Wnt/β-catenin Signaling Pathway Mediates The Impaired Osteogenic Differentiation Of Periodontal Ligament Stem Cells In Inflammatory Microenvironment

Periodontitis is a chronic inflammatory disease affecting the tissues thatsurround and support the teeth, including gingival inflammation and progressiveresorption of the alveolar bone. Most of the therapies which involve rootplanning and curettage under the gum margins, even the guided tissueregeneration (GTR), could not achieve complete regeneration of periodontaltissues. Periodontal ligament stem cells (PDLSCs) are a new population ofmesenchymal stem cells (MSCs) which has been isolated from the periodontalligament. Owing to the potential to generate a cementum/PDL-like structure invivo, PDLSCs have brought new light for reconstruction of complex tissuesdestroyed by periodontitis. In chronic inflammatory bone diseases, such asperiodontitis, bone regeneration is inhibited, which may be because theinflammatory microenvironment affects the MSCs differentiation. However, theinhibitory molecules repressing bone formation of PDLSCs in inflammatorymicroenvironment still remain unclear.The Wnt/β-catenin signaling pathway plays an essential role in boneformation and homeostasis. In our previous study, we found high levels of β-catenin in P-PDLSCs, which may be implicated in the impaired osteogenicdifferentiation of P-PDLSCs. GSK3β is a key negative regulator of theWnt/β-catenin pathway, which mediates the phosphorylation and subsequentdegradation of β-catenin via the ubiquitin-proteasome pathway. Some studiesindicated that the activity of GSK3β was involved in many human diseases,including inflammation. Based on these observations, we speculated that theinflammatory microenvironment may inhibit PDLSCs differentiation throughthe phosphorylation of GSK3β and subsequent activation of Wnt/β-cateninpathway.The study consists of two parts:1. The study of the biological characteristics of PDLSCs from periodontitispatients and their healthy controls.Objectives: To investigate the effects of inflammatory microenvironment onproliferative ability and multipotential differentiation of PDLSCs. Methods:1)We isolated stem cells from the periodontal ligament tissues of periodontitispatients (P-PDLSCs) and healthy controls (H-PDLSCs). Limiting dilutiontechnique was used to obtain clonal purification.2) Cell phenotype wasanalyzed by flow cytometry (FCM).3) MTT viability assay, clone-formingability, cell cycle and apoptosis analysis were used to study the proliferativeability.4) Osteogenic and adipogenic induction were performed. The osteogenicability was investigated by Alizarin red staining, ALP staining and ALP activity.Quantitative Real-time PCR determined the mRNA expression levels ofosteoblast marker genes, including BSP, ALP, Runx2and OCN. Theadipogenic ability was detected by Oil red O staining.5) Lipopolysaccharides(LPS) was used as a stimulant to induce the synthesis of inflammatory cytokines.After a further24hours of culture, TNF-α, IL-1β and IL-6in the culturesupernatant of H-PDLSCs, P-PDLSCs, and LPS-stimulated H-PDLSCs were measured by enzyme-linked immunosorbent assay (ELISA). Results:1) Wesuccessfully isolated PDLSCs from periodontitis patients and healthy controls.Single colonies of PDLSCs were formed7–10days after the cells were plated atlow density. In general, the two types of cells presented a thin and longfibroblastic spindle morphology. Both H-PDLSCs and P-PDLSCs expressedstem cell markers of MSCs and the overall expression patterns were similarbetween two groups.2) MTT viability assay, clone-forming ability and cellcycle analysis showed that the proliferative ability of P-PDLSCs was strikinglyhigher than that of H-PDLSCs. There were no significant differences betweenH-PDLSCs and P-PDLSCs in the apoptosis analysis. However, after osteogenicdifferentiation, the apoptosis of P-PDLSCs was significantly increased.3)Alizarin red staining, ALP staining and ALP activity indicated that theosteogenic differentiation of P-PDLSCs was lowed than that of H-PDLSCs; andthe mRNA expression of osteoblast marker genes decreased significantly inP-PDLSCs compared to H-PDLSCs after osteogenic induction. The impairedadipogenic differentiation was confirmed by Oil red O staining.4) LPSincreased the secretion of TNF-α, IL-1β and IL-6by monocytes, especiallyTNF-α. However, H-PDLSCs and P-PDLSCs do not have spontaneousexpression of these cytokines, as well as H-PDLSCs treated with LPS. Theresults of Alizarin red staining, ALP staining and ALP activity revealed thatTNF-α, rather than IL-1β and IL-6, could inhibit the osteogenic differentiationof PDLSCs significantly. Conclusion:1) PDLSCs from periodontitis patientshad increased proliferative ability but impaired differentiation capacity.2)TNF-α was a key perpetrator which inhibited the osteogenic differentiation ofPDLSCs in inflammatory microenvironment.2. The study of molecular mechanisms which mediates the impairedosteogenic differentiation of PDLSCs in inflammatory microenvironment. Objectives: To investigate the inhibitory molecules repressing osteogenicdifferentiation of PDLSCs in inflammatory microenvironment. Methods:1) Theexpressions of GSK3β/p-GSK3β and β-catenin in H-PDLSCs and P-PDLSCswere examined by Western blot.2) We used TNF-α-treated H-PDLSCs as apositive control and utilized Western blot to detect the levels ofGSK3β/p-GSK3β and β-catenin both in total and nuclear proteins.3) To studyβ-catenin/TCF transcriptional activity, we transfected H-PDLSCs withTOPFlash and FOPFlash. To normalize transfection efficiency, cells wereco-transfected with internal control reporter Renilla luciferase plasmid.24hoursafter transfection, luciferase assay was performed, using the Dual LuciferaseAssay System kit.4) We applied human recombinant Wnt3a and lithiumchloride (LiCl) during osteogenesis and used Alizarin red staining and ALPstaining to investigate the osteogenic differentiation of H-PDLSCs.5) To furtherconfirm the role of β-catenin in the osteogenic differentiation of PDLSCs, anRNA interference technique was used to suppress β-catenin levels in PDLSCs.Results:1) We found more strikingly enhanced p-GSK3β and β-catenin inP-PDLSCs than H-PDLSCs in the absence of changes in total GSK3β. Afterosteogenic differentiation, the expressions of p-GSK3β and β-catenin weredown-regulated in both two types of PDLSCs. In the TNF-α group, there was anincreased TOPFlash/FOPFlash activity ratio compared with the control group.2)After TNF-α-stimulation, the p-GSK3β levels in H-PDLSCs were increased indose-and time-dependent manners, while the GSK3β levels had no significantchange; and the expressions of β-catenin in both the total and nuclear proteins ofH-PDLSCs were increased with p-GSK3β.3) Both the ALP staining andAlizarin red staining showed that the osteogenic differentiation of PDLSCs wasdecreased significantly after LiCl or Wnt3a stimulation, which had the sameeffect as TNF-α.4) β-catenin mRNA levels were quantified in PDLSCs transfected with control (siCont) or β-catenin oligo (siβ-catenin) for48h byreal-time RT-PCR, and β-catenin protein levels were detected72h aftertransfection by Western blot to confirm RNAi effectiveness. ALP stainingrevealed that in the siCont group, TNF-α exposure striking decreased theosteogenic differentiation of PDLSCs, while in the siβ-catenin group, nostatistical difference was found between the osteogenic group and the TNF-αgroup. Conclusion: Inflammatory microenvironments could induce thephosphorylation of GSK3β and subsequent activation of Wnt/β-catenin pathway,which mediated the impaired osteogenic differentiation of PDLSCs.