A Comparative Evaluation of Pulpal Response to ACTIVA BioACTIVE Versus Mineral Trioxide Aggregate (MTA) As a Vital Pulpotomy Medications in Primary Teeth: an in Vivo Study

Sixty primary teeth in thirty children aged 7-9 years selected from Outpatient dental clinic Pedodontic and Endodontic Departments, Suez Canal University. divided into group I includes 30 teeth treated with ACTIVA BioACTIVE Base/Liner and group II includes 30 teeth treated by MTA with standard pulpotomy procedure Patients will be recalled after 15 and 30-days . 15 teeth from group I and 15 teeth from group II will be extracted after 15 days then subjected to decalcification then15 teeth from group I and 15 teeth from group II will be extracted after 30 days \& subjected to decalcification procedure in 20% EDTA at 4 °C for approximately 5 weeks then embedded in paraffin. Serial sections will be cut at 5 µm thickness. Deparaffinize the sections by 2 changes of xylene for 10 minutes each. Stain in Harris hematoxylin solution for 8 minutes. mount with xylene based mounting medium for conventional histological assessment using light microscope

1. Deparaffinization/Rehydration * Slides heated in an oven at 65C for 1 hour. * De-paraffinization 2. Antigen Retrieval * Immersion of slides into staining dish containing Antigen Retrieval Solution. * Placing the staining dish into rice cooker. * When turned to warm, unplug cooker * Allow to cool down for 20 min 3. Staining * Wash slides with TBST for 5 min on a shaker. * Inactivate endogenous peroxidase by 3% hydrogen peroxide for 10 min. * Block slides with blocking solution for 1 hour. * Dilute primary antibody in blocking buffer * Apply primary antibody to each section and incubate overnight in humidified chamber * Wash slides three times with TBST

will be carried out via avidin-biotin-peroxidase complex method using a VECTASTAIN ABC Kit . Sections will be deparaffinized by xylene and graded ethanol then treated with 20 µg/ml Proteinase K for 10 min. to prevent endogenous peroxidase activity, sections is incubated for 30 min in 0.3% H2O2/methanol then treated with 0.1% blocking serum albumin and incubated with primary antibody for 30 min. Rabbit polyclonal anti-osteopontin and goat polyclonal anti-RANKL will be used. dilution of primary antibodies used will be osteopontin (1 : 6000-8000). After being washed with phosphate-buffered saline several times, sections will be incubated with biotinylated IgG for 30 min and subsequently with streptavidin-horseradish peroxidase for 30 min. Following several washes with phosphate-buffered saline, 3,3'-diaminobenzidine substrate will be applied. As a negative control, non-immune serum will be used instead of primary antibody.