Mechanism Of A-PRF/i-PRF On Proliferation And Differentiation Of Human Gingival Fibroblasts

ObjectiveTo study the mechanism of A-PRF and i-PRF on the proliferation and differentiation of human gingival fibroblasts(HGFs),compare the release of A-PRF and i-PRF growth factors(PDGF-BB,TGF-β1,EGF)and A-The effect of PRF and i-PRF on the proliferation and migration of HGFs.Materials and Methods1.Preparation of A-PRF and i-PRF requires 10 ml of venous whole blood,centrifuged at 1500 r/min for 14 min to obtain A-PRF,and centrifuged at 700 r/min for 3 min to obtain i-PRF,growth factors in A-PRF and i-PRF.After one week of release,the growth factor-rich medium was collected and A-PRF,i-PRF conditioned medium was prepared.2.The primary HGFs were cultured by tissue block adherence method,and the cells with good growth phase in the logarithmic growth phase were selected.The morphology of the cells was observed under inverted phase contrast microscope for cell morphology identification,and the mouse anti-human vimentin staining and mouse anti-human Keratin staining was performed for cell source identification.3.The release of growth factors(EGF,PDGF-BB,TGF-β 1)by A-PRF and i-PRF at 1,3,and 7 days was detected by immunoenzyme-linked immunosorbent assay(ELISA).A-PRF and i-PRF conditioned medium were used respectively.HGFs were cultured for 7 days,and the expression of genes related to soft tissue healing(COL1a2,FN1,TGF-β,PDGF)were detected by qRT-PCR.A-PRF and i-PRF were explored by cell proliferation assay and DAPI immunofluorescence staining.Effects of 1,3,and 7 days on the proliferation of HGFs;Transwell prepared a cell migration model to observe the effects of A-PRF and i-PRF on the 24-hour migration of HGFs;and cultured HGFs using A-PRF and i-PRF conditioned medium,respectively.Theexpression of type I collagen was observed by immunofluorescence staining of type I collagen for 7 days.4.Statistical methods: using SPSS16.0 software for analysis,Shapiro-Wilk test results confirmed that the data conforms to the normal distribution,Bartlett test confirms that the data variance is homogeneous,the data is expressed as ± s,and the comparison between groups is one-way ANOVA The Dunnett’st test was used to compare the two groups.The test level was unilateral α=0.05,and the difference was statistically significant at P<0.05.Results1.A-PRF obtained after centrifugation can be clearly layered.The uppermost layer is a small cell slurry layer,the middle layer is A-PRF,which is jelly-like,and the lowermost layer is red blood cell layer.The i-PRF obtained after centrifugation is liquid.At the topmost layer;A-PRF and i-PRF were formulated into 15% L-DMEM conditioned medium for the culture of experimental HGFs with a volume fraction of15%.L-DMEM complete medium was used for the control group.Cultivation of HGFs.2.The primary HGFs were isolated and cultured by tissue block method.The primary HGFs were isolated from the gingival tissues for about one week.Under the microscope,the cells were long spindle-shaped,the cells were full,and the cells were adherent.After two weeks,the cells began to collect from the tissue blocks.It grows radially and spirally;the cells grow adherently and are long fusiform,which are morphologically preliminarily identified as HGFs,and the cells are positive by vimentin staining positive results and cytokeratin staining negative results.The source of the germ layer is HGFs.3.The results of ELISA showed that A-PRF and i-PRF contained a large amount of EGF,PDGF-BB and TGF-β 1,and the release from day 1 to day 7gradually decreased,and the release on day 7 remained at a higher level.Level,but there is a certain difference in the release of growth factors between A-PRF and i-PRF on days 1,3,and 7;qRT-PCR results show that COL1a2 is incubated after 7 days of culture of HGFs using A-PRF and i-PRF conditioned medium.The expressions of FN1,TGF-β and PDGF were up-regulated at the mRNA level.However,there were some differences in the up-regulation of A-PRF and i-PRF(P<0.05).The results of cell proliferation assay and DAPI immunofluorescence staining showed that:Compared with HGFs cultured in A-PRF and i-PRF conditioned medium,theproliferation was more significant(P<0.05),but there was no significant difference between A-PRF and i-PRF(P>0.05).Transwell cell migration results showed that HGFs were cultured in A-PRF and i-PRF conditioned medium,and the number of cell migration was significantly higher in 24 hours than in the control group(P<0.05).Cell migration was compared with A-PRF and i-PRF.There was no significant difference in the number(P>0.05).The results of immunofluorescence staining of type I collagen showed that A-PRF was used.After incubation of HGFs in i-PRF conditioned medium for 7 days,A-PRF and i-PRF significantly promoted the expression of type I collagen(P<0.05),and i-PRF was more potent than A-PRF(P<0.05).Conclusion1.A-PRF and i-PRF contain a large number of growth factors,and their release is a slow and continuous process.A-PRF and i-PRF conditioned medium have suitable growth factor concentration,which is obvious for the proliferation and differentiation of human gingival fibroblasts.Promote,non-toxic and inhibiting effects.2.A-PRF and i-PRF have different effects on the proliferation,migration,gene expression,and type I collagen of HGFs.