*2.2. Di*ff*erentiation Assays*

This study was performed in order to investigate the effect of the surface topography on the osteoblasts differentiation, using alkaline phosphatase (ALP) quantification assay as early marker, since it plays a critical role in bone formation [21,22], and alizarin red staining as late marker of osteoblast differentiation.

For the differentiation assays, control scaffolds (non-patterned) and patterned ones with 75 μm features (linear or hexagonal topographies) were tested. The differentiation in the two conditions was assessed in the absence (GM) and in the presence (DM) of the biochemical inducer for 21 days. The goal is to understand the physical effect, namely, the topography, of the patterned scaffolds on the cells' differentiation fate, with or without the cells being exposed to biochemical inducers.

After 7 days, ALP was evaluated in the different samples. Alkaline phosphatase is an enzyme mostly found in liver, kidney, and bones, and the measurement of its activity has been found to be suitable for monitoring changes in bone formation and thus in bone cells differentiation [23]. It was found that ALP activity levels are significantly higher for cells under DM, compared to GM (Figure 2). The physical influence of hexagons topographies on the improvement of differentiation can be confirmed by cells' ALP activity in 7 days of culture with GM, compared to cells over the control

and lines patterned scaffolds. These results demonstrating the positive influence of the hexagonal topography are further supported by the alizarin red staining results.

**Figure 2.** Osteogenic differentiation determined by relative alkaline phosphatase quantification assay expression after 7 days of culture (T7), using growth medium (GM) and differentiation medium (DM). The ALP expression was normalized against DNA content using CyQuant cell proliferation assay. \*\*\*\* *p* < 0.0001 (two-way ANOVA).

After 14 and 21 days of cell differentiation, the cell viability assay and alizarin red staining were performed (Figure 3).

**Figure 3.** (**a**) Cellular viability of pre-osteoblast cells in contact with control, linear, and hexagonal scaffold topographies, in a differentiation essay of 7, 14, and 21 days, with GM and DM. \* *p* < 0.05, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001 (two-way ANOVA); (**b**) Alizarin red staining for mineral deposition during osteogenesis induction, at day 14 and 21, with and without DM. Calcified areas are presented with pink color. The scale bar 500 μm is valid for all the images.

Cell viability results (Figure 3a) were compared to a cell mineralization assay (Figure 3b). The presence of DM induces differentiating pathways on cells, providing chemical stimuli to stop proliferating and begin differentiation processes. On the contrary, basal GM provides cells with all the nutrients necessary to proliferate continuously until reaching the confluency. In DM conditions, cells present lower viability for all topographies and dimensions, compared to GM (Figure 3a). Complementing the viability results with the alizarin red images, in order to evaluate the mineralization of bone cells, a lower osteogenic differentiation of cells over linear topographies can be seen, which was predictable from the proliferation assays. On the other hand, the degree of mineralization on isotropic hexagon patterned scaffolds is very similar both with GM and DM after both 14 and 21 days (Figure 3b). These results indicate that the provided physical effect is able to regulate cell fate and may activate differentiation signaling pathways, with no need of biochemical inducers.

In this way, it is concluded that pre-osteoblast cells can be differentiated into osteoblast by specific patterns that also support matrix mineralization.
