## 4 Conclusions  
In this work, NPTi surfaces were fabricated and adhesion, growth, and osteogenic differentiation of ADSCs were investigated. NPTi surfaces were characterized for their surface topography and surface chemistry using SEM and XPS, respectively. SEM displayed the presence of nano-petal-like features and a \"web\"-like structure in addition to the microporous structure. The XPS survey showed the presence of sodium, which was expected due to the formation of sodium titanate because of HT. The LDH assay was used to evaluate the cytotoxicity of the surfaces, which showed the surfaces were not toxic towards cells, and LDH activity was marginally lower than Ti. Cell viability, adhesion, proliferation, and differentiation on the surfaces were evaluated by seeding ADSCs on the surfaces. Cell viability for the surfaces was evaluated using a CellTiter-Blue assay, and it showed that  
Figure 8: (**a**) SEM and fluorescence microscopy images of ADSCs stained with DAPI (blue), osteoclast (green), and rhodamine phalloidin (red) after 7 days of differentiation induction. (**b**) Osteocalcin area coverage percentage normalized to the number of nuclei after 7 days of differentiation.  
even though the NPTi surface had lower cell viability on day 1, by day 7, it was equivalent to Ti surfaces. NPTi surfaces displayed significantly lowered cell adhesion and proliferation compared to Ti and PTi surfaces after days 4 and 7 of culture. After 7 days, osteogenesis was induced by the introduction of supplements in addition to growth media. Differentiation to osteogenic lineages was evaluated with immunofluorescence staining for a marker protein (OCN) along with protein assays to evaluate the ALP activity and calcium deposition. After 7 days of introducing differentiation media, NPTi surfaces displayed significantly higher OCN expression and calcium deposition amongst all groups. These results displayed that alkali-treated, nanostructured-micro-porous surfaces demonstrated enhanced osteogenic differentiation of ADSCs and have the potential to be used as surfaces for the fabrication of orthopedic implants.  
Conceptualization, A.V.S. and K.C.P.; methodology, A.V.S. and K.C.P.; validation, A.V.S. and E.H.; formal analysis, A.V.S.; investigation, A.V.S. and E.H.; resources, A.V.S. and K.C.P.; data curation, A.V.S.; writing--original draft preparation, A.V.S.; writing--review and editing, A.V.S. and K.C.P.; visualization, A.V.S.; supervision, K.C.P.; project administration, A.V.S.; funding acquisition, K.C.P. All authors have read and agreed to the published version of the manuscript.  
This research was funded by the National Heart, Lung, and Blood Institute of the National Institute of Health, grant number R21 EB033511.  
Not applicable.  
The datasets used and analyzed during the current study are available from the corresponding authors upon reasonable request.  
The authors acknowledge Analytical Resources Core (ARC, RRID: SCR_021758) at the Colorado State University, Fort Collins, for their help in SEM and XPS data acquisition and analysis.  
The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.