Hidroxiapatita modificada: estudos biológicos para avaliação da biossegurança e das propriedades osteogênicas

Abstract

Hydroxyapatite (HA) is a natural mineral compound found in bones and teeth and can be adapted as a widely used biomaterial in clinical and pharmaceutical applications. Studies show that ionic substitutes can be incorporated into HA, making it more similar to natural bone tissue and promising for bone regeneration. However, these modifications can alter the physicochemical characteristics of biomaterials, necessitating a comprehensive assessment of biosafety before large-scale production. This study aimed to investigate the safety and efficacy of HA-functionalized biomaterials for bone regeneration applications. Divided into five independent chapters, it addressed crucial aspects of biosafety and the regenerative potential of these materials. A systematic literature review was conducted to investigate whether nanostructured HA-based compounds are free of genotoxicity. In this study, it was found that genotoxicity of HA-based biomaterials concerning human health is underestimated in scientific literature. In general, needle-shaped HA nanoparticles tend to cause more DNA damage and chromosomal alterations compared to other forms of HA. Additionally, the results suggest that primary cells are more sensitive than cell lines in assessing the genotoxicity of these biomaterials. The biomaterials used in this study were characterized and produced at the Brazilian Center for Physical Research in Rio de Janeiro. In the second chapter, the cytotoxicity and mutagenicity of a biodegradable bone graft based on nHA, consisting of microspheres of HA nanoparticles encapsulated in alginate, with and without the incorporation of bioactive ions (CO3 2- , Zn2+, and Sr2+), were evaluated. The main results of these analyses were that cytotoxicity tests did not reveal significant adverse effects of HA microspheres in cell culture, and no relevant changes in chromosomal levels were observed in CHO-k1 cells after exposure to the different biomaterials. Moreover, there was no increase in the rate of reverse bacterial mutation, using Salmonella Typhimurium strains, after exposure to these biomaterials. In the third chapter, embryotoxicity tests were conducted in zebrafish to assess the safety of hydroxyapatite-based microspheres, including HA with substituted ions. There was no significant mortality (less than 20%), and no neurotoxic effects were detected. Furthermore, the biomaterials did not alter oxidative stress or negatively affect development. The fourth chapter focused on evaluating the biosafety and bone regeneration potential of HA nanoparticles. Preliminary results of the biosafety of HA nanoparticles at concentrations of 25, 50, and 100 µg/mL were investigated. We used mesenchymal stem cells obtained from umbilical cord tissue as a cellular model for this evaluation. Cytotoxicity was assessed through the MTT test, and osteogenic potential was analyzed by measuring alkaline phosphatase levels and alizarin red staining. In the final chapter, we assessed the biosafety of functionalized HA nanoparticles in zebrafish embryos and larvae at concentrations of 50, 100, and 500 µg/m. Additionally, we investigated the impact of these biomaterials on bone mineralization using techniques such as staining and fluorescence microscopy. Overall, the results obtained in the different chapters of this study highlight the biological safety of HA-based biomaterials, as well as their potential in osteogenesis at various concentrations.