## 3 Results and Discussion
Phosphate for bone mineral (hydroxapatite (HAp)) formation is provided by \(\beta\)-glycerophosphate. Ascorbic acid facilitates an increase in collagen type 1 secretion, which ensures osteogenic differentiation [37]. It is important for osteogenesis for successful osseointegration of implants [26]. ADSC differentiation to osteogenic lineages was evaluated through immunofluorescence microscopy and protein-based assays. Deposition of  
Figure 3: Normalized cell viability results after 4 and 7 days of culture (* denotes \(p<0.05\)). osteocalcin--a marker protein for osseointegration, was evaluated through immunofluorescence microscopy. Additionally, calcium deposition and ALP secretion were evaluated through assays to understand the differentiation level of cells. ALP plays a crucial role in the mineralization of the bone matrix and is an early indicator of immature osteoblast activity [38]. As it is an early indicator, ALP activity usually reaches its peak when differentiation is in its early stages and then decreases before increasing again [39]. The ALP results for different surfaces were normalized with the total protein (microBCA) content after 7 days of inducing differentiation (Figure 6). Normalized ALP activity was higher for Ti and PTi surfaces than for NPTi surfaces. The lower expression could be attributed to the cyclic nature of the ALP and might be due to the micro/nano topography, which alleviates the rate of differentiation to osteogenic lineages. Figure 4: **(a)** Fluorescence microscopy images showing ADSCs stained with DAPI (blue) and rhodamine phalloidin (red) on days 4 and 7 of culture. **(b)** Quantification of cell count on days 4 and 7 (* indicates \(p\) < 0.05). Hap is an integral inorganic component of the bone and is made up of two major components: calcium and phosphorous. Hap is released during the mineralization process; hence, it is necessary to quantify it to get an idea of the differentiation of cells. Phosphorous is released by cells in the form of ALP. Hap crystals are deposited on the surfaces by cells. HCl was used to dissolve calcium deposited on surfaces and quantified by a colorimetric assay, and the results were normalized with the microRNA content (Figure 7). Ti and Pti surfaces displayed similar calcium deposition; however, NPTi surfaces displayed significantly higher (almost three times) calcium deposition. As the differentiation of stem cells to osteogenic lineage begins, some matrix proteins that are required for mineralization are released. One of those proteins is osteoclast (OCN), a late marker in osteoblast differentiation that is involved in the formation of the bone matrix [40]. OCN deposition was evaluated on different surfaces using immunofluorescence microscopy 7 days after differentiation was induced (Figure 8). OCN was present on all surfaces, hence indicating the differentiation of ADSCs to osteoblasts. The OCN  
Figure 5: ADSC SEM images on surfaces after 4 and 7 days of culture at 500\(\times\) and 2500\(\times\) magnification. Figure 6: Normalized ALP activity on surfaces after 7 days of inducing differentiation. area was calculated using ImageJ software (Version 1.53) and normalized by the number of nuclei on the surfaces. Immunofluorescence images show the presence of OCN on all the surfaces. Despite a lower cell count, NPTi surfaces displayed a higher normalized OCN expression. These results, combined with calcium deposition results, demonstrate a higher differentiation of ADSCs to osteoblasts on NPTi surfaces. Lower cell proliferation yet higher differentiation has been observed in similar studies and is attributed to the promoted Runx2 on nanostructured surfaces [41; 42]. These studies also demonstrated increased OCN deposition and ALP activity [42]. The calcium deposition and OCN are higher for PTi surfaces than for the Ti surface, hence demonstrating that micro-porosity enhances differentiation. The higher differentiation between PTi and NPTi surfaces can be clearly attributed to HT. Stem cell morphology was evaluated using SEM (Figure 8).