Effect Of Liquid Phase Concentrated Growth Factors On Biological Behaviors Of Human Periodontal Ligament Cells

Background:Root conditioner and bio-modifiers are used to remove the smear layer formed on the roots because of making the root surfaces biologically acceptable,which has important significance for periodontal regeneration.Liquid phase concentrated growth factor(LPCGF)is a novel generation of platelet concentrate product after Platelet-Rich Plasma(PRP)and Platelet-Rich Fibrin(PRF)which is suggested to be a potent stimulator and a strong mitogenic agent for tissue.However,based on the present data,the effect of LPCGF as root bio-modifiers in the process of periodontal regeneration is not justified.Aim:This study aimed at assessing the effectiveness of LPCGF application on periodontal diseased root surfaces through adhesion,growth,migration and differentiation of periodontal ligament cells.Materials and methods:1.The hPDLSCs were isolated and cultured by enzyme digestion.The cell morphology was observed by inverted microscope.The cell clone formation rate and CCK-8 assay were used to evaluate the proliferation ability of hPDLSCs.Cell sources and surface markers were identified by cell immunocytochemistry and flow cytometry.Osteogenic,adipogenic and chondrogenic induction potentials of them were test.2.LPCGF and CGF gel were preparation from Collecting venous blood,then observation and contrast their character;Giemsa stain and SEM were used to observe structure of LPCGF;Cells were co-cultured with different concentrations of LPCGF in culture dish then assess cell skeleton and the ability of cell adhesion,proliferation,cell horizontal and vertical migration and cell differentiation.3.Dentin blocks were prepared from periodontitis teeth and made four groups: group I: control group;group II: 19%EDTA alone group;group III: 19%EDTA+20%LPCGF combine group;group IV: 20%LPCGF alone group.We measure the roughness and hydrophilicity of the surface of tooth block from different groups.Periodontal ligament cells were cultured on the above conditioned dentin blocks.Cell Counting Kit-8 assay were utilized to assess hPDLSCs adhesion and proliferation at.Migration of cells towards pre-treated dentine was assessed in a Transwell chamber assay.Expression of mineralization-associated genes(COL-1,ALP,BMP-2)in cells cultured on pre-treated dentine for 7 days was determined by RT-PCR.The samples were then subjected to routine preparation for SEM(With cells and cells free).4.The complex of cells-dentin blocks from four group were ectopicly transplanted into the subcutaneous tissue of nude mice after co-culture in vitro.After 8 weeks’ transplantation,samples were harvested.Routine fixation,decalcification,embedding and sectioning were performed.HE and Masson staining was used to observe the formation of periodontal tissues around dentin block.We analyzed the protein expression of COL-1 and BMP-2 by immunohistochemistry.Results:1.The primary cultures of hPDLSCs were clone-like growth.Most of them were short spindle and had a small volume.The colony formation rate showed that hPDLSCs was 8.5 %.The proliferation curve of them was in accordance with the logarithmic growth curve,showing a clear " S " shape.Immunocytochemistry showed that both cells positively expressed Vimentin,but not Cytokeratin.At the same time,they were positive for CD146,CD105 and CD90,while negative for CD35 and CD45.hPDLSCs had multipotent capability.2.The observation revealed that LPCGF is yellow and transparent or slightly turbid.The initial state of LPCGF is liquid.It well gradually translates into solidified or flocculent phase at room temperature or higher temperature.We observed a fibrin network constituted particularly by thin and thick fibrillary elements through Giemsa stain and SEM.Multiple blood cells such as red blood cells?platelets and white blood cells were trapped among the fibrin network.Adhesion experiments show that the control group(0%LPCGF)is less than that of the other five experimental group(P<0.01),while there was no statistically significant difference between other concentrations group(20%? 40% ? 60% ? 80% and 100% group).Different concentrations of LPCGF were demonstrated to have different capacity in enhancing the proliferation of hPDLSCs,and 20%CGF group was superior than other group(P<0.05)at day 7.We cannot found differences of cell morphological between the control group(0%LPCGF)and experimental group through phalloidine stain observation.The wound healing assay showed that 20%LPCGF group promote cell migration when compared to control group(0%LPCGF)in all time point(P<0.05).In Transwell vertical migration experiment,we found 20%LPCGF group cell make more cells migration than the control group in 24 h and 48 h two observation points(p<0.05).Immunofluorescence observations show that the 20%LPCGF group express more osteogenesis related protein(COL-1,BMP-2 and OPN)than the control group.Alizarin red staining and ALP staining results showed mineralization of 20%LPCGF group is better than that of control group.ALP activity staining presented similar results.3.Dentin block surface properties from four different group: Roughness of(EDTA+LPCGF)combined group and(LPCGF alone)group are higher than the control group and(EDTA alone)group.Hydrophilic experiment: hydrophilic of(EDTA alone)group is higher than the other three groups(p<0.05).Observation of tooth surface by Electron microscope,when compared with the control group,smear layer of(EDTA alone)group has been removed,and dentin holes exposed.Dentin block surface of(EDTA+LPCGF)group and(LPCGF alone)group are both covered by the fibrin network.(EDTA+LPCGF)group has more complete fibrin network than(LPCGF alone)group.It was found that combined groups showed significantly higher number of adhesion cells than that found in the control groups(p<0.05).Combined application of EDTA and LPCGF increased significantly hPDLSCs proliferation at day 5,7 and migration at hour24,48.Furthermore,combined groups showed significantly higher in up-regulated cell differentiation-related genes than control group.4.After transplantation 8 weeks,histological observation showed that periodontal ligament-like structures in all groups.Especially the EDTA+LPCGF group showed stronger ligament-like structures around dentin block surface.Furthermore,periodontal ligament fibers were organized neatly,there may be inserted fiber bundle of them in the cementum.Conclusions:Within the limitation of the study,we found LPCGF is a kind of high plasticity natural biological activity material for cells.EDTA and LPCGF application on the periodontitis-affected root surface seems to form a suitable surface for cells attachment,growth,migration and differentiation.Thus,as a root surface conditioner or surface biomodifier,LPCGF may have a promising role in clinical periodontics.Further studies to support these results are necessitated.