||Effects of Dental Restorative Materials on Human Dental Pulp Stem Cells
||Institute of Oral Medicine
Dental pulp stem cell
Glass isomer cement
Zinc oxide eugenol-based cement
Numerous studies have approved the significance of DPSC since it may differentiate into various cells to support tissue engineering and stem cell therapy. The source teeth are frequently filled with material including composite resin, glass ionomer, and zinc oxide eugenol-base materials, due to caries. Cytotoxicity and genotoxicity of these restorative materials have been well-documented in in vitro studies. However, only some of these compounds were also found to diffuse through the dentinal tubules and reach the pulp tissue, even in the presence of an intact dentin barrier. However, there is until now fragmentary information available on their biological effects on dental pulp stem cell (DPSC). Only few in vitro studies report that dental materials disturb the differentiation of DPSC. Therefore, it is important to develop further in vivo study in tissue engineering and stem cell therapy. The purpose of this study was to investigate the response of dental pulp stem cell after application of dental restorations in vivo.
In this study, volunteers with scheduled extracted human molar teeth were collected under informed consent. The cavity floor prepared on the occlusal surface of molars was restored with composite resin, glass ionomer and zinc oxide eugenol-base materials. The 36 molars teeth filled with dental restoration were extracted after 7 days and 30-60 days, then were processed for histopathological evaluation in order to examine the pulp response and the distribution of stem-cells. The histopathological finding showed mild pulp damage in teeth with restoration in cavities. The immunoreactivity of CD44 and α-SMA was obviously found in the pulp core of all groups. The stem cell percentage of the groups at day 30-60 decreased comparing with the groups at day 7, with the composite resin group showing the lowest value among groups. There was no significant difference among the tested groups and control group.
In established culture experiment, we investigated the morphology, proliferation, stemness, and differentiation potential of DPSCs. For each group, isolated DPSCs exhibited small, spindle-like, homogeneous morphology. In cell proliferation, all groups of cell formed adherent clonogenic cell cluster; however, there were similar CFU counts between all groups. In WST1 assay, no significant difference was observed among groups. Oct4 expression increased in resin-30days group comparing with control group. Mineralized nodules were formed after 2 and 4 weeks of osteogenic induction. The result suggested that resinous monomers may reduce the viability of DPSC but increased the stemness of the survival DPSCs under inflammatory stimulation. Additionally, the dental restoration caused mild damage of pulp tissue with little alteration in density of DPSCs, and the osteogenic potential was kept after dental restoration.
List of tables IX
List of figures X
Chapter 1.Introduction 1
1.1The Stem Cell 1
1.2 Mesenchymal Stem Cells 1
1.2.1 Dental Pulp Stem Cell 2
1.2.2 Tooth Regeneration of DSPC 3
1.2.3 Osteogenic Differentiation of DPSCs in Regenerative Medicine. 4
1.2.4 Neural Differentiation of Dental Stem Cells in Regenerative Medicine 4
1.3 Dental Restoration Material 5
1.3.1 Composite Resin and its Cytotoxicity 5
1.3.2 Glass Inomer Cements and its Cytotoxicity 6
1.3.3 Zinc Oxide-eugenol and its Cytotoxicity 7
1.4 Effects of the Dental Restoration Material on Stem Cell in Vitro 7
1.5 Motivation and Specific Aims 8
Chapter 2. Materials and Methods 9
2.1 Tooth Collection and Preparation 11
2.2 Immunohistochemical Stain 14
2.3 Primary Culture of Human Dental Pulp Stem Cells 15
2.4 Cell Morphology 15
2.5 Colony Forming Unit Fibroblast (CFU-F) Assays 16
2.6 WST-1 Assay 16
2.7 Osteogenic Differentiation Assays 17
2.8 RNA Isolation and Quantitative Real-time PCR (qRT-PCR) Analysis 17
2.9 Alizarin Red S Stain 18
2.10 Statistical Analysis 18
Chapter 3 Results 19
3.1 The inflammatory reaction of dental restorative material on DPSCs 19
3.2. The influence of the density and distribution of DPSCs with dental restorative material 20
3.3 Cell morphology 21
3.4 The proliferation of DPSCs with dental restorative material 21
3.5 mRNA expression of Oct-4, Nanog and CD44 in DPSCs of teeth with dental restorative material 22
3.6 Osteogenic differentiation of DPSCs of teeth with dental restoration material 22
Chapter 4. Discussion 24
Chapter 5. Conclusion 29
Appendix1. Reagents 62
Appendix2. Equipments 63
Curriculum Vitae 64
List of tables
Table 2.1 Experimental material used in experimental group 9
Table 2.2 Experimental and control groups 10
Table 2.3 Histopathologic event and scores 13
Table 2.4. Antibody 15
Table 2.5 Primers used in qRT-PCR 18
Table 3.1. The tissue response of experimental groups from histologic observations 20
List of Figures
Figure 1. Source of dental stem cells and potential regeneration. 40
Figure 2. Dental stem cell-based tissue engineering. 41
Figure 3. Chemical structures of monomers of dental resin material 42
Figure 4. Eugenol diffuses into the pulp. 43
Figure 5. The experimental scheme. 44
Figure 6. Adhesive system and restoration used in this study. 45
Figure 7. Flow chart 1 46
Figure 8. Flow chart 2 47
Figure 9. Post-operative radiography. 48
Figure 10. Pulp response. 49
Figure 11. Stro-1 expression in pulp tissue. 50
Figure 12.α-SMA expression in odontoblastic layer and pulp core. 51
Figure 13. CD44 expression in odontoblastic layer and pulp core. 52
Figure 14. CD44 (+) cell percentage. 53
Figure 15. Cell morphology. 54
Figure 16. Growth curves analyzed by WST1 assay 55
Figure 17. Colony formation in each group. 56
Figure 18. Colony forming unit fibroblast (CFU-F) assays. 57
Figure 19. mRNA expression of Nanog, Oct-4 and CD44 in DPSCs with dental restorative material 58
Figure 20. Osteogenic induction for 2 weeks in microscope. 59
Figure 21. Osteogenic induction for 2 weeks. 60
Figure 22. Osteogenic induction for 4 weeks. 61
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