Effects Of QK Combined With BFP-1 Polypeptide On The Proliferation And Osteogenic Differentiation Of Hpdlcs

Objective:In vitro,VEGF mimetic peptide(QK)and bone forming peptide-1(BFP-1)were applied to human periodontal ligament cells(hPDLCs)in different ways and evaluated the effects of their proliferation and osteogenic differentiation,in order to optimize growth factors and achieve better periodontal regeneration.Method:1.Primary hPDLCs were cultured with tissue block enzyme digestion method.Optical microscope were used to observe the cell morphology and colony formation.The stem cells surface markers(CD34,CD73,CD146 and STRO-1)were identified by flow cytometry.After directional induction of hPDLCs,alizarin red,alcian blue and oil red staining were performed to verify the multiple differentiation ability.The third passages of hPDLCs were used for subsequent experiment.2.The experiment was divided into 8 groups:(1)Control group;(2)QK(2?g/ml)group;(3)BFP-1(10?g/ml)group;(4)BFP-1(100?g/ml)group;(5)QK(2?g/ml)+ BFP-1(10?g/ml)group;(6)QK(5?g/ml)+ BFP-1(10?g/ml)group;(7)QK(10?g/ml)+ BFP-1(10?g/ml)group;(8)QK(50?g/ml)+ BFP-1(10?g/ml)group.3.The effect of each experimental group on the proliferation of hPDLCs was detected by CCK-8 at 1,3,5 and 7 days.4.The osteogenic induction of hPDLCs was grouped according to the above mentioned.After 7 days of culture in vitro,alkaline phosphatase(ALP)activity was measured and the expression levels of ALP,Runx2,and OCN mRNA were analyzed using real-time polymerase chain reaction(RT-PCR).ALP staining were performed after 14 days of culture.Alizarin Red staining and semiquantitative detection the formation of mineralized nodule after 21 days of culture.Result:1.Primary cultured periodontal ligament cells adherently grew in a typical fibroblast-like appearance.Flow cytometry showed that the expression of mesenchymal stem cell specific surface antigens was CD73: 99.99%,CD146: 85.01%,Strol-1: 1.03%;Hematopoietic cell surface antigen expression: CD34: 0.71%.After hPDLCs were were induced by osteogenesis,chondrogenesis and adipogenesis in vitro,alizarin red staining,alician blue staining and oil red O staining were positive.2.The results of CCK8 showed that compared with control group,the groups of BFP-1(10?g/ml),BFP-1(100?g/ml),QK(2?g/ml)+ BFP-1(10?g/ml),QK(5?g/ml)+ BFP-1(10?g/ml)and QK(10?g/ml)+ BFP-1(10?g/ml)could enhance the proliferative activity of hPDLCs(P <0.05).There was no significant difference in the proliferative activity of hPDLCs between all groups after 3 days and 5 days(P > 0.05).After 7 days of culture,the proliferative activity of hPDLCs was lower in the QK(50 ?g/ml)+ BFP-1(10?g/ml)group than in the control group(P <0.05).3.After 7 days of osteogenic induction,the ALP activity in BFP-1(100?g/ml)group and QK(5?g/ml)+ BFP-1(10?g/ml)group were significantly higher than that in control group(P<0.05).There was no significant difference in ALP activity between BFP-1(100?g/ml)group and QK(5?g/ml)+ BFP-1(10?g/ml)group(P> 0.05).The ALP activity in QK(5?g/ml)+BFP-1(10?g/ml)group was significantly higher than that in QK(50?g/ml)+ BFP-1(10?g/ml)group(P<0.01).RT-PCR results showed that ALP,Runx2 and OCN gene expression levels were significantly higher in BFP-1(100?g/ml)group and QK(5?g/ml)+ BFP-1(100?g/ml)group(P <0.001),no significant difference between the two groups.In addition,the expression of Runx2 and OCN gene in QK(2?g/ml)+ BFP-1(10?g/ml)group was also significantly higher than that in control group(P <0.05).After 14 d of culture,the ALP staining in the BFP-1(100?g/ml)group and QK(5?g/ml)+ BFP-1(10?g/ml)group was deeper than in the other groups,indicating the BFP-1(100?g/ml)group and QK(5?g/ml)+ BFP-1(10?g/ml)group had higher ALP activity than the other groups.The results of alizarin red staining(21 days)showed BFP-1(10?g/ml)group,BFP-1(100 ?g/ml)group,QK(2?g/ml)+ BFP-1(10?g/ml)group and QK(5?g/ml)+ BFP-1(10?g/ml)group could significantly promote the formation of mineralized nodules in hPDLCs(P<0.05),of which BFP-1(100?g/ml)group and QK(5?g/ml)+ BFP-1(10 ?g/ml)group was most effective in promoting mineralized nodules.On the other hand,the mineralized nodule in the QK(50?g/ml)+BFP-1(10?g/ml)group was significantly decreased compared with the control group(P<0.01).Conclusion:1.In this experiment,mesenchymal derived hPDLCs were successfully cultured,which had strong proliferation ability and multiple differentiation potential.It can be used for subsequent experimental studies.2.BFP-1 alone or the combination of BFP-1 with QK can promote the proliferation of hPDLCs in the early stage.When the ratio of QK/BFP-1 increased,the proliferation ability of hPDLCs decreased.3.The combined use of QK and BFP-1 showed a synergetic effect on osteogenic differentiation of hPDLCs.