Expression Of Hdpcs-derived Odontogenic Cell Marker Genes In Human Dental Pulp Cells-platelet Rich Fibrin (Hdpcs-prf) Complex

Objective:The aim of this study was to explore the biological behavior of hDPCs in the hDPCs-PRF complex and to assess the three-dimensional structure of the hDPCs-PRF complex and the migration and odontoblast differentiation of the hDPCs in vitro.The feasibility of the body was used as a dental pulp regeneration scaffold.Method:1 Experiment 1:The hDPCs were isolated and cultured by tissue block method.The expression of vimentin and keratin was detected by immunohistochemical staining.The PRF was prepared by one-step centrifugation with Choukroun.0.5 ml of 1×10~5/ml hDPCs was resuspended quickly before centrifugation.The h DPCs-PRF complex was then prepared by centrifugation.The complexes were isolated and cultured in 10%culture medium When the solution was changed every 3 days.Histology,immunohistochemistry and scanning electron microscopy were performed on the 7th,14th and 21th days of culture.2 Experiment 2:The preparation of hDPCs-PRF complex was the same withexperiment 1.Preparation of PRF:After collecting 5 ml ofblood,0.5ml of PBS was added befoer for 2700 rpm/min and centrifuged for 12 min.After centrifugation of the complex and PRF,the lower red blood cells was then discarded,leaving only the white film layer 10cm above as a research object.The experiments were divided into three groups:h DPCs-PRF complex group,PRF+cell group and cell culture alone group.The human dental pulp cells of the hDPCs-PRF complex group were embedded in the buffy coat of the PRF and grew.The human dental pulp cells of the PRF+cell group were dissociated from the PRF,and hDPCs of the pure cell culture group were cultured alone.The above three groups were cultured in a six-well plate supplemented with 4ml of10%mineralized solution and the medium was changed every three days.RT-PCR was used to detect the expression of ALP,DSPP and COL-I mRNA in human dental pulp cells on days 7 and 14.Result:1 The primary dental pulp cells cultured by the tissue block method have a single shape and long spindle shape.Immunohistochemical staining showed that the cells was positive staining for vimentin and negative staining for keratin,indicating that the cell was derived from mesenchymal original.2 The h DPCs-PRF complex is similar to PRF in macroscopic terms and consists mainly of yellow fibrin and terminal erythrocytes,with a leukocyte-rich buffy coat in the middle.SEM can be observed the complex in the wrinkled state of hDPCs and various white blood cells,red blood cells.Observed under the microscope,the hDPCs-PRF complex was a three-dimensional structure composed of soft,dense aggregated fibrin chains.The red part was mainly the normal shape of red blood cells entraining some immature fibrin;the middlelayer of the white tunica albuginea can see dense nuclear blue-stained white blood cells and platelets;irregularly shaped cells with larger volume than white blood cells;vimentin and keratin.The staining results showed that the larger cell vimentin staining was positive and the keratin staining was negative,suggesting that h DPCs were added when the complex was prepared.On the 7th,14th,and 21th day of culture,it was observed that as the culture time progressed,the buffy coat gradually dissolved and disappeared,and leukocytes and hDPCs gradually disappeared along the disappearance of the buffy coat or migrated along the edges of the complex.3 RT-PCR results showed that on the 7th day of mineralization induction,the expression of ALP and COL-I mRNA in the complex group was higher than that in the control and(PRF+cell)group;The expression on the 14th day of the DSPP and COL in the complex group was higher than the control group,and the difference was statistically significant.Conclusion:1 Human dental pulp cells were embedded in PRF to prepare hDPCs-PRF complexes,which can slowly release the dental pulp cells to the medium.2 The hDPCs-PRF complex provided a good environment for the growth and differentiation of human dental pulp cells.Under the mineralization-induced culture conditions,the expression of human dental pulp cell osteogenesis/dentin marker genes is enhanced.This suggests that the hDPCs-PRF complex is more conducive to the regeneration of pulp tissue.