Primary Study About BMP9Regulated Mice Stem Cells From Apical Papilla Odontoblast Differentiation And Mechanism

Part one The CD1mice stem cells from apical papilla extraction,culture,immortolizedObjective:The CD1mice stem cells from apical papilla primary culture and immortalized.Methods:The CD1mice were sacrificed by cervical,then extracted mouse mandibular central incisors, minced and digested in collagenase type Ⅰ after the root apical papilla was cut from the surface of the root gently, cells were Subcultured.The SV40T antigen expression cassette flanked by loxP sites retrovirus transfected primary cells and selected by hygromycin B, constructed the mouse immortalized stem cells lines from apical papilla.Results:The primary cells expressed as Spindle-shaped, polygonal, spindle-shaped, similar to fibroblasts after the root apical papilla digested in collagenase type Ⅰ, Adherent growth After the cells were plated rendered as polygons, bulky. transfected cells Successfully can survive after hygromycin B selected,and dead cells expressed as floating,the survival cells expressed as Adherent growth. And MAPs proliferative capacity will be stoped in a certain extent,whereas The proliferative capacity of iMAPs can be kept and become fusion slowly.There were significant difference between MAPs and iMAPs in doubling proliferative capacity (P<0.05)Conclusion:The root apical papilla can be digested effectively in collagenase type Ⅰ.The mice stem cells from apical papilla can be immortalized by SV40T antigen by retrovirus transfection after selecting by hygromycin B. Its was basis for the follow-up dental tissue regeneration Engineering Research. Part two Quantitative comparison biological characteristics inclouding proliferaion rate of MAPs and iMAPs cell linesObjective:Quantitiative comparison biological characteristics including proliferation rate of MAPs and iMAPs cell lines.Methods:Check the difference between the MAPs and iMAPs cells by cristal violet staining, MTT method, Trypan blue staining.Results:There are significant difference between the MAPs and iMAPs cells in proliferation rate by cristal violet staining and scan (P<0.05); There are significant difference between the MAPs and iMAPs cells in proliferation rate by MTT method (P<0.05); There are significant difference between the MAPs and iMAPs cells in proliferation rate and Survival rate by Trypan blue staining (P<0.05)Conclusion:The immortalized mice stem cells from the apical papilla had better survival rate and proliferation rate than the primary cells. Its had great significance for tissue engineering and regenerative medicine research. Part three Assay the iMAPs cells MSC marker expression by immunofluorescenceObjective:Assay the iMAPs cells MSC marker expression by immunofluorescence.Methods:The iMAPs cells were seeded into24-wells culture plate, Fixed with10%formalin at room temperature, Closed with10%BSA at room temperature. The first antibody was added to each well of the experimental group, the PBS was added to each well of the experimental group,followed by addition of fluorescent secondary antibody and DAPI, the cells were observed under different magnifications and image acquisition under inverted fluorescence microscope.Results:iMAPs cells express MSC markers BMPRⅡ,CD117/c-kit, CD29/Integrinβ1,CD44,CD73,CD90/Thy-1,CD105/Endoglin,CD133/Prom1,CD166/ALCAM.and can not express CD14,CD34,CD45.Conclusion:The iMAPS cells maintained the mesenchymal stem cells properties after immortalizing. Part four The BMP9regulate iMAPs and MAPs odontoblast differentiation in vitroObjective:ALP assay in early stage,OCN and OPN expression in later period,and matrix mineralization assay by Alizarin Red S staining after BMP9infected iMAPs and MAPs cells.RNA isolation and semi-quantitative RT-PCR analysis for odontoblast differentiation markers.To clarify its role in regulating the iMAPs and MAPs cells differentiation from the microscopic point of view.Methods:Amplified the Ad-BMP9and Ad-GFP,then infected iMAPs and MAPs cells and checked the expression of odontoblast differentiation markers.Those markers included ALP activity in early stage,OCN and OPN expression in later period,and matrix mineralization assay by Alizarin Red S staining.Total RNA was isolated in different time point,the RNA was used to generate cDNA templates by RT-PCR.To assay the target gene expression in odontoblast differentiation.Results:The Experimental amplified adenovirus Ad-GFP and Ad-BMP9having good titer, and can efficiently infect the iMAPs and MAPs cells; RT-PCR test results showed that:target gene expression was significantly up-regulated after iMAPs and MAPs cells infected by BMP9,especially odontoblast differentiation related genes marker:MEPE, DSPP and DMP1; ALP activity, staining and scanning results show that Ad-GFP can not induce iMAPs and MAPs the ALP activity increased. Ad-BMP9could significantly contribute to the ALP activity increased in three time points; Immunohistochemistry staining results showed that the Ad-GFP-infected cells was not detected the expression of osteopontin and osteocalcin. Ad-BMP9infected cells can be detected expression of osteopontin and osteocalcin. Alizarin Red S staining results indicate that the Ad-GFP group can not be detected the matrix mineralization,and matrix mineralization in the Ad-BMP9group was significantly higher than the Ad-GFP group.Conclusion:BMP9can induce iMAPs and MAPs cells ALP activity increased in early stage, OCN and OPN expression in later periond and increased matrix mineralization.Effective regulate the iMAPs and MAPs cells odontoblast differentiation, and upregulate of the target gene. Part five The BMP9regulate iMAPs and MAPs odontoblast differentiation in vivoObjective:To confirm the BMP9signal regulating mice stem cells from apical papilla by Stem cells transplantation experiments in vivo.To clarify its role in the regulation of odontoblast differentiation from macro perspective.Methods:We co-transduced iMAPs with Ad-GFP or Ad-BMP9in culture and injected the cells subcutaneously into anthymic nude mice and hind legs. Ectopic tissue massed were retrieved from mice killed by cervical after4weeks.To do Micro-computed tomography.To make three-dimensional reconstruction and statistical analysis for the results of the scan by Amira5.3Software.In addition, ectopic tissue masses paraffin-embedded, paraffin sections and histological analysis:H&E staining, Alcian Blue staining and Masson’s Trichrome staining.Results:Stem cells transplantation results indicate that the Ad-GFP control group can not formate ectopic dentin in athymic nude mice in vivo environment, whereas Ad-BMP9experimental group can do. Micro-CT scans showed that the average volume and the average density of the ectopic dentin of experimental groups were higher than the control . groups.There were significant difference between Ad-BMP9and Ad-GFP groups (P<0.05). H&E staining showed BMP9can fomate ectopic dentin,we can see the a partially matured matrix and collagen fibers into bundles embedded in the matrix, the ribbon-like shape, the frame structure, and we can see undifferentiatedearly cells in ectopic mass. And adipose tissue structure can be seen in ectopic mass. After the iMAPs cells infected by Ad-BMP9, we can see the expression of osteopontin and osteocalcin in ectopic tissue masses. We can see chondroitin sulfate calcium and cartilage matrix deposition by Alcian Blue staining. This conformed that BMP9can promote the differentiation of the stem cells from apical papilla, and there are multiple differentiation potential in different ways to form dentin. Masson’s Trichrome staining showed that the iMAPs cell infected by Ad-BMP9,dentin-like tissue masses can see the maturation and mineralization of the matrix, that is to say,BMP9can effectively induce and promote odontoblasts secretion of matrix mineralization.Conclusion:BMP9can effectively regulate the iMAPs cells Odontoblast differentiation in vivo, and can form ectopic dentin-like tissue masses in nude mice and hind leg muscles.