Establishment of a coculture in vitro model for assessing biomaterials for bone regeneration

Bone defects are mainly caused by inflammation, trauma and injury. The use of grafts is therefore necessary to replace damaged tissue. Nowadays, autografts remain the gold standard, despite their drawbacks in terms of availability. Therefore, the research into synthetic bone substitutes arises as a viable alternative. However, the use of a graft just a scaffold is limited to prevent or fight infections, due to the lack of antimicrobial properties that are needed to fight the infections cause. In this case, ideally, a bone substitute should be osteoconductive, osteoinductive and have antimicrobial properties. To correctly validate these new developed substitutes, research needs tools to test these new types of materials and evaluate their efficacy against pathogens, and cocultures seem to be one of the most promising solutions. The aim of this thesis work was to create a prokaryotic-eukaryotic coculture as an in vitro tool for the evaluation of biomaterials with dual-sequential release of antimicrobial and bone regeneration drugs. The bacterium chosen was Staphylococcus aureus because, according to statistics, it is the pathogen that most frequently causes infections during bone tissue repair and/or regeneration in humans. Given the differences observed in the literature papers, a workable protocol for creating the coculture was first implemented using MG-63 cells. Specifically, a bacterial concentration of 10^3 CFU/mL and a contamination time of 2 h were identified as important parameters for the coculture creation. Subsequently, the above protocol was adopted to create a coculture with MSCs, which was used to test porous ceramic microspheres loaded with gentamicin and alendronate, which confer antimicrobial and osteodifferentiation properties, respectively. For this purpose, resazurin cell viability assay and fluorescence microscopy were used to analyze the cells during this experiment. The possibility of applying these assays to the coculture and obtaining results has validated the coculture itself. This means that it has been proven that this coculture can be used as a useful and advantageous tool for the evaluation of biomaterials with antimicrobial and osteoregenerative purposes.