The Effect Of Hypoxia On Regulating The Proliferation And Osteogenic Differentiation Of Human Periodontal Ligament Cells Under Cyclic Tensile Stress

Part One Construct cultural model of human periodontal ligament cells under hypoxia as well as cyclic tensile stress stimulation in vitroObjective The purpose of this study was to construct the cultural model of human periodontal ligament cells(h PDLCs)under hypoxia and cyclic tensile stress(CTS)in vitro.Methods h PDLCs were isolated from non-carious human premolars extracting for orthodontic treatment from systemically and periodontally healthy patients with informed consents in Affiliated Stomatological Hospital of Nanjing Medical University.Periodontal ligament(PDL)tissues were gently scraped from the mid-third of teeth roots.The experiments were divided into four groups: group I,normoxia without CTS(control);group II,normoxia with CTS;group III,hypoxia without CTS;group IV,hypoxia with CTS.Cells were maintained at 37°C in a normoxic humidified atmosphere of 5% CO2,20% O2 and 75% N2 using a CO2 incubator with oxygen control.For hypoxic treatment,h PDLCs were incubated in the same incubator at 37°C in a hypoxic humidified atmosphere of 2% O2,5% CO2 and 93% N2.Cells at 3 to 6 passage were seeded onto six-well Bio Flex? culture plates coated with type I collagen.When cells had reached 80% confluence,the plates were subjected to an in-plane cyclic tensile deformation of 10% elongation with 0.5 Hz(30 cycles/min)for several loading time(12 h,24 h and 48 h)in a standard Bioflex baseplate linked to a Flexcell? FX-5000? Tension Unit.Cell morphology was observed by light microscope and cell structure was observed by transmission electron microscopy(TEM).Results After the third passage,h PDLCs stained positively for vimentin,but negatively for cytokeratin,confirming their mesodermal origin.A difference in cellular orientation was observed after the application of CTS.The number of cells in hypoxia was not less than that in normoxia.TEM analysis demonstrated that the mitochondria in normoxia with CTS groupwere slightly swollen compared with normoxia without CTS group.Moreover,in hypoxic groups,some mitochondria of h PDLCs demonstrated vacuolation and the rough endoplasmic reticulum exhibited dilatation.The degree of swelling and dilatation was the most obvious in hypoxia with CTS group than that in the other groupsConclusions The in vitro cultural model of h PDLCs under hypoxia and CTS was successfully constructed.Cell arrangement and cell structure were adapted to hypoxia and CTS.Part Two The effect of hypoxia on the osteogenic differentiation of human periodontal ligament cells under cyclic tensile stressObjective The present study was designed to investigate the effects of hypoxia on regulating the proliferation and osteogenic differentiation of h PDLCs under CTS.Methods h PDLCs in each group were cultured until 80% confluence,and then serum-starved for 24 h.DNA content was measured by FAC-Scan flow cytometer(FCM).Cell cycle fractions(G0/G1,S,and G2/M phases)were determined by FCM.The expressions of hypoxia-inducible factor-1?(HIF-1?)and osteogenic relative factors,i.e.,runt-related transcription factor 2(RUNX2),osterix(OSX),osteopontin(OPN)were assessed by Real-time PCR and Western blot.The experimental groups were the same as the first part.Results Flow cytometry assay revealed that the proliferation index(PI = S% +G2 M %)in hypoxia group was significantly higher than that in normoxia group.However,when h PDLCs were incubated under CTS,cell proliferation ability was reduced.The degree of the reduction was more obvious in normoxia with CTS group than that in hypoxia with CTS group.The expressions of HIF-1?,RUNX2,OSX and OPN were significantly increased in hypoxia with CTS group than that in the other groups for 24 h.Conclusions The proliferation of h PDLCs were enhanced under hypoxia,but were reduced by CTS.The osteogenic differentiation of h PDLCs was induced by CTS,and could be promoted under hypoxia.Part Three Hypoxia regulates the osteogenic differentiation of human periodontal ligament cells under cyclic tensile stress via MAPK PathwaysObjective To investigate whether mitogen activated protein kinase(MAPK)pathways were involved in the signal transduction of hypoxia regulating the osteogenic differentiation of h PDLCs under CTS.Methods MAPK pathways mainly consist of extracellular signal-regulated kinase1/2(ERK1/2),c-Jun NH2-terminal kinase(JNK),and P38 kinase.The involvements of MAPK signaling pathways were investigated by Western blot with specific inhibitor.The experimental groups were the same as the first part.Results In normoxic environment,the levels of phosphor-ERK1/2(p-ERK1/2)were increased under CTS.MAPK inhibitor(PD 98,059)significantly attenuated this increase.In hypoxic environment,the levels of p-ERK1/2,phosphor-JNK(p-JNK)and phosphor-P38(p-P38)were increased under CTS.PD 98,059 significantly attenuated hypoxia and CTS-induced phosphor-ERK1/2,phosphor-JNK and phosphor-P38 expressions.Moreover,the expressions of RUNX2,OSX and OPN were down-regulated by PD98,059,which were up-regulated in normoxia and CTS group.Conclusion Hypoxia regulates CTS-responsive changes in osteogenic differentiation of h PDLCs via MAPK pathways.Part Four Expressions of RUNX2,OSX and OPN in periodontal ligament at the tension side during orthodontic tooth movementObjective To examine the expressions of runt-related transcription factor 2(RUNX2),osterix(OSX)and osteopontin(OPN)in the PDL at the tension side during orthodontic tooth movement(OTM).Methods Twenty Sprague-Dawley male rats(average body weight 200 g)were used in OTM.The upper first right molar was moved mesially by means of a precalibrated 5-mm nickel-titanium closed coil spring delivering a constant force of 40 g with a stainless-steel ligature wire.Histological changes were evaluated to observe the location of osteogenic relative factors in the periodontal ligament(PDL)at the tension side in vivo.Results The width of PDL was approximately increased in a time-dependent manner and the blood capillaries were mostly detected near the alveolar bone.Positive cells and areas were seldom observed on day1 and day 3.RUNX2-positive cells were detected in the PDL on day 3 and day 7 and were lined up separately in the margin of the cementum and alveolar bone adjacent to the PDL on day 10.OSX-positive cells were strongly detected in the PDL,especially on the surface of the newly formed alveolar bone and cementum on day 7 and day 10.OPN-positive areas were clearly evident throughout the entire PDL,especially on the surface of the newly formed alveolar bone on day 7 and day 10.Conclusions The obvious expressions of RUNX2,OSX and OPN were detected in PDL at the tension side during OTM,indicating that OTM induced the osteogenic differentiation of PDL at the tension side.