Study On Differentiation And Polarization Of Dental Pulp Stem Cells Based On Dentinal Tubules And Inorganic Components

Promoting the differentiation and polarization of dental pulp stem cells(DPSCs)is important research work in tooth engineering.In the study of dentin regeneration,the differentiation and polarization of DPSCs determine the tissue structure of regenerated dentin,and then affect the physiological functions of regenerated dentin,such as mechanical properties and conducting stimulus signals.At present,there is no ideal biomaterial that can effectively promote the differentiation and polarization of DPSCs.In tissue engineering research,the micro-morphology of materials plays an important role in regulating the biological behavior of stem cells and determining the structure of regenerated tissue.Materials with tubular microstructures are used to regenerate tissues with similar structures because they contain open channels and can induce the growth and differentiation of stem cells.Since dentin is hard tissue containing densely arranged natural tubular structures(dentinal tubules),studying the functions of its tubular structures under physiological conditions and their effects on DPSCs' morphology and differentiation will hopefully help to form biomaterials which can regulate the fuctions of stem cells,and then produce important significance for dentin engineering.In chapter 2,we first studied the effect of dentin tubular structure on drug delivery in promoting the formation of restorative dentin.In this experiment,recombinant adenovirus connected with human bone morphogenetic protein 2(AdCMV-hBMP2)was absorbed into gelatin sponge and placed in the pulp cavity of rat molars to observe the ability of AdCMV-hBMP2 transmitting into the pulp through dentinal tubules and transfecting the pulp cells to promote the formation of restorative dentin.The results showed that AdCMV-EGFP could successfully transfect dental pulp cells and express green fluorescent protein at 1 day after transfection.After 3 days of transfection,almost all dental pulp cells could express green fluorescent protein.Histological observation showed that more restorative dentin was formed on the basis of the original dentin after transfection of AdCMV-hBMP2 than that in the control group.For pulp exposed specimens,no obvious reparative dentin was formed on the pulp surface during the experiment.The results show that dentinal tubules in physiological state can act as a natural channel for drug delivery in promoting the formation of restorative dentin.The tubular structure and composition of dentin may play an important role in the formation of restorative dentin.In order to study the effects of the tubular structure and inorganic components of dentin on the morphology and odontogenic differentiation of DPSCs,in chapter 3,we calcined the natural dentin to remove its organic components,and formed calcined dentin with tubules(CDT),and then studied its effect on the morphology and differentiation of DPSCs.The CDT was characterized by scanning electron microscopy,X-ray diffraction analysis and energy dispersive X-ray spectroscopy.The results showed that the CDT retained the structure of the natural dentin tubules well.The diameter of the tubules was 2.8(+0.3)um.CDT was a kind of tubular inorganic material with strong hydrophilicity.Its main component was hydroxyapatite.MTT results showed that PBS modified CDT had good biocompatibility.When DPSCs were co-cultured with CDT containing different numbers of dentinal tubules,it was found that with the increase of the number of dentinal tubules,the DPSCs tended to change their morphology from cubic to shuttle-shaped.Alizarin red staining and alkaline phosphatase(ALP)quantitative analysis showed that CDT could promote DPSCs to form calcium nodules and express ALP.Real-time PCR showed that CDT could promote DPSCs to express dentin sialophosphoprotein(DSPP),dentin matrix protein-1(DMP1),ALP and COL-1 genes.The results show that the tubular structure and inorganic components of dentin play an important role in promoting the transformation of DPSCs into odontoblasts.In order to make CDT a supporting material for promoting DPSCs polarization,in chapter 4,we explore the method of expanding the tubular structure of CDT.Calcined dentin with expanded tubules(CDET)were prepared by selective etching combined with ultrasound.After co-culture of CDET and DPSCs,it was found that with the enlargement of the tubular structure,DPSCs on the surface of CDET tended to form slender cell processes,similar to the morphology of odontoblasts.Scanning electron microscopy and histological observation showed that the enlarged tubular structure promoted the processes of DPSCs to grow into the tubular structure.Further immunofluorescence staining showed that the Golgi apparatus of DPSCs also entered the tubular structure after the process grew into the microtubule,but the nucleus remained on the surface of the material.These results showed the asymmetrical distribution of organelles in DPSCs,that is polarization.Real-time PCR showed that the expression of ECADHERIN and CD42,the polarization-related markers,were increased significantly when DPSCs were cultured on the surface of CDET.These results show that CDET prepared from natural dentin can promote DPSCs polarization in vitro.CDET is expected to be an ideal biomaterial for promoting polarization and differentiation of DPSCs.