Comparative evaluation of antimicrobial Efficacy, cytotoxicity, clinical and radiographic Outcomes of different Calcium Hydroxide–Iodoform Combinations in Primary Root Canals.

Pure strains of Enterococcus faecalis ATCC 29212, Streptococcus mitis ATCC 49456, and Porphyromonas gingivalis ATCC 33277 will be procured and cultured on selective media, which will be triplicated. Later the wells will be made on each plate and filled with four commercially available calcium hydroxide Iodoform combinations. These wells will be designated as the group 1 Metapex, group 2 Neopex, group 3 Iodotin, group 4 Dentocal. Further, each group will be subjected to antimicrobial testing against test microorganisms for 24, 48, and 72 hours. Antimicrobial activity will be evaluated by measuring the zone of inhibition using vernier calipers. The whole procedure will be carried out in an Ultraviolet chamber and laminar airflow under strict aseptic conditions.

For cytotoxic evaluation, human PDL cells are harvested from the roots of freshly extracted teeth and cultured by using an explants technique. Following extraction, teeth will be placed in Hank’s balanced salt solution to maintain PDL cells viability. Initially, these teeth will be rinsed in phosphate-buffered solution and then placed in 60 mm Petri dishes containing alpha modified Eagles medium aMEM and 3 microgram per ml amphotericin and 50 mg of streptomycin per ml. To avoid contamination from the gingiva, the periodontal ligament will be carefully removed from the middle third of the root using a scalpel. The fragments will grow in aMEM supplemented with 10% fetal calf serum (FCS) and antibiotics. Cultures will be maintained at 37 degree C in a humidified atmosphere of 5 percentage Carbon dioxide and 95 percentage air. Experiments with PDL fibroblasts will be conducted by using cells between the third and eighth passage. Fibroblasts will be seeded in 24 well culture plates and incubated for 24 hours. After overnight attachment, the culture medium will be replaced with fresh aMEM containing 5 percentage FCS and the Calcium hydroxide-Iodoform combinations. After incubation for 10 min at room temperature, the effect of the different materials on cultured cells will be evaluated by 0.2 percentage final concentration trypan blue dye exclusion analysis. Briefly, the cell number will be determined by counting the viable cells in a hemocytometer. The percentage of viable cells from each well after incubation with material extracts will obtain by applying the following equation percentage of viable cells  equal to VC divided by TC multiplied by 100. The values thus obtained will be tabulated and subjected to statistical analysis.

Before initiating the procedure, preoperative clinical and radiographic assessment will be done to assess the condition of the affected tooth. Informed consent will be obtained from the parents and guardians and then the selected tooth will be randomly assigned to four groups: Group 1 Metapex, Group 2 Neopex, Group 3 Iodotin, Group 4 Dentocal.

Topical anesthesia, 2 percentage lignocaine hydrochloride gel will be applied using a small cotton pellet on to the mucosa. The patients will then be administered with 2 percentage lignocaine given as inferior alveolar nerve block, lingual nerve block, and long buccal nerve block for mandibular molars and buccal and palatal infiltration for maxillary molars. Using rubber dam application isolation, access will be gained to canal orifices using no  4 round bur and non-end cutting bur in high-speed handpiece. Pulp extirpation will be done by using no  15 H files. Working length was estimated using radiographic method as described by Ingle. The root canals will be enlarged under copious irrigation. After adequate canal preparation, the canals will be thoroughly dried using sterile paper points to ensure complete moisture elimination. The canals will be then obturated using respective calcium hydroxide-based materials, ensuring proper sealing of the root canal system. Later Coronal seal will be done with suitable restorative material. Clinical and radiographic follow-up at intervals of 3, 6, and 9 months will be done to evaluate success criteria given by Subramanium P et al.