Effect of Currently Available Nanoparticle Synthesis Routes on Their Biocompatibility with Fibroblast Cell Lines

Abstract

Nanotechnology has acquired significance in dental applications, but its safety regarding human health is still questionable due to the chemicals utilized during various synthesis procedures. Titanium nanoparticles were produced by three novel routes, including Bacillus subtilis, Cassia fistula and hydrothermal heating, and then characterized for shape, phase state, size, surface roughness, elemental composition, texture and morphology by SEM, TEM, XRD, AFM, DRS, DLS and FTIR. These novel titanium nanoparticles were tested for cytotoxicity through the MTT assay. L929 mouse fibroblast cells were used to test the cytotoxicity of the prepared titanium nanoparticles. Cell suspension of 10% DMEM with 1 × 104 cells was seeded in a 96-well plate and incubated. Titanium nanoparticles were used in a 1 mg/mL concentration. Control (water) and titanium nanoparticles stock solutions were prepared with 28 microliters of MTT dye and poured into each well, incubated at 37 °C for 2 h. Readings were recorded on day 1, day 15, day 31, day 41 and day 51. The results concluded that titanium nanoparticles produced by Bacillus subtilis remained non-cytotoxic because cell viability was >90%. Titanium nanoparticles produced by Cassia fistula revealed mild cytotoxicity on day 1, day 15 and day 31 because cell viability was 60−90%, while moderate cytotoxicity was found at day 41 and day 51, as cell viability was 30−60%. Titanium nanoparticles produced by hydrothermal heating depicted mild cytotoxicity on day 1 and day 15; moderate cytotoxicity on day 31; and severe cytotoxicity on day 41 and day 51 because cell viability was less than 30% (p < 0.001). The current study concluded that novel titanium nanoparticles prepared by Bacillus subtilis were the safest, more sustainable and most biocompatible for future restorative nano-dentistry purposes.

Keywords: Bacillus subtilis; Cassia fistula; TiO2; cytotoxicity; nanoparticles.

Figure 1

Preparation of titanium nanoparticles by three routes: (a) Bacillus subtilis culture, (b) Titanium nanoparticles formed from Bacillus subtilis culture, (c) Cassia fistula plant extract, (d) Titanium nanoparticles formed from Cassia fistula plant extract, (e) Initial TiCl₄ solution, (f) Color change in TiCl₄ solution after heating and (g) Titanium nanoparticles formed from hydrothermal heating.

Figure 2

XRD image of titanium nanoparticles formed by: (a) Bacillus subtilis, (b) Cassia fistula and (c) hydrothermal heating.

Figure 3

SEM image of titanium nanoparticles formed by: (a) Bacillus subtilis, (b) Cassia fistula and (c) hydrothermal heating showing phase forms.

Figure 4

AFM image of titanium nanoparticles formed by: (a) Bacillus subtilis, (b) Cassia fistula and (c) hydrothermal heating showing surface roughness.

Figure 5

EDS image of titanium nanoparticles formed by: (a) Bacillus subtilis, (b) Cassia fistula and (c) hydrothermal heating showing peaks of titanium and oxygen.

Figure 6

FTIR images of titanium nanoparticles formed by: (a) Bacillus subtilis, (b) Cassia fistula and (c) hydrothermal heating showing different functional groups and Ti–O–Ti vibrations.

Figure 7

DRS scan of titanium nanoparticles formed by: (a) Bacillus subtilis, (b) Cassia fistula and (c) hydrothermal heating showing band-gap absorbance energy.

Figure 8

TEM image and histogram of titanium nanoparticles formed by: (a,d) Bacillus subtilis, (b,e) Cassia fistula and (c,f) hydrothermal heating showing size and shape.

Figure 9

DLS image of titanium nanoparticles formed by: (a) Bacillus subtilis, (b) Cassia fistula and (c) hydrothermal heating showing size and shape.

Figure 10

Cell Viability (%) of titanium nanoparticles formed by Bacillus subtilis, Cassia fistula and titanium tetrachloride at first day in comparison to control group (*** p < 0.001, error bars = S.E).

Figure 11

Cell Viability (%) of titanium nanoparticles formed by Bacillus subtilis, Cassia fistula and titanium tetrachloride at the 15th day in comparison to control group (*** p < 0.001, error bars = S.E).

Figure 12

Cell Viability (%) of titanium nanoparticles formed by Bacillus subtilis, Cassia fistula and titanium tetrachloride at 31st day in comparison to control group (*** p < 0.001, error bars = S.E).

Figure 13

Cell Viability (%) of titanium nanoparticles formed by Bacillus subtilis, Cassia fistula and titanium tetrachloride at 41st day in comparison to control group (*** p < 0.001, error bars = S.E).

Figure 14

Cell Viability (%) of titanium nanoparticles formed by Bacillus subtilis, Cassia fistula and titanium tetrachloride at 51st day in comparison to control group. (*** p < 0.001, error bars = S.E).

Figure 15

Mouse fibroblast’s cell morphology exposed to control group prepared by water on first day, 15th day, 31st day, 41st day and 51st day, showing normally large, elongated flat cells with cytoplasm (a,e,i,m,q). Mouse fibroblast’s cell morphology exposed to experimental group of titanium nanoparticles prepared by Bacillus Subtilus on first day, 15th day, 31st day, 41st day and 51st day showing normally large, elongated flat cells with cytoplasm (b,f,j,n,r). Mouse fibroblast’s cell morphology exposed to experimental group of titanium nanoparticles prepared by Cassia fistula on first day, 15th day, 31st day, 41st day and 51st day, showing initiation of pore formation (c), increased pore formation (g), increased pore formation and mild degradation (k), increased pore formation and mild degradation (o) and loss of normal spindle shape (s). Mouse fibroblast’s cell morphology exposed to experimental group of titanium nanoparticles prepared by hydrothermal heating on the first day, 15th day, 31st day, 41st day and 51st day, showing slight degradation (d), increased pore formation and degradation (h), greater disruption (l), complete loss of cell symmetry (p) and entire loss of normal size, shape and symmetry of cell (t).