Mechanism Of Action Of The Wnt Signaling Pathway In The BMP9-induced Osteogenic Differentiation Of Dental Follicle Stem Cells Under Inflammatory Microenvironment

Periodontitis is a chronic infectious disease with bacteria as the initial factors,which may induce tooth loosening and even loss in adults.Conventionally,periodontitis is treated through basis treatments?such as supragingival and subgingival scaling,and root planning?,ancillary drugs and laser therapy targeting local pathogenic factors,thus controlling the inflammation in clinic.In some cases,surgical methods like guiding bone/tissue regeneration are employed to restore the periodontal structure.However,it is discovered in clinic that,extremely limited periodontal regeneration can be achieved from these methods.Therefore,making great efforts to obtain the satisfactory periodontal bone defect repair and reconstruction of periodontal structure and function is the objective of current massive studies.In recent years,the tissue engineering technology has developed rapidly,which is gradually applied in oral disease research.Meanwhile,the combination of stem cells,growth factors and 3-dimensional support materials have provided a novel possibility for the regeneration of periodontal soft and hard tissues.Dental follicle stem cells?DFCs?derive from the loose connective tissues wrapping around the enamel organ and dental papilla,which eventually develop into cementum,periodontal ligament and alveolar bone.Research has verified that DFCs have strong self-renewability,as well as multi-lineage differentiation capacities in vivo and in vitro.Moreover,scholars have successfully induced the differentiation of DFCs into osteoblasts,neuroblasts and lipoblasts.As the precursor cells of periodontal tissue,DFCs show certain advantage as the periodontal tissue engineering stem cells.Therefore,intensive research on DFCs will certainly provide more possibilities for the breakthrough of technologies like clinical stem cell transplantation.Bone morphogenetic proteins?BMPs?are the growth factor family regulating skeletal development and remodeling.Among the 14 BMPs with pro-osteogenetic potentials,bone morphogenetic protein 9?BMP9?has the strongest osteogenesis inducing effect,which can greatly enhance the osteogenetic effect when being applied in periodontal tissue engineering as the biotic factors for periodontal bone regeneration.The periodontal tissues in periodontitis patients are in the inflammatory microenvironment,while inflammation will induce a series of changes of tissues,thus depriving them of the normal regeneration and repair capacities.As a result,only through investigating stem cell proliferation and differentiation in the inflammatory microenvironment can we truly understand its functional changes under the environment closer to the reality.Tumor necrosis factor-??TNF-??is one of the most important pro-inflammatory cytokines of periodontitis,which can result in alveolar resorption and collagenous fiber destruction.Consequently,TNF-?plays a vital role in the inflammatory changes of the periodontal microenvironment.In addition,our previous experiment confirmed that BMP9 could induce the osteogenetic differentiation of rat dental follicle stem cells?rDFCs?.However,the effects of inflammatory microenvironment on the BMP9-induced osteogenetic differentiation of rDFCs remain unclear at present.The stem cell differentiation process is under the complicated signaling pathway regulatory network.Nevertheless,the signaling regulatory mechanism involved in the osteogenetic differentiation of r DFCs under inflammatory microenvironment has not yet been illustrated,which has hindered the development of new technologies like applying tissue engineering technology in treating the periodontitis-induced bone defect.Additionally,research also proved that the Wnt signaling pathway was indispensable in bone development and bone regeneration.However,some research indicated that the Wnt signaling pathway played distinct regulatory roles in different stem cells under various environments.Currently,reports regarding the role of the Wnt signaling pathway in the BMP9-induced osteogenetic differentiation of rDFCS under inflammatory environment are not available.Thus,the current study was thereby carried out aiming to preliminarily investigate such problem.Objectives1.rDFCs cultured in vitro were harvested and purified to construct the rDFCs cell model infected with recombinant adenoviruses expressing BMP9?AdBMP9?.2.To explore the effects of the TNF-?-constructed inflammatory microenvironment on the BMP9-induced osteogenetic differentiation of rDFCs.3.To discuss the role of the Wnt signaling pathway in the BMP9-induced osteogenetic differentiation of rDFCs under inflammatory microenvironment.MethodsPart I:Dental follicle of mandibular molar was obtained from the 6-day-old SD rats,and the primary rDFCs were obtained through enzyme digestion and tissue explant and purified using differential passage.The purified third-generation rDFCs were infected with AdBMP9,and green fluorescence expression was observed at 24 h and 48 h.Part II:rDFCs were stimulated with TNF-?at various concentrations?0,2.5,5,10 and 50 ng/ml?,and rDFCs proliferative capacity was observed on 1 d,3d,5 d and 7 d,respectively,using CCK-8,so as to select the suitable TNF-?concentration as the stimulation concentration in subsequent experiments.To explore the inflammatory characteristics of rDFCs after stimulation upon inflammatory factors,rDFCs were stimulated with TNF-??10 ng/mL?.Afterwards,TNF-?secretion in rDFCs group,rDFCs+AdBMP9 group,rDFCs+TNF-?group,and r DFCs+AdBMP9+TNF-?group was detected using ELISA after 24 h of intervention followed by 72 h of conventional culture.To determine the effects of TNF-?-constructed inflammatory microenvironment on the BMP9-induced osteogenetic differentiation of r DFCs,osteogenetic induction was conducted in cells in rDFCs group,rDFCs+AdBMP9 group,rDFCs+TNF-?group,and rDFCs+AdBMP9+TNF-?group,respectively.Early osteogenesis was observed through alkaline phosphatase?ALP?staining after 7 days of osteogenetic induction,whereas advanced osteogenesis was observed with alizarin red S staining after 14 days of osteogenetic induction to detect calcium deposition.Finally,the expression levels of cell osteogenetic genes Runt-related transcription factor 2?RUNX2?,osteocalcin?OCN?,type 1collagen?COL-1?and ALP were detected using real-time qPCR?RT-PCR?.Part III:To investigate the role of the Wnt signaling pathway in the BMP9-induced osteogenetic differentiation of rDFCs under inflammatory microenvironment,the total proteins were extracted from rDFCs group,rDFCs+AdBMP9 group,rDFCs+TNF-?group,and rDFCs+AdBMP9+TNF-?group,respectively,after 7 days of osteogenetic induction.The expression levels of the Wnt canonical signaling pathway key protein?-catenin,glycogen synthase kinase-3??GSK-3??,the non-canonical signaling pathway key factor Ca²⁺/calmodulin-dependent protein kinase II?CaMKII?,and nemo-like kinase?NLK?were detected through Western blotting.In the meantime,expression of the downstream target gene of the Wnt canonical signaling pathway Cyclin D1 was determined through RT-qPCR.ResultsPart I:Construction of the Ad BMP9-infected rDFCs cell model1.The third-generation rDFCs successfully harvested and purified were dominated by the spindle shape.rDFCs had been successfully identified in the previous experiment of our research group.2.rDFCs could stably express the green fluorescence after infection with AdBMP9.Part II:Exploration of the effects of the TNF-?-constructed inflammatory microenvironment on the BMP9-induced osteogenetic differentiation of rDFCs.1.High concentration TNF-??50 ng/ml?could reduce the proliferative activity of rDFCs in a time-dependent manner.As a result,10 ng/ml was selected as the stimulation concentration to guarantee cell vitality.ELISA results indicated that,compared with the control group without inflammatory factor stimulation,the TNF-?secretion and synthesis amounts in rDFCs of the TNF-??10 ng/ml?group were increased.2.Compared with the control group without AdBMP9 induction,the AdBMP9 induction group had darker ALP staining,more calcium deposition,as well as higher expression levels of osteogenetic factors RUNX2,OCN,COL-1 and ALP.However,compared with the control group without TNF-?stimulation,the TNF-?stimulation group had lighter ALP staining,less calcium deposition,as well as lower expression levels of RUNX2,OCN,COL-1 and ALP.Part III:Exploration of the role of the Wnt signaling pathway in the BMP9-induced osteogenetic differentiation of rDFCs under inflammatory microenvironment.1.Western blotting results suggested that,the TNF-?-simulated inflammatory microenvironment would result in up-regulated?-catenin expression level,while the expression quantity of GSK3-?that could degrade?-catenin was lower than that in the control group without inflammatory factor stimulation.In addition,RT-qPCR results indicated that,the expression level of the downstream target gene of the Wnt canonical signaling pathway Cyclin D1 was increased.2.The expression quantities of the key proteins in the Wnt non-canonical signaling pathway CaMKII and NLK were lower in the TNF-?stimulation group than in the non-stimulation control group.ConclusionsTNF-?-stimulated rDFCs display inflammatory characteristics.The simulated inflammatory environment has inhibited the BMP9-induced osteogenetic differentiation of rDFCs.In addition,the Wnt canonical signaling pathway is activated during this process,while the non-canonical signaling pathway is weakened,which may be one of the key causes leading to the reduced osteogenetic capacity of rDFCs.These findings reveal that the Wnt/?-catenin pathway and Wnt/Ca²⁺pathway play respective important roles during the osteogenetic differentiation of rDFCs,and destruction of their dynamic balance will affect stem cell function.This has provided theoretical foundation for restoring stem cell function through regulating the Wnt signaling pathway in subsequent experiments.