The Effect Of Concentrated Growth Factors On Proliferation And Osteogenic Differentiation Of Human Periodontal Ligament Cells In Vitro

The ultimate aim of treatments for periodontal diseases is to make periodontal tissues which have been damaged regenerate.Periodontal ligament is a part of periodontal supporting tissues,which includes periodontal ligament cells(PDLCs).And periodontal ligament cell is a heterogeneous cell population which consists of several kinds of cells,including osteoblasts,osteoclasts,fibroblasts and undifferentiated mesenchymal cells.Periodontal ligament cells are considered to have the potential of proliferation and multilineage differentiation.The self-renewal capability of PDLCs plays an important role in the regeneration of periodontal tissues.Platelet-concentrated products,which produced from whole blood,act as one of the main source of autologous derived growth factors and are widely used in clinical therapies and experimental studies.Among many kings of platelet-concentrated products,platelet-rich plasma(PRP)and concentrated growth factor(CGF)are frequently used.As a kind of autologous platelet-concentrated products,PRP and CGF,riched in growth factors,can promote the aggregation,proliferation and differentiation of cells and further accelerate the bio-synthesis of extracellular matrix during wound healing,and may regulate the process of wound healing.To date,many studies have been carried out on PRP and PRP has also been used for clinical therapy for a long time.However,few experimental studies and clinical applications are related to CGF.Therefore,this artical focuses on the influence of PRP and CGF on the proliferation and osteogenic differentiation of hPDLCs.Part Ⅰ:Comparative studies of growth factors in whole blood,PRPand CGF.AimComparative study on the levels of growth factors:transforming growth factor-β1(TGF-β1),vascular endothelial growth factor(VEGF)and platelet-derived growth factor(PDGF)in whole blood,PRP and CGF.MethodEight young adults were recruited as volunteers for the test.25 mL cubital vein blood was collected from each volunteer.Then the blood samples were divided into three groups:(1)the whole blood group:5 mL blood added EDTA blood anticoagulation,(2)the PRP group:10 mL blood added EDTA blood anticoagulation,and(3)the CGF group:10 mL blood without any blood anticoagulation.Platelet count was carried out on each group respectively and ELISA test was adopted to detect the levels of TGF-β,VEGF and PDGF.Result1.Compared to whole blood,platelet count of PRP was more than six times over that of whole blood,and platelet count result of CGF was negative.2.Compared to whole blood,the levels of growth factors TGF-β1,VEGF and PDGF in PRP increased significantly(P<0.05);on the other hand,compared to whole blood,the levels of growth factor TGF-β1 and PDGF in CGF were slightly higher while the levels of VEGF decreased,but there was not significant difference.ConclusionCompared to whole blood,levels of growth factors:TGF-β1 and PDGF in PRP and CGF increased,the level of VEGF in PRP inceresed and which in CGF decreased.Especially,levels of growth factors in PRP increased significantly(P<0.05),and levels of growth factors in CGF increased without significant difference.Part Ⅱ:Culture human periodontal ligament cells in vitro and researches on the proliferation and differentiation potential of hPDLCs.AimTo master the isolation and culture method of human periodontal ligament cells in vitro and study their potential of proliferation and multilineage differentiation.Method1.Healthy third molars were collected(no caries,no periodontitis and no periapical periodontitis),and the combination method of enzymatic digestion and tissue block method was used to isolate and culture human periodontal ligament cells.When it came to the second generation,imunocytochemical staining cytokeratin and vimentin was conducted to identify the sources of human periodontal ligament cells.2.The fourth to the sixth generation of human periodontal ligament cells were selected to carry out the sbusequent experiments.Human periodontal ligament cells were inoculated with a density of 1 ×105/mL and 0.8 × 105/mL,methods of MMT and CCK-8 were adopted to study the proliferation capacity of human periodontal ligament cells.3.Human periodontal ligament cells were selected and carried out to induce mineralization and fat,and then they were stained with Alizarin Red and oil red O,respectively.The experiment were actually conducted to research the multi-directional differentiation potential of human periodontal ligament cells.Result1.Under microscopy,human periodontal ligament cells exhibited fusiform morphology,which were of plump body,uniform cytoplasm,and arranged spirally and palisadly.2.Immunocytochemical staining revealed positive results for vimentin and negative results for cytokeratin.3.Results draw from methods of MTT and CCK-8 showed that human periodontal ligament cells grew in a substantially S-shaped growth curve.4.Alizarin Red staining and oil red O staining of human periodontal liganment cells could observe mineralization nodules and lipid droplets after induction.ConclutionHuman periodontal ligament cells could be isolated and cultured in vitro by the method of combining enzymatic digestion and tissue block,and they have the potential for proliferation and multilineage differentiation.Part Ⅲ:The influence of PRP and CGF on the proliferation and osteogenic differentiation of hPDLCs.AimTo research the influence of PRP and CGF on the proliferation and osteogenic differentiation of hPDLCs.MethodHuman periodontal ligament cells of the fourth to the sixth generation were selected for the subsequent experiments.1.Cells were inoculated with a density of 1 × 105/mL into basic medium(DMEM medium containing 10%FBS),and cultured in the cultere dishes for 24h.Then a circle area with a diameter of 8mm without any cells was made in each culture dish.The experinment was divided into three groups:(1)Con group:cells cultured with basic medium,(2)PRP group:cells cultured with basic medium containing 5%PRP,and(3)CGF group:cells cultured with basic medium containing 5%CGF.At the first,fourth,seventh,tenth and thirteenth day after above step,one of each group was picked up to be stained with crystal violet solution for the detection of influence on human periodontal ligament cells proliferation caused by PRP and CGF.2.Cells were inoculated with a density of 1×105/mL into basic medium for 24h,and then changed to cultured with DMEM without FBS for 24h.And then the experinment was divided into three groups:(1)Control group:cells cultured with DMEM medium without FBS,(2)PRP groups:DMEM medium with PRP(1%,5%,10%),and(3)CGF groups:DMEM medium with CGF(1%,5%,10%)for different time(24h,48h,72h).Extracted total cellular protein in samples of 24h,48h,72h.Western blot was used to detect the expression of of osteogenesis-related transcription factors,including Runx2,Osx,DIx5 and Msx2.Result1.Compared with the Control group,PRP and CGF promoted the proliferation of human periodontal ligament cells.2,Western blot showed that when hPDLCs were cultured with the same concentration of PRP or CGF and stimulated with different stimulation of time,the protein expression of Runx2,Osx and DIx5 increased with increasing stimulation time in a time-dependent manner,and the propein expression peaked at 72h;Msx2 protein expressed relatively highest amount at the time of 24h.On the other hand,different concentrations of PRP,CGF stimulated hPDLCs with same time,the protein expression of Runx2,Osx and DIx5 increased with PRP and CGF concentration,protein expression reached highest at the concentration of 10%;and the protein expression of Msx2 reduced when PRP and CGF stimulating concentration increased.ConclutionPRP and CGF can promote the proliferation and osteogenic differentiation of human periodontal ligament cells,promoting the protein expression of Runx2,Osx and DIx5 and reducing the protein expression of Msx2.