Modern Approach to Testing the Biocompatibility of Osteochondral Scaffolds in Accordance with the 3Rs Principle─Preclinical In Vitro, Ex Vivo, and In Vivo Studies Using the Biphasic Curdlan-Based Biomaterial.

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The aim of this work is to provide a comprehensive set of biological tests to assess the biomedical potential of novel osteochondral scaffolds with methods proposed to comply with the 3Rs principle, focusing here on a biphasic Curdlan-based osteochondral scaffold as a promising model biomaterial. In vitro experiments include the evaluation of cytotoxicity, mutagenicity, and genotoxicity referring to ISO standards, the assessment of the viability and proliferation of human chondrocytes and osteoblasts, and the estimation of inflammation after direct contact of biomaterials with human macrophages. Ex vivo experiments include assessments of the response of the surrounding osteochondral tissue after incubation with the implanted biomaterial. In vivo experiments involve an evaluation of the toxicity and regenerative potential of the biomaterial in zebrafish (larvae and adults) and in osteochondral defects in dogs (veterinary patients). The applied set of tests allows us to show that the Curdlan-based scaffold does not induce cytotoxicity (cell viability close to 100%), mutagenicity (the level of reversion is not 2× higher compared to the control), and genotoxicity (it does not exhibit any change in chromosomal aberration; the frequency of micronuclei, micronucleated binucleated cells, and cytokinesis-block proliferation index is comparable to the control; moreover, it does not cause the formation of comets in cells). This biomaterial also promotes the viability and proliferation of chondrocytes and osteoblasts (the OD values between the fourth and seventh day of incubation increase by approximately 1.6×). The Curdlan-based scaffold stimulates only a transient inflammatory response in vitro and ex vivo. This biomaterial does not cause Danio rerio larvae malformation and also enables proper regeneration of the caudal fin in adults. Finally, it supports the regeneration of an osteochondral defect in veterinary patients. Thus, this is a proposal to use alternative methods for biological assessment of osteochondral scaffolds as opposed to commonly used tests using large numbers of laboratory animals.