Methodology

Patient with diabetes 2 with informed consent -convenience sampling over a one year period.The tooth isolated with a rubber dam and the correct disinfection procedures will be used. researcher will do the Access preparation Working length -electronic apex locator (Dentsply Propex II) as well as a radiographic approach.

After the canals will be obturated using ZOE sealer and gutta percha and post endodontic restoration is done using composite restoration. Patient is kept on follow up for 7days, 15 days, 30 days ,45 days and 60 days Clinical outcome is measure using periapical radiograph (RVG) Pain is measured using visual analog scale to mark the pain intensity. Data will be analysed statistically To enlarge the root canals (ISO size 20 file)

A sterile paper point inside the root canal for one minute until the apex of the root canal is reached

Using T.E Buffer, samples will be delivered to the School Of Basic Life Sciences, Sharda University.

The samples will be subjected to traditional PCR analysis for the detection of Peptostreptococcus spp. in the root canal.

Grouping Following instrumentation, 5% NaOCl and 17% EDTA irrigation will be done. Control group (n=15) The final irrigant will be saline Group 1 (n=15) Irrigation using 5% apple cider vinegar done The final irrigant will be saline Group 2 (n=15) Diode laser is used for disinfection in the presence of saline Group 3 (n=15) Gymnema Sylvestre Mother Tincture Q 1000 CH solution is used for disinfection in the presence of saline Group 4 (n=15) Diode laser is used for disinfection in the presence of 5% apple cider vinegar. The final irrigant will be saline Group 5 (n=15) Diode laser is used for disinfection in the presence of Gymnema Sylvestre Mother Tincture Q 1000 CH solution. The final irrigant will be saline

After cleaning and shaping, samples will be collected and sent through T.E. Buffer to the School Of Basic Life Sciences, Sharda University.

The samples will be subjected to traditional PCR analysis for the detection of Peptostreptococcus spp. in the root canal.

Study design and ethical consideration The present study will be a randomized controlled clinical study conducted over the period of one year; the study protocol will be reviewed and approved by the institutional; ethics committee , sharda university. All procedures will be performed in accordance with the ethical standards laid down in the declaration of Helsinki.

Patients diagnosed with type 2 diabetes mellitus will be recruited after obtaining written informed consent. Convenience sampling will be employed due to the clinical nature of the study and availability of eligible patient reporting to the out patient department during the study period. .Randomisation will be done using lottery method Patient selection and sampling method Type 2 diabetic patients fulfilling the inclusion criteria will be selected using a convenience sampling technique over a one year period.

Convenience sampling has been widely used in clinical endodontic trials where strict criteria limit patient availability.

After patient recruitment, the selected tooth will be isolated using the rubber dam to maintain asepsis and prevent salivary contamination, which is critical for accurate microbiological sampling.

Access cavity preparation will be performed using and access opening bur # 2 followed by refinement of the access wall using an endo z carbide bur to ensure straight line access to root canals.

Working length determination The working length was determined using the electronic apex locator (Dentsply propex II) and radiographic verification. The use of an electronic apex locator in conjunction with a radiograph has been shown to improve accuracy and reduce procedural errors.

Canal instrumentation and initial microbiological sampling Root canal enlargement was performed up ISO size 20 file , following standardized instrumentation protocol to ensure uniformity across all groups For predisinfection microbial sampling , a sterile paper point was inserted into the root canal and maintained for 1 min, ensuring it reached the apical 3rd of the canal. This technique has been validated for effective recovery of intracanal microorganisms.

The collected paper points were immediately transferred into strile vials containing TE buffer, which preserve bacterial DNA integrity during transport.

Transport and microbiological analysis All sample were transported to the school of basics life sciences , under sterile conditions The samples were subjected to PCR analysis for the detection of peptostreptococcus species, which are obligate anaerobics commonly associated with persistent endodontic infections and apical periodontitis.

PCR was chosen due to its high sensitivity and specificity in detecting low bacterial loads compared to culture methods

Grouping and Disinfection Protocol Following biochemical preparations, all canals were irrigated using 5% sodium hypochlorite (NaOCI) and 17% EDTA, which are considered the gold standard for organic tissues dissolution and smear layer removal, respectively

Patients were randomly allocated into six groups as follows:

Control Group (n=15) Final irrigation was performed using sterile saline

Group 1 - Apple Cider Vinegar (n=15) Irrigation was performed using 5% apple cider vinegar, followed by final irrigation with sterile saline. Apple Cider Vinegar has demonstrated antimicrobial efficacy due to its acetic acid content Group 2 - Diode Laser (n=15) A 940nm diode laser was used for canal disinfection in the presence of saline. Diode lasers have been shown to penetrate dentinal tubules and reduce anaerobic bacterial load effectively Group 3 - Gymnema Sylvestre (n=15) Disinfection was carried out using the Gymnema Sylvestre mother tincture Q 1000 CH solution in the presence of saline. Gymnema sylvestre exhibits documented antimicrobial and anit-infalamatory properties

Group 4 - Diode Laser + Apple Cider Vinegar (n=15) Disinfection was performed using a 940 nm diode laser in the presence of 5% apple cider vinegar followed by saline irrigation

Group 5 - Diode laser + Gymenam Sylvestre (n=15) Disinfection was performed using a 940 nm diode laser in the presence of Gynema Sylvestre mother tincture followed by saline irrigation.

Post-disinfection Sampling After completion of cleaning and shaping and final disinfection protocols, post-treatment microbial samples were collected using sterile paper points and transported in T.E buffer for PCR analysis to assess bacterial reduction.

Follow-up and Outcome Assessment Patients were recalled at 7, 15, 30, 45, 60 days for follow-up Clinical outcome was measured using RVG and PAI was calculated Post-operative pain was assessed using a VAS, a validated toll for pain assessment in endodontics studies Microbiological analysis using traditional polymerase chain reaction (PCR)

Inclusion Criteria:

• Using radiographs, intraoral periapical radiographs (RVG) primary endodontic infection will be selected.
• Multirooted teeth , root with periapical lesion will be selected
• Type 2 diabetics between the ages of 25 years and 60 years will be chosen; both male and female patients
• A fasting blood sugar level of 126 mg/dl and a random blood sugar level of less than 200 mg/dl
• HbA1c (glycated haemoglobin) ≥ 6.5%

Exclusion Criteria:

• Patients with systemic conditions other than type 2 diabetes
• Women who are pregnant
• Those patients that have used antibiotics within the previous three months,
• Those patients that have teeth with developmental defects, calcified canals,
• Teeth that cannot be isolated with rubber dams,
• Tortuous canals
• Roots that are fractured

PROTOCOL STRATERGIES, STATISTICAL ANALYSIS

one year

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