Effects Of Platelet-rich Fibrin On Proliferation And Differentiation Of Human Periodontal Ligament Cells In Vitro

Objects:To observe the effects of PRF on proliferation anddifferentiation of human periodontal ligament cells in vitro.Methods:1. The source of periodontal tissuesPeriodontal tissues were collected from healthy and noncariouspremolars for the purpose of orthodontic treatment and the age ofvolunteers range from13to18. Besides, there is no difference in gender.2. The culture of hPDLCsPeriodontal ligaments were gently scraped from the middle thirdtooth roots surface and were minced into1x1mm3little tissues. Then,these tissues were cultured by a tissue explant method. HPDLCs wereused at passages3to6for subsequent experiments.3. The identification of hPDLCs(1)The observation of cells morphology by a phase-contrastmicroscope.(2) Immunocytochemical staining for vimentin and cytokeratin.4. To observe different concentrations PRF on the effects of hPDLCsproliferation.(1) Cell proliferation was assayed with a CCK-8kit.(2) Cell cycle percentage was detected by a flow cytometry analysis.5. To observe different concentrations PRF on the effects of hPDLCsdifferentiation. (1) Assay of alkaline phosphatase activity.(2) Alizarin red S staining and semi-quantification of mineralizedmatrix nodules.(3) Analysis of Runx2protein relative expression with a WesternBlot technique.Results:1. Spindle-like shape cells were observed and some of them weredistributed as whirlpools with rapid proliferation. Cells displayed positiveexpression of vimentin and negative expression of keratin.2. Different concentrations PRF(4%、20%、100%) on the effects ofhPDLCs proliferation and differentiation.(1) human periodontal ligament cells proliferation:In the cck-8assay, there was significant difference between thecontrol group and experimental groups (P<0.05) at day1. On the day2and day3, except for4%PRF group, other group also remarkablystimulated cells proliferation.In the cell cycle test, the cell proliferative index(PI) of experimentalgroups were more higher than that of control group(P<0.05).(2) Assay of alkaline phosphatase activity:100%PRF group showed the highest level of ALP activity comparedwith other experimental groups and the control group, and the differencebetween the control group and experimental groups aresignificant(P<0.05). The AKP activity of hPDLCs in4%PRF group alsowere up-regulated, although the difference was not significant (P>0.05).(3) Alizarin red staining and semi-quantification of mineralizedmatrix nodules:The mineralized nodules were observed by an inverted phasecontrast microscope, and the amount of nodules was much more in MM+100%PRF group than that of MM group. However, it was hard todetect mineralized nodules in the control group and PRF group. Inaddition,the absorbance values at570nm reveled that extracellularcalcium deposition in100%PRF group and mineralmedium(MM)+100%PRF group both were higher significantly than thosecells treated simply with standard medium and osteogenic inductionmedium, respectively(P<0.05).(4) The relative protein expression of Runx2:Compared with the control group and MM group, the protein levelof RUNX2was upregulated in the100%PRF group and MM+100%PRFgroup respectively at different time-points.Conclusions:1. PRF can stimulate the proliferation of human periodontal ligamentcells in a dose-dependent way.2. PRF can prompt the differentiation of human periodontal ligamentcells.