Human Mesenchymal Stem Cell Proliferation onto Microtextured Zirconia To assess human mesenchymal stem cell (hMSC) proliferation around the microtextured Zirconia substrates, we next quantified the percentage of Ki-67+ nuclei at three days post seeding, using image cytometry (Determine 7a). osteogenic phenotype of hMSCs is usually obtained when produced onto isotropic grid/pillar-like patterns, showing an improved cell coverage and Ca/P ratio, with direct implications for BAHA prosthetic development, or other future applications in regenerating bone defects. test was used to compare the statistical significance of differences in area, perimeter, and elongation values for cells and nuclei for each proposed structure compared to the unprocessed flat control. (differences: * 0.05, ** 0.01). Since the data on orientation angles were not normally distributed, they were analyzed using the Kruskal-Wallis one-way analysis of variance followed by Dunns multiple comparison test (differences: * 0.05, ** 0.01, *** 0.001). 3. Results and Discussions 3.1. Design of Structure Arrangements Textured in Zirconia Ceramic Substrate and Surface Characterization When designing a bioinstructive mechanical microenvironment benefic to progenitor cell osteogenic commitments, an enhanced cytoskeleton stretching is essential [53,54,55,56,57]. Given the fact that Zirconia does not naturally form a direct bond with bone [58,59], improving its surface properties by laser texturing, and understanding cell behaviour to increase their pro-osteogenic properties, still represents a challenge. Moreover, it is known that cells behave differently on rectilinear versus curved surfaces, and suppression of cell adhesion and proliferation onto the concave microscaled structures was observed due to cell plasma membrane deformation and Sirt6 subsequent opening of membrane channels onto curved concave structures [60,61]. Within this context, our design entailed IRAK inhibitor 4 IRAK inhibitor 4 isotropic structures: i.e., micropillars with a curved top surface or rectangular micrometric flat tops, as well as their equivalent superimposed microridges/grooves anisotropic arrays. Thus, anisotropic arrays of lines/grooves were obtained, with the ridge top width of approximately 0.9 m (24 m stepwavy profileattenuating the abrupt profile characteristics of grooves and ridges and providing a surface curvature for cell surface interaction) and a 10 m ridge top width, respectively (for the 33 m step), as shown in Figure 2 and Table S1. Open in a separate window Figure 2 Scanning electron microscopy (SEM) and atomic force microscopy (AFM) images of isotropic (24 m || and 33 m ||) and anisotropic textured surfaces structures (24/24 m#, 33/33 m #, and 24/33 m #). When samples were translated in both the XY directions with a 33 m IRAK inhibitor 4 step (33/33 m #), pillars with a square top (side of 10 m) were developed, while a cross-step of 24 m (24/24 m #) led to a pillar with a 0.9 m top width IRAK inhibitor 4 (slightly curved). Alternating the two steps in the xy direction (24/33 m #) resulted in rectangular top pillars (sides of 5 m) ablated in a Zirconia substrate (Figure 2). Flat top structures were characterized by an approximately 4.5 m depth, while the wave-like structures were characterized by depths of 3.5 m (Figure S1). The double-crossing of the laser beam for creating grids/pillar-like structures led to a maximum depth of 8.2 m between the highest and lowest sites at the intersection points, as measured by atomic force microscopy (AFM) (Figure S1). The standard deviations were maintained below 1 m (Table S1, Supplementary Materials). Moreover, the resulting pillar-like structures resulting from crossing the lines and the height of these structures (~3.5C4.5 m) were designed as a hypothesis that the multiscaled surfaces could stimulate the membrane tension of the cells as a result of the.