Molecular insights into the role of Platelet Rich Fibrin and Biphasic Calcium Phosphate on Osteoclastogenesis in Chronic Periodontitis

Abstract

Background: The aim of this study was to elucidate the involvement of mitogen-activated protein kinases (MAPKs) and nuclear factor and#954;appa B (NF-and#954;B) signaling pathways on inflammation and osteoclast differentiation and their modulation by PRF/ BCP in chronic periodontitis. MaterialandMethods: To address this possibility, osteoclast differentiation was induced in-vitro using PBMCs derived from chronic periodontitis patients in the presence of PRF/BCP. We assessed osteoclasts generation by TRAP staining and TRAP activity by ELISA. Inflammatory mediators like pro-inflammatory cytokines and NF-and#954;B were investigated by ELISA and Immunoblot respectively. MAPK proteins and osteoclast transcription factors were studied by western blot analysis and transcriptional profile of genes related to osteoclastogenesis was assessed by RT-PCR. The mechanism of apoptosis was studied with reference to expression of Bcl-2, Bax, Bcl-xL, caspase 3/9 and DNA fragmentation. Results: The present results showed that PRF/BCP greatly suppressed osteoclast differentiation and also reduced TRAP activity, thus reducing the number of osteoclasts. The anti-inflammatory responses of PRF/BCP was confirmed by the down regulation of pro-inflammatory cytokines, IL-1and#946;, IL-6, TNF-and#945;, IKB and NF-and#954;B. In addition, the proteins associated with the MAPK pathway, p38, JNK and ERK1/2 were greatly expressed in chronic periodontitis which indicated the involvement of this pathway in osteoclast differentiation. However, PRF/BCP consistently reduced the expression of MAPK proteins. Furthermore, the potent inhibitory effect of PRF/BCP on osteoclastogenesis was evidenced by the decreased level of the adaptor protein, TRAF-6 and osteoclast transcription factors, NFATc1 and c-Fos. Finally, PRF/BCP was found to reduce the osteoclast resorptive activity marker genes, TRAP, Cathepsin-K and MMP-9. In PRF/BCP treated cells, an increased caspase 3/9 levels and caspase 3 activity was observed. Additionally, the protein expression and transcriptional profile of Bax was up-regulate