IntroductionDental composite resins have become a fundamental material in restorative dentistry due to their aesthetic appeal and adaptability1. However, despite significant advancements, conventional dental composites still face critical challenges that limit their long-term clinical success. These include polymerization shrinkage leading to marginal gaps, susceptibility of the restored tooth to secondary caries caused by bacterial colonization, and inadequate mechanical durability under oral stresses2,3. Such limitations often result in restoration failure, necessitating replacement procedures that increase patient discomfort and healthcare costs.To overcome these drawbacks, researchers have explored the incorporation of nanoparticles into dental resins to enhance their antimicrobial properties and mechanical performance4. Titanium dioxide nanoparticles (TiO₂-NPs) have attracted considerable attention due to their biocompatibility, photocatalytic, antimicrobial activity, chemical stability, and mechanical reinforcement potential4. TiO2 is widely used as a white pigment in paints, plastics, and cosmetics, as a UV blocker in sunscreens, and as a photocatalyst for self-cleaning surfaces and pollution control. It’s also used in solar cells, ceramics, food coloring, textiles, and some electronic devices due to its optical and chemical properties5,6,7,8. Nonetheless, traditional chemical and physical synthesis methods of TiO₂-NPs often involve toxic reagents, high energy consumption, and environmental concerns, which hinder their widespread biomedical application.Green synthesis of nanoparticles using plant extracts has emerged as a sustainable and eco-friendly alternative, offering advantages such as cost-effectiveness, reduced toxicity, and the presence of natural reducing and stabilizing agents. Among various plant sources, Vitis vinifera (grape) belongs to the Vitaceae family and is widely consumed globally. Grape seeds are a rich source of polyphenols, flavonoids, and other bioactive compounds, which have demonstrated potent antioxidants and antimicrobial properties9. These components act as free radical scavengers and show higher antioxidant activity compared to traditional antioxidants like vitamins C, E, and β-carotene9,10.Furthermore, Vitis vinifera has shown promise in the green synthesis of nanoparticles, acting as both a reducing and capping agent during the process10,11,12. Previous studies have demonstrated the successful green synthesis of TiO₂-NPs using Vitis vinifera extract, yielding nanoparticles with controlled size, enhanced stability, and promising photocatalytic properties13,14,15,16. However, the application of such green-synthesized TiO₂-NPs in dental composite resins remains underexplored.Most earlier studies on TiO₂-NPs incorporated dental composites have focused on commercially available nanoparticles or chemically synthesized by the authors. These studies reported significant antimicrobial activities especially against S. mutans and improvements in mechanical properties such as flexural strength and microhardness at various concentrations of TiO₂-NPs17,18,19. However, only a limited number have explored green-synthesized alternatives, and even fewer have directly compared plant-mediated nanoparticles in dental applications.A recent study by Ezzat et al.20 reported the green synthesis of TiO₂ nanoparticles mediated by grapefruit seed extract (GSE) and their incorporation into experimental dental composites. GSE-TiO₂-NPs enhanced antibacterial activity against S. mutans, improved flexural strength and modulus, and reduced polymerization shrinkage compared to unmodified composites.Building upon this foundation, the present study employs Vitis vinifera extract for the green synthesis of TiO₂-NPs and incorporates them into dental resin composites. While both grape and grapefruit seed extracts serve as effective green reducing agents, Vitis vinifera extract is distinguished by its unique phytochemical profile, which may influence nanoparticle characteristics such as size distribution, surface charge, and bioactivity. By comparing the present findings with those of GSE-mediated TiO₂-NPs composites, this study aims to highlight the advantages of Vitis vinifera-mediated synthesis in producing stable TiO₂-NPs that enhance the antimicrobial and mechanical performance of dental composites.This comparative approach underscores the potential of different plant extracts in tailoring nanoparticle properties for specific dental biomaterial applications and advancing the development of next-generation restorative materials. Thus, green-synthesized TiO₂-NPs, particularly those derived from Vitis vinifera extract, have the potential to overcome the most pressing clinical challenges of current dental composites by improving antimicrobial efficacy, mechanical strength, and reducing polymerization shrinkage in a sustainable manner.The novelty of this study lies in the use of Vitis vinifera extract, characterized by its unique phytochemical composition, as a green synthesis medium for TiO₂-NPs, the incorporation of these biogenic TiO₂-NPs into experimental dental composites with validated improvements in antimicrobial, mechanical, and polymerization properties, and the first-time application of molecular docking analysis to elucidate the mechanistic interaction between TiO₂-NPs and a key bacterial virulence enzyme (glucosyltransferase from S. mutans), linking physicochemical properties with biological function. This integrated approach offers a deeper understanding of bioactivity mechanisms and sets this work apart from prior studies relying solely on empirical observations.Materials and methodsMaterialsThe materials that used in this study are listed in Table 1.Table 1 List of materials and their suppliers used in this study.Full size tablePreparation of the Vitis vinifera extractGrape seeds were air-dried for two weeks to preserve their active components, followed by vacuum drying at 40 °C using calcium fluoride as a desiccant. The dried seeds were ground into a fine powder using an electric blender. Twenty grams of the powdered seeds were boiled in 200 mL of distilled water for 2 h. The extract was then cooled, filtered through multiple layers of filter paper to remove solid residues, and stored in a refrigerator until further use21.Green synthesis of titanium dioxide nanoparticles (TiO2-NPs)Titanium dioxide nanoparticles were synthesized by adding 100 mL of titanium isopropoxide solution (97%) dropwise to the grape seed extract while stirring vigorously at 80 °C. The mixture was aged for two hours to ensure uniform particle size distribution. The resulting yellowish-white precipitate was separated via centrifugation at 5,000 rpm for 10 min, washed with distilled water, and centrifuged again. The cleaned precipitate was dried overnight at 80 °C and calcined at 500 °C to yield TiO2-NPs22. The yield of TiO₂ NPs, determined from the dry weight following calcination, was found to be 95%.Preparation for experimental composite resinThe resin matrix comprised equal parts (50 wt%) of Bis-GMA and TEGDMA. Photoinitiators CQ and DMAEMA were added at 0.2 wt% and 0.8 wt%, respectively. The experimental composite was prepared by incorporating 28 wt% of the resin matrix with 72 wt% of fillers (Fumed silica and TiO2-NPs). fillers were salinized with 5 wt% MPTMS before being blended with the resin using a Speed Mixer (DAC 150 FVZK, Hauschild Engineering, Germany) at 3,500 rpm for one minute23,24. This mechanical mixing was followed by ultrasonication using a probe sonicator (Branson Ultrasonics, 40 kHz) for 10 min to break up any nanoparticle agglomerates and promote uniform distribution. The dispersion quality was visually inspected and further confirmed by scanning electron microscopy (SEM) imaging of the composite cross-sections, which showed well-distributed nanoparticles without significant aggregation.Sample preparation and polymerizationComposite resin samples were prepared by placing the nanoparticle-modified resin into Teflon molds of appropriate dimensions depending on the test. The resin was polymerized using an LED curing light (Bluephase, Ivoclar Vivadent) with an intensity of 1200 mW/cm² for 40 s on each side of the sample. Samples were stored in distilled water at 37 °C for 24 h before testing. Specimen groups were categorized as follows:1.Group I (Control): Unmodified composite containing only fumed silica fillers.2.Group II: Experimental composite containing 10% TiO2-NPs.3.Group III: Experimental composite containing 20% TiO2-NPs.The TiO₂-NP concentrations of 10 wt% and 20 wt% were selected based on prior studies that demonstrated significant improvements in antibacterial and mechanical properties of dental composites at these levels without adversely affecting the curing process or material integrity4,20.In all composite formulations, the total filler content was consistently maintained at 72 wt%. For the control group, the filler system consisted solely of fumed silica. In the experimental groups, part of the fumed silica was substituted with green-synthesized TiO₂ nanoparticles. Specifically, 10 wt% and 20 wt% of the fumed silica was replaced with TiO₂-NPs while keeping the overall filler loading constant at 72 wt%. This approach ensured that differences in composite performance could be attributed to the presence of TiO₂-NPs rather than variations in total filler content.Characterization techniquesThe Vitis vinifera extract was chemically analyzed using a GC-MS system equipped with a polar HP-5ms column. Extracts were diluted in acetone prior to injection. The analysis was conducted with helium as the carrier gas, an injector temperature of 200 °C, and a detector temperature of 250 °C25.The crystal structure and phase identification of the synthesized TiO2-NPs was observed using X-ray diffraction spectroscopy (XRD) utilizing SHIMADZU XRD-6000 (Shimadzu Corporation, Kyoto, Japan) technique under the conditions power diffraction system.The target was Cu-Ka x-ray tube with a wavelength (λ = 1.5406 Å). The X-ray scan was performed between 2θ equal to 4° and 80°. The voltage was 40 KV and the current was 30 mA, the scan mode was continuous, the speed was 5.0000 (deg. /min) and was handled by x-pert software26. The average crystallite size was determined using the Scherrer equations27,28,29. The modified composite resin was morphologically characterized using Scanning electron microscope Morphology was observed using a JEOL JSM-6510LV scanning electron microscope (JEOL Ltd., Tokyo, Japan)30.FTIR spectra were recorded using a PerkinElmer Spectrum Two FTIR spectrometer (PerkinElmer Inc., Waltham, MA, USA). Spectra were recorded with a resolution of 1 cm−1, comprising ten scans across the wavenumber range of 4000 to 400 cm−1.The average hydrodynamic size, particle size distribution and polydispersity were assessed using the Dynamic Light Scattering (DLS) technique with a ZS90 Zetasizer instrument from Malvern, UK. The Zeta potential, which indicates the colloidal stability, was measured through electrophoretic laser Doppler velocimetry31. Morphological characteristics of the nanoparticles were observed using a JEOL 2100 F TEM. Samples were prepared on copper grids after dispersion in distilled water and sonication32.Evaluation of experimental composite resinAntimicrobial activity measurementThe antimicrobial efficacy of composite resins was rigorously evaluated using a standardized agar diffusion method against three key oral bacterial strains: Streptococcus mutans (S. mutans, ATCC 25175), Streptococcus sanguinis (S. sanguinis, ATCC 10556), and Lactobacillus acidophilus (L. acidophilus, ATCC 4356). All bacterial strains were procured from VACSERA (Giza, Egypt)33.S. mutans and S. sanguinis were cultivated on Mitis Salivarius agar (Difco, USA) supplemented with 0.2 units/mL bacitracin to ensure selective growth. Cultures were incubated aerobically at 37 °C in a humidified atmosphere containing 5% CO₂ for 24 h. L. acidophilus was grown on de Man, Rogosa, and Sharpe (MRS) agar (Oxoid, UK) under strict anaerobic conditions (AnaeroPack System, Mitsubishi Gas Chemical, Japan) at 37 °C for 24 h. Following incubation, bacterial cells were harvested and suspended in sterile physiological saline solution (0.9% NaCl). The optical density of each bacterial suspension was adjusted to match a 0.5 McFarland standard, corresponding to approximately 10⁸ colony-forming units per milliliter (CFU/ml) for all strains. This standardization was verified spectrophotometrically at 600 nm and confirmed by plating serial dilutions on appropriate agar media.Mueller-Hinton agar (Oxoid, UK) plates, with a uniform thickness of 4 mm (approximately 25 mL per 90 mm diameter Petri dish), were prepared according to manufacturer instructions and sterilized by autoclaving. Each agar plate was inoculated with 100 µL of the standardized bacterial suspension (10⁸ CFU/ml). The inoculum was evenly spread across the entire surface of the agar using a sterile L-shaped glass spreader, ensuring a confluent lawn of bacterial growth. The inoculated plates were allowed to dry for 15 min at room temperature prior to disc placement34.Disc-shaped specimens of the composite resins, each measuring 4 mm in diameter and 2 mm in thickness, were prepared under aseptic conditions. All specimens were sterilized by exposure to ultraviolet (UV) light for 30 min on each side prior to placement on the inoculated agar plates. A total of five replicate specimens were used for each composite resin type against each bacterial strain. These specimens were aseptically placed on the surface of the inoculated agar, ensuring firm contact without embedding.For robust comparative analysis, two control groups were included on each agar plate. Sterile paper discs (6 mm diameter, Whatman No. 1 filter paper) were impregnated with 20 µL of 0.12% chlorhexidine (CHX) solution (Sigma-Aldrich, USA) and allowed to air dry for 10 min before placement. CHX is a well-established antimicrobial agent, serving as a benchmark for efficacy.Sterile paper discs (6 mm diameter, Whatman No. 1 filter paper) were impregnated with 20 µL of 10% dimethyl sulfoxide (DMSO) solution (Fisher Scientific, USA), which was used as the solvent for certain components in the composite resins. DMSO served as a vehicle control to ensure that any observed antimicrobial activity was attributable to the composite resin itself and not to the solvent.Both control discs were placed on the same agar plates as the composite resin specimens, maintaining adequate spacing to prevent overlapping zones of inhibition.All inoculated plates, including those with specimens and controls, were incubated at 37 °C under strict anaerobic conditions (AnaeroPack System, Mitsubishi Gas Chemical, Japan) for 24 h. This consistent anaerobic environment was maintained for all bacterial strains to ensure optimal growth conditions for the tested oral pathogens and to standardize the experimental setup. After 24 h of incubation, the plates were visually inspected for zones of inhibition around each specimen and control disc. The diameter of the clear zone of inhibition (in millimeters), including the diameter of the disc/specimen, was measured using a digital caliper (Mitutoyo, Japan). Measurements were taken at three different points for each zone, and the average diameter was recorded. The presence and size of the inhibition zone indicated the antimicrobial activity of the tested material. All measurements were performed in triplicate for each specimen, and the mean and standard deviation were calculated.Molecular docking measurementTo investigate the potential molecular mechanism underlying the antimicrobial activity of green-synthesized TiO₂ nanoparticles (TiO₂-NPs), molecular docking study was performed targeting the catalytic domain of S. mutans glucosyltransferase (GtfC), a key enzyme in biofilm formation and caries pathogenesis. The three-dimensional structure of GtfC was retrieved from the Protein Data Bank (https://www.rcsb.org/) with PDB ID : 8fjc35. The protein structure was prepared by removing water molecules and adding hydrogen atoms. Given the inorganic and particulate nature of TiO₂-NPs, a representative cluster model of anatase-phase TiO₂ was constructed based on the dominant crystallographic facets observed in TEM analysis, following established modeling approaches for metal oxide nanoparticles. The TiO₂-NPs was energy-minimized using the Universal Force Field (UFF) in Avogadro software to obtain a stable geometry suitable for docking36,37,38.Docking simulation was performed using AutoDock Tools 1.5.6., with the grid box centered on the active site of GtfC as defined by co-crystallized ligands and key catalytic residues. The binding affinity (ΔG, kcal/mol) and interaction profiles were analyzed, focusing on the interactions between the TiO₂-NPs cluster and the enzyme’s active site residues. During docking process, twenty different poses were created with the protein target site, Then the best-fitted pose with the active site was recorded and 3D figure was generated by the Discovery Studio 2024 visualizer.Flexural strength (FS) measurementBar-shaped specimens (2 × 2 × 25 mm) were tested using a three-point bending test on a universal testing machine (INSTRON, 3345 series, Norwood, MA, USA). Specimens were loaded at a crosshead speed of 0.75 mm/min until fracture occurred39. After the specimens were set, they were taken out, and the extra material was ground off using 800-grit silicon carbide paper. All specimens were then placed in distilled water and incubated at 37 °C for 24 h. The flexural strength was measured using a three-point bending method after the test assembly was attached to a universal testing machine (Instron 3345, Instron Corp., Norwood, MA, USA) with a crosshead speed of 1 mm/min and a span of 20 mm40. To calculate flexural strength, the following formula was used:$${\mathbf{FS}} = {\mathbf{3F}}{\text{ }}({\mathbf{l}})/{\text{ }}{\mathbf{2wh}}^{2}$$(1)where F is the load at fracture, l is the distance between the supports (20.0 mm), w is the specimen width, and h is the specimen height41.Vickers microhardness measurementFor Vickers microhardness testing, disc-shaped specimens (6 mm in diameter and 2 mm thick) were prepared. Once the specimens had set, they were carefully removed from their molds and polished with 800-grit silicon carbide paper to standardize the surface and eliminate any excess material. Following polishing, the specimens were stored in distilled water at 37 °C for 24 h. to simulate pre-testing storage conditions42. After the storage period, the specimens were air-dried and tested with a Digital Display (Model HVS-50, Laizhou Huayin Testing Instrument Co., Ltd., Laizhou, China) equipped with a Vickers diamond indenter and a 20× objective lens. A consistent load of 100 g was applied for 15 s. To ensure accuracy and reproducibility, three indentations were made on each specimen, evenly spaced by at least 0.5 mm. The Vickers hardness values were calculated using the standard formula43:$${\mathbf{VHN}} = {\mathbf{1}}.{\mathbf{854}}{\text{ }}{\mathbf{P}}/{\mathbf{d}}^{{\mathbf{2}}}$$(2)where VHN is Vickers hardness in Kgf/mm2, P is the load in Kg and d is the length of the diagonals in mm.Polymerization shrinkage measurementPost-gel polymerization shrinkage was measured using a strain gauge method. Composite specimens were cured in Teflon mold with strain gauges attached to a flat glass surface in biaxial configuration. The strain gauges (Kyowa, Ltd., Japan) were calibrated prior to testing using a standard reference material with known shrinkage characteristics to ensure accuracy and sensitivity. All measurements were conducted in a temperature-controlled environment at 23 ± 1 °C and 50 ± 5% relative humidity. The specimens were cured using an LED light source (1200 mW/cm²) for 40 s, consistent with the curing protocol used in mechanical testing44. The shrinkage strain was recorded continuously using a digital strain meter (Kyowa PCD-300 A, Japan), and average values were calculated from three replicates per group.The unmodified composite (Group I) served as a control to assess the baseline polymerization shrinkage associated with the fumed silica-based resin. Modified groups (10% and 20% TiO₂-NPs) were compared accordingly.Statistical analysisStatistical analysis was performed using IBM SPSS Statistics version 29.0 (IBM Corp., Armonk, NY, USA). Data distributions were evaluated for normality with the Shapiro–Wilk test and for homogeneity of variance with Levene’s test. When both assumptions were satisfied, inter-group differences were examined using one-way ANOVA with Tukey post-hoc comparisons; if variances were unequal, Welch’s ANOVA with Games–Howell post-hoc tests was employed. Datasets departing from normality were analysed with the Kruskal–Wallis test, followed by Bonferroni-adjusted pairwise comparisons. All results were considered statistically significant at p