## 4 Discussion  
In this study, the biological responses to different IAC, as well as those directly UID immersed in osteoblastic cell cultures using a novel direct-exposure technique (SAL), were evaluated. In this technique, the samples did not touch the cultured cells; therefore, the observed adverse cellular reactions were in fact responses to the substances being  
Figure 8: Pre- and post-immersion SEM images of implants (**A**,**C**) and their opposing abutments (**B**,**D**) after 21 days of immersion in culture media at \(\times\)150 and \(\times\)1000 magnification. Pitting areas and black spots were scattered post-immersion on the implant and abutment interfaces at different locations, some denoted with red arrows. released from the immersed samples. The current innovative in vitro model might fall into a category between the direct and indirect models described by [PERSON] [26]. It would be more accurately classified as a modified direct model. This model fits its intended purpose to test the biological responses of cultured cells to the degradation products released from the immersed portion of the hanging solid material. There was no barrier, such as agar or filters, which might influence the results [27] or interrupt the flow of the released products. The culture medium served as a conductive electrolyte, delivering the released substances to the cells from samples that were in the same well and exposed to the same incubation conditions as the cultured cells without direct physical contact with the cultured cells. Therefore, variables related to material surface energy, contact angle, roughness and other material characteristics were avoided [28]. Those variables, however, might have influenced the level of metal ion release and degradation products, which, in turn, might affect the cells. That is the main purpose of this technique: to test the biologic responses to the released substances and not to the material itself. The release of metal ions suggested the occurrence of corrosion phenomena, which may have developed as result of chemical dissolution in the presence of an electrolyte medium, the galvanic coupling of dissimilar metals of the implant and abutment, and crevices that were formed at the surface contact areas of the implants, abutments and abutment screws [22; 29]. The occurrence of corrosion was confirmed by the SEM images, which showed pitting areas and dark spots on the implant and abutment interfaces. The quantification of metal ions released from the IAC into the electrolyte immersion medium has been performed earlier [8], but was not performed using a cell culture medium, which was one of the limitations in the present study. Metal ions released from implant degradation do not exist in unbound ionic or colloidal forms in the serum, but rather specifically bound to proteins, forming metal-protein complexes that would enter the cell through the permeable cell membranes [10; 30]. Such metal-protein molecules require advanced digestion and protein-seperation methods, which should be considered in future research. It was evident that osteoblastic cell viability was influenced by the presence of these specimens, mainly the IAC. This observation was in line with the previous findings of [PERSON] et al., who demonstrated that epithelial cells, as well as fibroblasts cultivated with CoCr and Ti cylinders, showed reduced cell viability, with a stronger negative response to CoCr alloy [31]. However, it should be noted that the majority of published in-vitro biologic research has looked into the effect of prepared unconnected discs or cylinders and not IAC, as in the present study, except for the work of [PERSON] et al. [29], who investigated the effect of the coupling of Ti implants and dissimilar metal abutments on osteoblast differentiation. Therefore, the results of the present study could not be completely compared with other findings. However, it was clear that the UI were less cytotoxic than IAC.