The Effects Of Platelet-rich Fibrin Extract (PRFe) On Osteoblast

[Objective]To evaluate the early effect of platelet-rich fibrin extract (PRFe) on proliferation and differentiation and F-actin cytoskeleton of osteoblasts.[Methods]1. PRFe preparation:AT the aseptic conditions,10ml blood was taken from the abdominal aorta of healthy male SD rats, after centrifuge (400g,12min)., the platelet-rich fibrin was acquired, and then put them in-80℃and centrifugal (230g,10min), PRFe was acquired.2. Titanium plate preparation:The TA2pure titanium plates (10mm×1mm) were accepted the treatment of acid corrosion sandblasting (SLA).3. Grouping:Trials are divided into the experimental group (P) and the control group(D),group P use the a-MEM containing PRFe(10%FBS),while group D just use thea-MEM(10%FBS).4. MTT assay:To detect the number of the osteoblasts at1d、3d、5d; the activity of alkaline phosphatase (ALP) to detect the differentiation of osteoblast at1d、3d、5d、7d.5.Alizarin red dye:To observe the number of calcium nodus at14d、21d; the F-actin cytoskeleton were evaluated by CLSM observation at3h、6h、9h、12h.6. Real-time PCR:To detect the level of osteogenetic biomarkers Cbfal and OCN at3d、7d.7. Scanning electron microscope:To observe the cell morphology on the SLA titanium surfaces.[Result]1.MTT assay:At1d、3d、5d, a significant increase of absorbance were showed in group P than group D (P<0.05).2.ALP activity:At1d、3d、5d、7d, the absorbance of group P was significant higher than group D (p<0.05).3.Alizarin red dye:At14d、21d,the integrated absorbance of the calcium nodus in group P are significantly larger than group D (P<0.05).4.Cytoskeleton observation:With the time, the shape of the cytoskeleton from round to spindle and irregular polygon changes,meanwhile,F-actin become more spreaded, oriented paralled to another. At each time point, group P filaments were more well spread than group D.5. Realtime PCR:As the standardization in the group, the gene expression level of group D at3d were defined as1, at each time point, the Cbfal and OCN gene expression in group P are larger than group D (P<0.05).6.SEM observation:Over time and extend, short cell pseudopods gradually become long and grows wider, and also, the number of pseudopods were increased and interwoven into mesh, cells were rigid fixed on the SLA titanium surface, At each time point, group P filaments were more well spread than group D.[Conclusions]1.PRFe is useful in stimulating the proliferation and differentiation of osteoblasts;2.PRFe can significantly promote the spread of F-actin cytoskeleton.