Clinical trial NCT05744752: Effects of Allium Sativum and Moringa Oleifera Extract on Dental Enamel
Official title: Protective Effects of Allium Sativum and Moringa Oleifera Extract in Alleviating Lead Induced Defects on Dental Enamel of Rabbits
Study type: INTERVENTIONAL
Overall status: UNKNOWN
Phase: NA
Conditions: Lead Poisoning
Keywords: Moringa oleifera extract
Allium sativum extract
Dental Enamel
Rabbits
Lead sponsor: Rashid Latif Medical College
Sponsor class: OTHER
Collaborators: name: Baqai Medical University
First posted: 2023-02-27
Enrollment: 90
Sex: MALE
Minimum age (years): 3
Maximum age (years): 5
Standard ages: CHILD
Accepts healthy volunteers: no
Allocation: RANDOMIZED
Intervention model: FACTORIAL
Intervention model description: A total of 90 male rabbits of average weight 1-2 kg will be obtained from the animal house of COMSATS University, Lahore. These rabbits will be segregated into 06 groups, each group consisting of 15 rabbits. All rabbits will be kept safe in iron cages under controlled temperature (25±2 C), humidity (55 ± 5%), and 12-hour dark-light cycle. One week after their acclimatization into a new environment the trial will begin. All experimental protocols will be performed following the guidelines for the human use of laboratory animals established at COMSATS University, Lahore.
Primary purpose: PREVENTION

Brief summary: Primary objective is to observe the effects of lead on dental enamel of rabbit by measuring;

* Structural and morphological change by SEM, AFM
* Elemental composition by SEM-EDX
* Molecular composition and hydroxyapatite crystal changes by Raman Spectroscopy
* Blood parameters by serum analysis Secondary objectives includes
* To compare the protective effects of Allium sativum and Moringa oleifera on dental enamel defects due to lead
* To determine the beneficial effects of AS and MO extract in remineralization of dental enamel.

Detailed description: This Animal experimental study will be carried out in animal House, University of Health Sciences, Lahore (UHS) and COMSATS University, Lahore. For this study fifty four healthy male, white NewZeeland rabbits (9 to 10 weeks) will be taken for the study. Rabbits will be randomly divided into 6 groups. Sample size will be 9 rabbits in each group. Experimental animals will be assigned number 1,2,3,4, till 54 using the random number generator and the randomly divided into six groups (n=9) and will be assigned as group A, B, C, D, E, and F. Healthy male, white New Zeeland rabbits weighing 1000-1200 grams (Age 9-10 weeks) will be selected from University of Veterinary and Animal Sciences, Lahore. The animals will be shifted to animal house of UHS. Animals will be kept in experimental research laboratory at controlled room temperature (22-24°C) and humidity (45-65 %) under 12/12 hours natural light and dark cycle for 2 weeks to acclimatize in new environment. All experimental animals will be fed on vegetables, fruits and distilled water ad libitum. At the end of 12 weeks, all the animals will be sacrificed under deep anesthesia. Ketamine hydrochloride 45 mg/kg b.w and xylazine 5 mg/kg b.w I/M will be given to maintain deep anesthesia. Afterwards, 20 mg xylazine was injected into the pinna vein to sacrifice the rabbits while they were anesthetized.(35).

Blood Sampling: The Central Auricular Artery(36) or lateral saphenous vein will be selected to collect 4 ml blood on day 0, after 8 weeks and at the end of 12 weeks. The sample will be collected in serum separating tube for investigations like blood lead count, alkaline phosphatase, calcium and phosphate level.

Tissue Sampling Technique:

A portion of 4x5 mm2 from middle third of crown labial surface will be taken for investigations. The cutting will be done by diamond disc and high speed turbine with irrigation of water to ovoid over heating of sample. Right mandibular incisors of all the rabbits will be selected for SEM and AFM. The left mandibular incisors of all the rabbits will be selected SEM-EDX and RMS analysis. All samples will be labeled and stored in sterilized container having 12% formalin for 72 hours.

Laboratory Processing:

1. Scan Electron Microscopy: Right mandibular incisor will be selected for SEM analysis. The structure and morphological features of enamel will be seen by its images. The sample will be washed with distilled water and will be dried by hot air blower at slow speed. Sixteen samples can be fixed on a sample stab at a time. Samples will be fixed on stab by double copper tape. The exposed stab will also be covered by copper tape and only the sample part will be left uncovered.

   Gold coating will be done in sputter coater machine to make the surface conductor for fine details of images. The sample stab will be place in sputter machine chamber and lid will be closed. Inside the chamber, vacuum will be created and argon gas will be filled. Potential of 40 volts will be given. By this plasma will be formed inside the chamber and the electrons of argon gas will strike on gold plate. The tiny gold particles will start sputtering on sample stab. Once gold coating will be done, the procedure will be reversed. The chamber will be filled with air and lid will be opened. Now the sample will be processed in NOVA NANOSEM 450 for ultrastructural details.
2. Energy Dispersive Spectroscopy: EDX will be used to study elemental composition of mineral contents. A detector of OXFORD X-ACT, PENTA FET will be attached with NOVA NANOSEM 450. The same sample used for SEM will also be used for SEM-EDX analysis. This will give details on the surface up to 2 micron thickness.
3. Atomic Force Microscope: AFM (SPM-9500J3, Shimadzu Corp, Japan) will be used for the assessment of morphological features of enamel surface. It provides the three-dimensional surface topography without any sample preparation. This microscopy is designed to scan the sample with a probe. Sample will be attached on stub, which will be fixed and manually adjusted. Laser adjustment will be done after determining probe frequency. Finally, scanning and image processing will be done with WinSPM system processing. For one representative sample, micrographs at six different areas (2.0×2.0 μm scan area) will be taken using AFM (SPM-9500J3, Shimadzu Corp, Japan) and AFM software (SPM-Offline, Shimadzu Corp, Japan).

   AFM creates 3D images of a surface by scanning a micron-size cantilever across the surface, which is typically made of silicon or silicon nitride, with a tip whose radius of curvature is \~10nm. The cantilever is deflected by the forces between the tip and the surface, which are \~nN and include contributions from electrostatic forces, solvation forces, Vander Waals interactions, and chemical bonding. This deflection of cantilever is measured by a laser spot reflecting from the top surface of the cantilever into an array of photodiodes, which provides images on the computer.
4. Raman spectroscopy: It is a non-destructive chemical analysis technique which provides detailed information about chemical structure, crystallinity and molecular interactions. The RMS will give us the details of the entire thickness of enamel about the molecular composition and its interaction of the sample. No special sample preparation will be needed for this analysis and the sample can also be used for other investigations. InVia Raman Microscope by RENISHAW UK will be used for this study. The laser intensity 488nm will be used for 10 seconds. The RMS will be done on 6 different spots and the average reading will be used in study.

Eligibility criteria: Inclusion Criteria:

* Healthy rabbits
* Rabbits Age 9-10 weeks
* Weight 1000-1200 g will be selected

Exclusion Criteria:

* Diseased rabbit
* Age: More than 10 weeks and less than 9 weeks
* Weight more than 1200 g and less than 1000 g

Primary outcomes:
measure: Histological Outcomes
timeFrame: at the end of the study
Secondary outcomes:
measure: Mineral Concentration
timeFrame: at the end of the study
Other outcomes:
Locations:
city: Karachi
country: Pakistan
facility: Baqai Medical University
state: Sindh
Study officials:
affiliation: Baqai Medical University
name: Rashid Javaid, Ph.D scholar
role: PRINCIPAL_INVESTIGATOR
References:
citation: Du Toit N, Kempson SA, Dixon PM. Donkey dental anatomy. Part 2: Histological and scanning electron microscopic examinations. Vet J. 2008 Jun;176(3):345-53. doi: 10.1016/j.tvjl.2008.03.004. Epub 2008 Apr 18.
citation: Six N, Lasfargues JJ, Goldberg M. Differential repair responses in the coronal and radicular areas of the exposed rat molar pulp induced by recombinant human bone morphogenetic protein 7 (osteogenic protein 1). Arch Oral Biol. 2002 Mar;47(3):177-87. doi: 10.1016/s0003-9969(01)00100-5.
citation: Yu C, Abbott PV. An overview of the dental pulp: its functions and responses to injury. Aust Dent J. 2007 Mar;52(1 Suppl):S4-16. doi: 10.1111/j.1834-7819.2007.tb00525.x.