*3.4. Bioactivity Studies*

The bioactivity features of PCL/TiO2 nanocomposites are well standardized before. Figure 4 presents the SEM images of the cpTi, pure PCL coated and PCL/TiO2 nanocomposites with 2, 5 and 7 wt % TiO2 content after incubating in SBF for 21 days. The control substrates, i.e., cpTi (Figure 4a), and pure PCL (Figure 4b) exhibit very insignificant mineralization while substantial mineralization takes place in PCL/TiO2 nanocomposite samples. It was observed that PCL bio-nanocomposite with higher TiO2 concentration i.e., PCL/5TiO2 (Figure 4d) and PCL/7TiO2 sample (Figure 4e) have the highest nucleation rate of apatite formation after 21 days. It is worth mentioning that even after immersing in SBF for 21 days, the scaffold preserved its microstructure and showed better interconnectivity of the pores.

Development and growth of apatite layer on the substrate is a dynamic process where the material surfaces dissolve and the new bundle of layers precipitates on the surface [40]. When soaked in SBF, the high content of TiO2 in PCL/5TiO2 and PCL/7TiO2 composites leads to an increase of Ti–OH groups on the surface. The unique development of new Ti–OH groups can stimulus the formation of apatite nucleation. Once the apatite nuclei formed, the growth occurs spontaneously by consuming the positive ions of calcium (Ca2+) and negative ions of phosphate (PO3−) from the SBF fluid to form an amorphous phosphate, which impulsively transforms into hydroxyapatite [Ca10(PO4)6(OH)2] [HA] [41]. Based on the EDS analysis for PCL/5TiO2 (Figure 4f), the Ca/P molar ratio was estimated to be approximately 1.5 which is in close agreement with the chemical formulation of the biomineral HA [35].

**Figure 4.** SEM images of (**a**) An, (**b**) PCL (**c**) PCL with 2 wt % TiO2 (**d**) PCL with 5 wt % TiO2 and (**e**) PCL with 7 wt % TiO2 after immersing in SBF for 21 days. (**f**) EDS analysis results for the newly formed calcium and phosphate of PCL with 5 wt % TiO2.

### *3.5. Cytotoxicity-MTT Assay*

With the intention of using the PCL/TiO2 scaffolds in bone tissue engineering application, the influence of the prepared composites on the growth and viability with hFOB cell lines were investigated for day 1 and day 3 and presented in Figure 5. PCL has widely demonstrated polymer for bone tissue engineering application for its slow degradation kinetics and biocompatibility [42]. In vitro cell culture experiments in the current research manifested the importance of PCL as a function

of the TiO2 content. In fact, the presence of PCL has enhanced cell proliferation as a function of the TiO2 amount. The addition of TiO2 nanoparticles at low concentration (2 wt % and 5 wt %) to polymeric PCL scaffolds has favoured the proliferation of hFOB cells. Compared to cpTi and PCL fiber mat, initial attachment and proliferation of PCL/2TiO2 and PCL/5TiO2 nanocomposites supported the growth of the cells and mediated their proliferation by approximately 20% and 38% respectively. The results demonstrate the substantial and time-dependent growth in cell viability of TiO2 at lower concentrations.

**Figure 5.** hFOB Cell viability on An, Pure PCL coated, PCL/2TiO2 coated, PCL/5TiO2 coated and PCL/7TiO2 coated cpTi samples cultured for day 1 and day 3 (*p* < 0.05).

However, a noticeable reduction in the cell viabilities is being observed in the PCL/7TiO2 sample for both day 1 and day 3. At a higher content of TiO2, the TiO2 nanoparticles have fetched a significant cytotoxic effect with viabilities of 53 ± 4% and 35 ± 5% as observed on day 1 and day 3 respectively. As we know, the cellular behaviour particle cytotoxicity of TiO2 nanoparticles relay on numerous aspects which includes particle shape/size, chemical stability, and mechanical stimulation. Many studies conclusively showed that the effect of TiO2 nanoparticles concentrations on cellular behaviour at higher concentrations [25,43]. From the above analysis, it can be implied that the incorporation of TiO2 nanoparticles have a positive impact and suggests that PCL/5TiO2 can be considered as best suitable biocompatible material that can be employed as a tissue scaffold for the orthopedic application.
