Effect Of The Cyclic Stretch And Role Of Related Signal Pathway On The Osteogenic Differentiation Of MC3T3-E1and HPDL Cells

Mechanical strain stimulation is one of the important factors to regulatebone metabolism, maintain bone mass and bone structure.Periodontal tissueremodeling under stress state is the biological basis of orthodontic clinicaltreatment. As stress-sensitive cells, osteoblasts or periodontal ligament cells isnot only a mechanical and biochemical signaling sensor, but also an effector cellcould be stimulated by stress. In the process of osteoblasts response to themechanical strain stimulation, the signal transduction pathways activated bydifferent mechanical stimulation is not the same. That is just through theactivation, close or cross-talking of different signal transduction pathways, toregulate the expression of target genes of osteoblast and periodontal ligamentcells. At present, a lot of research about mechanical biological reaction ofosteoblasts and periodontal ligament cells has been done, including itsproliferation, differentiation, function state and signal pathway. But themechanism of recognition, signal pathway and biological reaction is not clear， and different signaling pathways is poorly researched. Extracellular signalregulated kinase1/2(ERK1/2) and Nuclear factor kappa B (NF-κB) signalpathways are both important in the process of signal transduction in osteoblasts.They played an important role in differentiation of osteogenic progenitor cellsand periodontal ligament cells. Therefore in this research we culturedMC3T3-E1osteoblasts and periodontal ligament cells, established the vitromodel of stretch stress to observe the differentiation of MC3T3-E1osteoblasticcells and human periodontal ligament cells. Then we could realize theinteraction between ERK1/2and the NF-kB signaling pathway under stretchstress.Experiment1: Effect of mechanical strain on osteogenic differentiationrelated genes expression in MC3T3-E1MC3T3-E1osteoblastic cells cultured in vitro, we observed the change ofalkaline phosphatase(ALP), COLⅠ, osteocalcin,and IL-6gene expression.The expression of ALP and OCN gene is reduced under the8%and12%cyclicstretch stress. ALP is a sign of early differentiation of osteoblasts. OCN is a signof mature osteoblasts. It is found that MC3T3-E1cells inhibit its ability todifferentiate into mature osteoblasts under the8%and12%cyclic stretch stress.The expression of COLⅠ and IL-6gene is increased after stretch stress for24hours. COL I is one of the bone extracellular matrix components. It isdemonstrated that stress stimulation can promote synthesis of extracellularmatrix in bone tissue. COL I is material base of remodeling bone tissue stress.IL-6, as a multifunctional cytokines, involved in conversion of periodontaltissue during orthodontic tooth movement. As an effective osteoclast-activating factor, it stimulates osteoclast formation and bone resorption activity.Experiment2: The effect of ERK1/2and NF-kB signal pathways onosteogenic differentiation related genes expression in MC3T3-E1undermechanical strainBy using the inhibitor of ERK1/2and NF-kB signal pathway, we knew theregulation of ERK1/2and NF-kB signal pathway to the differentiation ofMC3T3-E1cells at gene level. It was found that ALP, COLⅠ and IL-6wereregulated by ERK1/2signal pathway. The expression of OCN is not effected byERK1/2. The inhibitor of NF-kB signal pathway could inhibit the reduction ofALP and the expression of IL-6. The expression of COLⅠ and OCN genes werenot effected by NF-kB signal pathway.Experiment3: The effect of mechanical strain on ERK1/2and NF-kBsignal pathways in MC3T3-E1cells.We observed the effect and interaction of ERK1/2and NF-kB signal pathwayafter stretch stress in MC3T3-E1cells. It was found that ERK1/2and the NF-κb pathway were activated, p-ERK1/2, p-IKK and NF-kB（p65） level weresignificantly improved. It could not effect the activation of ERK1/2after usingPDTC. But when inhibiting ERK1/2signal pathway, NF–κB athway wasinhibited. It was showed that ERK1/2was at the IKK upstream. ERK1/2couldeffect the p-IKK.Experiment4: Effect of mechanical strain on osteogenic differentiationrelated genes expression in hPDLCsGene chip technology and Real-time RT-PCR were adopted to investigate theeffect of the cyclic stretch on the expression of osteogenic-related genes in human periodontal ligament cells (HPDLCs). The results show that after theHPDLCs were stimulated by12%cyclic stretch stress for24h, the expressionof21osteogenic-related genes was significantly upregulated, including10growth factor genes and their associated molecules,10extracellular matrixgenes and their associated proteins.These results indicate that cyclic stretch with12%deformation can stimulate or inhibit some genes expression whichassociated with the process of HPDLCs differentiation.