The research protocol was approved by the institutional experimentation and Animal Ethical Committee of Alexandria University, (IRBNO:00010556‐IORG0008839). It is a preclinical animal study and conformed to the updated ARRIVE 2.0 guidelines.
Six mongrel dogs (Canis familliaris) with inclusion criteria of being systemically healthy male dogs, 18-24 months old and weighing approximately 10- 18 Kg are included in the study. The dogs are matched regarding; defect dimensions, sex, age, weight, type of diet, and environmental housing conditions. Exclusion criteria are dogs included in previous experimental study and dogs with any apparent illness or wounds.
Sample size is calculated based on previous study aimed to investigate the management of Class II furcation defects in dogs using Guided Tissue Regeneration and Df granules(29). Based on their results, adopting level of significant 95%(α=0.05), the minimum required sample size is found to be 6 defects per group (number of groups 4) (Total sample size =24 defects). Software Sample size is calculated using Gpower version 3.1.9.2.
Grouping: The study included twenty-four surgically induced critical sized class II furcation defects in the buccal surface of the right and left mandibular premolars (P3, P4). Twelve defects were assigned in the experimental group and were managed using Angelica sinensis*, ß‐TCP allograft† and collagen membrane‡. The other twelve defects acted as the control group with ß‐TCP and collagen membrane only. Angelica sinensis is supplied in 1 bottle of 100 g of concentrated granules extracted from 330 g of the raw herbs. The size of the granule ranges from 3 to 4 μm. In order to minimize the risk of cofounding, random allocation was done, by using a computer-generated random sequence of numbers to assign treatment status. Randomization was done by giving each defect used in this study a number from 1 to 24 and using computer assisted software, a number of 12 defect was selected in each group
Surgical procedure
Proper sterilization of all the surgical instruments used in the procedures was ensured. The animals were generally anesthetized by sodium thiopental§ intravenous injection (13 mg/kg).1:100,000 epinephrine. Sulcular incisions at the buccal aspect of the mandibular third (P3) and fourth (P4) premolars in the right and left quadrants were then followed. Mucoperiosteal flaps were fully reflected. A rotary 0.5 mm rounded carbide bur was used to create Grade II critical sized furcation defects of 3 × 4 at P3 and P4 for each dog (29, 30). The inter-radicular bone was removed under copious irrigation using sterile saline.
For the future histological examination, a reference point of the bone level surrounding the created defects at the time of the surgery was essential, therefore two reference notches were created on the mesial and distal root surfaces at the base of each defect (31). Root planing by universal curette and root conditioning using Ethylenediaminetetraacetic Acid (EDTA)ǁ were carried out to the exposed root surfaces. The defects of control group were filled with β‐TCP alone, while defects of the experimental group were filled with Angelica sinensis granules mixed with β‐TCP (1:1). Trimming of the collagen membranes to the proper size extending 2-3 mm all around the created defects was followed by the application of the membranes to cover the graft filled defects. The flap was repositioned coronally to have a complete coverage of the defect area. The wound closure was achieved with simple interrupted suturing using 3-zero silk sutures¶ (Figure 1)
Postoperative care
The dogs were given intramuscular injection of ampicillin (1 g)# and Ibuprofen 600 mg[*] in the first day, postoperatively. For the following week, the dogs received the medication mixed with their food. Throughout the study period, the dogs were fed soft diet to minimize the possibility of local trauma at the site of operation. Sutures were removed after 10 days.
Animal euthanasia
Three dogs were euthanized at 1 month postoperatively and the other three dogs after 2 months. The euthanasia involved the use of overdose intravenous injection of concentrated thiopental sodium4.
Histological procedure
After euthanasia, the mandibles were dissected out and the jaw segments containing the operated teeth were separated and fixed in 10٪ neutral buffered formalin. They were decalcified and processed to obtain 5 microns thickness mesiodistal serial sections which were stained with Hematoxylin and Eosin (H&E) and Gomori trichrome and examined by light microscope to evaluate the regenerative potential in the defects and perform the morphometric analysis.
Histomorphometric quantitative analysis
Histologic photomicrographs were quantitatively analyzed using image J 1.46 r software (32). Measurements were preformed in mm or fractions of mm.
Three variables were analyzed:
- Height of newly formed bone in the interradicular region. This is the distance from the most coronal point of the formed bone till a line crossing between the two notches on the mesial and distal roots at the most apical end in mm. Standardized magnification of x40.
- Mean percentage of the newly formed bone surface area. Standardized magnification of x100.
- Thickness of newly formed bone trabeculae in mm. Standardized magnification of x100.
From each of the 24 specimens of the defects, two standardized depths were selected from which two sections were obtained and hence a total of 48 sections were examined (24 sections from the control group ,12 for each observation period and the other 24 sections from the experimental groups, 12 for each observation period). From each of the chosen sections, one standardized image for specific same structures needed for quantification was taken then measurements were recorded and statistically analyzed.
All the morphometric analysis was carried out by one investigator. Each measurement was taken 2 times, two days apart, and the mean was then recorded.
Statistical analysis
Data obtained from the histomorphometry were statistically analyzed. Quantitative data were analyzed using median, mean ± standard deviation and range (minimum -maximum). The level of Significance of the results was calculated at the 5% level. Comparison between the results of the study and experimental groups was done using two-tailed students t-test (GraphPad software.Inc.).
Footnote:
* Active Herb company, Suite E, San Diego
† Med bone Biomaterials Sentra, Portugal.
‡ Bioimplom GmbH, Giessen, Germany
§ Sandoz GmbH Biochemiestraße, Österreich, Austria
ǁ META BIOMED CO, LTD, Korea
¶ Ethicon silk suture, Johnson & Johnson, Somerville, NJ
# Eipico, 10th of Ramadan City, Egypt
* Brufen 600 mg Abbot, Germany