• Establishment of a sheep model, which allows a maximum amount of sample evaluations with a minimal burden on the animal (Pobloth et al., Establishment of a preclinical ovine screening model for the investigation of bone tissue engineering strategies in cancellous and cortical bone defects. BMC Musculoskelet Disord. 2016 Mar 1;17:111. doi: 10.1186/s12891-016-0964-4.)

Refine:

• Our pre-clinical models are supervised by our specialist veterinarians in laboratory animal science.
• Protocols are standardised and have been carefully developed
• We work closely with RISystem (https://risystem.com) to provide clinically relevant fixation methods for small rodents.

• We collaborate with specialists to optimise our protocols, e.g. optimising our pain management (Jirkof et al., Administration of Tramadol or Buprenorphine via the drinking water for post-operative analgesia in a mouse-osteotomy model. Sci Rep. 2019 Jul 24;9(1):10749. doi: 10.1038/s41598-019-47186-5.)

Regeneration is the ultimate goal of healing: the tissue should return to its "healthy" state in terms of form and function. Therefore, bone is an ideal model system because it is capable of regeneration. In the ideal case, injuries can heal in such a way that form and function are fully restored.

•  We have documented bone healing over time for our models:

FIGURE 1a

Movat Pentachrome Histology: Bone yellow, muscle light red, bone marrow dark red, cartilage blue-green, connective tissue light blue.

FIGURE 1b

Safranin/ van Kossa Histology:

Mineralised tissue black, rest red

• We work primarily with female animals, as bone healing is slower here and interventions to improve healing can therefore be better investigated.

FIGURE 2

In the case of a small defect, cartilage (blue-green) can still be found in the gap in the female animals 6 weeks after the osteotomy, while yellow mineralised tissue is already visible in the male animals. In the case of a critical defect, the bone marrow cavity is closed up in the female animals, an indication that the healing process has come to a standstill. In the male animals, on the other hand, you see cartilage tissue between the ends of the bones, a sign that endochondral healing is still active and bridging can still take place.

• Our animals are characterised immunologically. Since the immune system plays a crucial role in healing and bone formation, it is essential to know the immune cell composition of the experimental animals (Bucher et al., Experience in the Adaptive Immunity Impacts Bone Homeostasis, Remodeling, and Healing. Front Immunol. 2019 Apr 12;10:797. doi: 10.3389/fimmu.2019.00797.; Schlundt et al, The multifaceted roles of macrophages in bone regeneration: A story of polarization, activation and time. Acta Biomater. 2021 May 8:S1742-7061(21)00298-1. doi: 10.1016/j.actbio.2021.04.052.)

Healing worsens as the proportion of effector T cells increases (Reinke et al. Terminally differentiated CD8⁺ T cells negatively affect bone regeneration in humans. Sci Transl Med. 2013 Mar 20;5(177):177ra36. doi: 10.1126/scitranslmed.3004754.)

FIGURE 3

Legend: µCt healing outcome after 21 days of a 0.7 mm osteotomy in female Bl6n mice, 16 weeks old, LEFT - SPF: animals kept under specific pathogen free (SPF) conditions and having a naïve immune system; RIGHT - non-SPF: animals exposed to environmental pathogens for at least 4 weeks prior to osteotomy (FELASA health-monitored), resulting in effector T cell production (MIDDLE - flow cytometry analysis of CD8+ T cells).

• In other models, the "immune age", i.e. the experience of the adaptive immune response, also plays a crucial role in the course of healing and therapeutic efficacy:

Front Immunol. 2018 May 28;9:1069. doi: 10.3389/fimmu.2018.01069. eCollection 2018. Cytometry A. 2017 Jan;91(1):85-95. doi: 10.1002/cyto.a.22906. epub 2016 Jul 12. Am J Transplant. 2012 Sep;12(9):2384-94. doi: 10.1111/j.1600-6143.2012.04143.x. Epub 2012 Jun 15.

FIGURE 4

By changing the husbandry of the experimental animals, it is possible for the animals to build up immune experience, making them more robust to pathogens and at the same time better reflecting the patient situation. Therefore, an immunological characterisation of our laboratory animals is part of our standard examination.

The legal framework for animal experiments can be found at

charite3r.charite.de/en/research/animal_research/legal_framework/

The following key points below also provide a few additional point about the use of animal models in research.

• Basic Law Article 2: The right of every human being to life and physical integrity.
• Nuremberg Code 1947: Binding principles laid down for experiments on humans
• World Medical Association 1964: Guideline for biomedical research on humans, binding worldwide as the "Declaration of Helsinki" - here it is stipulated that research on humans may only take place when all other possibilities for knowledge (including animal experiments) have been exhausted.
• Animal experiments are expressly provided for in the guidelines for the testing of medicinal products and in the Medicinal Products Act.
• Basic Law Article 5 para. 3 GG: freedom of science is enshrined as an unconditionally guaranteed basic law
• Animal protection was incorporated into the Constitution as a state objective in 2002: Article 20a "The State shall, also in responsibility for future generations, protect the natural foundations of life and animals within the framework of the constitutional order by legislation and, in accordance with the law and justice, by executive power and the administration of justice."

As a consequence of these provisions, a central element of each individual animal experiment is the balancing of the obligation to maintain and improve human health, scientific freedom and the protection of animals from pain and suffering.

Legal frameworks applicable to this consideration are listed below (note text is only available in German):

• EU Directive on the protection of animals used for scientific purposes
• Animal Protection Act
• Animal Protection Experimental Animal Ordinance