3D-coculture Of DPSCs And EPCs Enhances Odontogeinc And Angiogenic Potential

BackgroundEndodontics and periapical disease are the common diseases in dentistry.Due to the characteristics of the dental pulp tissue, root canal treatment is the main treatment for these diseases, the procedure of which is removing the pulp tissue and filling artificialmaterials with the original closure the space occupied by the pulp tissue. With the development of materials and technology, root canal treatment can obtain higher success rate, but because the teeth lost the nutritional supportby the dental pulp tissue, the teeth is easy to root fracture and fold. Therefore, the ideal treatment of the infected dental pulp is dental pulp regeneration.ObjectivesIn this research, an co-culture system constructed with umbilical cord blood derived endothelial progenitor cells (EPCs) and dental pulp stem cells (DPSCs) were seeded onto CAM model. EPCs would be infected with the VEGF overexpression lentivirus and/or the SDF-1overexpression lentivirus. The efficiency of DPSCs and the combination of VEGF and SDF-1on the angiogenesis of umbilical cord blood EPCs, and the EPCs and the combination of VEGF and SDF-1on the odontogeinc of DPSCs were dynamic monitored both in vitro and in CAM model.Part1Isolation, culture and identification of human dental pulp stem cells and endothelial progenitor cellsMethods1、Isolation and culture of human dental pulp stem cells and endothelial progenitor cells:DPSCs isolated from the impacted third molar of healthy adult or orthodontic bicuspid pulp with tissue predigest cell culture. EPCs isolated from umbilical cord blood with Ficoll density gradient centrifugation were highly purified.2、Identification of DPSCs and EPCs:DPSCs would be indentified by three factors: a. the cell morphological characteristics, b. BMSCs cell surface markers (CD44, CD71and CD90), and c. the ability of multiple differentiation potential (osteogenic, chondrogenic and adipogenic). EPCs would be indentified by three factors:a. the cell morphological characteristics, b. cell cycle, and c. cell surface markers (endothelial cell surface marker CD31, KDR and CD105, hematopoietic cell surface markers CD45and CD14, stem cell surface markers CD34and CD133).Results1、Human dental pulp stem cells:DPSCs mostly demonstrated long fusiform and fibroblast-like morphology, expressed BMSCs cell surface markers CD44+, CD71+and CD90+(99.58%,99.51%and99.79%), and had the ability of multiple differentiation potential (osteogenic by alizarin stain, chondrogenic by alcian blue stain and adipogenic by nile red stain).2、Endothelial progenitor cells:EPCs mostly demonstrated long fusiform, were slow-cycling and expressed endothelial cell surface marker CD31+, CD105+and KDR+CD105+(99.82%,98.91%and99.82%), hematopoietic cell surface markers CD45-and CD14-(0.06%and1.1%), stem cell surface markers CD34-and CD133-(7.51%and0%). Part2VEGF and/or SDF-lstably overexpressioninEPCsMethods1、Obtain VEGF or SDF-1overexpression lentivirus:preparation of VEGF/SDF-1lentivirus expression vector, overexpression of VEGF/SDF-1lentivirus packaging.2、Identification of the overexpression lentivirus:the overexpression of VEGF/SDF-1lentivirus transfection into293T cells, fluorescence microscope cell fluorescence or Puromycin drug screening, collecting cells after extracting protein Werstern Blot detection of expression of VEGF/SDF-1.3、The experiment of the lentivirus transfected to EPCs:confirm endothelial precursor cells infected with two kind of lentivirus infection condition and the infection parameter.4、The characteristics of EPCs stably transfected VEGF and/or SDF-1overexpression lentivirus:the overexpression of VEGF/SDF-1positive/negative lentivirus after stable transfection into endothelial progenitor cells, fluorescence microscope cell fluorescence or Puromycin drug screening, using qRT-PCR detection mRNA expression level of VEGF/SDF-1, Werstern Blot detection protein expression level of VEGF/SDF-1. In vitro tube formation assay test cells after transfection.Results1、Obtain VEGF or SDF-1overexpression lentivirus, and to compare the positive cloning sequencing, the sequence is consistent with the gene pool. Overexpression of VEGF/SDF-1lentivirus drops degrees are2E+9TU/ml.2, After transfection VEGF overexpression lentivirus of293T cells, fluorescence microscope is more than90%of the cells with green fluorescence, the fluorescence intensity is moderate. After expressing lentivirus transfection SDF-1293T cells, there are still a lot of cell survivals after Puromycin drug screening, and cell proliferation ability is strong. Werstern Blot results of two kinds of cells show that both VEGF protein band/SDF-1protein band.3, After transfection VEGF overexpression lentivirus into endothelial precursor cells, fluorescence microscope is more than80%of the cells with green fluorescence, the fluorescence intensity is moderate. After transfection SDF-1express lentivirus of endothelial progenitor cells, there are still a lot of cell survivals after Puromycin drug screening, and cell proliferation ability is strong. After transfection SDF-1overexpression lentivirus into endothelial progenitor cell transfection again VEGF overexpression lentivirus, fluorescence microscope is more than80%of the cells with green fluorescence, the fluorescence intensity is moderate. qRT-PCR results showed that VEGF expression of VEGF overexpression of lentivirus cells quantity increased about three times as much as the negative control cells, cells of the two kinds of overexpression lentivirus VEGF expression quantity increased about2.5times than negative control cells; Infection of SDF-1overexpression of lentivirus cells expressing a negative control cells SDF-1about15times, two kinds of overexpression of lentivirus cells express a negative control cells increase SDF-1about17times. Werstern Blot results showed that protein bands are visible VEGF/SDF-1. Tube formation assay showed that24h after all visible monolayer cells are linked together into a tubular structure, the EPCs(VEGF＋) and EPCs(VEGF+SDF-l+) cells formed small tube is negative lumen density is bigger, more branches, lumen diameter smaller.Part3The characteristics of3D-DPSCsMethods1、3D culture of human dental pulp stem cells:take2to5generations of human dental pulp stem cells, according to the inoculation cells was divided into three groups, three groups of cells are administered to3D culture plate, every day in liquid,2,3,4, and5days to collect3D speroid cells. 2、The cell viability of3D-DPSCs:from the following three aspects of3D human dental pulp stem cells were analyzed:a. measuring the diameter of the3D cell speroid; b. on3days, the3D cell speroid by HE stain to observe internal structure; c. PI/Calcein-AM fluorescent dyes were used to detect the activity of the3D cells speroid.32Cell size of3D-DPSCs:the same cell for2D and3D culture, collect3days of culture, flow cytometry analysis of the cell relative size.4、The osteo/odontogeinc ability of3D-DPSCs:the same cell for2D and3D culture, collect3days of culture, induced by osteo/odontogeinc medium after21d alizarin red staining, using qRT-PCR to detect the mRNA level of ALP, DSPP, OPN and DMP-1expression.5、The anti-inflammatory ability of3D-DPSCs:the same cell for2D and3D culture, collect3days of culture, using qRT-PCR to detect the mRNA level TSG-6, IL8and STC-1expression; After collecting culture supernatant, using ELISA to detect the contents of TSG-6in the supernatant.Results1,3D-DPSCs speroid cell diameter:The higher the cell concentration, the bigger the diameter.2, HE staining:cells arranged closely in three groups of3D-DPSCs speroid, the third group of cells (2.5×105cell/drop) speroid centralliquefied necrotic phenomenon in a visible, the other two groups were not observed significant phenomenon of liquefaction necrosis.3, Cells activity of3D-DPSCs speroid:with the increase of cell inoculation density,3D-DPSCs cells formed by dead cells in the speroid (show the red fluorescent cells) ratio increased gradually, the third group central area can be observed a significant concentration of dead cells.4, Flow cytometry to detect the cell relative size:The volume of a3D culture cell is about half of the2D culture cells.5, The osteo/odontogeinc ability of3D-DPSCs:after induced osteo/odontogeinesis by alizarin red staining, the cells cultured in2D visible size reddish-brown mineralized nodules, and the cells cultured3D speroid visible overall darker red-brown is not tempted group; qRT-PCR in ALP, DSPP, OPN and DMP-1expression in3D-DPSCs speroid in a2D-DPSCs in about4,4,2and3times respectively.6, Anti-inflammatory properties of speroid:The expression of TSG-6, IL8and STC-1in3D-DPSCs speroid are higher than in a2D-DPSCs about29,13and1.5times respectively; ELISA in TSG-6in3D-DPSCs speroid content is67.28pg/ml, in2D-DPSCs cells content is30.88pg/ml.Part4The osteo/odontogeinc and angiogenic potential of3D-coculture of DPSCs and EPCsMethods1,3D co-culture of human dental pulp stem cells/endothelial progenitor cells in:take2to5generations of human dental pulp stem cells and5-10generations of endothelial progenitor cells in cell count the ratio of1:1, to a1×105cell/drop cell density inoculated to3D culture plate, every day in liquid, in3days to collect3D cell speroid.2, Osteo/odontogeinc potential:induced in the osteo/odontogeinc medium for21d, a. alizarin red staining; b. using qRT-PCR to detect the mRNA level of ALP, DSPP, OPN and DMP-1expression; c. the cellular immune fluorescence detection of DMP-1protein expression.3, Angiogenic potential:a. Tube formation assay; b. using qRT-PCR to detect the mRNA level of CD34, Flk, CD117and vWF expression; c. Werstern Blot test vWF expression in protein level.Results 1, Alizarin red staining:6groups, speroids are visible reddish brown on the whole.2, qRT-PCR:with ALP, DSPP, OPN and DMP-lgene expression level in comparison,3D-DPSCs/EPCs (VEGF+SDF-1＋) is the highest grop.3, Immunofluorescence:6groups of cell speroids in both cytoplasm area clear expression of DMP-1,3D-DPSCs/EPCs (VEGF+SDF-1＋) and3D-DPSCs/EPCs (VEGF+SDF-1＋) are stronger than the others.4, Tube formation assay:after joining6groups of cells24h, the tube structures of3D-EPCs and3D-DPSCs/EPCs almost disappeared; after48h, the tube structures of3D-DPSCs/EPCs (VEGF＋),3D-DPSCs/EPCs (SDF-1＋) and3D-DPSCs/EPCs (VEGF-SDF-1-) almost disappeared, expect3D-DPSCs/EPCs (VEGF+SDF-1+).5, Werstern Blot:Compared with3D-EPCs, vWF expression in3D-DPSCs/EPCs (VEGF+) increased about1.23times, in3D-DPSCs/EPCs (SDF-1＋) increased about1.27times and in3D-DPSCs/EPCs (VEGF+SDF-1＋) increased about1.70times.Part5The angiogenic potential of3D-coculture of DPSCs and EPCs in CAM modelMethods1、3D coculture of DPSCs and EPCs:take2to5generations of human dental pulp stem cells and5-10generations of endothelial progenitor cells (VEGF+SDF-1+) according to the number of cells the proportion of1:1,1×105cell/drop cell density inoculated to3D culture plate, every day in liquid, in3days to collect3D cell speroid.2、CAM angiogenesis experiment:in7days of age fertilized chicken eggs, the preparation of chicken embryo villus allantois membrane angiogenesis model, the cultivation of3d cell culture into the serosa,6,7,8,9, and10days for taking photos and collecting3D cell speroid. From the following two aspects to analyze using gross observation combined with HE staining,1,angiogenesis around the speroid;2. angiogenesis inside the speroid.Results1、Confirm the cell spheroid:3D-DPSCs/EPCs (VEGF+SDF-1+) cell speroid in cultivating five days later on the CAM model microscopically visible two white relatively dense clumps, visible two briquette were observed under fluorescent green fluorescent expression, the fluorescence intensity moderate.2、Vascular growth in the surrounding area and around the3D speroid:train6days later, the surrounding area and around the3D speroid no obvious cell angiogenesis; Training in7days, the vessels in the surrounding area and around the3D speroid incrseaed; with the increase of incubation time, the vessels in the surrounding area and around the3D speroid corresponding incrseaed, especially around the3D speroid region.3、Vascular growth inside of the spheroid:train after7days, inside of the3D cell speroid have not observed phenomenon of liquefaction necrosis, and have new blood vessels in the region contacting the CAM. On10days, the speroid is full of the new blood vessels and can be observed clearly blood flow, by chicken embryo allantoic chorionic blood vessels to the flow of speroid.Conclusion1、DPSCs isolated from the impacted third molar of healthy adult or orthodontic bicuspid pulp with tissue predigest cell culture, which are from mesenchymal organization and have the ability of multiple differentiation potential. EPCs isolated from umbilical cord blood with Ficoll density gradient centrifugation were highly purified and no induction needed. DPSCs and EPCs are optimal models of follow-up tests in this experiment.2、EPCs could be infected with the VEGF overexpression lentivirus and/or the SDF-1overexpression lentivirus efficiently in vitro. The VEGF and/or the SDF-1can be efficient stably oversecreted in EPCs. Then VEGF and/or SDF-1enhance EPCs into the capillary-like structures in vitro. 3、In3D cultures of spheroids of1×105DPSCs for3days, almost of the harvested cells were viable and the size of spheroids was almost biggest.The spheroid DPSCs were about half of the volume of DPSCs from adherent cultures. DPSCs into3D spheroids enhance their odontogeinc and antiinflammatory properties.4、The odontogeinc of3D-DPSCs could be promoted by umbilical cord blood derived EPCs and the combination of VEGF and SDF-1. The angiogenic of3D-EPCs could be promoted by the combination of VEGF and SDF-1. Considering the odontogeinc and angiogenic properties, the optimal combination is3D-DPSCs/EPCs (VEGF+SDF-1＋), which is3D coculture of DPSCs and EPCs infected with the VEGF overexpression lentivirus and the SDF-1overexpression lentivirus.5、On the CAM model, the spheroid of3D-DPSCs/EPCs (VEGF+SDF-1+) enhances the angiogenic properties in and around the spheroid, which provide some theoretical basis for farther animalexperimental research.