Study Design
The study was a randomized controlled animal study.
Study setting
The present study was conducted at the animal house of the Faculty of Dentistry, Alexandria University, Egypt. The histological procedures were carried out in the histological unit of the department of Oral Biology, Faculty of Dentistry, Alexandria University, Egypt.
Sample size
The minimal sample size is calculated based on a previous study aimed to evaluate the effect of antimicrobial photodynamic therapy (aPDT) on the endodontic treatment of apical periodontitis.(21) Silva et al. (2019)(21) reported that there was statistically significant differences in blood vessel count between groups (p<0.0001). The sample size was calculated to detect the difference in blood vessel count between the two interventions. Based on Silva et al. (2019)(21) results, adopting a power of 80% (b=0.20) to detect a standardized effect size in blood vessel count (primary outcome) of 1.583, and level of significance 5% (α error accepted =0.05), the minimum required sample size was found to 8 teeth per group (number of groups=2) (Total sample size=16 teeth) (22). Any sample withdraws from the study due to any reason will be replaced to maintain the sample size (23).
Animals and ethical statement
The experiment was approved by the ethical committee on the guidelines of Alexandria University – Institutional Animal Care and Use Committee approval number (IORG008839). This study was carried out in accordance with the ARRIVE guidelines for reporting animal research (24). Sixteen healthy male rabbits 4 months old with approximate weight of 1.25-1.5kg were used in this study. Animals were obtained from the animal house of Medical Research Institute, Alexandria University. They were kept under suitable environmental conditions in the experimental animal house. They were kept in specially designed cages with wire mesh bottoms, having unrestricted access to standard food and water, in the same controlled laboratory settings of temperature (22-25°C), good ventilation, and regular light/dark cycle (12/12 h). The cages were cleaned daily. As for the inclusion Criteria, healthy rabbits having all maxillary and mandibular incisors free from caries and fracture and rabbits less or more than 25-1.5kg were not included in the study.
Animal grouping
The upper labial incisor teeth of the 16 rabbits were used.
• In each rabbit: each side was specific for one material.
• Group MTA: The right upper central incisor teeth were treated with MTA as a direct pulp capping. (n=16 teeth).
• Group SIM-CH: The left upper central incisor teeth were treated with (Simvastatin –calcium hydroxide) (SIM-CH) as direct pulp capping (n=16 teeth)
Eight rabbits were euthanized after one week and the others were after 2 weeks.
Pulp capping
The rabbits were anesthetized with intramuscular injection of xylazine ( Xyla-ject® by Adwia co. S.A.E., Egypt) (3 mg/kg), and ketamine (Ketamine10%® by Alfasan, Holland )(25).
The teeth were disinfected with 2.5% tincture of iodine and 70% isopropyl alcohol., and then isolated with rubber dam. On the labial surface of each tooth a class V cavity with the dimension of 1.5 mm 2 mm was done using a sterile round bur (0.10 ISO standards). Copious irrigation with sterile distilled water irrigation was obligatory during the cutting to prevent heat form inducing pulpal damage. When the pulp was almost reached, the pulp horn was exposed using the tip of an explorer till a drop of blood appears. Controlling the bleeding was achieved by applying sterile cotton pellets with 0.9% normal saline on the exposed pulp (26).
-Group MTA: MTA material (Root dent, TehnoDent” Co.Ltd.Belgord region, Russia. ) was mixed with distilled water according to the manufactured structure.
-Group SIM-CH: Simvastatin powder (EGPI (Egyptian Group for Pharmaceutical Industries), Egypt.) was mixed with calcium hydroxide (Prevest Denpro Limited, Bari Brahmana, Jammu - 181133 India.) with ratio (1:1). The concentration was (100 mg simvastatin: 100 mg Calcium hydroxide/ 25microliter saline). Saline was measured using the Micro-Pipette, then it was added to form a creamy mix. Finally, for better handling properties of the new material, 4 spoons using Excavator (Excavators double ended, Dentsply Maillefer, code B 0095, ISO SIZE 6x 57 58) of catalyst of calcium hydroxide paste cement (TechNew HYDCAL, Made in Brazil).
Pulp capping materials were applied using hand pluggers No.2 (Dentsply, Maillefer). After that, cavities were sealed by Glass ionomer GC Fuji - LC CAPSULE, GC corporation, Tokyo, Japan.).
All surgical procedures were done using 3.5× magnifying dental loupes (Zumax Medical Co Ltd, Suzhou New DisTtrict, China).
Follow up
After the restorative procedure, rabbits were monitored for any symptoms and the operative sites were monitored daily to check the sealing of the filling, abscess formation, or presence of infection. To alleviate pain, subcutaneous injection with a non-steroidal anti-inflammatory drug at Carprofen (2 to 4 mg/kg), was be administered daily for 3 consecutive days., Animal health monitoring was performed by observing their normal diet consumption, sleeping hours, and weight (25, 26).
Animal euthanasia
Euthanasia was done after 1 and 2 weeks of direct pulp capping via the injection of an overdose of barbiturates intravenously (Nembutal, pentobarbital sodium, Akorn Pharmaceuticals Co.) (27) . The upper jaws were dissected out and cleaned of soft tissues. The anterior teeth and the surrounding bone were dissected in buccolingual direction for histological evaluation, and histomorphometric analysis.
Histological processing
The samples were fixed in 10% buffered formalin with pH of 7.1 for 48 h then they were subjected to decalcification in a mild acid (5% solution of formic acid) for 14 day Demineralized specimens were washed under running tap water for 1 h. Histological processing was done then blocks were cut in buccolingual serial sections of 3–4 μm thickness and sections were stained with hematoxylin and eosin stain (H&E) (Jones H&E staining kint ® by Venatana Medical systems, USA)(28).
Histological analysis
Histological analysis was done by oral biologist who was blind to the groups under light microscope (Eclipse E1000; Nikon, Tokyo, Japan). Images were captured using optika pro view digital camera at different magnification powers. After 7 days the analysis was repeated to confirm the results.
For descriptive analysis, pulp region was evaluated including:
- Odontoblast cell.
- Pulp core.
- Vasculature.
For quantitative assessment,
1-Morphologic changes in the odontoblastic layer were given grading in which:
(0): odontoblasts were polarized and arranged in palisade pattern along the dentin surface in the pulp.
(1): odontoblasts did not show any polarization and/or palisade arrangement in the pulp (20).
2- The width of the odontoblastic layer was measured in mm under ×100 magnification power(29) using image J 1.46r software.
3- For angiogenesis analysis (blood vessel count ),The number of blood vessels in the medial region of the pulp were count in an area of approximately 0.002 mm2 under ×100 magnification as a selected region of interest (ROI)(21) using image J 1.46r software.
Statistical analysis
Data were collected and analyzed using Statistical Package for Social Science (SPSS) program (ver 25).(30) Data were entered as numerical or categorical, as appropriate. Kolmogorov-Smirnov test of normality of the distribution of the variables revealed significance, so the non-parametric statistics was adopted.(31) Data were described using minimum, maximum, median, 95% Confidence Interval of the median, 25th to 75th percentiles. Comparisons were carried out between two studied independent not-normally distributed subgroups using Mann-Whitney U test.(32) Comparisons were carried out between more than two studied dependent not-normally distributed subgroups using Wilcoxon signed-rank test.(33) Pearson Chi-square test was used to test association between qualitative variables.(34) . Fisher's exact test is used when indicated.(35) During sample size calculation, beta error accepted up to 20% with a power of study of 80%. An alpha level was set to 5% with a significance level of 95%. Statistical significance was tested at p value <.05(36)