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

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.


Introduction
Nanotechnology has quickly gained importance in medical and dental applications due to its quality production and prompt response to host tissues interaction by crossing tissue barriers [1,2]. Several metal nanoparticles have recently attracted interest as a consequence of their unique qualities, which include optical, mechanical, biological and physical properties [3]. Titanium is the most preferred material among them because it has many additional compelling features and characteristics that make it superior, e.g., high electrical conductivity, high thermal diffusivity, malleability, low thermal conductivity and wear and corrosion (scratching) resistance [4]. Moreover, titanium has also become the material of choice due to its cost-effectiveness [5], non-allergic nature, low toxicity, fatigue resistance and biocompatibility [6,7]. Titanium has achieved great success in dentistry as a result of its accountable biological reaction with human tissues [8]. There are multiple applications of commercially available titanium nanoparticles in medicine and dentistry [9], such as cell imaging, biosensors of biological assays, drug delivery systems, photodynamic therapy for cancer and genetic engineering in medicine [10]. The vast utilization of titanium nanoparticles in clinical dentistry includes composite adhesives and bonding agents [11], glass ionomer cement restorations [11][12][13], dental implants [14], bleaching and whitening agents [15], irrrigants in root canal treatment, mouth washes, tooth pastes and polishing pastes [16]. Previously, titanium nanoparticles enhanced the antimicrobial properties and bond strength of composites in orthodontics [11]. In the application of glass ionomer cements, these nanoparticles significantly increased the flexural strength, compressive strength, micro-hardness and shear bond strength to both enamel as well as dentin to a large extent [12,13]. In addition, their usage as dental implants have improved the osteoblast proliferation, phosphate activity, bone matrix deposition and adhesion [14]. The bleaching and whitening products that employed titanium nanoparticles in dentistry has imparted the utmost aesthetics to the teeth [15]. The increased efficacy of titanium nanoparticles as irrigants, toothpastes and mouthwashes in dentistry has been due to their increased antibacterial activity as compared to the chlorohexidine used previously. Titanium nanoparticles are also used in manufacturing orthodontic wires, crowns, maxillary obturators, bridges and files [17,18]. The biocompatibility of titanium nanoparticles is the main feature that makes them unique and extensively utilized in the field of dentistry [16].
The factors responsible for the biocompatibility of titanium nanoparticles are synthesis routes, surface topography, properties such as phase form, particle size, band gap energy, elemental composition and functional groups [19]. The most significant factor responsible for making these nanoparticles cytotoxic and non-biocompatible are the routes involved in their synthesis. The nanoparticles are synthesized by either conventional methods (physical and chemical) or biological methods (microorganisms and plants) [20]. For years, several different popular conventional methods used for synthesis of metal oxide nanoparticles have employed different chemicals as reducing and capping agents. These chemicals form toxic by-products in the production of titanium nanoparticles, resulting in the cytotoxicity of these newly formed nanoparticles [21][22][23]. Many completely natural resources have been manipulated by biological synthesis, such as algae, plants, bacteria, viruses and fungi. These organisms utilize their natural biomolecules as reducing and capping agents. These natural biomolecules do not produce any toxic byproduct, resulting in non-cytotoxic behavior of these nanoparticles [24]. Thus, the stability and sustainability of these nanoparticles is enhanced, which leads to their superior behavior in clinical performance [25,26]. The conventional methods including both physical and chemical processes for production of nanoparticles in dentistry are very common because of their purity, uniformity and quick production. The major drawbacks associated with these nanoparticles is their low yield; high temperature, pressure and energy consumption; potent chemical accelerators utilization; and release of toxic by-products. All these factors are responsible for adversely affecting living beings, as well as our environment on the larger scale [25,27,28]. Although, titanium nanoparticles synthesized by physical and chemical processes, microorganisms and plants have been widely used in various dental applications, insufficient data are available on the cytotoxicity of these nanoparticles regarding synthesis protocols. Still, hazardous side effects of titanium of nanometer size have already been reported in the literature [29]. The reason behind this could be that chemically synthesized titanium nanoparticles had been used previously to enhance the mechanical properties of dental materials without focusing on the most important aspect in the health conditions referred as biocompatibility and biosafety [12,13]. There is an urgent need to evaluate human health and environmental safety regarding the use of nanoparticles. The biocompatibility and biosafety of few commercial titanium nanoparticles have been investigated, which showed different levels of cytotoxicity against various cell lines [19]. The current study is performed to find out the cytotoxic nature of novel titanium nanoparticles produced by Bacillus subtilis, Cassia fistula and hydrothermal heating of titanium tetrachloride methods in order to ascertain the most biocompatible titanium nanoparticles obtained that could be utilized in future restorative nanodentistry without any fear of failure.

Materials
Titanium chloride-IV (Sigma-Aldrich, Darmstadf, Germany) was purchased from the Pakistan Institute of Engineering and Applied Sciences (PIEAS). The strains of Bacillus subtilis having accession no. ATCC ® 6633 TM were acquired from the National Institute of Health (NIH), Islamabad, Pakistan. The leaves of Cassia fistula plant were taken from Public Park of I/8 sector, Islamabad, and were dried. The titanium chloride-IV, strains of Bacillus subtilis and leaves of Cassia fistula were used for the synthesis of titanium nanoparticles. The L929 mouse fibroblast cell line ('ATCC, Manassas, VA, USA) was used to test the cytotoxicity of Ti nanoparticles using the MTT assay [30].

Preparation of Titanium Nanoparticles by Three Routes
The methodology for synthesis of biogenic titanium nanoparticles incorporating Bacillus subtilis was produced according to Kirthiet et al. (2011) [31]. Fresh culture of Bacillus subtilis was incubated at 28 • C and centrifuged at 150 rpm into 100 mL of nutrient broth to form the bacterial culture solution. After 24 h, 20 milliliters of 0.025M Ti(OH) 2 solution (American Elements, 10884-Weyburn Ave, Los Angeles, CA, USA) was inserted into the bacterial culture solution at 60 • C for 10 min to obtain newly formed titanium nanoparticles, which were annealed at 80 • C and calcinated at 450 • C to obtain a fine powder. The method for the formation of green titanium nanoparticles using Cassia fistula leaves was taken from a previous study [32]. One milligram of dried Cassia fistula leaves was mixed with 100 mL of water, which was heated at 100 • C for 5 min to form a plant extract. Then, 1 mL of Ti(OH) 2 (American Elements, 10884-Weyburn Ave, Los Angeles, CA, USA) was poured into 80 mL of water to obtain a Ti(OH) 2 stock-solution. Afterwards, 20 mL of plant extract solution and 80 mL Ti(OH) 2 stock-solution was kept at 28 • C and centrifuged at 150 rpm for 24 h to obtain titanium nanoparticles. The nanoparticles were dried at 80 • C and then calcinated at 450 • C for fine powder formation. The procedure used to synthesize titanium nanoparticles through hydrothermal heating of titanium tetrachloride salt (TiCl 4 ) was conducted according to the study conducted previously [33]. Firstly, 1 mL TiCl 4 salt (Sigma-Aldrich, Merck KGaA, Darmstadf, Germany) was poured into 100 mL of deionized water to obtain a 1M salt solution. After this, the salt solution was heated at 80 • C under continuous stirring in order to attain titanium nanoparticles. Then, these nanoparticles were annealed at 110 • C and calcinated at 450 • C into fine powder form.

Characterization Techniques
The characterization techniques used for confirming the shape, phase state, size, surface roughness, elemental composition, texture and morphology of the novel titanium nanoparticles formed by Bacillus Subtilis, Cassia fistula and hydrothermal heating was carried out by XRD cells were maintained in the Dulbecco modified eagle medium (DMEM) (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, New York, NY, USA) and 1% penicillin-streptomycin antibiotics (Life Technologies, Auckland, NZ, USA). L929 cells from passage P4-P8 at 70-80% confluence was seeded directly onto a 96 well plate (1 × 10 4 cells), which was incubated for 24-48 h to obtain a confluent culture [34,35]. All types of titanium nanoparticles prepared by three routes were utilized as 1 mg/mL concentrations to form stock solutions for these nanoparticles, which was 100 ug/mL. Deionized water was used as the control. When cells attained 70-80% confluence, the cells were exposed to titanium nanoparticles (50ul/well) for the first 24 h. Then, twenty-eight microliters of MTT dye (Sigma-Aldrich, Merck CT01-5,KGaA, Darmstadf, Germany) (2mg/mL) was added to each well, and incubated at 37 • C for 2 h. The culture medium was changed after every two weeks throughout the experiment to prevent its contamination by bacteria and fungi. The mouse fibroblast cells were checked after every 2-h and were incubated at 37 • C in 5% CO 2 in humidified atmosphere on regular intervals for the growth. After the multiplication of cells in the flask, their splitting was performed, and they were detached from the base to float easily. After the increase in the number of cells, they were seeded in a 96-well plate (1 × 10 4 cells) and were cultured after every two days for the whole duration of the experiment. Before cytotoxicity testing, 50 ul/well of titanium nanoparticles were exposed to freshly prepared cell culture containing the maximum number of cells prepared for each analysis at different days. Later on, the fluorescence reader 'BIORAD' (Thermo-Fisher, New York, NY, USA) was utilized to measure fluorescence at 490 nm wavelength for day 1. Similarly, the readings were obtained in triplicates for the remaining days, i.e., day 15, day 31, day 41 and day 51, with the reader [36]. The cytotoxicity (cell viability) was measured as [37]: The cell viability refers to the cytotoxicity status of nanoparticles by showing the percentage of alive or dead fibroblast cells exposed to them. The cytotoxicity status of titanium nanoparticles is declared as "non-cytotoxic in case of cell viability = >90%", "mildly cytotoxic in case of cell viability = 60-90%", "moderately cytotoxic in case of cell viability = 30-60%" and "severely cytotoxic in case of cell viability = 30% or less" [38].

Cell Morphology Assessment
An inverted fluorescence microscope (OPTO-EDU, A-16.0910, Beijing, China) was used to investigate the extent of the cytotoxicity status through fibroblasts cell morphology exposed to titanium nanoparticles produced by different routes in this study. The abnormal changes in the cell morphology of these fibroblasts were demonstrated via images taken with this microscope. These abnormal changes were witnessed with respect to the fibroblasts' size, shape, structure and organelles after being exposed to the titanium nanoparticles [34].

Statistical Analysis
All statistical analyses were conducted using statistical analysis software PRISM (GraphPad Prism 6, San Diego, CA, USA). Data for the experiments are expressed as mean ± standard deviation (SD). One-Way ANOVA test was used to determine the statistically significant differences. Once differences were obtained, then a Post Hoc Tukey test was conducted for multiple differences at a confidence interval of 95% (p < 0.05).

Preparation of Titanium Nanoparticles by Three Routes
The fabrication of titanium nanoparticles from Bacillus subtilis culture, Cassia fistula plant and titanium tetrachloride salt was confirmed by change in the color of their solutions used during the preparation process. The initial color of Bacillus subtilis culture solution was yellowish, whereas that of the Cassia fistula plant was green and titanium tetrachloride salt was purplish black (Figure 1). The color of these solutions turned white initially, followed by the formation precipitates at the bottom of each flask containing the titanium nanoparticles.

Preparation of Titanium Nanoparticles by Three Routes
The fabrication of titanium nanoparticles from Bacillus subtilis culture, Cassia fi plant and titanium tetrachloride salt was confirmed by change in the color of their tions used during the preparation process. The initial color of Bacillus subtilis cultur lution was yellowish, whereas that of the Cassia fistula plant was green and titanium rachloride salt was purplish black (Figure 1). The color of these solutions turned w initially, followed by the formation precipitates at the bottom of each flask containin titanium nanoparticles.

XRD
The XRD investigated the phase form and particle size of the Titanium nanopart The XRD of the titanium nanoparticles generated by Bacillus subtilis were mixed an and rutile phases, whereas those formed by Cassia fistula and hydrothermal heating pure anatase phase. The particle sizes of the titanium nanoparticles were calculated b Debye-Scherer formula and were found to be 63.13 nm for nanoparticles prepared b Bacillus subtilis, while those formed by Cassia fistula and hydrothermal heating were nm and 11.29 nm, respectively ( Figure 2, Table 1).

XRD
The XRD investigated the phase form and particle size of the Titanium nanoparticles. The XRD of the titanium nanoparticles generated by Bacillus subtilis were mixed anatase and rutile phases, whereas those formed by Cassia fistula and hydrothermal heating were pure anatase phase. The particle sizes of the titanium nanoparticles were calculated by the Debye-Scherer formula and were found to be 63.13 nm for nanoparticles prepared by the Bacillus subtilis, while those formed by Cassia fistula and hydrothermal heating were 15.79 nm and 11.29 nm, respectively ( Figure 2, Table 1). cles prepared by the Bacillus subtilis, while those formed by Cassia fistula and hydrothermal heating were 15.79 nm and 11.29 nm, respectively ( Figure 2, Table 1).

SEM
The SEM images confirmed the shape and particle size of the titanium nanoparticles. The SEM image of titanium nanoparticles formed by Bacillus subtilis were spherical and

SEM
The SEM images confirmed the shape and particle size of the titanium nanoparticles. The SEM image of titanium nanoparticles formed by Bacillus subtilis were spherical and 63.13 nm in diameter. On the other hand, the titanium nanoparticles prepared by Cassia fistula were 15.79 nm in diameter, having a mixture of spherical and irregularly shaped nanoparticles. The titanium nanoparticles prepared by hydrothermal heating were predominantly irregular in shape and 11.29 nm in diameter. The results of SEM and XRD were in agreement with each other ( Figure 3, Table 1).

AFM
The AFM determined the surface roughness of the titanium nanoparticles. The AFM image of the titanium nanoparticles prepared by the Bacillus subtilis showed the minimum surface roughness of about 4.11 Rms while the titanium nanoparticles formed by Cassia fistula revealed moderate surface roughness of about 7.96 Rms. Additionally, the titanium nanoparticles formulated by hydrothermal heating depicted severe surface roughness of about 11.31 Rms ( Figure 4).

SEM
The SEM images confirmed the shape and particle size of the titanium nanoparticles. The SEM image of titanium nanoparticles formed by Bacillus subtilis were spherical and 63.13 nm in diameter. On the other hand, the titanium nanoparticles prepared by Cassia fistula were 15.79 nm in diameter, having a mixture of spherical and irregularly shaped nanoparticles. The titanium nanoparticles prepared by hydrothermal heating were predominantly irregular in shape and 11.29 nm in diameter. The results of SEM and XRD were in agreement with each other ( Figure 3, Table 1).

EDS
The EDS images were utilized to check the titanium and oxygen peaks in the titanium nanoparticles. The EDS image of the titanium nanoparticles formed by Bacillus subtilis showed large quantities of titanium and less oxygen, whereas the EDS image of the titanium nanoparticles prepared by Cassia fistula and hydrothermal heating revealed comparatively lesser quantities of titanium and greater quantities of oxygen. The amount of titanium in the hydrothermal heating was much less than those formed by Cassia fistula (Figure 5).

EDS
The EDS images were utilized to check the titanium and oxygen peaks nium nanoparticles. The EDS image of the titanium nanoparticles formed subtilis showed large quantities of titanium and less oxygen, whereas the ED the titanium nanoparticles prepared by Cassia fistula and hydrothermal heatin comparatively lesser quantities of titanium and greater quantities of oxygen. T of titanium in the hydrothermal heating was much less than those formed by C ( Figure 5).  3.2.6. DRS DRS scans were taken to confirm the size of the titanium nanoparticles through band gap absorbance energy using a standard value of 3.2 EV. The greater value showed the small particle size of the nanoparticles while the smaller value revealed the larger particle size. The DRS scan of the titanium nanoparticles formed by Bacillus subtilis depicted a larger particle size with a lesser calculated band-gap absorbance energy of 2.7 EV. The titanium nanoparticles fabricated by the Cassia fistula and hydrothermal heating confirmed the smaller particle size with greater calculated band-gap absorbance energies of 3.6 EV and 3.9 EV, respectively ( Figure 7).

TEM
The diameter of particles measured by TEM images (DTEM) was found to be 63 nm for the titanium nanoparticles prepared by Bacillus subtilis, whereas the DTEM values for titanium nanoparticles fabricated by Cassia fistula and hydrothermal heating were calculated to be 15 nm and 11 nm, respectively ( Figure 8). The sharp, elongated and narrow peak confirming the large particle size were depicted by titanium nanoparticles fabricated by Bacillus subtilis (Figure 8d). On the other hand, shallow and broad peaks confirming a small particle size was revealed by titanium nanoparticles prepared by Cassia fistula whereas the most shallow and broadest peak confirming the smallest particle size was revealed by titanium nanoparticles produced by hydrothermal heating (Figure 8e,f, Table  1). 3.2.6. DRS DRS scans were taken to confirm the size of the titanium nanoparticles through band gap absorbance energy using a standard value of 3.2 eV. The greater value showed the small particle size of the nanoparticles while the smaller value revealed the larger particle size. The DRS scan of the titanium nanoparticles formed by Bacillus subtilis depicted a larger particle size with a lesser calculated band-gap absorbance energy of 2.7 eV. The titanium nanoparticles fabricated by the Cassia fistula and hydrothermal heating confirmed the smaller particle size with greater calculated band-gap absorbance energies of 3.6 eV and 3.9 eV, respectively ( Figure 7). 2.2.6. DRS DRS scans were taken to confirm the size of the titanium nanoparticles through band gap absorbance energy using a standard value of 3.2 EV. The greater value showed the small particle size of the nanoparticles while the smaller value revealed the larger particle size. The DRS scan of the titanium nanoparticles formed by Bacillus subtilis depicted a larger particle size with a lesser calculated band-gap absorbance energy of 2.7 EV. The titanium nanoparticles fabricated by the Cassia fistula and hydrothermal heating confirmed the smaller particle size with greater calculated band-gap absorbance energies of 3.6 EV and 3.9 EV, respectively ( Figure 7).

TEM
The diameter of particles measured by TEM images (DTEM) was found to be 63 nm for the titanium nanoparticles prepared by Bacillus subtilis, whereas the DTEM values for titanium nanoparticles fabricated by Cassia fistula and hydrothermal heating were calculated to be 15 nm and 11 nm, respectively ( Figure 8). The sharp, elongated and narrow peak confirming the large particle size were depicted by titanium nanoparticles fabricated by Bacillus subtilis (Figure 8d). On the other hand, shallow and broad peaks confirming a small particle size was revealed by titanium nanoparticles prepared by Cassia fistula whereas the most shallow and broadest peak confirming the smallest particle size was revealed by titanium nanoparticles produced by hydrothermal heating (Figure 8e,f, Table 1).

TEM
The diameter of particles measured by TEM images (D TEM ) was found to be 63 nm for the titanium nanoparticles prepared by Bacillus subtilis, whereas the D TEM values for titanium nanoparticles fabricated by Cassia fistula and hydrothermal heating were calculated to be 15 nm and 11 nm, respectively ( Figure 8). The sharp, elongated and narrow peak confirming the large particle size were depicted by titanium nanoparticles fabricated by Bacillus subtilis (Figure 8d). On the other hand, shallow and broad peaks confirming a small particle size was revealed by titanium nanoparticles prepared by Cassia fistula whereas the most shallow and broadest peak confirming the smallest particle size was revealed by titanium nanoparticles produced by hydrothermal heating (Figure 8e,f

DLS
The hydrodynamically calculated sizes (DH) of the titanium nanoparticles matched with the results of DTEM as hydrodynamic sizes (DH) are always greater than XRD, SEM and TEM as per standards. The diameter of particle size measured by DLS data (DH) was 200 nm for the Titanium nanoparticles prepared by Bacillus subtilis, whereas DH value for titanium nanoparticles fabricated by the Cassia fistula and hydrothermal heating was calculated to be 37 nm and 29 nm (Figure 9). The longest sharp peak confirmed the hydrodynamically calculated large size of the titanium nanoparticles prepared by the Bacillus subtilis which was greater than DTEM (Figure 9a). On the other hand, broad peak confirmed the hydrodynamically calculated small sizes of the titanium nanoparticles formed by the Cassia fistula and hydrothermal heating completely, which was again greater than DTEM (Figure 9 b-d, Table 1).

DLS
The hydrodynamically calculated sizes (D H ) of the titanium nanoparticles matched with the results of D TEM as hydrodynamic sizes (D H ) are always greater than XRD, SEM and TEM as per standards. The diameter of particle size measured by DLS data (D H ) was 200 nm for the Titanium nanoparticles prepared by Bacillus subtilis, whereas D H value for titanium nanoparticles fabricated by the Cassia fistula and hydrothermal heating was calculated to be 37 nm and 29 nm (Figure 9). The longest sharp peak confirmed the hydrodynamically calculated large size of the titanium nanoparticles prepared by the Bacillus subtilis which was greater than D TEM (Figure 9a). On the other hand, broad peak confirmed the hydrodynamically calculated small sizes of the titanium nanoparticles formed by the Cassia fistula and hydrothermal heating completely, which was again greater than D TEM (Figure 9 b-d, Table 1).

Cytotoxicity (Cell Viability %) of Prepared Titanium Nanoparticles by Three Routes:
The titanium nanoparticles synthesized by Bacillus subtilis, Cassia fistula and titanium tetrachloride were compared with each other at day 1, day 15, day 31, day 41 and day 51. The control group (water) depicted 100% non-cytotoxic behavior at all the days investigated. The titanium nanoparticles prepared by Bacillus subtilis were in close collaboration with control group revealing a non-cytotoxic behavior in comparison to titanium nanoparticles fabricated by Cassia fistula revealing moderate cytotoxicity and titanium tetrachloride revealing severe cytotoxicity, which was significant at day 51 of cytotoxicity analysis (Figures 10-14).  The titanium nanoparticles synthesized by Bacillus subtilis, Cassia fistula and titanium tetrachloride were compared with each other at day 1, day 15, day 31, day 41 and day 51. The control group (water) depicted 100% non-cytotoxic behavior at all the days investigated. The titanium nanoparticles prepared by Bacillus subtilis were in close collaboration with control group revealing a non-cytotoxic behavior in comparison to titanium nanoparticles fabricated by Cassia fistula revealing moderate cytotoxicity and titanium tetrachloride revealing severe cytotoxicity, which was significant at day 51 of cytotoxicity analysis (Figures 10-14).

Cytotoxicity Analysis (Cell Viability %) at First Day
The titanium nanoparticles formed by Bacillus subtilis revealed slight reduction in fibroblast cell lines viability% on the first day as compared to the control group but these nanoparticles remained non-cytotoxic as cell viability was > 90%. The titanium nanoparticles prepared by Cassia fistula and titanium tetrachloride depicted more reduction in fibroblast cell lines viability % as compared to the control group. They fell in the mildly

Cytotoxicity Analysis (Cell Viability %) at First Day
The titanium nanoparticles formed by Bacillus subtilis revealed slight reduction in fibroblast cell lines viability% on the first day as compared to the control group but these nanoparticles remained non-cytotoxic as cell viability was > 90%. The titanium nanoparticles prepared by Cassia fistula and titanium tetrachloride depicted more reduction in fibroblast cell lines viability % as compared to the control group. They fell in the mildly cytotoxic status, as the cell viability was between 60 and 90%. The linear decrease in cell viability % was observed in titanium nanoparticles formed by Bacillus subtilis, Cassia fistula and titanium tetrachloride as compared to the control group which was significant. The mean differences between titanium nanoparticles formed by Bacillus subtilis, Cassia fistula and titanium tetrachloride was also significant on the first day (p < 0.001) ( Figure 10).

Cytotoxicity Analysis (Cell Viability %) at 15th Day
The titanium nanoparticles formed by Bacillus subtilis displayed more reduction in fibroblast cell lines viability % 15th day as compared to control group, but these nanoparticles were non-cytotoxic as cell viability was still >90%. The titanium nanoparticles prepared by Cassia fistula and titanium tetrachloride depicted comparatively more reduction in fibroblast cell lines viability % when compared with the control group. They were found to be in the range of mild cytotoxicity as cell viability was between 60 and 90%. The 15th day also revealed linear pattern decrease in cell viability % in titanium nanoparticles prepared by Bacillus subtilis, Cassia fistula and titanium tetrachloride in comparison to control group which was significant. The mean differences between titanium nanoparticles formed by Bacillus subtilis, Cassia fistula and hydrothermal heating was also significant at 15th day (p < 0.001) (Figure 11).

Cytotoxicity Analysis (Cell Viability %) at 31st Day
The titanium nanoparticles formed by Bacillus subtilis again revealed a slight reduction in fibroblast cell lines viability % at the 31st day as compared to the control group, but these nanoparticles remained non-cytotoxic as cell viability was >90%. The titanium nanoparticles prepared by Cassia fistula displayed moderate reduction in fibroblast cell lines viability % at the 31st day when compared to the control group and fell within range of mild cytotoxicity, as cell viability was between 60 and 90%. The titanium nanoparticles prepared by titanium tetrachloride depicted maximum reduction in fibroblast cell lines viability % as compared to control group. They fell in moderately cytotoxic status as cell viability was found to be between 30 and 60%. The linear pattern decreases in cell viability % was observed in titanium nanoparticles formed by Bacillus subtilis, Cassia fistula and titanium tetrachloride, as compared to control group which was significant. The mean differences between titanium nanoparticles formed by Bacillus subtilis, Cassia fistula and hydrothermal heating was also significant at the 31st day (p < 0.001) (Figure 12).

Cytotoxicity Analysis (Cell Viability %) at 41st Day
The titanium nanoparticles formed by Bacillus subtilis again revealed a slight reduction in fibroblast cell lines viability % at the 41st day as compared to the control group, but these nanoparticles remained non-cytotoxic, as cell viability was >90%. The titanium nanoparticles prepared by Cassia fistula displayed moderate reduction in fibroblast cell lines viability % at the 41st day when compared to the control group and fell within the range of mild cytotoxic as cell viability was between 60 and 90%. The titanium nanoparticles prepared by titanium tetrachloride depicted a maximum reduction in fibroblast cell lines viability % as compared to the control group. They fell in severely cytotoxicity standard because cell viability was found to be 30% or less. The linear decrease in cell viability % was observed in titania nanoparticles formed by Bacillus subtilis, Cassia fistula and titanium tetrachloride as compared to the control group, which was significant. The mean differences between titanium nanoparticles prepared by Bacillus subtilis, Cassia fistula and titanium tetrachloride was also significant at the 41st day (p < 0.001) (Figure 13).

Cytotoxicity Analysis (Cell Viability %) at 51st Day
The titanium nanoparticles formed by Bacillus subtilis again revealed a slight reduction in fibroblast cell lines viability % at 51st day as compared to control group, but these nanoparticles remained non-cytotoxic as cell viability was > 90%. The titanium nanoparticles prepared by Cassia fistula displayed moderate reduction in fibroblast cell lines viability % at the 51 st day when compared to control group and fell within range of moderate cytotoxic as cell viability was between 30 and 60%. The titanium nanoparticles prepared by titanium tetrachloride depicted maximum reduction in fibroblast cell lines viability % as compared to control group. They fell in severely cytotoxicity standard because cell viability was found to be 30% or less. The linear decrease in cell viability % was observed in titanium nanoparticles formed by Bacillus subtilis, Cassia fistula and titanium tetrachloride as compared to the control group, which was significant. The mean differences between titanium nanoparticles prepared by Bacillus subtilis, Cassia fistula and titanium tetrachloride was also significant at the 51st day (p < 0.001) (Figure 14).

Cell Morphology of Fibroblasts Exposed to Titanium Nanoparticles
The fibroblasts are normally large, elongated and flat cells possessing branched cytoplasm surrounding nucleus having two or more nucleoli.

Cell Morphology at First Day
The normal characteristic morphology of fibroblast cell lines was observed when they were exposed to titanium nanoparticles prepared by Bacillus subtilis on the first day (Figure 15b), which was quite similar to the control group. The initiation of pore formation was revealed by fibroblast cell lines exposed to titanium nanoparticles formed by Cassia fistula and titanium tetrachloride (Figure 15c,d), leading to slight degradation in the fibroblast's cell morphology as compared to the control group (Figure 15a).  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).

Cell Morphology at 41st Day
Normal characteristic morphology of fibroblast cell lines was observed when they were exposed to titanium nanoparticles prepared by Bacillus subtilis at the 41st day ( Figure  15 n), which was quite similar to the control group (Figure 15m). There was pore formation and mild degradation revealed by fibroblast cell lines exposed to titanium nanoparticles formed by Cassia fistula at the 41st day (Figure 15o) after comparing it with the 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).

Cell Morphology at 15th Day
The normal morphology of the fibroblast cell lines was revealed after exposing them to titanium nanoparticles formed by Bacillus subtilis at the 15th day (Figure 15f), similar to the control group (Figure 15e). The titanium nanoparticles prepared by Cassia fistula and titanium tetrachloride (Figure 15g,h) manifested increased pore formation and degradation in the fibroblast cell lines in comparison to the control group (Figure 1e).

Cell Morphology at 31st Day
The fibroblasts of titanium nanoparticles prepared by Bacillus subtilis (Figure 15j) displayed normal morphology without any change after comparing it with the control group (Figure 15i). There was mild degradation in the fibroblast cell lines at the 31st day when they were exposed to titanium nanoparticles prepared by Cassia fistula (Figure 15k). On the other hand, greater disruption of fibroblast cell lines was depicted on exposure to titanium nanoparticles prepared by titanium tetrachloride at the 31st day (Figure 15l) in comparison to the control group (Figure 15i).

Cell Morphology at 41st Day
Normal characteristic morphology of fibroblast cell lines was observed when they were exposed to titanium nanoparticles prepared by Bacillus subtilis at the 41st day (Figure 15 n), which was quite similar to the control group (Figure 15m). There was pore formation and mild degradation revealed by fibroblast cell lines exposed to titanium nanoparticles formed by Cassia fistula at the 41st day (Figure 15o) after comparing it with the control group ( Figure 15m). The titanium nanoparticles prepared by titanium tetrachloride (Figure 15p) displayed entire fibroblast cell line disruptions on exposure where these cells lost their complete symmetry as compared to the control group on the 41st day (Figure 15m).

Cell Morphology at 51st Day
Fibroblasts of titania nanoparticles prepared by Bacillus subtilis (Figure 15r) displayed normal morphology without any change after comparing them with the control group at the 51st day (Figure 15q). There was comparatively greater degradation in the fibroblast cell lines revealed at the 51st day when they were exposed to titanium nanoparticles prepared by Cassia fistula. These fibroblasts showed signs of losing their normal spindle shape (Figure 15s). On the other hand, maximum disruption of fibroblast cell lines was depicted on exposure to titanium nanoparticles prepared by titanium tetrachloride at the 51st day ( Figure 15t) in comparison to the control group (Figure 15q). These fibroblasts completely lost their normal size, shape and symmetry (Figure 15a-t).

Discussion
There is a dire need to carry out cytotoxicity testing on a large scale before declaring nanoparticles safe for use in medical and dental applications [39]. The MTT assay is the most reliable test utilized to investigate the toxicity of nanoparticles [40]. There is a universal standard used for assessing the cytotoxicity of nanoparticles depending on cell viability %, which is given as: "non-cytotoxic as cell viability > 90%", "mildly cytotoxic as cell viability between 60-90%", "moderately cytotoxic as cell viability between 30-60%" and "severely cytotoxic as cell viability of 30% or less [38]. The cytotoxicity analysis of titanium nanoparticles prepared with the help of Bacillus subtilis demonstrated a non-cytotoxic nature when exposed to fibroblast cell lines because of cell viability > 90% at all days investigated in comparison with control group. This titanium nanoparticle justified higher biocompatibility and higher cell viability as compared to other nanoparticles synthesized by Cassia fistula and titanium tetrachloride. The cytotoxicity analysis of titanium nanoparticles prepared through Cassia fistula displayed mild cytotoxicity on the first day, 15th day, 31st day and 41st day because the cell viability was between 60 and 90%. These nanoparticles turned moderately cytotoxic at the 51st day due to cell viability being between 30 and 60% as compared to the control group. The cytotoxicity analysis of titanium nanoparticles prepared through titanium tetrachloride revealed their mild cytotoxic nature on the first day and 15th day because of their cell viability between 60 and 90%; they were moderately cytotoxic at the 31st day due to their cell viability being between 30 and 60%; and they eventually turned severely cytotoxic at the 41st day and the 51st day as the cell viability was less than 30% in comparison to the control group (Figures 10-14). The most significant factor responsible for the cytotoxicity of the nanoparticles is their mode of synthesis [41]. Bacteria are considered as the best option for synthesizing metal oxide nanoparticles because of their outstanding biocompatibility, flexible nature, high growth rate, high yield, cost-effectiveness and ease of culturing and manipulation. The biocompatibility of titanium nanoparticles produced by bacteria is due to presence of natural biomolecules which are used during their synthesis, such as enzymes [42]. These biomolecules might have produced safe, strong, stable and sustainable layers around these nanoparticles during the synthesis, thus rendering them biocompatible without producing any toxic by-product. The results in the current study regarding titanium nanoparticles synthesized by Bacillus subtilis matched the literature [43]. Different phytochemicals such as terpenoids, glycosides and alkaloids present in the plants are responsible for inducing toxicity in nanoparticles. This is due to the fact that they adversely affect the biological functions of the cells to which they are exposed [44]. The titanium nanoparticles produced by Cassia fistula might have become toxic due to the presence of residual phytochemicals that might have been released during the synthesis. These residual phytochemicals might have become entrapped and accumulated on the surfaces of these titanium nanoparticles, thus making them cytotoxic. Eventually, these nanoparticles, after coming in contact with cell lines, might have initiated a toxic reaction, thus reducing the cell viability [45]. The cytotoxicity results of titanium nanoparticles prepared by plants in the current study are in accordance with previous studies. The nanoparticles prepared by hydrothermal conventional method utilize toxic chemicals and high temperature during their synthesis [41]. These two factors might have released toxic byproducts that might have been adsorbed on the surface of these nanoparticles, bringing them into an unstable state. Thus, these titanium nanoparticles became cytotoxic and non-biocompatible by reducing the cell viability [46]. The nanoparticles synthesized by conventional methods revealed enhanced cytotoxicity compared to those synthesized by biological processes as a result of utilizing the toxic chemicals [47].
Other factors that play a key role in the cytotoxicity of nanoparticles are predominantly dependent on their physico-chemical properties, time-duration of exposure and concentration used. The important physico-chemical properties that take part in the cytotoxicity include their shape, size, phase state, texture, elemental composition, band gap absorbance energy, functional groups and surface roughness, which are measured by standard characterization techniques such as XRD, SEM, TEM, EDS, AFM, DRS, DLS and FTIR. The size and shape of nanoparticles are the key factors involved in the cytotoxicity, where the results of XRD, SEM, TEM and DLS are in accordance with each other as per standard protocols in the current study. The XRD and SEM data gave the exact particle size of all the fabricated titanium nanoparticles by Bacillus Subtilis, Cassia fistula and hydrothermal heating, which was in close proximity with EDS, AFM, DRS and FTIR. There was a visible collaboration in the particle size of titanium nanoparticles fabricated by Bacillus Subtilis, Cassia fistula and hydrothermal heating measured by TEM and DLS that confirmed the formation of both the actual physical size and hydrodynamic size of these nanoparticles. The particle sizes of all the titanium nanoparticles calculated by D TEM images taken through TEM were comparatively smaller than D H data calculated via DLS, which revealed larger particle sizes. This is due to the fact that TEM calculates the actual physical particle size of nanoparticles, which is always smaller as compared to DLS, which calculates the hydrodynamic size and is always larger. This particle size difference between TEM and DLS values in the current studies was in accordance with the already reported literature that confirmed their significant role in cytotoxicity [48]. Previous research has reported that nanoparticles of large size and spherical shape are non-cytotoxic and vice versa. The large size and spherical shape have smaller surface area to volume ratios, due to which they cannot be penetrated easily. The mixed phase states of anatase and rutile are safer compared to the pure phase sate of anatase because anatase is a highly reactive state. The minimal surface roughness supports the non-cytotoxic behavior of nanoparticles as compared to the moderate and maximum surface roughness depicted by the nanoparticles. The standard value for the band gap absorbance energy is 3.2 eV, where a calculated value greater than this shows a smaller particle size, whereas a calculated value lesser than this depicts a larger nanoparticle size [41]. The titanium nanoparticles prepared by Bacillus Subtilis were large in size, spherical and contained a large quantity of Ti, a band-gap absorbance energy of 2.7 eV, minimum surface roughness, few functional groups and a mixed phase state of anatase and rutile. The titanium nanoparticles formulated by Cassia fistula were small in size, had mixture of spherical and irregular shapes, contained a smaller quantity of Ti, a band-gap absorbance energy of 3.6 eV, moderate surface roughness, more functional groups and a pure phase state of anatase. On the other hand, the titanium nanoparticles produced by hydrothermal heating were very small in size, dominantly irregular in shape and contained the lowest quantity of Ti, had a band-gap absorbance energy of 3.9 eV, maximum surface roughness, a large amount of functional groups and a pure phase state of anatase. The titanium nanoparticles prepared by Bacillus Subtilis were the most biocompatible because of their large size and spherical shape, which might have prevented their absorption in fibroblast cell lines in large quantities. Additionally, the titanium nanoparticles formed by Cassia fistula and hydrothermal heating were of smaller size and irregular shape, which might have increased their absorption in the fibroblast cell lines, leading to their cytotoxicity. The minimum roughness, mixed phase state of anatase and rutile, and few functional groups present in the titanium nanoparticles formed by Bacillus Subtilis might have resulted in providing stability and sustainability to these nanoparticles, thus preventing them from becoming cytotoxic. On the other hand, moderate and maximum roughness, pure phase state of anatase and more functional groups observed in the titanium nanoparticles prepared by Cassia fistula and hydrothermal heating might have reduced their stability and sustainability, resulting in their cytotoxicity. The presence of a large amount of Ti in the elemental composition of the titanium nanoparticles formed by Bacillus Subtilis might have prevented them from becoming cytotoxic as compared to the lesser amount of Ti in the elemental composition of the titanium nanoparticles formulated by Cassia fistula and hydrothermal heating.
The increased concentration and time duration of nanoparticles' exposure to cell lines also reduces cell viability with every passing day and makes them cytotoxic eventually [49,50]. The titanium nanoparticles synthesized by Bacillus subtilis were exposed to fibroblast cell lines in large concentrations and longer duration but remained non-cytotoxic even after a month. This made possible the formation of biologically stable and uniform capping layer around these nanoparticles that imparted them biocompatibility and safety without affecting their cell viability. The titanium nanoparticles synthesized by Cassia fistula exposed to fibroblast cell lines become moderately cytotoxic after a month because residual phytochemicals adsorbed on the surfaces of these nanoparticles became more toxic, reducing their cell viability with the progression of time. The titanium nanoparticles synthesized by hydrothermal heating when exposed to fibroblast cell lines became severely cytotoxic after a month. A plausible explanation could have been that toxic by-products released during the synthesis might have made these nanoparticles unstable and reduced the cell viability with every passing day. The possible mechanisms responsible for generating cytotoxicity in titanium nanoparticles are apoptosis, inflammation and oxidative stress, which in turn results in rapid and excessive generation of ROS (reactive oxygen species), leading towards a reduction in cell viability followed by the death of cells exposed to the nanoparticles. The titanium nanoparticles formed by Bacillus subtilis did not produce any abnormal morphological changes in fibroblast cell lines in comparison to the control group. The fibroblasts were elongated, large, flat, elongated and spindle-shaped, with special processes coming out. This shows that all the fibroblast cells exposed to titanium nanoparticles formed by Bacillus subtilis were mostly viable (alive) and normal because of their normal size and shape. The titanium nanoparticles formed by Cassia fistula produced slight abnormal changes with pore formation and degradation in the cell's structure when compared to control group. These nanoparticles decreased the number of viable (alive) fibroblasts by changing their size and shape in turn, disintegrating them. The titanium nanoparticles formed by titanium tetrachloride produced increased number of pores with entire disruption of cell's structure resulting in their irregular sizes and shapes. Thus, these nanoparticles severely declined the number of viable (alive) fibroblast cells. The mor-phological changes, including pore formation and degradation, were similar to previous research on these materials ( Figure 15) [51].

Conclusions
The present study concluded that routes of synthesis greatly influence the cytotoxicity of nanoparticles. The titanium nanoparticles synthesized by Bacillus subtilis remained non-cytotoxic with enhanced cell viability > 90%, even after their exposure to L929 mouse fibroblast cell lines for more than one month as compared to titanium nanoparticles produced by other routes such as Cassia fistula and hydrothermal heating, which depicted reduced cell viability in the ranges of cytotoxicity. Moreover, characterization techniques, including XRD, SEM, DRS, TEM and AFM, are the most important tools for measuring the cytotoxic behavior of TiO 2 nanoparticles. These tools purely supported the TiO 2 nanoparticles prepared by Bacillus subtilis as a result of their large size, spherical shape, mixed anatase-rutile phase form and minimum surface roughness in comparison to TiO 2 nanoparticles fabricated by Cassia fistula and hydrothermal heating, which revealed smaller particle size, irregular shape, pure anatase phase form and maximum roughness. Additionally, EDS and FTIR depicted the presence of increased content of titanium and decreased level of functional groups in the TiO 2 nanoparticles synthesized by Bacillus subtilis as compared to those formed by Cassia fistula and hydrothermal heating, which showed decreased content of titanium and increased content of functional groups. This showed that the synthesis of titanium nanoparticles through Bacillus subtilis is favorably and is an easy, sustainable and biocompatible route for not only the safe production of nanoparticles but also their utilization in the advancements of nanomaterials production in dentistry. This study accomplishes the concept of environmental sustainability and supporting green dentistry.