Method Article
Time dependent effect of Chitosan nanoparticles used as final irrigant on smear layer removal and dentine microhardness in extracted human permanent mandibular premolars with single canal. A randomized in vitro study.
https://doi.org/10.21203/rs.3.pex-2275/v1
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The aim of this study is to compare the efficacy of chitosan nanoparticle and EDTA solutions as final irrigants on smear layer removal and dentine microhardness for one and three minutes contact time in extracted human permanent mandibular premolars with single canal.
Calculated sample size for each experiment
Samples used in this study will be divided into two experiments, namely smear layer removal and dentine microhardness.
A power analysis was designed to have adequate power to apply a statistical test of the null hypothesis that there is no difference between different tested groups regarding dentine microhardness reduction. By adopting an alpha (α) level of (0.05), a beta (β) of (0.2) (i.e. power=80%), and an effect size (f) of (0.974) calculated based on the results of a previous study1; the sample size (n) was found to be a total of (16) samples (i.e. 4 samples per group). Sample size was increased by (25%) to compensate for possible drop-out during different testing procedures to be a total of (20) samples (i.e. 5 samples per group). Sample size calculation was performed using G*Power version 3.1.9.72.
The sample size of each experiment will be increased to be a total of (40) samples (i.e. 10 samples per group) to increase the power of the study. So, both experiments will use 80 samples.
8. Description of study sample
Human permanent single rooted teeth extracted due to orthodontic, periodontal or prosthodontics reasons will be collected from the Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Cairo University and National Institute Of Diabetes And Endocrinology.
Eligibility criteria:
Inclusion criteria:
• Extracted human permanent mandibular premolars with single canal.
• Mature root apice
•Average teeth length 20-25 mm
Exclusion criteria:
• Teeth with root caries, root irregularities or other anomalies.
• Teeth with calcified root canals.
• Teeth with internal or external root resorption.
• Cracked teeth.
• Teeth with previous endodontic manipulation.
Sample preparation:
1) External root surfaces of teeth will be debrided with a curette to remove calculus and periodontal tissues, and then will be placed in Sodium hypochlorite (NaOCl) for 30 min. to remove soft tissue debris. The extracted teeth will be autoclaved for 30 minutes at 121℃ .
2) Conventional radiographs with different angulations will be taken to confirm that each tooth has a single canal with no internal calcifications, irregularities or any other anomalies.
3) All teeth will be inspected under microscope to exclude any tooth with cracks, caries or fracture.
4) Crowns will be decoronated using a diamond disk under copious irrigation to obtain approximately 15 mm uniform root lengths.
5) Patency of the canals will be established using k file #10 (MANI, Matsutain Seisakusho Co., Tochigi-Ken, Japan).
6) The root canals will be prepared using Rogin rotary system (Shenzhen Rogin Medical Co., China) starting with orfice opener (#25/.09), (#20/.04), (#25/.04) (#30/.04) (#35/.04) and finally (#40/.04) using an Endomotor (E-connect pro; Chanzhou Eghteeth Medical Technology Co.,Ltd) with speed of 250 RPM and 1.5 N.Cm torque for all the files. The canal will be irrigated with 3 ml of 2.5% NaOCl between each file size using a disposable plastic syringe with a 30-G needle to reach 1–2 mm short from the working length without binding. Irrigation with 5 ml of saline will be done at the end of instrumentation then , the canal will be then dried using paper points #40
Preparation of CNPs irrigation
First, a solution of 0.2% chitosan solution (2mg/ ml) will be prepared by dissolving chitosan (CS) in 2% acetic acid solution in deionized water under magnetic stirring. This will be followed by constant stirring for 30 minutes. Afterwards, Tripolyphosphate (TPP) solution will be added under stirring to form the nanoparticles. Finally, the CNPs will be stored in refrigerator and ultra-sonication of the suspension will bedone immediately before its usage.
9. Intervention for each group
Eighty samples will be used in this study and will be divided between two experiments, namely smear layer removal and dentine microhardness. Each experiment will use 40 mandibular premolars. These Fourty mandibular premolars will then be divided randomly into four groups of ten teeth each.
Group 1 (control): final irrigation using 5 ml of 17% ethylenediaminetetraacetic acid (EDTA) using a disposable plastic syringe with a 30-G needle to reach 1–2 mm short from the working length without binding for one minute contact time .
Group 2: final irrigation using 5 ml of 17% ethylenediaminetetraacetic acid (EDTA) using a disposable plastic syringe with a 30-G needle to reach 1–2 mm short from the working length without binding for three minutes contact time .
Group 3: final irrigation using 5 ml of with 0.2% chitosan nanoparticles using a disposable plastic syringe with a 30-G needle to reach 1–2 mm short from the working length without binding for one minute contact time.
Group 4: final irrigation using 5 ml of with 0.2% chitosan nanoparticles using a disposable plastic syringe with a 30-G needle to reach 1–2 mm short from the working length without binding for three minutes contact time.
A final flush with 5 ml sterile saline will be followed.
Scanning electron microscopic assessment of smear layer removal
After the application of final irrigation solutions, each root will be longitudinally split using double faced diamond disc at low speed under copious irrigation without penetrating into the lumen. Splitting of the root into halves will then be completed using a chisel and mallet. Each sample will be prepared to be examined at the cervical, middle and apical thirds using Environmental Scanning Electron Microscope. The apical, middle and coronal thirds of each specimen will be examined at 3, 7, and 12 mm, respectively from the apex.
The micrographs of each third will be coded and evaluated by two blinded well trained observers using a scoring method for evaluating smear layer. The 4 level score system described by Takeda., et al. (1999).
Score 1: No smear layer and debris evidence on dentinal tubules.
Score 2: Few dentinal tubules covered with a smear layer and debris.
Score 3: Most dentinal tubules covered with smear layer and debris, a few tubules cleaned and opened.
Score 4: Dentinal tubules completely covered with smear layer and debris
Microhardness test:
After the application of final irrigation solutions, each root for the micro-hardness test will be sectioned vertically using a cutting saw to the axis of tooth . Each root half will be horizontally embedded in auto polymerized acrylic resin exposing the most dentin surface . The micro_hardness of the root dentin will be assessed using Vicker’s tester for microhardness. The indentations will be made at three separate locations on the cervical, middle, and apical dentin using a vicker microhardness tester with a vicker’s diamond indenter. Each measurement will be oriented perpendicular to the root surface. By using a built-in scaled micrometer, the diagonal lengths of the indentations will be measured, and the measurements acquired for each indentation mark area will be converted into Vickers hardness values (VHIN).
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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