2.4.3. Cell Culture Studies

The cytotoxicity, cell adhesion and proliferation of surface coated PCL/TiO2 nanocomposites were determined by using human fetal osteoblastic cell lines (hFOB 1.19, ATTC CRL 11372) for day 1 and day 3. Five substrates (cpTi, PCL with 2, 5 and 7 wt % of TiO2 content) were adopted to evaluate the potential of using them for biomedical applications. In brief, cells were cultured in base medium of hFOB cell lines i.e., Dulbecco's Modified Eagle's Medium/Ham's Nutrient Mixture F12 (1:1 DMEM/F12) and complemented with 10% fetal bovine serum (FBS), 1% of non-essential amino acids (MEM) and antibiotics (penicillin G, and streptomycin). For the entire duration of experiment, the culture medium was replaced every alternative day and was preserved in a humidified incubator at 37 ◦C under an atmosphere of 5% CO2. After attaining about 80% confluence, the cells were trypsinated and digested at a final concentration of 5 × 104 cells/cm2 onto the substrates in 24-well plates. Prior to cell seeding, the substrates with PCL/TiO2 coatings were sterilized by immersing in 70% ethanol for 1 h followed by washing 3 times with sterilized phosphate buffered saline solution (pH = 7.4, PBS).

MTT assay for toxicity connected with cell viability and proliferation were also observed on surface coated cpTi substrates for day 1 and day 3. Pure cpTi without any coating is used as the control. After removing the culture medium, hFOB cells were quantitatively assessed by seeding 4 mg mL−<sup>1</sup> MTT 3-(4,5-dimethylazol-2-yl)-2,5-diphenyltetrazolium bromide (yellow) reagent on to the substrates and determined at day 1 and day 3. In brief, both days of incubation, culture medium was removed and washed with 400 μL of prewarmed PBS. Then 400 μL of culture medium accompanied with 60 μL of MTT solution was added to each well-plate containing samples and incubated at 37 ◦C in a 5% CO2 humidified atmosphere. After an incubation period of 4 h, 100 μL of the resulting supernatant was added to each well of 96-well ELISA plate. The plates were gently agitated for 3 min to establish complete crystal dissolution. Percentage cell viability was determined by recording optical absorbance at 570 nm with reference to 690 nm using a microplate reader (Bio TEK Instrument, Winooski, VT, USA, EL307C). To ensure reproducibility, tests were carried out by performing triplicates of samples.
