## 5 In Vitro Biological Performance of HEA
developed novel TiZrHfCr\({}_{0.2}\)Mo and TiZrHfCo\({}_{0.07}\)Cr\({}_{0.07}\)Mo HEAs by a combination of Ti-Nb-Ta-Zr-Mo alloy system and Co-Cr-Mo alloy system as accepted biomaterial for surgical implants and dental alloys [70]. The in vitro cytocompatibility of these BioHEAs with mouse primary osteoblasts isolated from neonatal calvariate (cell  
Figure 4: Stages of the cell-material surface interactions investigated during the process of evaluation of HEA in vitro biological performance. density, cytomorphological features, actin cytoskeleton organization and focal adhesions maturation) was equivalent to CP-Ti and superior to SUS-316 and Co-Cr-Mo commercial alloys, demonstrating their application as bone implants with multiple functions. As already presented, [PERSON] et al. developed a Ti\({}_{28.33}\)Zr\({}_{28.33}\)Hf\({}_{28.33}\)Nb\({}_{6.74}\)Ta\({}_{6.74}\)Mo\({}_{1.55}\) BioHEA and studied its behavior in vitro in comparison to CP-Ti, SUS316L stainless steel and Co-Cr-Mo as reference metallic biomaterials [69]. Cell adhesion and densities of the primary osteoblasts isolated from neonatal mouse calcural bone and incubated for 24 h in contact with the analyzed samples were significantly lower on SUS316L and Co-Cr-Mo substrates than on CP-Ti and TZHNTM-3 surfaces, as assessed by Giemsa staining. Furthermore, microscopic images acquired after fluorescent labeling of actin cytoskeleton and vinculin (protein enriched in focal adhesions) evinced that the osteoblasts in contact with the surfaces of SUS316L and Co-Cr-Mo alloys exhibit reduced spreading and altered cytoskeletal organization. On the contrary, the cells that adhered to the TZHNTM-3 surface exhibited a widespread morphology and densely packed actin filaments quite similar to the osteoblasts grown on CP-Ti substrate. Altogether, these results demonstrated that the developed BioHEA is significantly more suitable than SUS-316 L and the Co-Cr-Mo alloy for bone tissue regeneration and possesses similar cytocompatibility to CP-Ti. This finding is also supported by its low cytotoxicity derived from high corrosion resistance and the formation of a protective and bioactive oxide layer. In another study performed on MC3T3-E1 mouse pre-osteoblasts seeded on the surface of the equimolar Ti\({}_{20}\)Zr\({}_{20}\)Hf\({}_{20}\)Nb\({}_{20}\)Ta\({}_{20}\) HEA, [PERSON] et al. investigated the substrate cellular colonization by using MTT assay and expressing the percentage of cell viability relative to negative control over a culture period of 7 days [61]. In addition, the number of attached cells was semiquantitatively evaluated by their staining with live/dead kit. For comparative purposes, the cellular response to the conventional Ti6 Al4V alloy was assessed. It was found that on both alloys, the cells expressed high and similar cell viability levels and proliferation rates. Moreover, SEM images indicated that these alloys support the good adhesion and the healthy state of MC3T3-E1 pre-osteoblasts, suggesting the similar biosafety of the TiZrHfbNtHa HEA and the Ti6 Al4V alloy. In a more recent paper, [PERSON] et al. comparatively evaluated in vitro pre-osteoblast response to this HEA bioalloy (Alloy-I), Ti\({}_{25}\)Zr\({}_{25}\)Hf\({}_{25}\)Nb\({}_{12.5}\)Ta\({}_{12.5}\) (Alloy-II) and Ti\({}_{27.78}\)Zr\({}_{27.78}\)Hf\({}_{27.78}\)Nb\({}_{83.33}\)Ta\({}_{83.33}\) (Alloy-III) [67]. These HECs exhibited an increased number of adhered MC3T3-E1 cells and high cellular viability and proliferation, which were comparable to those displayed by the conventional Ti6 Al4V alloy, demonstrating their great potential for biomedical applications. [PERSON] et al. investigated the in vitro behavior of human Wharton's jelly-derived mesenchymal stem cells (HW-MSCs) and MC3T3-E1 pre-osteoblasts (ATCC CRL-2593) grown in contact with the equiatomic MoNbTaTiZr HEA [76].