Assessment of Biphasic Calcium Phosphate mixed with injectable platelet rich fibrin (i-PRF) on healing of surgically created bone defects in a sheep animal model (A Histological Analysis)

Aims: Assessment of Biphasic Calcium Phosphate mixed with injectable platelet rich fibrin (i-PRF) on healing of surgically created bone defects in a sheep animal model. Materials and Methods: In each tibia / radius of five sheep, three defects each measuring 7mm in diameter and 4mm in depth were created. The defects were filled with study materials and in the following order: from a proximal to distal orientation; first defect was filled with biphasic calcium phosphate alone, second left empty to be filled by physiological clot and the third with i-PRF mixed with biphasic calcium phosphate. Histological examination of bone defects was made to assess bone formation at fourtime intervals (two, four, six and eight weeks) post-surgically. Results: Regarding bone formation, histological findings showed the presence of a significant difference within the time intervals in the BCP+i-PRF group and in the BCP group when compared with control group with the highest mean being at eight weeks post-surgery in the BCP+i-PRF group. Conclusions: Both BCP and i-PRF, enhanced bone formation when compared to the control group and throughout the period of study and as disclosed by histological findings.


Preparation of i-Platelet Rich Fibrin:
The jugular vein was used to collect blood in sheep.Two -10 ml blood samples were The aspirate is a partially active injectable form of PRF.Once the injectable PRF is obtained, it can be mixed with any particulate bone graft.

Surgical procedure:
Surgery was carried out under sterile conditions and under general anesthesia.
An intramuscular injection of a mixture of

RESULTS
Healing was uneventful in all animals and no complications were observed up to the day of sacrifice.A total of 60 sample were evaluated.Table (2

Groups Two weeks interval
of study was from the Scientific Research Committee / Department of Oral and Maxillofacial Surgery / College of Dentistry / Mosul University.Five male sheep of local breed, aged 1.5 to 2 years, weighing 40-45 kg (mean= 42.5kg) were included in the study.The animals were acclimated for two weeks before surgery and their overall health was evaluated to ensure there were no general or infectious diseases.Each sheep model was divided into four observation subgroups, with each of the five animals undergoing surgery.The tibias and radiuses of each sheep were operated on at random intervals of two weeks between surgeries.
collected from each sheep and promptly centrifuged in plastic tubes without any coatings.The centrifuge cycle for preparing an injectable PRF is 700 rpm for 3 minutes, according to the preparation technique20 .At completion of cycle, the tubes are removed out of the centrifuge, the blood has been divided into two parts.Red blood cells make up the bottom layer, while plasma, platelets, and coagulating components in their uncoagulated state make up the top layer.The separated plasma and platelets form a light-yellow colored layer in liquid form.The top layer is aspirated using a syringe.This is done by placing the tip of the syringe just above the junction of the 2 layers and carefully aspirating the top layer.

(
10mg/ml/kg) Ketamine hydrochloride general anesthetic agent (Hameln / Germany) and (2mg/ml/kg) Xylazine sedative analgesic solution (Intercheme / Holland) was used for general anesthesia (induction and maintenance).With a no.15scalpel blade, any residual fine fleece at the operation site was carefully scraped off after the animal was anesthetized.The surgical region was disinfected with a 10% povidone iodine (Iraq) solution.Local anesthetic with epinephrine 1:80,000 (New Static / Colombia) was infiltrated at the operative site for hemostasis.A 5cm long longitudinal incision was created in the skin and periosteum along the lateral surface of the tibia/radius bone.A trephine bur (Dentium Implant Systems / South Korea) with a 7 mm width and 4 mm depth level set on a straight angle handpiece (speed of 1000 rotations per minute) was used to create three standardized monocortical bone defects.During the preparation of the defects, the trephine bur was positioned perpendicular to the long axis of the bone surface.Three conventional bone defects of 7 mm width and 4 mm depth, about 6mm apart, were created in each tibia or radius under copious irrigation with cooled 0.9 % normal saline (Haidylena / Egypt).From proximal to distal, the defects were filled in the following order: first with biphasic calcium phosphate alone, second is left empty to be filled with physiological clot , and third with i-PRF mixed with biphasic calcium phosphate.For standardization, a pre-weighed amount of BCP (using an electronic weight scale (A&D GX-200)) was placed into the allocated defects using a small head sized spoon excavator.The flap is closed using 3.0 black silk suture.Intramuscular injection of antibiotic Oxytetracycline 20 mg/ml / 10 kg B.W (Alamycin 10 / Norbrook / UK) was given immediately after surgery.The animals were kept in the animal house for the first week and then were free to eat and drink and checked by a veterinarian on a regular basis.Until the sutures were removed (at the tenth post-operative day), the bandage covering the incision was changed every three days and the wound was monitored for any signs of infection.Histological examination: Histological examination was performed by 2 blinded examiners.All harvested specimens were fixed in 10% buffered formalin (PH 7.3) for 2 weeks, then rinsed with water.After fixation was completed, the tibias/radius that included artificial bone defects were cut using automated a minimicrotom (Struers minitom, Denmark).Decalcification was performed by immersion of the bony specimen in 50% formic acid and 20% sodium citrate solution.The solution was changed on an alternate day basis for 6 weeks.All samples were retransferred into formalin for 48 hours before final preparation for sectioning.Dehydration of samples was achieved in ascending series of ascending ethanol concentration from 70%, 80%, 95%, and absolute alcohol and then were placed into xylol to substitute alcohol.In the infiltration step, samples were cut in 5µm thickness in serial sections with microtome, and hematoxyline and eosin staining was performed for microscopic examination.Then they were submitted to histopathological examination under light microscope (Light microscope/Optica / Italy).A histological evaluation of the bone defects at four-time intervals (two, four, six and eight weeks) in each bone defect were performed following the completion of the surgical operations and as a foundation for comparing the three groups.Statistical analysis: All histological scoring variables to be assessed were considered non-parametric and hence the following tests were used: Kruskal -Wallace test: to show the significance between each interval at 2,4,6 and 8 weeks in the same group.The Mann-Whitney U test was used to show significance between groups for histological analysis at the scheduled intervals.Significance was set at p≤ 0.05.

Table ( 2
): Comparisons of histological scores of each group at four-time intervals.Values are Mean.