Investigation of the cytotoxic and genotoxic effects of different forms of nanohydroxyapatites

Biomaterials are used in orthopedic applications, facial and maxillofacial surgery, artificial heart parts, metal parts and implantable devices. These materials are widely used in the field of medicine because they are compatible with the organism, not allergic, and resistant to physical and chemical factors. Hydroxyapatite (HA) is bioactive calcium phosphate ceramics that is compatible with tissue. In this study, the effects of spherical and rod forms of nano sizded Has (nHA) were investigated in Drosophila melanogaster in vivo and in two different cell lines (human embryonic kidney epithelium (293T) and human vascular endothelial (HUVEC) cell lines in vitro). Potential genotoxicity of nHAs was investigated with Drosophila Wing Somatic Mutation and Recombination Assay (SMART) and Single Cell Gel Electrophoresis (COMET) methods in in vivo studies. Caspase activities were evaluated in Drosophila hemocytes to examine the activation in cell death pathways. In order to investigate gene expressions in Drosophila, the expressions of 36 genes involved in DNA repair were investigated with the Real Time Polymerase Chain Reaction (RT2-PCR) method. Within the scope of in vitro studies, cytotoxicity with WST-1 cell viability test and trypan blue test and genotoxicity potential with KOMET were evaluated in 293T and HUVEC cell lines. According to the results obtained, the potential genotoxic effects of nHA sphere and rod forms In the SMART test with D. melanogaster somatic cells, genotoxicity was detected in spherical form in small single-type, total mwh clones and total clones at concentrations of 50, 100 and 200 ppm. No genotoxic effect was observed at any dose studied in rod-form nHA applications. In the KOMET test with Drosophila hemocytes, spherical and rod-shaped nHAs at 100 ppm concentration caused genotoxicity in Drosophila hemocytes in terms of tail density. In the caspase activities studies, it was determined that 200 ppm concentration in spherical form increased the caspase-3/7, caspase-8 and caspase-9 activities. It has been determined that rod-formed nHA activates the intrinsic pathway signal cascades (caspase-9 and caspase-3/7) at 25 and 50 ppm doses, while it activates the extrinsic apoptotic pathway (caspase-8) at high concentrations. In the RT2-PCR analysis in which gene activation was examined, an increase in Mlh1 and Brca2 genes was observed, while a decrease in spel1, Rad51C, dap, MCPH1, Rad51D, Rad1, Pms2, mu2, Sir2 genes were observed in the application of 25 ppm in spherical form. At 200 ppm concentration, an increase was observed in the expression of only the Mlh1 gene, while the expression of the Fen1, Rad9, Fancd2, Rad51C, Hus1-like, dap, DNA-ligl, MCPH1, Rad51D, Ercc1, Rev1, Pms2, tefu, RpLP2 genes decreased. A decrease in 25 ppm concentration and an increase in Mlh1 and Brca2 gene expressions were observed in rod form. In addition, there was a decrease in CG9601/PNKP, Rev1, Dap, mu2, RpLP2, Pms2, spel1, Sir2, Rad51C, PCNA, stg, Rad51D, Fen1, MCPH1 genes. There was no increase in the expression of genes operating at 200 ppm concentration, but a decrease was detected in Xpac, spel1, Fen1, Rad9, Tbp, Cdk7, DNApol-eta, Fancd2, XRCC1, Rad51C, Hus1-like, Ogg1, dap, DNA-ligl, CG9601, mei-41, MCPH1, put, Rad51D, Rev1, Rad17, Rad1, Pms2, mu2, tefu, RpL32, Sir2, RpLP2, stg, SdhA genes. Within the scope of the thesis study, in the evaluation made with the WST-1 test on the 293T cell line, it was observed that the cell viability increased in both spherical and rod form nHA applications in 24, 48 and 72 hours incubation periods depending on the dose. In the test performed with trypan blue, a dose-dependent decreased cytotoxic effect was observed in spherical form. In stick form, however, no cytotoxic effect was detected. In the HUVEC cell line WST-1 test, it was observed that cell viability increased in both spherical and rod form in a dose-dependent manner. In the evaluation made with trypan blue, cytotoxic effects were observed only at concentrations of 1000, 800, and 600 ppm in spherical form in 24 hours of incubation. In stick form, cytotoxic effect was observed in 48 and 72 hour incubation periods. According to in vitro comet results, it was observed that the genotoxic effect on tail density parameter in spherical nHA 293T cell line was higher at low doses in all three incubation periods. The genotoxic effects of rod-form nHA in 293T cell line were observed at tail density at doses other than 200 ppm in the 24-hour incubation period, while DNA damage was observed at 200 and 400 ppm in the 72-hour incubation period at a dose of 400 ppm in the 48-hour incubation period. The genotoxic effect of spherical nHA on the HUVEC cell line was observed at all doses except 200 ppm in the 24-hour incubation period, 800 ppm in the 48-hour incubation period, and 1000 ppm in the 72-hour incubation period in the tail density parameter. Genotoxic effects were observed in the rod-formed nHA HUVEC cell line at doses of 200 and 400 ppm in the 24-hour incubation period, and 400 and 800 ppm in the 48-hour incubation period in the tail density parameter. In addition, DNA damage was not observed in any of the working doses during the 72 hour incubation period., According to the results obtained from the study, it was observed that the rod form caused less toxic effects than the spherical form in both D. melanogaster and experiments with 293T and HUVEC cell lines. It is thought that the spherical form may have induced toxicity responses in the cells due to its morphological structure and its smaller size compared to the rod form.