Effect of Plasma Rich in Growth Factors and Platelet-Rich Plasma on Bone Formation in Rat Calvaria

Eda, Takashi

Plasma Rich in Growth Factors (PRGF) and Platelet-Rich Plasma (PRP), which were prepared from autologous blood from the patient, had been reported in bone regeneration for dental implant and trauma therapeutics ^{1, 2)}. PRGF and PRP are concentrated platelets containing several growth factors to promote bone regeneration. The aim of this study is to evaluate the therapeutic effects of PRGF and PRP for bone healing in rat calvalia.

PRGF or PRP were prepared by centirifugation of rat whole blood (WB). PRGF was prepared from WB centrifuging at 460 g for 8min according to Anita’s protocol. PRP was prepared from WB using double spin method by Marx’s protocol, first spin is 1,000 g for 4 min, and then second spin is 800 g for 9 min. PRP and PRGF was activated using 10% calcium chloride solution. The activated PRP or PRGF within a PTFE tube. Periosteum of rat calvaria was carefully removed with a raspatorium after elevation of the skin-periosteum flap. The calvarial bone area including the PTFE tube was extirpated en blocat 2, 4 and 8 weeks after operation. Semi-serial sections were obtained in a sagittal orientation. The sections were stained by hematoxylin and eosin (H-E). Immunohistochemical stainings were performed using antibodies of Runx2, Osterix, ALP, Osteocalcin or VEGF. Newly formed bone was evaluated using micro-computed tomography (micro-CT). The number of platelets, leukocytes, and red blood cells (RBC) in PRGF and PRP were examined using Celltac a MEK-6358. Levels of TGF-β, PDGF-BB and IGF-1 were measured by each ELISA kits. All values were statistically analyzed with a two-way analysis of variance, and independent group variables were compared using the Mann- Whitney U- test with Bonferroni correction.

The platelet concentration is 2.5-fold higher in PRGF, and is 3.7-fold higher in PRP compared to WB. PRGF is leukocyte free, although PRP included leukocytes. PRGF contained the higher levels of TGF-β, PDGF-BB than serum and PRP. In the implantation in rat calvaria, PRGF group was observed trabecular structure of a low immature calcification degree from cortical bone side for 2 weeks after implantation, but other groups cannot detect it. In addition, a number of inflammatory cells were appeared in PRP samples. The newly formed bone was observed in all groups for 4 and 8 weeks. Immunohistochemistry showed positive for Runx2, Osterix, ALP and osteocalcin in the newly formed bone. VEGF involved in vascularization were expressed in osteoblast and vascular endothelial cells. We next analyzed using micro-CT. bone mineral content and bone volume value of PRGF group was significantly higher than that of PRP group and control after 4 and 8weeks. Although bone mineral density was not different among the three groups after 2, 4 and 8 weeks.

This study showed that PRGF significantly promoted new bone formation compared to PRP. It may affect that PRGF include leukocyte free and the high levels of growth factors.

This result suggested that PRGF could be proven to be a useful method for bone regeneration treatment.

Robert E. Marx, Arunk. Garg: Dental and Craniofacial Applications of Platelet – Rich Plasma. Quintessence Publishing Co, Inc 2005.
Anitua E, Piñas L, Begoña L, Orive G: Long-term retrospective of short implants in the posterior areas: clinical results after 10-12 years. J Clin Periodontal. 2014: 41: 404-414.