## 5 In Vitro Biological Performance of HEA
Thus, on this surface, HW-MSCs exhibited a high cell viability percentage (89%) and a larger cellular body when compared to the control surface (tissue culture polystyrene (TCPS)), suggesting its high potential to support cell adhesion and growth. To investigate the compatibility of this HEA with pre-osteoblast cells, a parallel study was carried out on the SS304 stainless steel. The cellular morphological examination by SEM and fluorescence microscopy revealed smaller cells on this surface with elongated shapes and relatively fewer filopodia extensions when compared to MC3T3-E1 cells in contact with MoNbTaTiZr HEA. On this last material, a cellular monolayer represented by elongated and spindle-shaped pre-osteoblasts was evinced, exhibiting a migratory phenotype with leading and trailing edges and several filopodial extensions as well as actin filament bundles that are crucial for the cell adhesion and proper functioning of cells. Altogether, these in vitro results confirmed the highly biocompatible nature of the MoNbTaTiZr bioHEA. It is worth noting that several members of the same research team previously reported on the behavior of HW-MSCs cultured in the conditioned media from dual-phase HEA comprising the same elements (MoNbTaTiZr) in comparison to conditioned media derived from conventional metallic biomaterials, namely, SS316L and Ti6 Al4V [54]. This alloy underwent extensive surface deformation using stationary friction processing (SFP) and friction stir processing (FSP) to homogenize its microstructure. MTT assay performed on cells cultured in the conditioned media from HEA, HEA-FSP, and HEA-SFP revealed that higher cell viability was induced by the processed HEAs when compared to its as-cast counterpart, SS316L and Ti6 Al4V conventional materials. It was found that all alloys are biocompatible with a cell viability of 85% and over; e.g., the HEA-SFP sample exhibited better cell viability (95%), comparable to that of the cells grown in complete media (control cells). Furthermore, fluorescence microscopy images revealed the exclusive presence of live cells. Thus, it was concluded that the HEA, HEA-FSP and HEA-SFP do not exhibit acute toxicity, all of these samples demonstrating good biocompatibility as implant biomaterials. These results are not surprising since they are in accordance with previous data reported by [PERSON] et al. and [PERSON] et al. regarding the MoNbTaTiZr bioHEA's in vitro biocompatibility following elemental homogenization through 168 h furnace annealing [75; 78]. The superior biological performance of the SFP sample was ascribed to the characteristics of the oxide layer at cell-metallic substrate interface. Specifically, the oxide layer for this specimen was enriched in ZrO\({}_{2}\) and was already presented to promote cell viability and proliferation as well as more homogeneous and stable. This last study was based on indirect contact cell-based tests. The only available study on the in vitro behavior of human gingival fibroblasts (HGFs) on HEAs was reported by [PERSON] et al. [68]. They comparatively investigated the biocompatibility of the single-phase body-centered cubic (BCC) structured Ti\({}_{40}\)Zr\({}_{20}\)Hf\({}_{10}\)Nb\({}_{20}\)Ta\({}_{10}\) BioHEA and single-phase hexagonal close-packed (HCP) equiatomic TiZrHf and CP-Ti conventional reference biomaterial. The fibroblasts in contact with the HEA alloy exhibited enhanced adhesion when compared to TiZrHf and, especially, CP-Ti surfaces, as shown by the increased number of the attached cells, cytoplasmic and filopodial extensions, and more rapid and wider cellular spreading and the genetic expression levels of multiple cell adhesion factors like vascular endothelial growth factor A (VEGFa), matrix metalloproteinase-9 (MMP-9), collagen type 1a (COL1a), collagen type 5a (COL5a) and fibronectin (FN1) indicating the reduction in wound healing times.