The Pilot Study Of The Effect Of Hedgehog Signaling Pathway In The Osteogenic Differentiation Of Human Periodontal Ligament Stem Cells Under Cyclic Tension

Orthodontic teeth movement occurs when the appropriate forces are applied to theteeth by orthodontic appliances, which is mediated by coupling alveolar bone resorption inpressure side and deposition in tension side of the periodontal ligament, respectively.Recently, periodontal ligament cells have been found to potentially differentiate intoosteoblast-like cells and influence the deposition process of tension side of periodontaltissues under mechanical strain conditions, which plays a critical role in the process of teethmovement. However, this osteogenic process under mechanical strain is complex andinvolved in a variety of pathways including bone morphogenetic protein(BMP), Wnt, andHedgehog(Hh) signaling with interaction and the molecular mechanism has remainedunclear.The Hh gene has been considered as a key regulator in the proliferation anddifferentiation of stem cells and progenitor cells. The Hh genes are essential highlyconserved in Drosophila melanogaster and mammals. In mammals, the hedgehog geneshave at least three homologs: Sonic hedgehog(Shh)，Indian hedgehog(Ihh) and Deserthedgehog(Dhh). Recently, the Hh pathway has been shown to play an important role ininducing the mesenchymal cells to differentiate into osteoblasts.In our previous experiment, we successfully separated and identified humanperiodontal ligament stem cells (PDLSC) and confirmed that PDLSC differentiated intoosteoblast under cyclic tension. However, the molecular mechanism is still unknown. Sincethe Hh signaling pathway has been shown to associate closely with the osteogenicdifferentiation in many cells, we hypothesized that the Hh signaling pathway plays animportant role in the osteogenic differentiation of human PDLSC under cyclic tension.Purmorphamine (the Hh signaling pathway agonist) and cyclopamine (the Hh signalingpathway antagonist) were applied to change the expression of Hh to detect its role in the osteogenic differentiation of human PDLSC. These results will provide an avenue toestablish a new experimental model for future insight into elucidating the molecularmechanism of orthodontic teeth movement. A better understanding of the cellular andmolecular responses of periodontal cells is a prerequisite for further improvements oftherapeutic approaches in orthodontics。ObjectiveTo examine the role of Hedgehog signaling pathway in the osteogenic differentiationof human periodontal ligament stem cells under cyclic tension, and to explore theosteogenic regulatory mechanism of PDLSC under mechanical stress.Methods1Isolation and identification of human PDLSC: premolars extracted for orthodonticreasons and impacted third molars with good localization and general healthy conditionwere collected from patients aged12-24years, at the Department of Stomatology ofSouthwest Hospital, the Third Military Medical University. Informed consent was obtainedbefore extraction. Teeth were immediately washed by phosphate buffered saline (PBS)supplemented with300units/mL penicillin and300μg/mL streptomycin repeatedly for atleast fifteen minutes in a clean bench. Periodontal ligament (PDL) tissues were then gentlyscraped from the middle third of the root with a scalpel, and then digested with collagenasetype I at37℃for about forty minutes. After centrifugation, the tissue explants wereplated onto dishes with alpha minimal essential medium (α-MEM) with10%FBS forprimary culture incubated at37℃in a5%CO2incubator. When the primary PDL cells wereobtained, single-cell suspensions were prepared and seeded into96-well plates. Then thecells in the sole colony were selected to passage. By colony-forming assay,immunofluorescence for vimentin and pan-cytokeratin, flow cytometry for themesenchymal stem cell surface markers STRO-1and CD146, osteogenic and adipogenicinduction were used to identify PDLSC.2To evaluate whether Hh signaling pathway is involved in the osteogenicdifferentiation of PDLSC under cyclic tension, tensile strains (12%-0-12%equiaxialstretching,5sec stretch/5sec relaxation) were applied to PDLSC in osteogenic inductioncondition with Flexcell FX-4000T Tension Plus System for24h. PDLSC were subcultured into six-well BioFlex plates especially with sinica membrane bottom coated with type Icollagen. Control cells were cultured into BioFlex plates just in the incubator statically.Then the mRNA expressions of Runx2, ALP, GLI1, PTCH1and SMO were detected byreal-time PCR, respectively.3In order to confirm the positive regulatory role of the Hh signaling pathway in theosteogenic differentiation of PDLSC under cyclic tention, tensile strains (12%-0-12%equiaxial stretching,5sec stretch/5sec relaxation) were applied to PDLSC in osteogenicinduction conditions with Flexcell FX-4000T Tension Plus System for24h. PDLSC weresubcultured into six-well flexible-bottomed plates added with Hh pathway agonistpurmorphamine (1μM,2μM,4μM). Control cells were cultured in osteogenic inductionconditions added DMSO of10-2M. Then the mRNA expressions of Runx2, ALP, GLI1,PTCH1and SMO were detected by real-time PCR, respectively.4In order to further confirm the positive regulatory role of the Hh signaling pathwayin the osteogenic differentiation of PDLSC under cyclic tention, tensile strains (12%-0-12%equiaxial stretching,5sec stretch/5sec relaxation) were applied to PDLSC in osteogenicinduction conditions with Flexcell FX-4000T Tension Plus System for24h. PDLSC weresubcultured into six-well flexible-bottomed plates added with Hh pathway antagonistcyclopamine(5μM). Control cells in osteogenic induction conditions were added DMSO of10-2M. Then the mRNA expressions of Runx2, ALP, GLI1, PTCH1and SMO weredetected by real-time PCR, respectively.Results1By using single-cloning methods, we successfully obtained PDLSC which mostlyshowed the classic long spindle shape similar to the PDLC but a little smaller in size,arranged in swirling or radial with high colony-forming efficiency by13.87%.Immunofluorescence staining showed that PDLSC were positive for vimentin and negativefor pan-cytokeratin. Flow cytometry revealed that the PDLSC were positive formesenchymal stem cell markers STRO-1(89.9%) and CD146(91.3%). Alizarin redstaining had calcium deposits and oil red O staining showed lipid droplets under the lightmicroscope.2Runx2, ALP, GLI1, PTCH1and SMO mRNA expressions were up-regulated in thetwo groups (P＜0.05) after24h. The group under cyclic tension showed significantly higher expression (P＜0.05) than the control. The results indicated that the Hh signalingpathway was activated in the osteogenic differentiation process under tension.3The mRNA expressions of GLI1, PTCH1and SMO were up-regulated in the threegroups added different concentration purmorphamine after24h (P＜0.05), and the mRNAexpressions of Runx2, ALP were also enhanced in a dose-dependent manner(P＜0.05).So for the first time we proved that Hh signaling pathway had a positive effect on theosteogenic differentiation under tension.4To further confirm the positive regulation of Hh signaling pathway on PDLSC undertension, we used the Hh signaling antagonist cyclopamine to inhibit the activity of Hhsignaling. After using quantitative cyclopamine, the mRNA expressions of GLI1,PTCH1and SMO were a little increased, and the expressions of Runx2and ALP were alsoslightly increased, but lower than the control group. The results confirmed that the Hhsignaling pathway played a positive role in the osteogenic differentiation of PDLSC undertension.Conclusions1It is still a bottleneck to obtain sufficient seed PDLSC for our cyclic tensionexperiment, however, limiting dilution single-cloning assay has been proved to be a simple,effective method to isolate PDLSC, which showed the self-renewal and multi-lineagedifferentiation properties in vitro.2The present results showed that the Hh signaling pathway was activated for the firsttime in the osteogenic differentiation process of PDLSC under cyclic tension. When the Hhsignaling pathway was activated, the osteogenic differentiation was strengthened, and theosteogenic differentiation was weakened while the Hh signaling pathway was inhibited. Soall the results in our experiment confirmed that the Hh signaling pathway was activated andhad a positive effect on the osteogenic differentiation process of PDLSC under cyclictension.In our previous experiment, we successfully separated and characterized PDLSC andconfirmed that PDLSC differentiated into osteoblast under cyclic tension. This finding laida solid platform for the study of the molecular mechanism of the osteogenic source formalocclusion. The purpose of the present experiment was to explore the mechanism of theosteogenic differentiation of PDLSC under cyclic tension. Our results will provide a theoretical basis to improve the orthodontic clinical treatment, and is useful for theperiodontal disease from the perspective of enhancing the osteogenic repair.Since several signal pathways are involved in the osteogenic differentiation, and thesignal pathways interact network-like, we can’t evaluate the accurate molecular mechanismof Hh signaling pathway in the osteogenic differentiation. However, the model under cyclictension we have established will provide a solid basis for the investigation of the molecularmechanism of orthodontic tooth movement.