*3.6. Cell Morphology (Cell Adhesion and Proliferation) Studies*

Cell morphology (cell adhesion and proliferation) of An, PCL and PCL/TiO2 (2, 5 and 7 wt %) scaffolds were carried out using hFOB, a human osteoblast-like cell lines were presented in Figure 6a–j. Integration of TiO2 nanoparticles into PCL, thereby surface coatings on cpTi has significantly increased cell adhesion on day 1 as hFOB cells spread well over the surface and showed the normal morphology of phenotype. Form the SEM analysis, when compared to An (Figure 6a–b) and PCL (Figure 6c–d), it is evident that higher cell proliferation is been observed on the scaffolds containing TiO2 nanoparticles. This early interaction of biomolecules and cells with the material is strongly dependent on the

PCL/TiO2 surface properties, among which hydrophilicity is a key factor. After just 3 days of culture, hFOB cells on PCL/5TiO2 nanocomposites showed an excellent homogeneous structure with a clear evidence of cells penetrating into the scaffolds through their pores (Figure 6g–h). The results are well in connection with the wettability studies (Section 3.3). It is evident that the added TiO2 nanoparticles increase the surface area and surface hydrophilicity, thus by favoring cell adhesion on day 1 and cell proliferation on day 3.

**Figure 6.** SEM images of hFOB cells seeded on various substrates after day 1, and day 3.

Interestingly, when compared to other scaffolds, cell behavior to higher TiO2 content i.e., with 7 wt % demonstrated little attachment and proliferation (Figure 6i–j). This is probably due to some leachables and an inhomogeneous mixture of TiO2 nanoparticles (Figure 1e). Also, it has been stated in many reports that higher TiO2 content leads to toxicity into the substrates [25,43,44]. As found in this study, for a given PCL concentration, depending on type of cells used, seeding densities and cell viability test method, it must be definite and established that at given polymeric concentration, the amount of TiO2 nanoparticles in scaffolds should not surpass a higher activation level, which should be of the order of 5 wt % in the present case. However, optimized TiO2 nanoparticle concentration (5 wt %) will help to engineer the PCL/TiO2 based scaffold favoring new tissue formation with an antimicrobial property.
