An evaluation and comparison of microbial colonization property of Orthodontic bands luted with Chitosan CH and Chlorhexidine CLX modified Glass Ionomer Cement GIC: An In Vivo Study

This will be a randomized control trial. 60 patients undergoing orthodontic treatment will be selected for the study. Each participant to be treated with fixed pre-adjusted appliance with 0.022 x 0.028” slot metal MBT bracket system will have banded molars.

Modification of Glass Ionomer Cement

Mixing of Chitosan Nanoparticles to GIC

20 milligrams of Chitosan in powder form will be taken which will be combined with 1.8 milliliters of glacial acetic acid, completed with 100 milliliters of distilled water. One milliliter of this Chitosan solution will be mixed with 9 milliliters of GIC liquid to produce a 10% Chitosan modified glass ionomer solution.

Chitosan nanoparticles will be bought from Nanoarray Biosciences Pvt. Ltd., Valsad, Gujarat, India.

Mixing of Chlorhexidine to GIC

To obtain Chlorhexidine modified GIC, 18 % of CLX solution will be mixed with GIC liquid at the time of manipulation, by adding one drop of CLX solution for every drop of GIC liquid, both dispensed using the same dropper. Subsequently, the mixture will be spatulated with the cement powder until it formed a luting consistency.

18% Chlorhexidine digluconate will be provided by SafeEndo Dental India Pvt. Ltd., Vadodara, India.

Allocation Method:

Patient selection will be done as per inclusion and exclusion criteria. Participants will be randomly divided into 3 groups by lottery method.

(1) Molar bands will be cemented using Conventional GIC

(2) Molar bands will be cemented using Chlorhexidine (CLX) modified GIC

(3) Molar bands will be cemented using Chitosan (CH) modified GIC

The experiment will be a single blinded study conducted by keeping the participants and technician blinded about the groups to prevent the bias.

Patients to be treated with fixed pre-adjusted appliance with 0.022 x 0.028” slot metal MBT bracket system will have banded molars. These patients will be divided randomly into 3 groups- for cementing orthodontic bands on mandibular molars; Conventional Glass Ionomer Cement will be used in 1st group, Chitosan modified GIC will be used in 2nd group and Chlorhexidine modified GIC will be used in 3rd group. The patients will be checked for Oral Hygiene Index- Simplified (OHI-S) given by John C. Greene and Jack R. Vermillion. For OHI-S scoring, 6 surfaces, i.e., facial surfaces of 16, 11, 26, 31 and lingual surfaces of 36, 46 will be examined. These teeth will be examined for debris index-simplified (DI-S) and calculus index-simplified (CI-S).

OHI-S = DI-S + CI-S

Before the patients start undergoing treatment with fixed appliances, the first molars in both the arches will be banded with orthodontic bands. These bands in the mandibular arch will be cemented with Conventional GIC, with Chitosan (CH) modified GIC (10% v/v) and with 18% Chlorhexidine digluconate modified GIC as per the allocated group.

Following instructions will be given to all the patients:

Brush after every meal.

All patients will be instructed not to use any chemical plaque control method. e.g. Chlorhexidine Mouthwash etc.

Sample collection:

After 4 weeks of banding, plaque sample will be collected from the cervical area of the banded molar with the help of a scaler. Sample will be placed in a test tube containing saline before being sent to the Genexis Biotech Pvt. Ltd, Vadodara, Gujarat, India, for a microbiology test.

To check the microbial colonization: Plaque sample will be collected in a container and will be inoculated in BHI Broth for isolation of S.mutans. Grown culture will be used for serial dilution, plated on chrome agar plates for specific identification of bacterial growth. The different colour colonies will be further inoculated in BHI Broth and incubate overnight at 37°C for growth.  Bacterial isolate will be used for genomic DNA extraction, G DNA will be isolated using Genomic DNA extraction kit, followed by 16srdna using universal primers for 16srDNA, PCR samples will be confirmed on (0.8%) agarose gel electrophoresis. PCR sample will be purified using PCR clean up kit. PCR cleanup product will be sent for sequencing.  Protocol used for pcr amplification will be followed accordingly. Thermocycle conditions are 94°C for 3 minutes, 94°C for 45 seconds, 50°C for 60 seconds, 72°C for 90 seconds, Repeat steps 2-4 35 times, 72°C for 10 minutes, 4°C HOLD

For Results – Colony Forming Units (CFU) will be counted.