# Lithographically-Fabricated HA-Incorporated PCL Nanopatterned Patch for Tissue Engineering
## 3 Results
### Cellular Behaviors on the DPSCs in the Equine Bone Coated Nanopatterned Patch  
To evaluate the initial cellular behaviors, we cultured DPSCs on the substrates for 12 h (Figure 3). The DPSCs on the patterned substrates were aligned along the nanogroove direction, whereas DPSCs attached to flat substrates were not arranged in a particular direction (Figure 3a). EBP coating affected the cell morphology of DPSCs in nanopattern and flat patches. DPSCs in the flat patch had smaller cell adhesion areas than other groups, whereas DPSCs in the F-PCL-C had a larger cell area than flat patches and DPSCs formed clusters around the coated EBP powder. DPSCs attached to the surface formed with the nanopatterns were observed to be elongated along with the nanogroove direction. The samples which treated with EBP coating, and more cells were attached to stray from the direction of the pattern (Figure 3a). A 12-hour cell viability evaluation experiment confirmed that all four groups had similar values (Figure 3b). Figure 2: Measurement of wettability of PCL flat and nano-patterned patches with or without equine bone powders. (**a**) Morphology of water droplet, (**b**) elongation factor of the water droplets (n=7) (the ratio of long/short axis length of the water droplet), (**c**) schematic of observation direction of water contact angle. (**d**) The water contact angle on the flat patches (F-PCL and F-PCL-C) was constant regardless of the observation direction, and in the nanogroove pattern (NG-PCL, NG-PCL-C), there was a difference in the observation direction; the water contact angle in both cases was measured. (**n** = 7) (**e**) The results of calculated surface energy based on the Owen–Wendt method. Orange bars mean the surface energy by polar component and blue bars mean the surface energy by non-polar component. It was observed that the total surface energy was decreased by equine bone powder coating, and the surface energy by polar components was increased. To observe the time-dependent morphologies of DPSCs on the substrate, we cultured the DPSCs on the nanopatterned matrix with EBPs for 1 and 7 days (Figure 4) and the images were obtained through FE-SEM and CLSM (Confocal laser scanning microscopy). The DPSCs morphology on NG-PCL-C at day 7 was especially well-oriented along with the nanogroove pattern compared to day 1. In addition, we can observe the expanded cellular shape on the EBPs coated substrate. Although DPSCs on F-PCL and F-PCL-C densely populated, we observed that the cellular morphologies on the substrate were randomly aligned at day 7. Figure 4: DPSCs viability for 7 days of cell culture. (**a**) Observation of the DPSCs morphologies 1 and 7 days of cell culture. (**b**) The results of DPSCs cell viability at 7 days for the cell culture (n =5). Bars with the same letter are not significantly different according to [PERSON]’s multiple range test at 5% probability. Figure 3: Observation of cellular behavior of DPSCs cultured for 12 hours. (**a**) Representative fluorescence images of DPSCs on the substrate and red arrows indicate the EBPs (upper row), FE-SEM images of DPSCs on the substrate and pink color indicate DPSCs (lower row). (**b**) The result of DPSCs cell viability at 12 hours of cell culture (n = 5). Bars with the same letter are not significantly different according to [PERSON]’s multiple range test at 5% probability. To evaluate the combined effect of nanopatterns and EBPs, we cultured DPSCs using the osteogenic conditioned media for 2 weeks. The results of the ALP activity (Figure 5a), the initial osteogenic differentiation marker, showed significant differences in NG-PCL-C compared to F-PCL-C group. As shown in Figure 5c, differences in calcium mineral deposition were observed by Alizarin Red S (ARS) staining. The DPSCs cultured on F-PCL-C and NG-PCL-C showed more accumulated calcium deposition than those on the uncoated substrate (i.e., without EBPs) (Figure 5b).