*3.4. Di*ff*erentiation Study*

Osteogenic differentiation of hMSCs cultured on the 3D printed scaffolds was studied for up to 21 days. Figure 6 shows the results from alizarin red (AR) staining and assay. The scaffolds with wavy designs showed more staining (Figure 6A) and higher values of mean calcium deposition (Figure 6B). The value of the mean calcium deposition in wavy groups was in the range of 2.5 to six times higher than that of the orthogonal group. Specifically, the average calcium deposition was equal to 9.33 ± 0.98 mM for A0.75W4, 8.14 ± 2.86 mM for A0.5W2, 7.60 ± 1.65 mM for A1W4, 6.12 ± 3.07 mM for A0.5W4, 3.96 ± 2.06 mM for A0.5W3, and 1.53 ± 0.10 mM for orthogonal scaffolds, in descending order. ALP activity assay results, at Culture Days 14 and 21, are given in Figure 7. Our results showed an increase in ALP activity for all sample groups from Day 14 to Day 21, and the ALP activity of the wavy scaffolds was higher than that of the orthogonal group at both Day 14 and Day 21 (Figure 7). At Day 14, A0.5W3 (13.16 ± 3.17 a.u.) was significantly higher than the orthogonal group (5.96 ± 1.58 a.u.). At Day 21, A0.5W2 (46.83 ± 7.90 a.u.) and A0.5W3 (45.51 ± 4.20 a.u.) were much higher than that of the orthogonal group (32.31 ± 0.89 a.u.). Representative fluorescent images showing vinculin staining at Day 7 are shown in Figure 8. We observed more pronounced vinculin fibers that were aligned with the wavy struts for wavy scaffolds as compared to diffused and randomly oriented vinculin for the orthogonal scaffold. Figure 9 shows the representative confocal images of the hMSCs cultured on 3D printed scaffolds, in which cells were stained for osteocalcin (OC, green), F-actin (red), and nuclei (blue) at Culture Day 14 and 21. Osteocalcin staining was more pronounced on curved struts as compared to linear struts.

**Figure 6.** (**A**) Optical microscopy images of the hMSCs stained for alizarin red (red) after culture in osteogenic induction media for 21 days. Scale bars are 200 microns. (**B**) Alizarin red concentration indicating calcium deposition at Day 21. (\* *p* < 0.15, \*\* *p* < 0.05, for *n* = 3).

**Figure 7.** Alkaline Phosphatase (ALP) activity assay results for: (**A**) Day 14 and (**B**) Day 21 (\* *p* < 0.15, \*\* *p* < 0.05, for *n* = 3).

**Figure 8.** Multiphoton confocal images of hMSCs that are stained for vinculin (green) at Day 7.

**Figure 9.** Multiphoton confocal images for hMSCs that were cultured in osteogenic induction media for 14 (**top** row) and 21 days (**bottom** row). Cells were immunostained for osteocalcin (green) and stained for F-actin (red) and cell nuclei (blue). Scale bars are 200 microns.
