The current in vitro study began after obtaining clearance from the Institutional Ethics Committee, Manipal College of Dental Sciences Mangalore (Protocol Reference Number: 20072).

Sample size: It was estimated assuming a relative difference of 30% in the antibacterial activity (Matalon et al. 2009) and surface microhardness (Chen et al. 2020) between the unmodified HVGIC and the nisin-modified HVGIC, using ClinCalc.com (Calculator and ClinCalc 2019). At 95% confidence interval and 80% power, each group's sample size was calculated to be 9. Thus, the sample size for the antibacterial activity tests (agar disc diffusion method and direct contact test) and surface microhardness was 9 in each group.

The sample size for assessing net setting time and compressive strength was determined according to the ANSI/ADA Standard No. 96 (which is a modified adoption of ISO 9917:1991) for dental water-based cements (n = 3 and n = 5, respectively).

The groups included in the study were: Commercially available, unmodified HVGIC (GC Fuji IX Gold Label, GC Corporation Tokyo, Japan) was the control group (C). The test groups were Group 1(G1) and Group 2 (G2), and nisin was added at concentrations of 1% and 3% (w/w), respectively.

Sample preparation: All weight measurements were done using a micro-measuring balance (SAB 103L Scaletec, Pune, India) with a measuring accuracy of 0.001 g. The weight of the mixing paper pad was deducted during measurements. One drop of the HVGIC liquid (GC Fuji IX Gold Label, GC Corporation Tokyo, Japan) was dispensed onto the pad and weighed. For every 1 g HVGIC liquid, 4.6 mg and 13.8 mg nisin powder (2.5%w/w pure) (Sigma-Aldrich, St Louis, USA) was added to obtain 1% and 3% (w/w) concentrations for Groups 1 and 2, respectively. The nisin powder was mixed into the HVGIC liquid just before each sample preparation using a plastic spatula until a clear liquid was obtained. Premeasured HVGIC powder (GC Fuji IX Gold Label, GC Corporation Tokyo, Japan) was added to the nisin-containing liquid using a plastic spatula, maintaining a powder–liquid ratio (p/l) of 3.6:1(w/w), following the manufacturer’s instructions, in two equal parts at room temperature. The mix was then packed into petroleum jelly-coated moulds, whose dimensions followed the requirement of each experiment, to obtain test samples. For the control group (Group C), liquid without nisin was used and was mixed per the manufacturer's instructions and packed into the moulds in the same manner as the test groups. The moulds were packed in incremental layers to avoid voids. The specimens were then covered with polyester sheets (Ecodent, Gurgaon, India), flattened with a microscopic glass slide, and allowed to set for one hour. They were finished with 400-grade silicon carbide paper under constant water irrigation. The dimensions of the specimens were verified with Vernier callipers, coated with petroleum jelly for surface protection, and stored in Borosil® glass containers (Borosil Limited, Mumbai, India) with distilled water for 24 h before testing for all physical properties. The specimens for antibacterial activity tests were sterilised for 12 h in the UV Light Chamber (Sentinal Gold, ESCO Singapore).

Agar disc diffusion test (ADD) It measures the cement's antibacterial activity based on the solubility and diffusability of the antibacterial component (Matalon et al. 2009). Nine discs per group were prepared using a 6 mm-depth and 4 mm-diameter brass circular disc-shaped mould. All microbiological experiments were performed in the Biosafety level II (BSL II) lab, using ESCO class II type A biosafety cabinet, maintaining sterile conditions. A loopful of the lyophilised S. mutans (MTCC 497, Microbial Type Culture Collection and Gene Bank, Chandigarh, India) was transferred to 5 ml Brain Heart Infusion (BHI) broth with 0.5% bacitracin, incubated for 24 h in a 5% CO2 incubator (Nuaire, Plymouth USA) at 37 ± 0.5 °C. The bacterial growth was adjusted to 0.5 Mcfarland’s standard [108 colony forming units (CFU) per ml]. A lawn culture of S. mutans was prepared by swabbing the surface BHI agar supplemented with 5% sheep blood in a 4 mm-depth Petri dish. The set disc-shaped specimens were placed on the medium, maintaining a 24 mm distance between the two discs. The plates were then incubated at 37 ± 0.5 °C for 48 h using a 5% CO2 incubator (Nuaire, Plymouth, USA). The diameter of inhibition zones around the specimens was measured using Vernier Callipers at three different points, and an average value was obtained at 24 and 48 h. The absence of bacterial growth in the halo region was confirmed under the light microscope at 10× magnification. All measurements were done by two independent examiners blinded to the group type.

Direct contact test (DCT) evaluates the effects of direct and close contact between the test microorganism and the substance under test (Matalon et al. 2009). Using a flat-ended instrument, the bottom of the 9 wells of a 96-well round-bottomed microtiter plate (HiMedia Pvt Ltd, Mumbai, India) was equally coated with the cement mix from each group. All the samples were then sterilised for 4 h using UV Light Chamber (Sentinel Gold, ESCO Singapore). The surface of each sample was then covered with 10 μl of S. mutans suspension (108 CFU/ml) and incubated at 37 °C for 1 h. Further, 250 ml of BHI broth containing 0.5% bacitracin was added to each well after the suspension liquid had evaporated for direct contact between the bacteria and the test material's surface. The identical inoculums placed at the bottom of four microwells not coated with the cement formed the positive control (PC). The uninoculated medium added to four microwells coated with the cement at the bottom formed the negative control (NC). The microtiter plate was then incubated at 37 ± 0.5 °C in a 5% CO2 incubator. The Optical Density values (OD) were recorded spectrophotometrically (ELISA Reader ELX-800, Biotek Vermont USA) at 650 nm at 3, 6, and 24 h after inoculation of the broth to measure the bacterial growth. Test samples in all three groups were done in duplicates, and the average OD values (9 values per group) were obtained (Matalon et al. 2009; Chen et al. 2020).

Scanning electron microscopy (SEM): was used to observe the morphology of the biofilms on the tested samples.

Preparation of biofilm (Chen et al. 2020).

Four disc-shaped specimens for each group were prepared similarly as the ADD and were placed in 24-well flat-bottomed tissue culture plates (HiMedia Pvt Ltd, Mumbai India) containing 2 ml BHI medium with 1% sucrose and S. mutans (108 CFU/ml) in each well. After incubation for 24 h in a 5% CO2 incubator, a gentle rinse with 0.5% phosphate buffered saline (PBS) removed the non-adherent cells from the samples.

SEM observation Gram staining the smear obtained by scraping one disc in each group confirmed biofilm formation. The remaining three samples in each group went through the SEM observation for further exploratory analysis of the S. mutans biofilm. The samples were fixed with 2.5% glutaraldehyde and dehydrated through ascending series of 50%, 70%, 85%, 90%, and 100% ethanol for 15 min each. After drying, the samples were mounted on aluminium stubs, coated with 6 nm gold film, and examined under SEM at 2000×, 5000×, and 10,000× magnification. The images were analysed qualitatively for the S. mutans biofilm.

Measurement of bacterial cells and surface area For quantitative analysis of the S. mutans, the Image J software (Version 1.50b-2015, National Institutes of Health, Bethesda, USA) was used. Grids of 100 µm2 were drawn on the 10,000× SEM images. In each image, two adjacent grids (100 µm2 each) with high cell numbers were selected based on visual assessment. The bacterial cells were counted in the two grids using the measuring tool of the software, expressed as the number of cells per 200 µm2 surface of the specimen (Rahim and Thurairajah, 2011). The surface area occupied by bacteria per 200 μm2 was calculated using the magic wand tool of the software.

The stainless-steel rectangular moulds of dimensions (10 × 8 × 5 mm) and rounded internal corners, per ADA specifications were filled with mixed cement according to the mixing protocols described earlier. Mixing time was maintained between 15 and 20 s per manufacturer (GC Fuji IX Gold Label, GC Corporation Tokyo, Japan). Sixty seconds after the end of mixing, the unset specimens were indented with a Gilmore needle apparatus [needle mass 400 g, length 5 mm, and tip diameter 1 mm, per ANSI/ADA specification No. 96 (ISO 9917) at room temperature (30 °C)]. For a trial run to determine the approximate setting time, 90 s after the end of mixing, the indentations were repeated at 30-s intervals until the needle failed to make a complete circular indentation in the cement. Indentations were viewed at 2 × magnification using a magnifying lens. The needle was cleaned between indentations. To determine the net setting time, the indentations began 30 s before the approximate setting time, as defined by the trial run, making indentations at 10s intervals. The time was recorded using a stopwatch as the time elapsed from the end of mixing until the needle failed to make a complete circular indentation in the cement. Two independent examiners blinded to the group allocation repeated the test thrice in each group.

Nine samples were prepared for each group as described earlier using brass moulds, 10 mm diameter, and 2 mm depth. To evaluate microhardness, the surface of each specimen was divided into four quadrants using a #11 scalpel blade. Vickers hardness number (VHN) was measured with a Digital Micro-Hardness tester (Matsuzawa MMT-X, Toshima Japan) using a 50gf load for 30 s. Two indentations were made in each quadrant with a 100 μm distance between them (visualised using a magnifying lens), giving eight values for each specimen, from which the mean was obtained (Marti et al. 2014). The investigator who conducted the assessment was blinded to the experimental groups.

Five specimens were prepared in each group, using brass split moulds, 4 mm diameter and 6 mm depth, per ANSI/ADA specification No. 96 (ISO 9917). One kg weight was placed on the top of the mould for one hour to standardise the pressure during the setting of the material, and the excess material was allowed to flow out. Twenty-four hours after mixing, an investigator unaware of the groups estimated the compressive strength using the universal testing machine (Instron 3366, Massachusetts USA). A compressive load at a crosshead speed of 0.75 mm/min was applied along the long axis. The compressive strength in megapascals (MPa) was calculated based on the maximum force applied and the surface area of the specimen.

Data were analysed using IBM SPSS Statistics for Windows, Version 23 (IBM Corp., Armonk, NY, USA). Descriptive statistics were tabulated, and mean values were obtained. The test for normality (Shapiro–Wilk test) was not significant, and hence, all analysis was done using parametric tests. One-way ANOVA with post hoc Tamhane’s T2 test was applied to analyse the difference in net setting time, surface microhardness, compressive strength values, and the S. mutans count between the groups. As the control group values were zero, the Student’s t test was used to analyse the difference in the inhibition zones obtained by ADD. Repeated-measures ANOVA was used to check the difference in the OD values at different time intervals between the groups. For all the tests, the significance level was at a 95% confidence interval (p < 0.05).