The Study Of The Effect Of Plasmatrix On Inflammation Regulation And Periodontal Ligament Cells’ Behavior

Part IObjective:The purpose of this part was to investigate PRF’s antibacterial property.Materials&methods:H-PRF and L-PRF were prepared with horizontal centrifugation and fixed-angle centrifugation respectively.CFU-forming unit and inhibition ring test were performed to evaluate the antibacterial effect of both PRF;both PRF were divided into 5 parts to compare the antibacterial property of the solid and exudate components of PRF.FACS were performed to evaluate the leukocyte of different layers of both PRF.Results:There were no differences between the weight of both PRF.H-PRF show better antibacterial property than L-PRF,especially for E.coli.And the exudate of H-PRF showed higher inhibition rate,especially in E.coli.The FACS result indicate that H-PRF contains more leukocyte than L-PRF.Conclusion:H-PRF exhibited better antibacterial activities against S.aureus and E.coli,and immune cells in H-PRF contribute to its better antibacterial property.Part IIObjective:The purpose of this part was to investigate the immune regulation of i-PRF Materials&methods:First,low-speed centrifugation technology is used to obtain i-PRF,and SEM is used to detect its morphology.Then use scissors to create a 5 mm deep muscle defect in the rat gastrocnemius muscle,and GS+SA or GS+SA+i-PRF were implanted in the defect area respectively.Three days later,the samples were taken,and the changes of dendritic cells and macrophages were detected by histological methods.In vitro,macrophages and dendritic cell lines were co-cultured with i-PRF under LPS stimulation for three days,and the expression of RAW264.7 polarization and DC2.4 maturation markers were detected by immunofluorescence,RT-PCR and Western blot.Results:H&E staining showed less infiltration of inflammatory cells around muscle defects treated with i-PRF.IHC staining revealed a decrease in CD11b~+cells around the defect after i-PRF treatment,suggesting decreased inflammatory cells such as neutrophils and macrophages.Immunofluorescent staining showed that i-PRF can reduce the macrophage M1 polarization marker i NOS.And through RT-PCR and Western blot,it was detected that i-PRF can promote the expression of M2 polarization markers in macrophages compared to whole blood.It also inhibits the activation of NF-κB pathways in macrophages.In dendritic cells,immunofluorescence staining showed that i-PRF reduced the expression of maturation markers CD11c and CD86,and RT-PCR showed that cells treated with i-PRF expressed lower levels of Cd86,Mhc II,Tlr4and Traf6.Western blot in DC2.4 shows that the NF-κB pathway in cells is inhibited by i-PRF.Conclusion:Injectable-PRF inhibited classical inflammatory related NF-κB pathway which indicated its potential anti-inflammatory role during regeneration and restoration.PartⅢObjective:To compare the biological effect of PRP and liquid-PRF on periodontal ligament cells(PDLCs)in vitro.Materials&methods:Migration and proliferation assay were performed by wound healing/Transwell assay and CCK-8 assay respectively.To investigate PDLCs’osteogenic differentiation,ALP staining assay,alizarin red staining and semi-quantification were done.Also,mineralization-related genes expression levels were detected by RT-PCR.Furthermore,cells cultured with lipopolysaccharide(LPS)to induce an inflammation condition were utilized to investigate the impact of liquid-PRF on inflammatory resolution.Results:Either PRP or liquid-PRF can promote proliferation,migration of PDLCs,and osteogenic differentiation of PDLCs.It was noteworthy that liquid-PRF demonstrated a significantly higher ability to promote the biological differentiation and mineralization of PDLCs compared with PRP.Lastly,when PDLCs were incubated with LPS,cells cultured with liquid-PRF showed significantly lower m RNA expression levels of inflammatory genes.Conclusion:Liquid-PRF notably promoted PDLCs activity and attenuated the inflammatory state induced by LPS.