Proportion of platelet-rich plasma/autogenous bone: immunohistochemical study in rat calvaria

Objectives: This immunohistochemical study evaluated the influence of the proportion of particulate autogenous bone (AB) graft/platelet-rich plasma (PRP) on bone healing in surgically created critical-size defects (CSD) in rat calvaria.

Methods: 50 rats were divided into 5 groups: Group C (control), Group AB, Group AB/PRP-50, Group AB/PRP-100 and Group AB/PRP-150. A 5 mm diameter critical-size defect was created in the calvarium of each animal. In Group C, the defect was filled by blood clot only. In Group AB, the defect was filled with 0.01 ml of AB graft. In Groups AB/PRP-50, AB/PRP-100 and AB/PRP-150, the defects were filled with 0.01 ml of AB graft combined with 50, 100 and 150 µl of PRP, respectively. All animals were euthanized at 30 days post-operative. Serial longitudinal sections 6 µm thick were obtained and subjected to immunohistochemical reactions using the immunoperoxidase detection method. Osteocalcin and osteopontin were used as primary antibodies and biotinylated rabbit anti-goat as the secondary antibody. The StreptABComplex/HRP was then used to amplify the signal of the reaction developed with Diaminobenzidine as the chromogen.

Results: Positive immunolabels against osteocalcin and osteopontin were observed in all groups. The highest expression of osteocalcin and osteopontin occurred in Group AB/PRP-100 while the lowest expression was observed in Group C. When compared to Group AB, Groups AB/PRP-150 and AB/PRP-50 presented lower expression of osteocalcin. With regard to osteopontin, Groups AB and AB/PRP-50 demonstrated similar expression of this protein which was greater than that seen in Group AB/PRP-150.

Conclusions: The proportion AB graft/PRP influences bone healing in CSD in rat calvaria. The highest expression of osteocalcin and osteopontin was found in Group AB/PRP-100 indicating a more developed healing process that resulted in earlier calcification of newly formed bone than in the other groups. Natália de Campos received a scholarship from FAPESP (06/58094-7).