Novel microbial synthesis of titania nanoparticles using probiotic Bacillus coagulans and its role in enhancing the microhardness of glass ionomer restorative materials

Poor oral hygiene adversely affects the health of an individual and causes dental caries as reported by the World Health Organization (WHO) [1]. Invasive restorative treatments are frequently used to treat dental caries. Glass ionomer cements (GIC) have long been used for restoration purposes due to their good adherence potential to teeth. The binding ability of these materials to tooth structure enables them to be placed in cavities without creating any gaps, thus preventing the unnecessary tooth structure loss [2]. Moreover, these cements have the capability of releasing fluoride that helps in remineralization of teeth, consequently preventing secondary caries. Additionally, the color of these restorative materials is quite close to that of human teeth [3]. These materials display lower strength; therefore, efforts have been made to improve their mechanical properties in the past resulting in modifications of these cements such as resin-modified glass ionomer cements (RMGIC) and compomer [4]. These cements have higher mechanical strength as compared to conventional glass ionomer cements, but lack biocompatible behavior with adjacent biological tissues. The GIC is a potential bioactive dental material because of its higher favorable biocompatibility with oral tissues [5]. However, these materials need to be strengthened to act as long-term restorative materials due to their inferior mechanical qualities.

The composition of GIC may be responsible for its reduced strength. It is made of ion-leachable fluoroaluminosilicate glasses and polyacrylic acid solution that form a composite gel phase matrix eventually. An acid–base reaction forms this phase matrix, which does not bind all glass particles together and leaves some unreacted glass particles in the structure of set fluoroaluminosilicate glass and polyacrylic acid. These unreacted glass particles in the GIC matrix are responsible for the hydrolysis of the remaining tightly bound set glass structure [6]. This process disturbs bonding in the established glass structure of the GIC matrix, resulting in the release of calcium and aluminum ions. Cross-linking of the polymer chains in the GIC matrix is maintained by these ions, which are responsible for the ordered arrangement of the restorative material [6, 7]. The cross-linking in the GIC matrix is attributed to its microhardness properties. The loss of cross-linking in the GIC matrix by release of calcium and aluminum ions create voids in its structure and allows ingress of oral fluids and saliva dissolving its structure and compromising its mechanical properties [8]. Therefore, improving the mechanical properties of the GIC materials is one of the important aspects of future research and development. Nanotechnology-based approaches have significant contributions in modifying the quality of medical and dental materials. The incorporation of NPs in restorative materials and endodontic materials, mouthwashes, dentifrices, implants and luting cements has led to an enhanced performance of these materials [9‐11].

Therefore, these NPs can be integrated in glass ionomer cements to improve their properties and strength, consequently improving their longevity. Various NPs such as silver (Ag), stainless steel, TiO₂, tin and carbon have been used to enhance the properties of these restorative materials. Importantly, metallic NPs have gained exceptional success in dentistry due to their magnetic, mechanical, optical and strength properties [9‐11]

TiO₂ NPs have gained significant commercial attraction for various applications because of their chemical stability, high electrical conductivity, high thermal diffusivity and low thermal conductivity [12]. In dentistry, commercially available TiO₂ NPs are considered as cost-effective, attrition controlling, fatigue resistant and corrosion-resistant materials for their application along with conventional restorative substances, but their purity is questionable as these NPs have displayed toxicity [8, 12]. Their added advantages and excellent mechanical properties make them ideal candidates for the formulation of improved dental-restorative materials. In recent years, incorporation of TiO₂ NPs in dental restorations has immensely increased owing to their physical, chemical, mechanical and strength properties [13]. Many contemporary researchers have suggested that TiO₂ NPs display potent anti-bacterial, anti-parasitic, anti-inflammatory, anti-fungal and anti-cariogenic properties [14]. TiO₂ NPs are capable of producing desirable changes in the composition, structure, texture and topography of the GIC cement through strong bonding with its constituents. Therefore, modified GIC could attain various important properties, which are lacking in the absence of NPs [15]. One of the major limitations of the NPs could be the toxicity of the metals, which has been reported previously. Moreover, synthetic synthesis of these NPs has harmful effects on the environmental safety as a result of chemical by-product formation [16‐18]. Therefore, an eco-friendly route for the fabrication of these NPs would be a step toward a safe and healthy environment. In this view of environmental biosafety, compatibility and stability, biological synthesis has gained ample interest in the current era. Therefore, Bacillus coagulans was used to produce TiO₂ NPs for this study. Bacillus coagulans is an ideally effective probiotic that is easily available, highly safe and stable in nature. This microbially oriented synthesis would produce eco-friendly, pure, sustainable and environmentally safe NPs [19]. It displays strong antimicrobial activity against multiple pathogenic bacteria, as a result of its enhanced potency of releasing bacteriocin and acetic and lactic acid, which are taken as strong inhibitory compounds to kill microorganisms [20]. The current research focuses on the synthesis and characterization of novel TiO₂ NPs via Bacillus coagulans, which were introduced into the conventional GIC cement to produce a novel compatible TiO₂ GIC oral restorative material. The formulated GIC material was investigated for its microhardness and surface topography for its applications as a mechanically better and more durable treatment option.

TiO₂ NPs used in this study were fabricated by a novel microbial route using Bacillus coagulans. These TiO₂ NPs were added to conventional dental glass ionomer cements to produce a novel compatible dental cement (5% TiO₂ GIC) with increased microhardness. TiO₂ NPs produced via microbial synthesis were considered biocompatible, sustainable, and environmentally friendly, as no toxic chemicals and artificial ingredients were involved in their synthesis. They were prepared at very low pressure and temperature. The production of TiO₂ NPs is cost-effective, and thus they can be easily incorporated in the commercial restorative products and medicines used in dental and medical fields [24]. The oxidation reaction between the microbial culture solution and Ti(OH)₂ was mainly responsible for the production of TiO₂ NPs. The X-ray diffraction pattern revealed the TiO₂ NPs were 100% pure anatase phase with a particle size of 21.84 nm. The 100% pure anatase phase of TiO₂ NPs was produced due to their calcination at 500 °C in the furnace, resulting in their active and reactive phase. SEM analysis revealed spherical shape in agglomerates and clustered form of TiO₂ NPs, while DLS measurement indicated the hydrodynamic diameter of 34 nm.

The formation of TiO₂ NPs was observed at 320 nm, which was in accordance with the previous study, where formation of TiO₂ NPs was confirmed between 200 and 600 nm [25]. The standard band-gap energy value (standard value = 3.2 eV) also confirms the formation of TiO₂ NPs. Values higher than 3.2 eV demonstrate a smaller particle size of NPs, while values lesser than the recommended value demonstrated a larger particle size of NPs. The band-gap energy of 3.5 eV NPs could be due to the slow secondary reaction by the biomolecules (capping and reducing agents).

EDX analysis demonstrated prominent peaks of Ti and O with no other impurity in its composition due to the presence of Bacillus coagulans culture solution and Ti precursors [26]. FTIR analysis demonstrated the evident peak of Ti–O–Ti bending at 581.17 cm^-1 ensuring the presence of TiO₂ NPs [27]. The absence of C–H stretching peak, particularly at 2900 cm⁻¹, confirmed the absence of any organic compound responsible for producing impurities that might lead to cytotoxicity. The beneficial effect of pure amine linkages might have occurred as a result of involvement of the proteins present in TiO₂ NPs [28]. These novel TiO₂ NPs prepared from microbial route revealed cell viability percentage > 90%, thus confirming their toxicity-free biocompatible nature. Previous studies reported the nontoxic behavior of the TiO₂ NPs when synthesized from microbes [29, 30]. The strong biomolecular linkages during the fabrication might have generated a dominant overlapping aggregation process that prompted the stable nucleation and binding of the NPs, thus attributing to their characteristically safe, sustainable and impurity-free nature. The production of impurity-free TiO₂ encourages their usage in restorative materials applied adjacent to the oral biological tissues.

The hardness of a dental material is a significantly essential property that may be used to validate its strength and longevity in the oral cavity [31, 32]. The current study showed maximum microhardness in the novel experimental group E-3 as compared to the conventional control group E-1, E-4 and E-5 (Table 3). This could be possible due to the fact that TiO₂ NPs occupied the voids available in the glass ionomer matrix. Eventually, this could have resulted in strong bonding and cross-linking between pure and small-sized 5% TiO₂ NPs and dental glass ionomer powder particles. The strong binding prevented propagation of cracks through the GIC when a force is applied to these NPs. The increase in microhardness of the E-3 group did not correspond to that in the literature [16] due to the high incorporation of TiO₂ NPs. These NPs created a powerful meshwork to fill each void in the glass ionomer matrix by strongly entangling the matrix in an orderly arranged manner to prevent the loss of cross-linking and bond around the void.

The other experimental groups E-4 and E-5 revealed reduction in the microhardness, which were not evaluated previously [13] The reason behind the declination of microhardness could be the weak attractive forces between 7% TiO₂ NPs and the GIC matrix in the E-4 and E-5 groups. The SEM analysis revealed a minor crack formation in the surface of group E-3, because of its maximum microhardness as compared to conventional control group E-1 that depicted major crack formation with minimum microhardness. This clearly indicated that any alteration in the composition of conventional dental GIC by smart NPs might have a significant effect on its microhardness and surface topographical structure. The amount of NPs added, particle size, distribution, cross-linking, chemical reaction and chemical bonding between TiO₂ NPs and dental glass ionomer matrix played a potent role in enhancing the microhardness and surface topographical structure of group E-3.

TiO₂ NPs were prepared using probiotic Bacillus coagulans in an environmentally friendly manner. TiO₂ NPs possessed 100% pure anatase phase with the particle size ranging between 21 and 34 nm. NPs were spherical in shape and arranged in agglomerates and clusters showing their smooth texture. TiO₂ NPs were potently stable, sustainable and reproducible enough to enhance the microhardness strength of 5% TiO₂ GIC dental glass ionomer cement. TiO₂ incorporated cement displayed strong surface topographical structure with minimal pores that ensured its compatible biological nature with better hardness strength and negligible crack propagation.