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

Aims: To assess bone density means with Biphasic calcium phosphate alone or mixed with injectable platelet rich fibrin in bone defects created surgically in sheep’s tibia / radius using standard radiographic assessment. Materials and Methods: Three bony defects of 4mm full and 7 mm in diameter were created in every tibias/radius of five sheep. The defects were stuffed within the following order: from a proximal to distal orientation; 1st defect was full of biphasic calcium phosphate alone, second left empty to be filled by physiological clot and also the third with i-PRF mixed with BCP and covered with absorbable collagen membrane for tissue guiding and then sutured. Densitometric analysis of bone defects was made to measure density of bone at four-time intervals (two, four, six and eight weeks) post-surgically. Results: Densitometric analysis results showed a major distinction within the BCP+i-PRF group and BCP group compared with the control one and at all-time intervals with the best density being in the 1st group followed by the BCP alone group and least in the control group. Conclusions: BCP and i-PRF, both of them increased and sustained bone density (synergistic effect) throughout the study period and as revealed by densitometric data.


Statistical analysis:
Because all of the variables to be evaluated were non-parametric, the following tests were used: The Kruskal-Wallace test is used to determine the significance of each interval within the same group.For densitometry analysis at scheduled intervals, the Mann-Whitney test is used to show significance between groups.

DISCUSSION
Due to the propensity of bone to regenerate, under optimal physiological conditions, the vast majority of bone defects can heal on their own.On the other hand, the repair of a bone defect takes time and the formation of new bone is delayed due to a lack of blood flow to the fracture site and a lack of Ca and Ph to reinforce and harden the new bone 10  showed that the usage of (i-PRF) as a biomaterial is well documented in the literature, particularly in dentistry.In all cases where it has been used, authors have found it to be a good alternative to PRP in dermatology 9 .

CONCLUSIONS
Within the limits of the present study, and at the end of experiment, the study concludes that throughout the study period and as revealed by densitometric results, both BCP and i-PRF increased and sustained bone density.In its current variants, PRF seem to be a well-accepted minimally invasive procedure with positive clinical outcomes.
of study was from the Scientific Research Committee / Department of Oral and Maxillofacial Surgery / College of Dentistry / Mosul University.Five healthy local breed male sheep (age 1.5 -2 years), weighing 40-45 kg (mean= 42.5kg) were used.Prior to any surgery, the animals were acclimated for two weeks and their general health was checked to assure the absence of any general or infectious disease.Each sheep model served as four observation subgroups; two, four, six and eight weeks with all five animals being operated on.Each sheep's tibias and radiuses were operated on at random intervals of two weeks between each surgery.Preparation of i-Platelet Rich Fibrin: The site of blood collection in sheep was the jugular vein.From each sheep, two -10 ml blood samples were drawn in plastic tubes without any coatings, and immediately centrifuged.According to the preparation protocol, the centrifuge cycle for the preparation of an injectable PRF is 700 rpm for 3 minutes.Retrieve the tube from the centrifuge and ensure that the blood has separated into 2 fractions.The lower layer fraction consists of red blood cells, and the upper layer consist of plasma, platelets, and coagulating factors still in the uncoagulated form.In liquid form, the separated plasma and platelets form a lightyellow tinted layer using a syringe aspirate the top layer with the head of the syringe slightly above the intersection of the two layers.The aspirate is a partially active injectable version of PRF that can be blended with any particulate bone transplant once it is received.Surgical procedure: It was performed under general anesthesia and under sterile conditions.IM injection of a mixture containing (10mg/ml/kg) Ketamine hydrochloride general anesthetic agent (Hameln / Germany) and (2mg/ml/kg) Xylazine solution for sedation and analgesia (Intercheme / Holland) was given for general anesthesia (induction and maintenance).With a no.15-scalpel 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.For hemostasis, local anesthesia with epinephrine 1:80,000 (New Static / Colombia) was placed by infiltration at the site of the operation prior to the incision.A longitudinal incision of at least 5cm was made separately in skin and periosteum along the lateral surface of bone of the tibia / radius.A trephine bur with 4 mm depth and 7 mm width level mounted 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 holes, the trephine bur was positioned perpendicular to the long axis of the bone surface.Three typical bone defects of 7 mm width and 4 mm depth, more than 6mm apart, were produced in each tibia or radius under abundant irrigation with chilled 0.9 percent normal saline (Haidylena / Egypt) as shown in Figure (1).The defects were filled in the following order: from a proximal to distal orientation; first defect was to be filled with biphasic calcium phosphate alone, second left empty to be filled by physiological clot, third with i-PRF mixed with biphasic calcium phosphate as shown in Figure (2).For standardization, a pre-weighed amount of BCP (using an electronic weight scale) was instilled into the defect.Animal post-/10kg) 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 allowed to eat and drink as they pleased, with the veterinarian inspecting them on a regular basis.Until the sutures were removed, the bandage covering the incision was changed every three days and the wound was monitored for any signs of infection (at the tenth post-operative day).

Figure ( 1 ):
Figure (1): Three standard bone defects of 7 mm width and 4 mm depth, more than 6mm apart made in each tibia or radius.

Figure ( 2 )
Figure (2) : A-Defect filled with biphasic calcium phosphate alone, B-Defect left empty to be filled by physiological clot, C-Defect filled with i-PRF mixed with biphasic calcium phosphate.

Figure ( 3 ):
Figure (3): Red line represent Focal of Interest (FOI) for densitometric analysis of bony at the upper (A), middle (B) and lower regions (C).
of the animals' healing ran smoothly, with no difficulties.There was a total of 60 samples analyzed.Descriptive and Kruskal-Wallis test analysis for mean gray scale values in the control surgical bone defect group, BCP alone group and BCP+i-PRF group at four intervals showed a statistical significant difference among them, Mann-Whitney test between intervals revealed a highly significant difference between mean gray scale values in two week interval control bone defects and BCP alone group when compared to the other three intervals, a significant difference in four weeks surgical bone defects versus six weeks and eight weeks intervals, and a significant difference in four weeks surgical bone defects versus six weeks and eight weeks intervals.There was also a significant difference between sixand eight-week intervals.The results of the BCP+i-PRF group demonstrated a significant difference between mean gray scale values in the two-week interval when compared to the four-and eight-week intervals, but not with the six-week period.Furthermore, there was a significant difference in surgical bone abnormalities at four weeks, six weeks, and eight weeks intervals.There was also a significant difference between six-and eight-week intervals.When compared to the control defect group and at each time interval, Mann-Whitney test findings for mean gray scale values demonstrated statistical significance across the three groups, with means higher in the BCP + i-PRF group and BCP alone group correspondingly.

Table 1 :
Mean gray scale value comparison of each group at four-time intervals.Vale are Mean (Standard Deviation SD).

Table 2 :
Mean gray scale value comparison among the three groups at four-time intervals.