Tissue Engineering Of Tooth-like Structures By Human Dental Epithelial And Mesenchymal Cells

Tooth loss due to periodontal disease, dental caries, trauma, or a variety of genetic disorders continues to affect most adults adversely at some time in their lives. A biological tooth substitute that could replace lost teeth would provide a vital alternative to currently available clinical treatments, such as the commonly used but not so efficient denture restoration therapy. To design and manufacture it becomes a highlight for many researchers. So far, little breakthroughs have been seen in this research field because of lacking related knowledge and techniques. Recently, it has been found that human dental pulp stem cells could generate dentin-pulp complex-like structures in nude mice, but studies on tissue engineering of tooth-like structures by cultured human dental epithelial and mesenchymal cells isolated from tooth germ are still unreported.Tooth is an organ of multi-germinal layer origin, which developed through the complicated reciprocal interaction between dental epithelium and mesenchyme precisely modulated by signal network constituted by a series of signal molecules. Dental enamel is formed by dental epithelial cells, while dentin-pulp complex is generated by the mesenchymal cells around them. These two kinds of cells show strong proliferation capability and differentiationpotential. The interaction between them plays an important role in tooth development. In this study, simulating the natural mechanism of tooth development, in vitro cultured human dental epithelial and mesenchymal cells were implanted as seed cells onto selected scaffolds, and then cell/scaffold constructs were transplanted s.c. into immunocompromised mice in order to bioengineer tooth-like structures. The results of this study will operate a new direction for treating periodontal disease, dental caries and tooth loss in clinic, lay a good experimental foundation for tooth regeneration and its clinical application, and provide a novel approach for engineering other tissues and organs of multi-germinal layer origin. Following experiments were involved in this study:1. In vitro culture of human dental epithelial and mesenchymal cellsTooth germ tissues were isolated, and then the dental epithelium as well as mesenchyme were separated microscopically from which dental epithelial and mesenchymal cells were prepared by tissue explant culture technique. The cultured cells were purified using several methods together. Their morphology and growth were observed under phase-contrast microscope and their origin were identified by immunofluorescent staining. The results showed that primary cultured human dental epithelial cells grew well but mixed with a few mesenchymal cells. After purification, the dental epithelial cells had a morphology of typical epithelial cells and expressed cytokeratin and ameloblastin. Cultured human dental mesenchymal cells were in good shape and expressed vimentin. Thus, in vitro culture system of human dental epithelial and mesenchymal cells has been established successfully, which will provide a reliable source of seed cells for tooth engineering.2. Induction of odontoblast differentiation of dental mesenchymal cellsThe cultured human dental mesenchymal cells were induced in two-dimensional culture model or in both two-dimensional culture model and subsequent three-dimensional culture model in collagen gel. Cell growth incollagen gel and change of cell morphology after induction were observed. The expression of human DSP protein and DSPP mRNA in the induced cells were detected by immunofluorescent staining and RT-PCR methods. Mineralization capability of the induced cells was evaluated using Von Kossa staining. It was found that some of the induced cells showed a long single process and expressed DSP protein as well as DSPP mRNA, forming mineralized nodule spontaneously. The induced cells exhibited morphological and functional characteristics of typical odontoblasts in both two and three-dimensional culture model, suggesting above-mentioned growth factors could induce dental mesenchymal cells to...