The study will be carried out in the Department of periodontia and community Dentistry, ZADC, AMU in collaboration with Interdisciplinary Biotechnology Unit, AMU. All experimental protocols will be approved by the Research Ethics Committee of the Faculty of medicine, Aligarh Muslim University, Aligarh, India. The study will be carried out following the institutional and international guidelines mentioned in the Declaration of Helsinki for the use of human body material in medical research. The methodology will follow the checklist for reporting in vitro studies (CRIS Guidelines). Written and verbal informed consent will be obtained from all participants.

In this in vitro study, the antibacterial efficacy of two topical agents, namely SDF and prepared Selenium nanoparticles infused hydroxyapatite will be evaluated against S. mutans. Also staining potential of SDF will be compared with that of selenium nanoparticles infused hydroxyapatite.

Preparation of SeNPs infused hydroxyapatite

Biogenic preparation of material will be carried out in the department of biotechnology, AMU. We shall be using leaf/flower extracts of certain indigenous plants for synthesis of nanoparticles. In this regard we shall be using leaf extracts of Azadirachta indica(neem) or Acacia arabica (babul).

Microwave–assisted biomimetic Hydroxyapatite nanoparticles will be synthesized using calcium and phosphorus sources.

100gm leaf powder/ 100 ml water boiled till the volume of the water gets half of the original volume. The solution will be decanted and filtered to get clear solution.

Evaluation of Antibacterial efficacy via disc diffusion method:

The bacterial sensitivity will be tested using the Disc Diffusion method where the diameter of the inhibition zone specifying the relative susceptibility of bacteria (S mutans) to SDF and SeNPs infused hydroxyapatite will be evaluated.

To implement this method, the fresh culture of S. mutans will be prepared. The sterile paper discs coated with the SDF and other disc coated with selenium nanoparticles infused hydroxyapatite will be transferred on plates containing the microbes. In the next step, the culture media in an anaerobic jar containing gasp will be used to drain the culture medium for S mutans. Finally, the anaerobic jar containing the culture media will be incubated at 37°C for 24 h. Subsequently, the sensitivity of S. mutans strain to SDF and selenium nanoparticles infused hydroxyapatite will be evaluated by measuring the diameter of the inhibition zone.

Evaluation of staining potential:

For evaluation of staining property 20 extracted noncarious teeth and 20 extracted carious teeth will be collected from both genders with no history of systemic diseases. Extracted teeth will be allocated into two groups, group 1 and group 2 containing extracted carious and noncarious teeth respectively which will be further divided randomly into two groups. Two independent observers will assess the outcomes. Each group will be associated with a specific objective as described below:

Group 1a: staining potential following application of SDF on carious teeth.

Group 1b: staining potential following application of SeNPs infused Hydroxyapatite on carious teeth.

Group 2a: staining potential following application of SDF on non-carious teeth

Group 2b: staining potential following application of SeNPs infused Hydroxyapatite on non-carious teeth.

Tooth preparation and Addition of the test materials:

The teeth will be clean using deionized water and will be mounted to standardized photographic set-ups. Teeth in Group 1a will receive 38% SDF application (n = 10) and Teeth in Group 1b will receive SeNPs infused hydroxyapatite application (n = 10). similarly, Teeth in Group 2a will receive 38% SDF application (n = 10) and Teeth in Group 2b will receive SeNPs infused hydroxyapatite application (n = 10) As per the manufacturer’s instructions, SDF will be applied using a micro brush to the clinically carious lesions only with the surrounding tooth structure left untouched. All teeth in Groups 1a,1b, 2a and 2b will be selected such that they will match by tooth type, size and location of the carious lesion to reduce selection bias.

First, plaque will be thoroughly clean from the teeth using a polishing brush and a nonfluoridated prophylactic paste in a low-speed handpiece. Then, the teeth will be mounted in acrylic resin. Afterwards, they will be stored in distilled water at room temperature.

Image analysis and statistical methods

All teeth will be imaged in the same room, under the same controlled lighting conditions and positions with a digital VR lens reflex camera (Nikon Z50 Mirrorless Optical Zoom Camera with Z DX 16-50mm f/3.5-6.3 VR Lens, Japan). Images of each tooth surface will be taken at baseline and at regular intervals (2, 5, 15, 60 minutes and 6, 24, 48, 72 h) for a period of 3 days post-application. Subsequently, the images will be imported and calibrated using image analysis software where each carious lesion will be isolated and the mean grey values and their standard deviations will be calculated.