# Lithographically-Fabricated HA-Incorporated PCL Nanopatterned Patch for Tissue Engineering
## 4 Discussion
In general, it has been hypothesized that surfaces with nanotopography can improve the cell adhesion by increasing the surface area of the substrate because cells could recognize the morphological features of the surface such as grating, pit, hole, etc.. In our study, nanogrooves also influenced cell adhesion and morphology, as demonstrated by 12-hour cell culture [9]. In this work, DPSCs both recognized the surface topography and EBPs distributed on the substrate. Our interpretation was supported by the results of cellular behavior which include the aligned cellular morphology on nanogrooves and the cluster formed on the EBPs. Moreover, this cellular behavior also influenced the osteogenic differentiation of DPSCs. No significant difference was found in the adhesion ability of DPSCs in all groups, however, the morphology was affected by the surface characteristics of the substrate. The DPSCs cultured on the nanogroove patches were found to have a longer shape than DPSCs on F-PCL and F-PCL-C, with the elongation (major/minor axis) level of cells approximately four times higher. As cells reach confluence, MSCs become bigger and had mostly spindle-like, fibroblastic-like shape [40]. The DPSCs became fibroblastic-like shape and more aligned according to nanogroove. In this study, the ALP activity was observed to evaluate the early stage of osteogenesis on day 7. the ALP activity results showed a significant difference between F-PCL-C and NG-PCL-C, but the difference is not much great. However, in the mineral deposition study, the significant differences in the substrate were found. Previous reports have shown that, in the case of a substrate composed of calcium phosphate (CaP) cement, CaPs in the substrate were stained in red, and it gives background color. After the cell seeding, the stained color became darker when the calcium mineral was deposited in the cells. This trend was also observed in our experiments and confirmed that higher levels of calcium minerals were deposited at NG-PCL-C than F-PCL-C. Given that the mineral deposition of DPSCs in 2 weeks is higher than that of 1 week. We could observe that our substrate affected the promotion of DPSCs' osteogenic differentiation. According to our observation, we had developed an index that represents the degree of osteogenic differentiation of DPSCs using the quantification results of mineral deposition in 2 weeks. The effect of the nanogroove was defined as about 1.28 times, and the effect by EBP was defined as about 1.86 times. In order to achieve successful tissue regeneration, applying a scaffold that stimulates stem cells to improve treatment efficiency is one of the ultimate goals in tissue engineering and regenerative medicine. Bioceramics, calcium phosphate-based materials such as hydroxyapatite, beta-tricalcium phosphate, and xenograft-derived bone grafts, have been used for human bone regeneration. In addition, nanogrooved surface is a platform that is constantly being explored as a new approach for effective implants. No studies have yet been reported on the behavior of human pulp-derived stem cells on nanogrooves and bioceramics materials through in vitro experiments. Here, we suggest a nanogroove/bioceramics integrated platform through the simple dip-coating method that can synergistically enhance the cell-substrate interaction. In summary, the nanogroove substrate coated with EBPs provides an adequate environment for DPSCs compared to the F-PCL group, facilitating the osteogenesis of DPSCs. This explanation was supported through the analysis of morphological features, and our insight according to these observations are briefly shown in Figure 6f. In our platforms, EBP coating could provide a site for cells to be attached and nanogroove could induce cell elongation. This novel combined effect of each cue may enhance cell differentiation.Since endothelin and vitamin have been known as an important role of ECM, it would be efficient strategies to improve our PCL/EBP patches to alleviate the inflammatory response [30].