The antibiofilm effect and mechanism of silver nanowire-modified glass ionomer cement against multi-species oral biofilm
Dental caries is a continuing destruction of tooth hard tissue attributable to the acid produced by bacterial fermentation . It is one of the most prevalent chronic diseases affecting billions of people worldwide . Untreated caries may cause pain and infection, significantly influencing oral health . Teeth missing due to dental caries can lead to impaired dentition and eventually cause a reduction of patients’ chewing ability and nutrition intake . In addition, a high level of oral pathogenic organisms may spread to other parts of the body and correlate with the incidence of systemic diseases including cardiovascular diseases and bacterial pneumonia . Dental plaque is recognised as a complex consortium of microorganisms ensconced within extracellular polymeric substances adhering to dental hard tissue . It is a biofilm that constitutes a diverse microbial community, including Streptococcus and Lactobacilli , which are believed to be the specific bacterial species leading to dental caries . Streptococcus mutans (S. mutans) is one of the key cariogenic bacteria and is responsible for producing substantial quantities of extracellular polysaccharides (EPS) and lactic acid . Streptococcus sobrinus (S. sobrinus) is considered a primary bacterial etiologic on smooth surfaces of dental caries. It was discovered in 43–60% of plaque cultures from children with caries . Lactobacillus fermentum (L. fermentum) was associated with severe caries . Lactobacillus rhamnosus (L. rhamnosus) isolated from caries patients was identified as the microorganism responsible for the high production of EPS . Because biofilm is composed of bacteria ensconced within a compact matrix mainly built by EPS, showcasing distinctive microarchitecture and characteristics, the biofilm always exhibits greater resilience to environmental challenges than planktonic bacteria . Studies have reported that the presence of dental plaque is a significant risk factor for dental caries . Glass ionomer cement (GIC) is a commonly used material in restorative dentistry. It usually consists of a powder phase typically based on fluoroaluminosilicate glass and a liquid phase composed of polyacrylic acid . One of the key advantages of GIC is its ability to release fluoride ions , which can help prevent dental caries and promote remineralisation of the teeth. In addition, GIC exhibited good biocompatibility and bonding well to the tooth structure, making it suitable for various dental applications such as fillings, liners, and cementation . Despite possessing several advantageous properties, conventional GIC has been associated with its relatively high failure rate and susceptibility to secondary caries. Studies have shown that the mechanical properties and survival rate of GIC restorations are lower compared to other dental materials. GIC restorations may exhibit signs of wear, fracture, or dislodgement, leading to restoration failure over time . Apart from this, secondary caries is also a common issue associated with GIC restorations. Carious lesions develop at the margins of existing GIC restorations due to microbial accumulation and subsequent demineralisation of the adjacent tooth structure , highlighting the limited antimicrobial efficacy of traditional GIC. Hence, there is a recognised necessity for modifying GIC to augment its properties and ultimately achieve superior clinical outcomes. Silver has been extensively utilised as an antimicrobial agent in various medical applications, including wound dressings , catheters, surgical implants, and surgical instruments . Its broad-spectrum antimicrobial properties , along with low toxicity to mammalian cells  make it an attractive option for combating infections and promoting wound healing. Nanotechnology, with its ability to manipulate matter at the nanoscale, offers remarkable advantages such as enhanced mechanical properties and increased surface area . Exhibiting potent antimicrobial activity due to their high surface area-to-volume ratio and ability to release silver ions, nanosilver has been widely used in dentistry . Silver nanoparticle (AgNP) have been incorporated into various dental materials such as implants, orthodontic adhesives, and types of cement to prevent peri-implant infection, the development of white spot lesions, and secondary caries . In addition, nanosilver can offer a potential solution to combat antimicrobial resistance in dental care . Recently, we added silver nanowire (AgNW) into GIC and the preliminary results showed that AgNW-GIC has an antibacterial effect against S. mutans . However, the antibiofilm properties against multi-species biofilm of AgNW-GIC are unknown. Therefore, the aim of this study is to investigate the antibiofilm effect and mechanism of AgNW-GIC against multi-species oral biofilm consisting of S. mutans , S. sobrinus , L. fermentum , and L. rhamnosus . In addition, we evaluated the material properties of the newly developed AgNW-GIC using different AgNW concentrations. 

Preparation of the GIC specimens We used Ketac™ Molar Easymix (3 M-ESPE, St Paul, USA) as the base GIC. Commercially available AgNW (diameter: 120 nm, length: 45 μm, Shanghai Aladdin Biochemica Technology Co., Ltd., Shanghai, China) and AgNP (diameter: 60 to 120 nm, Shanghai Macklin Biochemical Co., Ltd., Shanghai, China) were used to cooperate with the base GIC. The structure of AgNW was observed using a transmission electron microscope (TEM, JEOL JEM-2100plus, JEOL, Beijing, China), and the pictures are shown in Appendix Figure S1. AgNW and AgNP were mixed with the liquid phase of GIC at different concentrations. Then, the nanosilver-modified liquid phase was hand-mixed with the GIC powder phase according to the product manual. Teflon moulds were utilised to shape the GIC specimens for different assessments. After the specimens were fully set, they were hand polished by a hand polisher (XH142, Xinhean, Wuhan, China) and autoclaved at 121 ℃ for 15 min by autoclave sterilisation (NE75, GER IRM, Beijing, China). Based on the weight and configuration of nanosilver integration, the GIC samples were categorised into six groups: Group A – GIC without nanosilver (negative control), Group B – 0.05 wt% AgNW-GIC, Group C – 0.1 wt% AgNW-GIC, Group D – 0.3 wt% AgNW-GIC, Group E – 0.5 wt% AgNW-GIC, and Group F – 0.5 wt% AgNP-GIC (positive control). Biofilm formation S. mutans (ATCC 700,610), S. sobrinus (BNCC 353,916), L. fermentum (ATCC 7,469), and L. rhamnosus (ATCC 14,931) were obtained from BeNa Culture Collection, Beijing, China. The bacteria were cultured in brain–heart infusion (BHI) broth (ELITE-MEDIA, Shanghai, China) and incubated in 5% CO 2 at 37 ℃ until the OD 600 = 0.5. 10 μL of each of these four bacteria was added to a 24-well plate containing 1 mL BHI with 1% sucrose. Then, sterilised GIC discs (5 mm in diameter and 2 mm in height) were put into the well and incubated in 5% CO 2 at 37 ℃ for 24 h. Following this, the specimens were transferred into a new 24-well plate, with each well containing 1 mL BHI with 1% sucrose, and incubated for another 24 h. The transferring process of the specimens and cultivating the biofilm was iterated once more to form a 72-h multi-species biofilm. After biofilm formation, the medium was aspirated, and the multi-species biofilm was gently rinsed three times with phosphate-buffered saline (PBS) to eliminate non-adherent bacteria. We have conducted a pilot test to study the feasibility of the multi-species biofilm formation. A fluorescent in situ hybridisation (FISH) experiment was used to observe the distribution of Streptococcus and Lactobacillus on the surface of conventional GIC . After establishing the biofilm on the GIC surface, the biofilm was fixed with 4% paraformaldehyde (P39200, Acmec, Shanghai, China), followed by cell wall disruption using lysozyme (MF 108–01, Mei5bio, Xiamen, China) and dehydration through a gradient ethanol (E99943, Acmec, Shanghai, China) series (50%, 80%, and 96% for three minutes each). Fluorescent probes were performed to label different bacteria. The oligonucleotide probes used were purchased from GENERAL BIOL (Anhui, China) and the specific probes are listed in Appendix Table S1. Then, confocal laser scanning microscopy (CLSM, FV1000MPE-B, Olympus, Japan) was employed to observe the biofilm distribution under a 40 × magnification. By confirming the feasibility, the aforementioned protocol of biofilm formation was adopted in the following experiments. Scanning electron microscope (SEM) imaging SEM (Supra 55, ZEISS, Jena, Germany) was used to observe biofilm morphology on the prepared discs. The SEM images of GIC surfaces before bacterial culture are shown in Appendix Figure S2. The GIC discs with 72-h multi-species biofilm were gently dip-washed and fixed with 2.5% glutaraldehyde at 4 ℃ overnight. After that, the GIC discs were washed with PBS and dehydrated by a graded procedure using 30% to 100% ethanol. Then, the specimens were critical point dried and sprayed with a layer of gold. We examined the biofilm at 1,000 × and 10,000 × magnification under electron high tension conditions set at 3 kV, with an aperture size of 30 μm. Live/dead bacterial viability assay The LIVE/ DEAD™ BacLight™ Bacterial Viability Kit (Invitrogen™, Thermo Fisher Scientific, Waltham, USA) was used to detect the bacterial viability of the biofilm formed on GIC surfaces. A homogeneous mixture of SYTO-9 and propidium iodide (PI) was prepared in a 1:1 ratio in a 24-well plate. Then, we added 3 μL of this mixture into 1 mL PBS and placed the GIC disks into the mixture. The specimens were kept in the dark for 15 min at room temperature following the manufacturer’s instructions. We gently dip-washed the discs with 1 mL PBS and observed the amount of biofilm on the discs using a confocal laser scanning microscope (CLSM, FV1000MPE-B, Olympus, Japan). Live bacteria were stained green by SYTO-9 (excitation/emission channel are 480 nm/500 nm), and dead bacteria were stained red by PI (excitation/emission channel are 490 nm/635 nm). Three specimens were set per group; each sample was acquired at three randomly selected images with a 40 × objective lens. The ratio of live to total bacteria was analyzed with ImageJ software (National Institutes of Health, Maryland, USA). Three-dimensional (3D) reconstruction of biofilms was performed with the software Imaris 10.0.0 (Bitplane, Oxford, England). Extracellular polysaccharides (EPS) assay EPS and bacterial cells of the multi-species biofilm were labelled with Alexa Fluor 647-labelled dextran conjugate (2.5 μM; Invitrogen™, Thermo Fisher Scientific, Waltham, USA) and SYTO-9 (2.5 μM; Invitrogen™, Thermo Fisher Scientific, Waltham, USA), respectively . Alexa Fluor 647-dextran conjugate was supplemented into the culture media after every BHI replacement. After incubating for 72 h to form a multi-species biofilm, the discs were gently rinsed. SYTO-9 was added and kept for 15 min in the dark at room temperature. The specimens were gently dip-washed again with PBS and observed under a CLSM (FV1000MPE-B, Olympus, Japan). Bacteria were stained green by SYTO-9 (excitation/emission channel are 480 nm/500 nm), and EPS were stained red by Alexa Fluor 647-dextran conjugate (excitation/emission channel are 650 nm/668 nm). Three specimens were set per group; each sample was acquired at three randomly selected images with a 40 × objective lens. The ratio of EPS to bacteria was analysed using ImageJ software (National Institutes of Health, Maryland, USA). Three-dimensional (3D) reconstruction was performed with the software Imaris 10.0.0 (Bitplane, Oxford, England). Lactic acid assay The production of lactic acid was measured by the D/L-Lactic Acid Assay Kit (Megazyme, Ireland). After incubating 72 h of multi-species biofilm, we detected the amount of lactic acid in the medium. The sample, distilled water, buffer, nicotinamide-adenine dinucleotide (NAD + ), D-glutamate-pyruvate transaminase (D-GPT), D-lactate dehydrogenase (D-LDH), and L-lactate dehydrogenase (L-LDH) were added into a plastic cuvette according to the manufacturer’s instructions. A spectrophotometer (V-1100D, MAPADA, Shanghai, China) was used to observe the absorbances at 340 nm (A 340 ). The data for each group is in triplicate. Mechanical properties Compressive strength The compressive strength of the GIC specimens was determined following the ISO 9917:2007 standard. Six replicates of each group were prepared, each GIC cylinder measuring 4 mm in diameter ( d ) and 6 mm in height. The specimens were then submerged in deionised water at 37 ℃ for 24 h, followed by removal of excess water before analysis. The GIC specimens were placed between the platens of the Universal Testing Machine (CTM5105, MTS SANS, Shenzhen, China), and a compressive load was applied along their longitudinal axis at a cross-head speed of 0.2 mm/minute. The maximum force exerted upon fracture ( p ) was recorded, and the compressive strength ( C ) was calculated using the equation: \documentclass[12pt]{minimal}
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				\begin{document}$$C= \frac{4p}{\pi {d}^{2}}$$\end{document} C = 4 p π d 2 Surface roughness The surface roughness of GIC specimens was evaluated using an atomic force microscope (AFM, Oxford Instruments Asylum Research Cypher ES, Oxford, England). Each GIC disc measures 5 mm in diameter and 2 mm in height. 3D images were obtained in tapping mode through AFM. The data was presented by mean surface roughness values (Ra). Microhardness The GIC discs were prepared at 5 mm in diameter and 2 mm in height. The specimens were placed under the objective lens of the microscope of the Vickers microhardness tester (1-1000gf, Beijing, China) for measurement, with a magnification of 40x, a load of 1.962N, and a pressing time of 15 s. Each sample was tested at three sites and the average value was taken. Each group had three replicates. Wettability The wettability of GIC specimens was measured using a contact angle meter (LSA100, Beijing, China). The GIC specimens are disks with a diameter of 5 mm and a height of 2 mm. For each specimen, 5 μL deionised water is dropped onto the surface. Subsequently, a contact angle meter is used to measure the contact angle (CA). Each specimen is measured three times at three different time points, and the averages are taken. There are three replicates in each group. Colour stability GIC specimens were prepared for three replicates of each group, measuring 20 mm in diameter and 2 mm in height. The colour of each sample was assessed immediately after preparation at two randomly selected sites. The colour assessment was performed using VITA Easyshade® V (VITA Zhnfabrik, Bad Säckingen, Germany), with parameters including L* for whiteness or blackness, a* for green–red opponent colours, and b* for blue-yellow opponent colours. The colour difference was calculated as ΔE based on the Commission Internationale de l’Eclairage L*a*b* (CIELAB) colour space based on the equation: \documentclass[12pt]{minimal}
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				\begin{document}$$\Delta E={[{\left({L}^{*}x-{L}^{*}0\right)}^{2}+{\left({a}^{*}x-{a}^{*}0\right)}^{2}+{\left({b}^{*}x-{b}^{*}0\right)}^{2}]}^\frac{1}{2}$$\end{document} Δ E = [ L ∗ x - L ∗ 0 2 + a ∗ x - a ∗ 0 2 + b ∗ x - b ∗ 0 2 ] 1 2 In the equation, L*0 , a*0, and b*0 represent the colour parameters of the negative control group; L*x , a*x, and b*x represent the colour parameters of AgNW-GIC and AgNP-GIC. Silver distribution Three samples of each group were prepared with each GIC cylinder measuring 15 mm in diameter and 1 mm in height. The silver distribution on the surfaces of the GIC samples was evaluated using time-of-flight secondary ion mass spectrometry (TOF–SIMS: M6, IONTOF, Muenster, Germany). Images were captured within a 100 μm × 100 μm region. Fluoride ion release Six GIC discs of 5 mm in diameter and 2 mm in height were prepared for each group to evaluate the amount of fluoride ion release. Each specimen was submerged in 10 mL of deionised water within a centrifuge tube, followed by agitation at 60 rpm on a shaker at 37°C. We prepared four batches of GIC samples and tested the fluoride release at 1, 3, 7, and 14 days, respectively. The release of fluoride ions was measured using an ion chromatograph (IC: ICS-6000, Thermo Fisher, Scientific, Waltham, USA). Sodium fluoride was used as a standard solution, with concentrations of 0.01, 0.02, 0.04, 0.06, 0.08, and 0.1 g/L. Cytotoxicity analysis GIC discs were prepared with a diameter of 5 mm and a height of 2 mm. The specimens were placed in a 24-well plate and soaked in 885 µL of Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12, Procell, Wuhan, China) supplemented with 10% fetal bovine serum (FBS, A3161001C, Grand Island, America), following the surface area/volume ratio of 1.25 cm 2 /mL recommended by ISO 10993–5. The GIC was incubated at 37 °C with 5% CO₂ and the medium was replaced daily. Extracts were collected on days 1, 4, and 7 into 1.5 mL centrifuge tubes, stored at -20 °C, and subsequently subjected to cytotoxicity analysis. Human gingival fibroblasts (HGFs, HUM-iCELL-m005, Shanghai, China) were cultured in DMEM/F12 supplemented with 10% FBS, 100 IU/mL penicillin, and 100 IU/mL streptomycin (S110JV, Shanghai, China). We seeded 100μL of HGFs at a density of approximately 5 × 10 4 cells/mL into a 96-well plate, with three replicate wells for each group. The plates were incubated in a 37 °C incubator with 5% CO₂ for 24 h. The medium was then aspirated, and each well was replenished with 90 µL of the supplemented medium and 10 µL of the extract. The supplemented medium served as the negative control, while wells containing only the supplemented medium but no cells were set as the blank group. PBS was added to the edge wells to minimise evaporation effects. The HGFs were incubated for 24 h at 37 °C with 5% CO₂. After incubation, the medium in the wells was aspirated, and 100 µL of a 10% cell counting kit-8 (CCK8, GK10001, Montclair, California) solution was added to each well. The plates were incubated in the dark at 37 °C for 2 h, and the absorbance was measured at 450 nm using a microplate reader (Mutiskan Sky, Massachusetts, America). Cell viability was then calculated, with the averages of replicate wells being taken, and each group was repeated three times. Statistical analysis One-way analysis of variance (ANOVA) was employed for the statistical analysis. Tukey’s multiple comparison tests were used for pair-wise comparisons in cases where ANOVA yielded significance. The level of significance was established at p ≤ 0.05 for all tests. 

We used Ketac™ Molar Easymix (3 M-ESPE, St Paul, USA) as the base GIC. Commercially available AgNW (diameter: 120 nm, length: 45 μm, Shanghai Aladdin Biochemica Technology Co., Ltd., Shanghai, China) and AgNP (diameter: 60 to 120 nm, Shanghai Macklin Biochemical Co., Ltd., Shanghai, China) were used to cooperate with the base GIC. The structure of AgNW was observed using a transmission electron microscope (TEM, JEOL JEM-2100plus, JEOL, Beijing, China), and the pictures are shown in Appendix Figure S1. AgNW and AgNP were mixed with the liquid phase of GIC at different concentrations. Then, the nanosilver-modified liquid phase was hand-mixed with the GIC powder phase according to the product manual. Teflon moulds were utilised to shape the GIC specimens for different assessments. After the specimens were fully set, they were hand polished by a hand polisher (XH142, Xinhean, Wuhan, China) and autoclaved at 121 ℃ for 15 min by autoclave sterilisation (NE75, GER IRM, Beijing, China). Based on the weight and configuration of nanosilver integration, the GIC samples were categorised into six groups: Group A – GIC without nanosilver (negative control), Group B – 0.05 wt% AgNW-GIC, Group C – 0.1 wt% AgNW-GIC, Group D – 0.3 wt% AgNW-GIC, Group E – 0.5 wt% AgNW-GIC, and Group F – 0.5 wt% AgNP-GIC (positive control). 

S. mutans (ATCC 700,610), S. sobrinus (BNCC 353,916), L. fermentum (ATCC 7,469), and L. rhamnosus (ATCC 14,931) were obtained from BeNa Culture Collection, Beijing, China. The bacteria were cultured in brain–heart infusion (BHI) broth (ELITE-MEDIA, Shanghai, China) and incubated in 5% CO 2 at 37 ℃ until the OD 600 = 0.5. 10 μL of each of these four bacteria was added to a 24-well plate containing 1 mL BHI with 1% sucrose. Then, sterilised GIC discs (5 mm in diameter and 2 mm in height) were put into the well and incubated in 5% CO 2 at 37 ℃ for 24 h. Following this, the specimens were transferred into a new 24-well plate, with each well containing 1 mL BHI with 1% sucrose, and incubated for another 24 h. The transferring process of the specimens and cultivating the biofilm was iterated once more to form a 72-h multi-species biofilm. After biofilm formation, the medium was aspirated, and the multi-species biofilm was gently rinsed three times with phosphate-buffered saline (PBS) to eliminate non-adherent bacteria. We have conducted a pilot test to study the feasibility of the multi-species biofilm formation. A fluorescent in situ hybridisation (FISH) experiment was used to observe the distribution of Streptococcus and Lactobacillus on the surface of conventional GIC . After establishing the biofilm on the GIC surface, the biofilm was fixed with 4% paraformaldehyde (P39200, Acmec, Shanghai, China), followed by cell wall disruption using lysozyme (MF 108–01, Mei5bio, Xiamen, China) and dehydration through a gradient ethanol (E99943, Acmec, Shanghai, China) series (50%, 80%, and 96% for three minutes each). Fluorescent probes were performed to label different bacteria. The oligonucleotide probes used were purchased from GENERAL BIOL (Anhui, China) and the specific probes are listed in Appendix Table S1. Then, confocal laser scanning microscopy (CLSM, FV1000MPE-B, Olympus, Japan) was employed to observe the biofilm distribution under a 40 × magnification. By confirming the feasibility, the aforementioned protocol of biofilm formation was adopted in the following experiments. 

SEM (Supra 55, ZEISS, Jena, Germany) was used to observe biofilm morphology on the prepared discs. The SEM images of GIC surfaces before bacterial culture are shown in Appendix Figure S2. The GIC discs with 72-h multi-species biofilm were gently dip-washed and fixed with 2.5% glutaraldehyde at 4 ℃ overnight. After that, the GIC discs were washed with PBS and dehydrated by a graded procedure using 30% to 100% ethanol. Then, the specimens were critical point dried and sprayed with a layer of gold. We examined the biofilm at 1,000 × and 10,000 × magnification under electron high tension conditions set at 3 kV, with an aperture size of 30 μm. 

The LIVE/ DEAD™ BacLight™ Bacterial Viability Kit (Invitrogen™, Thermo Fisher Scientific, Waltham, USA) was used to detect the bacterial viability of the biofilm formed on GIC surfaces. A homogeneous mixture of SYTO-9 and propidium iodide (PI) was prepared in a 1:1 ratio in a 24-well plate. Then, we added 3 μL of this mixture into 1 mL PBS and placed the GIC disks into the mixture. The specimens were kept in the dark for 15 min at room temperature following the manufacturer’s instructions. We gently dip-washed the discs with 1 mL PBS and observed the amount of biofilm on the discs using a confocal laser scanning microscope (CLSM, FV1000MPE-B, Olympus, Japan). Live bacteria were stained green by SYTO-9 (excitation/emission channel are 480 nm/500 nm), and dead bacteria were stained red by PI (excitation/emission channel are 490 nm/635 nm). Three specimens were set per group; each sample was acquired at three randomly selected images with a 40 × objective lens. The ratio of live to total bacteria was analyzed with ImageJ software (National Institutes of Health, Maryland, USA). Three-dimensional (3D) reconstruction of biofilms was performed with the software Imaris 10.0.0 (Bitplane, Oxford, England). 

EPS and bacterial cells of the multi-species biofilm were labelled with Alexa Fluor 647-labelled dextran conjugate (2.5 μM; Invitrogen™, Thermo Fisher Scientific, Waltham, USA) and SYTO-9 (2.5 μM; Invitrogen™, Thermo Fisher Scientific, Waltham, USA), respectively . Alexa Fluor 647-dextran conjugate was supplemented into the culture media after every BHI replacement. After incubating for 72 h to form a multi-species biofilm, the discs were gently rinsed. SYTO-9 was added and kept for 15 min in the dark at room temperature. The specimens were gently dip-washed again with PBS and observed under a CLSM (FV1000MPE-B, Olympus, Japan). Bacteria were stained green by SYTO-9 (excitation/emission channel are 480 nm/500 nm), and EPS were stained red by Alexa Fluor 647-dextran conjugate (excitation/emission channel are 650 nm/668 nm). Three specimens were set per group; each sample was acquired at three randomly selected images with a 40 × objective lens. The ratio of EPS to bacteria was analysed using ImageJ software (National Institutes of Health, Maryland, USA). Three-dimensional (3D) reconstruction was performed with the software Imaris 10.0.0 (Bitplane, Oxford, England). 

The production of lactic acid was measured by the D/L-Lactic Acid Assay Kit (Megazyme, Ireland). After incubating 72 h of multi-species biofilm, we detected the amount of lactic acid in the medium. The sample, distilled water, buffer, nicotinamide-adenine dinucleotide (NAD + ), D-glutamate-pyruvate transaminase (D-GPT), D-lactate dehydrogenase (D-LDH), and L-lactate dehydrogenase (L-LDH) were added into a plastic cuvette according to the manufacturer’s instructions. A spectrophotometer (V-1100D, MAPADA, Shanghai, China) was used to observe the absorbances at 340 nm (A 340 ). The data for each group is in triplicate. 

Compressive strength The compressive strength of the GIC specimens was determined following the ISO 9917:2007 standard. Six replicates of each group were prepared, each GIC cylinder measuring 4 mm in diameter ( d ) and 6 mm in height. The specimens were then submerged in deionised water at 37 ℃ for 24 h, followed by removal of excess water before analysis. The GIC specimens were placed between the platens of the Universal Testing Machine (CTM5105, MTS SANS, Shenzhen, China), and a compressive load was applied along their longitudinal axis at a cross-head speed of 0.2 mm/minute. The maximum force exerted upon fracture ( p ) was recorded, and the compressive strength ( C ) was calculated using the equation: \documentclass[12pt]{minimal}
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				\begin{document}$$C= \frac{4p}{\pi {d}^{2}}$$\end{document} C = 4 p π d 2 Surface roughness The surface roughness of GIC specimens was evaluated using an atomic force microscope (AFM, Oxford Instruments Asylum Research Cypher ES, Oxford, England). Each GIC disc measures 5 mm in diameter and 2 mm in height. 3D images were obtained in tapping mode through AFM. The data was presented by mean surface roughness values (Ra). Microhardness The GIC discs were prepared at 5 mm in diameter and 2 mm in height. The specimens were placed under the objective lens of the microscope of the Vickers microhardness tester (1-1000gf, Beijing, China) for measurement, with a magnification of 40x, a load of 1.962N, and a pressing time of 15 s. Each sample was tested at three sites and the average value was taken. Each group had three replicates. Wettability The wettability of GIC specimens was measured using a contact angle meter (LSA100, Beijing, China). The GIC specimens are disks with a diameter of 5 mm and a height of 2 mm. For each specimen, 5 μL deionised water is dropped onto the surface. Subsequently, a contact angle meter is used to measure the contact angle (CA). Each specimen is measured three times at three different time points, and the averages are taken. There are three replicates in each group. 

The compressive strength of the GIC specimens was determined following the ISO 9917:2007 standard. Six replicates of each group were prepared, each GIC cylinder measuring 4 mm in diameter ( d ) and 6 mm in height. The specimens were then submerged in deionised water at 37 ℃ for 24 h, followed by removal of excess water before analysis. The GIC specimens were placed between the platens of the Universal Testing Machine (CTM5105, MTS SANS, Shenzhen, China), and a compressive load was applied along their longitudinal axis at a cross-head speed of 0.2 mm/minute. The maximum force exerted upon fracture ( p ) was recorded, and the compressive strength ( C ) was calculated using the equation: \documentclass[12pt]{minimal}
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				\begin{document}$$C= \frac{4p}{\pi {d}^{2}}$$\end{document} C = 4 p π d 2 

The surface roughness of GIC specimens was evaluated using an atomic force microscope (AFM, Oxford Instruments Asylum Research Cypher ES, Oxford, England). Each GIC disc measures 5 mm in diameter and 2 mm in height. 3D images were obtained in tapping mode through AFM. The data was presented by mean surface roughness values (Ra). 

The GIC discs were prepared at 5 mm in diameter and 2 mm in height. The specimens were placed under the objective lens of the microscope of the Vickers microhardness tester (1-1000gf, Beijing, China) for measurement, with a magnification of 40x, a load of 1.962N, and a pressing time of 15 s. Each sample was tested at three sites and the average value was taken. Each group had three replicates. 

The wettability of GIC specimens was measured using a contact angle meter (LSA100, Beijing, China). The GIC specimens are disks with a diameter of 5 mm and a height of 2 mm. For each specimen, 5 μL deionised water is dropped onto the surface. Subsequently, a contact angle meter is used to measure the contact angle (CA). Each specimen is measured three times at three different time points, and the averages are taken. There are three replicates in each group. 

GIC specimens were prepared for three replicates of each group, measuring 20 mm in diameter and 2 mm in height. The colour of each sample was assessed immediately after preparation at two randomly selected sites. The colour assessment was performed using VITA Easyshade® V (VITA Zhnfabrik, Bad Säckingen, Germany), with parameters including L* for whiteness or blackness, a* for green–red opponent colours, and b* for blue-yellow opponent colours. The colour difference was calculated as ΔE based on the Commission Internationale de l’Eclairage L*a*b* (CIELAB) colour space based on the equation: \documentclass[12pt]{minimal}
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				\begin{document}$$\Delta E={[{\left({L}^{*}x-{L}^{*}0\right)}^{2}+{\left({a}^{*}x-{a}^{*}0\right)}^{2}+{\left({b}^{*}x-{b}^{*}0\right)}^{2}]}^\frac{1}{2}$$\end{document} Δ E = [ L ∗ x - L ∗ 0 2 + a ∗ x - a ∗ 0 2 + b ∗ x - b ∗ 0 2 ] 1 2 In the equation, L*0 , a*0, and b*0 represent the colour parameters of the negative control group; L*x , a*x, and b*x represent the colour parameters of AgNW-GIC and AgNP-GIC. 

Three samples of each group were prepared with each GIC cylinder measuring 15 mm in diameter and 1 mm in height. The silver distribution on the surfaces of the GIC samples was evaluated using time-of-flight secondary ion mass spectrometry (TOF–SIMS: M6, IONTOF, Muenster, Germany). Images were captured within a 100 μm × 100 μm region. 

Six GIC discs of 5 mm in diameter and 2 mm in height were prepared for each group to evaluate the amount of fluoride ion release. Each specimen was submerged in 10 mL of deionised water within a centrifuge tube, followed by agitation at 60 rpm on a shaker at 37°C. We prepared four batches of GIC samples and tested the fluoride release at 1, 3, 7, and 14 days, respectively. The release of fluoride ions was measured using an ion chromatograph (IC: ICS-6000, Thermo Fisher, Scientific, Waltham, USA). Sodium fluoride was used as a standard solution, with concentrations of 0.01, 0.02, 0.04, 0.06, 0.08, and 0.1 g/L. 

GIC discs were prepared with a diameter of 5 mm and a height of 2 mm. The specimens were placed in a 24-well plate and soaked in 885 µL of Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12, Procell, Wuhan, China) supplemented with 10% fetal bovine serum (FBS, A3161001C, Grand Island, America), following the surface area/volume ratio of 1.25 cm 2 /mL recommended by ISO 10993–5. The GIC was incubated at 37 °C with 5% CO₂ and the medium was replaced daily. Extracts were collected on days 1, 4, and 7 into 1.5 mL centrifuge tubes, stored at -20 °C, and subsequently subjected to cytotoxicity analysis. Human gingival fibroblasts (HGFs, HUM-iCELL-m005, Shanghai, China) were cultured in DMEM/F12 supplemented with 10% FBS, 100 IU/mL penicillin, and 100 IU/mL streptomycin (S110JV, Shanghai, China). We seeded 100μL of HGFs at a density of approximately 5 × 10 4 cells/mL into a 96-well plate, with three replicate wells for each group. The plates were incubated in a 37 °C incubator with 5% CO₂ for 24 h. The medium was then aspirated, and each well was replenished with 90 µL of the supplemented medium and 10 µL of the extract. The supplemented medium served as the negative control, while wells containing only the supplemented medium but no cells were set as the blank group. PBS was added to the edge wells to minimise evaporation effects. The HGFs were incubated for 24 h at 37 °C with 5% CO₂. After incubation, the medium in the wells was aspirated, and 100 µL of a 10% cell counting kit-8 (CCK8, GK10001, Montclair, California) solution was added to each well. The plates were incubated in the dark at 37 °C for 2 h, and the absorbance was measured at 450 nm using a microplate reader (Mutiskan Sky, Massachusetts, America). Cell viability was then calculated, with the averages of replicate wells being taken, and each group was repeated three times. 

One-way analysis of variance (ANOVA) was employed for the statistical analysis. Tukey’s multiple comparison tests were used for pair-wise comparisons in cases where ANOVA yielded significance. The level of significance was established at p ≤ 0.05 for all tests. 

Antibiofilm effects The results of FISH are shown in Appendix Figure S3. The findings indicate that both Streptococcus and Lactobacillus successfully colonised the surface of GIC, demonstrating the successful establishment of the multi-species biofilm. The morphology of multi-species biofilm on nanosilver-modified GIC is shown in Fig. . Aggregated bacteria clumps and extracellular matrix can be observed in the negative control group (Fig. A). The bacteria were less but still clumped in the 0.05% AgNW-GIC group (Fig. B). In 0.1% and 0.3% AgNW-GIC groups, the bacteria were no longer clumped, and scattered distributed cells were observed (Fig. C and D). In the 0.5% AgNW-GIC and 0.5% AgNP-GIC groups, bacterial growth on the GIC surface was notably limited, with isolated instances of cells rarely observed (Fig. E and F). The results of the vitality test of bacteria in the biofilm are shown in Figs. and . In the negative control group, the biofilm appeared thick with green-stained live bacteria almost occupying the image (Fig. A). However, with increasing concentrations of AgNW and AgNP, the biofilm became visibly thinner. In addition, the number of live bacteria decreased and the dead bacteria increased significantly (Figs. B, C, D, E, and F). The ratio of live to total bacteria was 100% in the negative control group, and 95.77%, 91.72%, 56.74%, 5.80% and 7.76% in the groups of 0.05% AgNW-GIC, 0.1% AgNW-GIC, 0.3% AgNW-GIC, 0.5% AgNW-GIC, and 0.5% AgNP-GIC, respectively (Fig. ). The proportion of live bacteria in the biofilm grown on the surface of nanosilver-modified GIC was significantly decreased compared to the negative control group ( p < 0.0001). Antibiofilm mechanism The results of EPS and bacteria staining are shown in Fig. . It can be observed that the bacteria and EPS are widespread in the negative control group. As the concentration of AgNW increased, the quantity of bacteria and EPS gradually decreased. The accumulation of bacteria and EPS in the 0.5% AgNP-GIC group is also significantly lower than that in the negative control group. The ratio of EPS/bacteria was quantified and presented in Fig. . The EPS/bacteria ratio in nanosilver groups is significantly lower than that of the negative control group. The lactic acid production of multi-species biofilm is presented in Fig. . There was no difference between all groups regarding lactic acid production on the third and seventh days of incubation. Mechanical properties Compressive strength The compressive strength of GIC specimens is shown in Fig. . The average compressive strength of the negative control group was 45.83 ± 3.13 MPa. The compressive strength of 0.05% AgNW-GIC, 0.1% AgNW-GIC, 0.3% AgNW-GIC, 0.5% AgNW-GIC, and 0.5% AgNP-GIC were 40.00 ± 5.76 MPa, 40.00 ± 4.05 MPa, 42.67 ± 2.34 MPa, 41.67 ± 1.51 and 44.33 ± 2.42 MPa respectively. There was no statistical significance between all the groups. Surface roughness The surface roughness of GIC specimens is shown in Fig. . The Ra of the negative control group was 18.104 nm. The Ra of 0.05% AgNW-GIC, 0.1% AgNW-GIC, 0.3% AgNW-GIC, 0.5% AgNW-GIC, and 0.5% AgNP-GIC were 19.724 nm, 18.036 nm, 22.663 nm, 16.782 and 26.006 nm respectively. Microhardness The Vickers Hardness (HV) of GIC specimens is displayed in Fig. . The average HV of the negative control group was 541.2 ± 5.8 kg/mm 2 . The average HV of 0.05% AgNW-GIC was 529.5 ± 6.6 kg/mm 2 with no significant difference compared with the control group. The average HV of 0.1% AgNW-GIC and 0.3% AgNW-GIC were 492.6 ± 9.7 and 487.0 ± 1.2 kg/mm 2 , respectively. There was no significant difference between these two groups but there were significant differences compared to both the control group and the 0.05% AgNW-GIC group. The average HV of 0.5% AgNW-GIC was 470.9 ± 5.3 kg/mm 2 . No significant difference was observed between 0.5% AgNW-GIC and 0.3% AgNW-GIC groups but there were significant differences between 0.5% AgNW-GIC and negative control, 0.05% AgNW-GIC, and 0.1% AgNW-GIC groups. The average HV of 0.5% AgNP-GIC was 457.8 ± 2.7 kg/mm 2 with no significant difference compared with the 0.5% AgNW-GIC group but there were significant differences with the other groups. Wettability The wettability of GIC specimens is displayed in Fig. . The average CA of the negative control group, 0.05% AgNW-GIC, 0.1% AgNW-GIC, and 0.3% AgNW-GIC were 86.0 ± 2.3°, 86.6 ± 2.0°, 88.9 ± 2.2°, and 91.7 ± 0.5°, respectively. There were no significant differences between these groups. The average CA of the 0.5% AgNW-GIC was 96.1 ± 1.4° with no significant difference compared with the 0.3% AgNW-GIC; but there were significant differences between 0.5% AgNW-GIC and negative control, 0.05% AgNW-GIC, and 0.1% AgNW-GIC groups. The average CA of the 0.5% AgNP-GIC was 90.9 ± 3.5° with no significant difference compared with other groups. Colour stability Figure shows the visible colour contrast among the six groups. Compared to the negative control group, the colour change in the 0.05% AgNW-GIC and 0.1% AgNW-GIC groups is not significant, while the colour of the 0.3% AgNW-GIC and 0.5% AgNW-GIC groups becomes darker and greyish. However, the colour of the 0.5% AgNP-GIC group visibly turns black. The quantified colour difference ( ΔE ) is shown in Fig. . The ΔE between 0.05% AgNW-GIC group and the negative control group was 1.50 ± 0.47, the ΔE between 0.1% AgNW-GIC group and the negative control group was 4.67 ± 0.97, the ΔE between 0.3% AgNW-GIC group and the negative control group was 8.65 ± 1.05, the ΔE between 0.5% AgNW-GIC group and the negative control group was 12.07 ± 0.70. As the concentration of AgNW increased, the ΔE value gradually rose, but it remained significantly lower than that of the 0.5% AgNP-GIC group. The ΔE value between the 0.5% AgNP-GIC group and the negative control group was 54.97 ± 0.83. Silver distribution The distribution of silver ions on the surface of GIC specimens is shown in Fig. . Yellow dots and clusters represent the presence of silver ions. No silver was exhibited in the negative control group (Fig. A). For 0.05% AgNW-GIC and 0.1% AgNW-GIC groups, minimal amounts of silver ions were presented (Fig. B and C). The distribution of silver ions on the sample surfaces in 0.3% AgNW-GIC and 0.5% AgNW-GIC groups increased with the concentrations of AgNW (Fig. D and E). However, they were still not as extensive as that of the 0.5% AgNP-GIC group (Fig. F), which is nearly ubiquitous across the entire sample surface. Fluoride release The release of fluoride ions from the GIC specimens after being agitated in deionized water for 1, 3, 7, and 14 days is shown in Fig. . There was no significant trend or variation regarding the release of fluoride ions, either over time or among the different groups. Cytotoxicity analysis The cell viability results of the GIC extracts from days 1, 4, and 7 are shown in Fig. . The results indicate that there was no significant difference in cell viability among the groups at any of the time points. 

The results of FISH are shown in Appendix Figure S3. The findings indicate that both Streptococcus and Lactobacillus successfully colonised the surface of GIC, demonstrating the successful establishment of the multi-species biofilm. The morphology of multi-species biofilm on nanosilver-modified GIC is shown in Fig. . Aggregated bacteria clumps and extracellular matrix can be observed in the negative control group (Fig. A). The bacteria were less but still clumped in the 0.05% AgNW-GIC group (Fig. B). In 0.1% and 0.3% AgNW-GIC groups, the bacteria were no longer clumped, and scattered distributed cells were observed (Fig. C and D). In the 0.5% AgNW-GIC and 0.5% AgNP-GIC groups, bacterial growth on the GIC surface was notably limited, with isolated instances of cells rarely observed (Fig. E and F). The results of the vitality test of bacteria in the biofilm are shown in Figs. and . In the negative control group, the biofilm appeared thick with green-stained live bacteria almost occupying the image (Fig. A). However, with increasing concentrations of AgNW and AgNP, the biofilm became visibly thinner. In addition, the number of live bacteria decreased and the dead bacteria increased significantly (Figs. B, C, D, E, and F). The ratio of live to total bacteria was 100% in the negative control group, and 95.77%, 91.72%, 56.74%, 5.80% and 7.76% in the groups of 0.05% AgNW-GIC, 0.1% AgNW-GIC, 0.3% AgNW-GIC, 0.5% AgNW-GIC, and 0.5% AgNP-GIC, respectively (Fig. ). The proportion of live bacteria in the biofilm grown on the surface of nanosilver-modified GIC was significantly decreased compared to the negative control group ( p < 0.0001). 

The results of EPS and bacteria staining are shown in Fig. . It can be observed that the bacteria and EPS are widespread in the negative control group. As the concentration of AgNW increased, the quantity of bacteria and EPS gradually decreased. The accumulation of bacteria and EPS in the 0.5% AgNP-GIC group is also significantly lower than that in the negative control group. The ratio of EPS/bacteria was quantified and presented in Fig. . The EPS/bacteria ratio in nanosilver groups is significantly lower than that of the negative control group. The lactic acid production of multi-species biofilm is presented in Fig. . There was no difference between all groups regarding lactic acid production on the third and seventh days of incubation. 

Compressive strength The compressive strength of GIC specimens is shown in Fig. . The average compressive strength of the negative control group was 45.83 ± 3.13 MPa. The compressive strength of 0.05% AgNW-GIC, 0.1% AgNW-GIC, 0.3% AgNW-GIC, 0.5% AgNW-GIC, and 0.5% AgNP-GIC were 40.00 ± 5.76 MPa, 40.00 ± 4.05 MPa, 42.67 ± 2.34 MPa, 41.67 ± 1.51 and 44.33 ± 2.42 MPa respectively. There was no statistical significance between all the groups. Surface roughness The surface roughness of GIC specimens is shown in Fig. . The Ra of the negative control group was 18.104 nm. The Ra of 0.05% AgNW-GIC, 0.1% AgNW-GIC, 0.3% AgNW-GIC, 0.5% AgNW-GIC, and 0.5% AgNP-GIC were 19.724 nm, 18.036 nm, 22.663 nm, 16.782 and 26.006 nm respectively. Microhardness The Vickers Hardness (HV) of GIC specimens is displayed in Fig. . The average HV of the negative control group was 541.2 ± 5.8 kg/mm 2 . The average HV of 0.05% AgNW-GIC was 529.5 ± 6.6 kg/mm 2 with no significant difference compared with the control group. The average HV of 0.1% AgNW-GIC and 0.3% AgNW-GIC were 492.6 ± 9.7 and 487.0 ± 1.2 kg/mm 2 , respectively. There was no significant difference between these two groups but there were significant differences compared to both the control group and the 0.05% AgNW-GIC group. The average HV of 0.5% AgNW-GIC was 470.9 ± 5.3 kg/mm 2 . No significant difference was observed between 0.5% AgNW-GIC and 0.3% AgNW-GIC groups but there were significant differences between 0.5% AgNW-GIC and negative control, 0.05% AgNW-GIC, and 0.1% AgNW-GIC groups. The average HV of 0.5% AgNP-GIC was 457.8 ± 2.7 kg/mm 2 with no significant difference compared with the 0.5% AgNW-GIC group but there were significant differences with the other groups. Wettability The wettability of GIC specimens is displayed in Fig. . The average CA of the negative control group, 0.05% AgNW-GIC, 0.1% AgNW-GIC, and 0.3% AgNW-GIC were 86.0 ± 2.3°, 86.6 ± 2.0°, 88.9 ± 2.2°, and 91.7 ± 0.5°, respectively. There were no significant differences between these groups. The average CA of the 0.5% AgNW-GIC was 96.1 ± 1.4° with no significant difference compared with the 0.3% AgNW-GIC; but there were significant differences between 0.5% AgNW-GIC and negative control, 0.05% AgNW-GIC, and 0.1% AgNW-GIC groups. The average CA of the 0.5% AgNP-GIC was 90.9 ± 3.5° with no significant difference compared with other groups. 

The compressive strength of GIC specimens is shown in Fig. . The average compressive strength of the negative control group was 45.83 ± 3.13 MPa. The compressive strength of 0.05% AgNW-GIC, 0.1% AgNW-GIC, 0.3% AgNW-GIC, 0.5% AgNW-GIC, and 0.5% AgNP-GIC were 40.00 ± 5.76 MPa, 40.00 ± 4.05 MPa, 42.67 ± 2.34 MPa, 41.67 ± 1.51 and 44.33 ± 2.42 MPa respectively. There was no statistical significance between all the groups. 

The surface roughness of GIC specimens is shown in Fig. . The Ra of the negative control group was 18.104 nm. The Ra of 0.05% AgNW-GIC, 0.1% AgNW-GIC, 0.3% AgNW-GIC, 0.5% AgNW-GIC, and 0.5% AgNP-GIC were 19.724 nm, 18.036 nm, 22.663 nm, 16.782 and 26.006 nm respectively. 

The Vickers Hardness (HV) of GIC specimens is displayed in Fig. . The average HV of the negative control group was 541.2 ± 5.8 kg/mm 2 . The average HV of 0.05% AgNW-GIC was 529.5 ± 6.6 kg/mm 2 with no significant difference compared with the control group. The average HV of 0.1% AgNW-GIC and 0.3% AgNW-GIC were 492.6 ± 9.7 and 487.0 ± 1.2 kg/mm 2 , respectively. There was no significant difference between these two groups but there were significant differences compared to both the control group and the 0.05% AgNW-GIC group. The average HV of 0.5% AgNW-GIC was 470.9 ± 5.3 kg/mm 2 . No significant difference was observed between 0.5% AgNW-GIC and 0.3% AgNW-GIC groups but there were significant differences between 0.5% AgNW-GIC and negative control, 0.05% AgNW-GIC, and 0.1% AgNW-GIC groups. The average HV of 0.5% AgNP-GIC was 457.8 ± 2.7 kg/mm 2 with no significant difference compared with the 0.5% AgNW-GIC group but there were significant differences with the other groups. 

The wettability of GIC specimens is displayed in Fig. . The average CA of the negative control group, 0.05% AgNW-GIC, 0.1% AgNW-GIC, and 0.3% AgNW-GIC were 86.0 ± 2.3°, 86.6 ± 2.0°, 88.9 ± 2.2°, and 91.7 ± 0.5°, respectively. There were no significant differences between these groups. The average CA of the 0.5% AgNW-GIC was 96.1 ± 1.4° with no significant difference compared with the 0.3% AgNW-GIC; but there were significant differences between 0.5% AgNW-GIC and negative control, 0.05% AgNW-GIC, and 0.1% AgNW-GIC groups. The average CA of the 0.5% AgNP-GIC was 90.9 ± 3.5° with no significant difference compared with other groups. 

Figure shows the visible colour contrast among the six groups. Compared to the negative control group, the colour change in the 0.05% AgNW-GIC and 0.1% AgNW-GIC groups is not significant, while the colour of the 0.3% AgNW-GIC and 0.5% AgNW-GIC groups becomes darker and greyish. However, the colour of the 0.5% AgNP-GIC group visibly turns black. The quantified colour difference ( ΔE ) is shown in Fig. . The ΔE between 0.05% AgNW-GIC group and the negative control group was 1.50 ± 0.47, the ΔE between 0.1% AgNW-GIC group and the negative control group was 4.67 ± 0.97, the ΔE between 0.3% AgNW-GIC group and the negative control group was 8.65 ± 1.05, the ΔE between 0.5% AgNW-GIC group and the negative control group was 12.07 ± 0.70. As the concentration of AgNW increased, the ΔE value gradually rose, but it remained significantly lower than that of the 0.5% AgNP-GIC group. The ΔE value between the 0.5% AgNP-GIC group and the negative control group was 54.97 ± 0.83. 

The distribution of silver ions on the surface of GIC specimens is shown in Fig. . Yellow dots and clusters represent the presence of silver ions. No silver was exhibited in the negative control group (Fig. A). For 0.05% AgNW-GIC and 0.1% AgNW-GIC groups, minimal amounts of silver ions were presented (Fig. B and C). The distribution of silver ions on the sample surfaces in 0.3% AgNW-GIC and 0.5% AgNW-GIC groups increased with the concentrations of AgNW (Fig. D and E). However, they were still not as extensive as that of the 0.5% AgNP-GIC group (Fig. F), which is nearly ubiquitous across the entire sample surface. 

The release of fluoride ions from the GIC specimens after being agitated in deionized water for 1, 3, 7, and 14 days is shown in Fig. . There was no significant trend or variation regarding the release of fluoride ions, either over time or among the different groups. 

The cell viability results of the GIC extracts from days 1, 4, and 7 are shown in Fig. . The results indicate that there was no significant difference in cell viability among the groups at any of the time points. 

This study demonstrated that AgNW is effective in inhibiting the growth of multi-species biofilm on the GIC surfaces. The antibiofilm mechanism is related to obstructing the production of EPS. In addition, adding AgNW would not influence the mechanical properties and fluoride release of GIC. These results suggest that AgNW-GIC has the potential to serve as a novel dental restorative material with an antibiofilm effect for clinical use. Dental caries is a prevalent oral health issue characterised by the demineralisation of tooth hard tissue and subsequent destruction of tooth structure . The primary cause of dental caries is the bacterial biofilm known as dental plaque, which forms on tooth surfaces and harbours acid-producing bacteria . Within dental plaques, the complex communities were formed by various microorganisms engaging in interactions and metabolic activities . These bacteria metabolize sucrose, producing lactic acid that demineralizes enamel and creates cavities over time . Cariology research usually includes a single bacterial strain in the study design . Some research utilised multi-species models to study dental caries but there is no agreement on the bacteria strains used in the model . Verspecht et al. developed a biofilm composed of 14 bacterial species, with S. mutans and S. sobrinus being used as representative cariogenic pathogens . Oliva et al. constructed polymicrobial biofilms of caries using S. mutans , L. rhamnosus , and Candida albicans to investigate the effects of Lippia origanoides on these biofilms . Edlund et al. used the methods of metagenomics and metatranscriptomics and revealed several mechanisms underlying the growth of cariogenic species L. fermentum in a saliva-derived biofilm model . To imitate the complex environment of dental plaques and study the cariogenic characteristics, we chose four representative bacterial strains, i.e., S. mutans , S. sobrinus , L. fermentum , and L. rhamnosus to form a multi-species biofilm in this study. We have pilot-tested the feasibility of using these four strains to culture on the material surface. The results showed that these four types of bacteria successfully colonised the surface of GIC, forming a multi-species biofilm. GIC is a commonly used dental restoration material. It is capable of self-adhering to teeth and releasing fluoride ions. However, residual bacteria between the teeth and GIC may lead to secondary caries. Therefore, the addition of antimicrobial agents to GIC is crucial . AgNW has been reported to exhibit significant antibacterial activity against several bacteria, including Staphylococcus aureus , Bacillus cereus , Micrococcus luteus , and bacteriophage MS2 . We developed AgNW-GIC and found it had an antimicrobial effect against S. mutans . However, research on the antibiofilm effect of this new material on multi-species bacteria is still lacking. Therefore, this study aims to investigate the impact of AgNW-modified GIC on multi-species biofilm. In the prior investigation, we found that both 0.05% and 0.5% AgNW-GIC exhibited antibacterial activity, with 0.5% AgNW-GIC showing more significant effects . However, since 0.5% AgNW-GIC showed a certain level of colour change compared to traditional GIC, we established four incremental concentration gradients of AgNW, which were 0.05%, 0.1%, 0.3%, and 0.5% in this study to explore a more suitable concentration. Traditional GIC and 0.5% AgNP-GIC were set as negative control and positive control respectively. SEM and CLSM assessments demonstrated the excellent antibiofilm performance of AgNW-GIC, which was consistent with other studies researching the antimicrobial effect on nanosilver-modified GIC . The antibiofilm mechanism may be related to the production of EPS and lactic acid by bacteria. EPS is primarily produced by Streptococcus and Lactobacillus through the fermentation of dietary carbohydrates . These bacteria possess enzymes that convert sucrose into glucans, fructans, and other polysaccharides, which are then secreted into the extracellular environment to form the sticky matrix known as EPS. EPS helps bacteria adhere to tooth surfaces and each other, facilitating biofilm formation and plaque accumulation . EPS also contributes to the formation of acidic microenvironments within the biofilm, facilitating enamel demineralisation and the development of dental caries . In this study, we found that the addition of nanosilver to GIC results in a significant reduction in EPS production. Reduction in EPS production has been shown to diminish the virulence of cariogenic bacteria, leading to decreased caries formation . Lactic acid is produced through carbohydrates metabolised by bacteria in dental plaque. The acidic environment created by lactic acid disrupts the balance of minerals in the tooth enamel, contributing to its breakdown over time and finally causing dental caries . One study has shown that incorporating nanosilver into GIC can reduce the production of lactic acid, potentially mitigating its harmful effects on dental health . However, the results of this study indicated that there were no significant differences in lactate production among the groups on either the third or seventh day of incubation. The reason for these inconclusive results may be that Ding et al. utilised only S. mutans , while we developed a biofilm consisting of four different bacterial species. The lactobacilli we used may produce more compensatory lactate when the number of surviving bacteria decreases significantly . Nevertheless, due to the limited evidence, further research is needed to validate the specific reasons for the observation regarding lactate production. The mechanical properties are crucial for the longevity of dental restorations. GIC demonstrates high water sorption, which can lead to increased dissolution, low wear resistance, and fracture toughness, contributing to potential restoration failures . Studies have tried some antimicrobial add-ons such as chlorhexidine, antibiotics, and silver nanoparticles to be incorporated with GIC and found that the incorporations would not diminish the mechanical properties of GIC . Little impact on the mechanical properties was identified by incorporating AgNW into GIC. This study found that incorporating nanosilver into GIC slightly decreased the compressive strength but the results were not significant. This result was consistent with a previous study . However, other studies found that adding silver nanoparticles to GIC can enhance compressive strength [ – ]. The reason for this inconclusive result may be attributed to the variations in the manufacturing protocol including differences in concentration of nanosilver added. Overall, incorporating nanosilver into GIC did not weaken its compressive strength. AFM was utilised to obtain 3D images of the GIC surface and to measure the Ra value for assessing surface roughness. It was observed that the incorporation of AgNP into GIC significantly roughened the material, while the addition of AgNW had a less pronounced effect compared to AgNP. Notably, the group with 0.5% AgNW exhibited the lowest Ra value. A rougher surface tends to accumulate more dental plaque and undergo faster material wear compared to a smoother surface . This finding suggests that the stronger antibacterial effect of incorporating AgNW into GIC compared to AgNP may partly be attributed to differences in surface roughness. Adding a concentration of nanosilver greater than 0.05% into GIC significantly reduces its microhardness. El-Wassefy et al. also found that the incorporation of AgNP into GIC decreased microhardness, although the difference was not statistically significant . By observing the surface of GIC without bacterial culture, as shown in Figure S2, we can see that the addition of nanosilver to GIC results in more surface cracks, which may be the reason for the decrease in microhardness. The contact angles of the control group, the 0.05% AgNW group and the 0.1% AgNW group were all less than 90°, indicating that deionised water could easily wet these surfaces, resulting in better wettability compared to the other three groups. However, their average contact angles were all above 85°, differing by no more than 10° from the highest value in the 0.5% AgNW group. In general, the addition of nanosilver to GIC did not significantly influence its wettability, and consequently, there was also no significant change in solubility. The results of compressive strength, surface roughness, microhardness and wettability suggested that the addition of nanosilver to GIC has minimal impact on its mechanical properties. Colour stability is crucial for the aesthetic appeal of dental restorative materials. Our previous research found that incorporating AgNW into GIC resulted in a more stable colour compared to incorporating AgNP-GIC . In this study, various concentrations of AgNW were introduced to investigate their colour stability. The results showed that as the concentration of AgNW increased, the specimens became progressively darker, while the addition of 0.5% AgNP resulted in an almost black discolouration. To explore the reasons for the colour changes after the addition of nanosilver, TOF–SIMS was employed to demonstrate the distribution of silver ions on the GIC surface. It was observed that with increasing concentration of AgNW, the area of silver ion distribution gradually expanded, whereas the surface with 0.5% AgNP was abundant with silver ions. Therefore, the colour changes of nanosilver-modified GIC were due to the silver ions expressed on the GIC surfaces. As the concentration of AgNW increases, the distribution area of silver ions gradually expands, which corresponds with the gradual decrease of bacterial distribution on the material’s surface. CLSM images also showed a decrease in bacteria amount with the increased concentrations of AgNW, together with the increased ratio of dead bacteria, which is consistent with the aforementioned tendency. Consistent fluoride release is regarded as a significant benefit of GIC . This characteristic will aid in preventing dental caries and preserving the mechanical property of the GIC over extended durations . Fluoride ions can reduce enamel dissolution and enhance the remineralisation of teeth by promoting the deposition of minerals back into the enamel . Studies reported that 1) fluoride ions can be diffused through the pores of GIC material during surface rinsing or 2) microfractures on the GIC surface can facilitate the leaching of fluoride ions . Therefore, adding other materials into GIC may influence the release of fluoride. We found that the fluoride release from nanosilver-modified GIC was higher compared to the negative control group within one week. After two weeks, there is no significant difference between groups regarding fluoride release. This result suggests that adding nanosilver to GIC does not influence its fluoride release, which is consistent with a previous study . As being used as a dental restorative material, nanosilver-modified GIC may have indirect contact with oral cells , and the silver ions released from GIC may diffuse through the dentinal tubules, potentially affecting the function of normal oral cells . Therefore, it is crucial to assess the cytotoxicity. Our results showed that there are no significant differences regarding cytotoxicity between groups or over time, indicating that the addition of nanosilver to GIC would not affect cell viability. These findings are consistent with the research results of Siqueira et al. . There are several strengths of this study. First, this study is the first one to utilise a multi-species biofilm to study the antibiofilm effect of nanosilver-modified GIC, which aimed to simulate a more oral ecological environment. Second, most previous studies used AgNP to incorporate with GIC but we found that AgNP would lead to a black colouration of GIC [ , , ]. Therefore, we utilised a novel type of nanosilver material, i.e., AgNW, to modify GIC. The results showed that AgNW-GIC had an equivalent antibiofilm effect as AgNP-GIC but significantly less discolouration than AgNP-GIC. The limitation of this study is that we only performed an initial exploration of the antibiofilm mechanism of nanosilver-modified GIC regarding EPS and lactic acid production. Further research is needed to investigate other possibilities related to the antibiofilm mechanism of this novel material. 

AgNW-GIC exhibited an excellent antibiofilm effect against multi-species oral biofilm. In addition, AgNW-modified GIC showed comparable mechanical and biochemical properties and did not significantly affect the colour stability of conventional GIC. Moreover, incorporating AgNW into GIC would not influence the cell viability. The antibiofilm mechanism of AgNW-GIC may be related to inhibiting the viability and EPS production of bacteria. 

Supplementary Material 1. 

