Sequential Application Of BFGF And BMP-2 Facilitates Osteogenic Differentiation Of Human Periodontal Ligament Stem Cells In Vitro

ObjectivesPeriodontitis is a common chronic inflammatory disease characterized by,the progressive destruction of periodontal support tissues.Current treatment strategies can only control the progression of the disease,but cann't achieve complete regeneration of the damaged periodontal tissue.Recruitment enough functional cells to the periodontal defect regions and promotion of their committed differentiation is expected to become a new strategy for periodontal tissue regeneration according to the principle of in sirtu tissue engineering.As one of the most potential growth factors in in situ tissue engineering,our previous studies found that basic fibroblast growth factor(bFGF)could promote proliferation and migration of a variety of mesenchymal stem cells(MSCs).Bone morphogenetic protein-2(BMP-2)has great osteogenic potential.The current application of bFGF and BMP-2 tends to release these two factors at the same time,which is not appropriate for the real state of tissue regeneration.Therefore,the sequential application of bFGF and BMP-2 will simulate the real state of tissue regeneration.engraft enough functional cells by bFGF in the early stage of wound healing and promote their committed osteogenesis differentiation by BMP-2 at the late stage.In this present study,sequential application system of bFGF and BMP-2 in vitro was constructed and investigate the effects on the proliferation,migration and osteogenic differentiation of human periodontal ligament stem cells(PDLSCs).The results will provide a new strategy for periodontal tissue regeneration.MethodsThe healthy premolars that extracted due to orthodontics were collected and human PDLSCs were isolated and cultured by limiting dilution method.The sternness was examined by colony formation assay and the expression of surface markers for human PDLSCs were quantified by flow cytometry.The optimal concentration of bFGF on human PDLSCs was determined by cell counting kit(CCK8)and transwell assay.The optimal concentration of BMP-2 on human PDLSCs was detected and the sequential application system of bFGF and BMP-2 was initially constructed by alkaline phosphatase(ALP)activity assay.The effects of different sequential application systems on ALP activity,calcium deposition,osteogenic related gene and protein expression of human PDLSCs were determined by Alizarin red staining,cetylpyridinium chloride(CPC)colorimetric method,quantitative real-time polymerase chain reaction(qRT-PCR)and western blot analysis.The experimental data were processed by GraphPad Prism 6.0 software package.The data between groups were compared by one-way ANOVA and the results were expressed as mean±standard deviation(SD).Statistical probability of P<0.05 was considered significant.Results1.Isolation and identification of human PDLSCs:Human PDLSCs were successfully isolated and cultured by limiting dilution method.The primary cells which cultured in vitro had typical characteristics of fibroblasts and good morphology.The results of colony formation experiments indicated that human PDLSCs had the colony-forming abilities.By flow cytometry analyses,human PDLSCs were positive for MSCs associated makers CD29,CD44 and CD73,and were negative for haematopoietic stem cell markers CD45.2.bFGF promoted the proliferation of human PDLSCs:The results of CCK8 showed that 50 ng/mL bFGF had a significant promotive effect on the proliferation of human PDLSCs compared with the control group(P<0.001).Transwell assay indicated that each concentration of bFGF enhanced the migration capacity of human PDLSCs compared with control group(P<0.001)and 50 ng/mL bFGF exhibited the maximal chemotactic capability(P<0.001).3.BMP-2 enhanced ALP activity of human PDLSCs:ALP activity results showed that 50 ng/ml BMP-2 significantly up-regulated ALP activity of human PDLSCs compared with osteogenic induction group(P<0.001).4.The sequential application of bFGF and BMP-2 promoted the osteogenic differentiation of human PDLSCs:bFGF inhibited ALP activity of human PDLSCs in a time-dependent manner compared with osteogenic induction group(P<0.001).The best sequential application modality of these two methods was 25 ng/mL bFGF pretreated for the first 3 days and BMP-2 cultured for the following 9 days,which could significantly increase ALP activity of human PDLSCs(P<0.001).Alizarin red staining and CPC colorimetric method results showed that 25 ng/mL bFGF pretreated for the first 3 days and BMP-2 cultured for the following 18/25 days could maximally promote the formation of mineralized nodules in human PDLSCs(P<0.001).qRT-PCR results showed this sequential application modality could significantly up-regulate the expression of gene expression of osteogenesis-related markers such as ALP,runt-related transcription factors 2(Runx2),osteopontin(OPN),bone sialoprotein(BSP)and osteocalcin(OCN)(P<0.05).Western blot analysis showed that 25 ng/mL bFGF pretreated for the first 3 days and BMP-2 cultured for the following 18/25 days could significantly enhance protein expression of osteogenesis-related markers such as ALP and Runx2(P<0.001).Conclusions1.Human PDLSCs were successfully isolated and cultured by limiting dilution method.2.50 ng/mL bFGF significantly promoted the proliferation and migration of human PDLSCs but inhibited ALP activity of human PDLSCs in a time-dependent manner.50 ng/ml BMP-2 significantly enhanced ALP activity of human PDLSCs.3.The sequential application of bFGF and BMP-2 promoted osteogenic differentiation of human PDLSCs by up-regulating ALP activity,calcium deposition,gene and protein expression of osteogenesis-related markers.Results indicated that sequential application of these two growth factors could engraft sufficient functional cells by 25 ng/mL bFGF pretreated for the first 3 days and promote their committed osteogenesis differentiation by 50 ng/mL BMP-2 for the following days.Therefore,this approach had a good application prospect.