The Effect Of Huscs On The Proliferation And Osteogenesis Differentiation Of Cementoblasts

Objective: Root resorption is one of the most common complications during orthodontic treatment and severe absorption of the teeth will occur loose or even fall off.One of the main cells of bone reconstruction in the process of root resorption is cementoblasts,.Therefore,this study used the methods of common non-contact culture of USCs and cementoblasts to conduct the proliferation and osteogenesis of cementoblast.Part one: Effect of hUSCs on the proliferation of cultured dental cementoblasts(OCCM-30).Methods: 1.The High speed centrifugation method was used to extract hUSCs from human fresh urine and observe its morphology under microscope and detect the expression of surface markers by flow cytometry.22.The expression of surface antigen(CD29,CD34,CD73,CD90,CD133 and CD146)were observed by flow cytometry to divide hUSCs.3.OCCM-30 in4 groups according to different proportions(G1:1:1,G2:2:1,G3: 3:1 and OCCM-30 control group),two kinds of cells were non-contact co-cultured,and after 12 h and 24 h,the growth of cementoblasts was observed under microscope,and the proliferation of dental cementoblasts was detected by CCK8 technique after non-contact co-culture of 1d,3d and 7d.Results: 1.A technique of extracting hUSCs from human fresh urine is used successfully: hUSCs began to adhere to the wall 3-7d after the centrifugation,and began colony-like growth 7-10 d after the centrifugation.The number of cells at 10-14 d reached 50%-60% and capable of generation,with most shaped grainy and a few were oval or short-spindle.After generation passed to around P3,the shape of the cells were basically maintained in the form of rice grains,and begin apoptosis when cultured to P8 cells;2.Biological characteristics of hUSCs: flow-test the hUSCs surface markers.proving that hUSCs belongs to one of the MSCs;3.Growth and proliferation of cementoblast under the influence of hUSCs: the cells were observed under the microscope 12 h and 24 h after non-contact co-cultivation,which indicates that wall-adherent growth and proliferation occurred in cementoblast of each group,among which the G3 cementoblasts had the fastest proliferation rate and highest coverage up to 70%,100% respectively.After test of CCK8,the results showed : OD value with the increase in culture time,the OD value of each experimental group is significantly greater than the control group(p<0.05),OD value in G3 significantly higher than other experimental groups(p<0.05),will HUSCs and OCCM-30 to 3 : 1 non-cantact co-culture can significantly promote the proliferation of cementoblasts.Part two: The cementoblasts were induced osteogenesis under the influence of hUSCs.Methods: 1.Directional induct the cementoblasts under the influence of different proportions of hUSCs using osteogenic inducing medium;2.Perform ALP staining of the indicators and measure the activity of the early osteogenic;3.Alizarin red staining of late osteogenic markers;4.Inspect the expression change of OPN and OCN by real-time quantitative PCR(q-PCR).Results: 1.ALP staining results indicated that the intracellular stained granules of indirect co-culture group were more obvious than the control group,and the ALP activity in each group increases with the induction time,which reaches the peak in 7 days,the experimental group is significantly larger than the control group(p<0.05),the G3 group is significantly higher than the other experimental group(p<0.05).2.The alizarin red staining results showed that the red staining of the cementoblasts group was most obvious when it has the highest proportion of hUSCs when in co-culture.3.q-PCR results showed that the expression of OPN and OCN was the highest in the adult-bone cells with the highest proportion of Huscs in the Huscs-induced bone cells under the influence of different number ratio of the cultured osteoblasts after 7d.(p<0.05)shows that Huscs can help to promote the osteogenesis of bone cells.Conclution: hUSCs can promote the proliferation and osteogenic of osteoblast significantly,and provide a theoretical basis for the clinical application of h USCS to promote the restoration of root resorption.