In vitro compatibility investigations of new coatings on biomaterials

Purpose. To investigate the cytotoxicity of new biomaterial surfaces using standardized procedures based on human gingival fibroblasts evaluations of cell viability and cell morphology plus additional analysis of scanning electron (SEM), immunofluorescence and supravital microscopy methodologies. Materials and methods. Human gingival fibroblasts were cultured and all coated and uncoated discs were cleaned and sterilized. Some discs were left in PBS for 30 days for the elution test and the extractant solution was transferred to confluent cell layers, observations were made after 1, 24 and 72 hours. Direct cell contact test, discs were placed in 24-wells and cells seeded onto the discs and observed at 1, 24 and 72 hours for any change of cell morphology, lysis, proliferation and differentiation. Cell viability was measured by adhesion after 24 hours with crystal violet using a spectrophotometer at 595nm wavelength. Cell adhesion was observed using SEM. Samples were fixed and stained with fluorescent dyes and observed under a fluorescent microscope for immunofluorescence. Supravital microscopy was done by staining the living cells with Acridine Orange and observed using EPI illuminescence. Results. The elution test after 1, 24 and 72 hours continued cell proliferation was observed. Direct cell contact test at 1, 24 and 72 hour observations showed no signs of toxicity. The SEM showed cell attachment with all the materials, 48 hours of incubation was better than 24 hours. Immunofluorescence showed normal external cell morphology with no degeneration in the nuclei. Supravital microscopy showed that is a viable method to stain a living cell and the internal morphology. Conclusions. Direct contact test between the new biomaterial coatings and titanium alloy did not show a contact inhibition zone in any of the time periods. Cell viability between the materials was constant, increasing the number of cells with time. The elution test the extractant solution did not produce cell lysis. The SEM, in situ immunofluorescence and supravital microscopy were reproducible and informative tests. SEM showed good cell attachment with all materials. In situ immunofluorescence showed that cells retained normal cell morphology. Supravital microscopy showed the possibility to directly observe the internal structures of cells when in contact with the biomaterials. Overall, these tests showed similar biocompatibility for the boron, diamond and standard titanium alloy surfaces.