Effects Of Tooth Germ Microenvironment On Differentiation Of Dermal And Gingival Derived Adult Stem Cells

The crucial point of tooth engineering is to obtain plenty candidate cells that have odontogenic potential and capable of development and differentiation in suitable microenvironment. Tooth development follows the concept of epithelial–mesenchymal interactions which require dental epithelial cells to differentiate into enamel as well as dental mesenchymal cells to develop into dentin-pulp complex. However, the limitation of dental-derived stem cells obscured the approach of the tooth regeneration. What is evident is that work in this field is urgently needed as the ability to isolate large numbers of adult stem cells which biopsied easily, proliferate efficiently, and have distinct identity without ethic conflicts would be of great interest scientifically particularly with respect to future therapeutic applications and the developing discipline of tooth engineering. In this paper, the recombined microenvironment from developmental teeth was used to provide odontogenic niche for candidate cells. Using the mimic odontogenic environment, the potential of the differentiation and morphogenesis of skin dermal-derived multipotent stem cells (DMSCs) and gingival epithelial stem cells (GESCs) was assessed. Meanwhile, the results provide an effective strategy for the odontogenic induction of non-dental adult stem cells and present evidences of sorting candidate cells for tooth regeneration research.Part 1. Potential of tooth germ tissues from different development stagesWe investigated the ectopic growth and development of 14-day embryonic and one-day postnatal molar germ-related tissues from Sprague-Dawley rats in renal capsules. The HE results demonstrated that the tooth germ-related tissues at cap stage that could form regular tooth structures developed much better than those at late bell stage. To further confirm the development potential of dissociated cells of tooth germs at the two stages, we implanted tooth cells and dental mesenchymal cells of the two stages of molar germs in renal capsules, respectively. The results shows that dissociated tooth germ cells at cap stage formed better tooth structures than those at late bell stage; and the mesenchymal cells at cap stage produced dentin-pulp complex. However, tooth germ cells and papilla cells at late bell stage generated irregular bone-dental-like structures.In conclusion, dissociated dental epithelial and mesenchymal cells could recapitulate the embryonic tooth germ environment to favour normal tooth development, indicating that the dissociated single dental cell retained genetic signaling network which is necessary for odontogenesis.Part 2. Differentiation of tooth germ microenvironment induced skin dermal multipotent stem cellsIn present study we sought to explore the possibility of utilizing DMSCs easily available from skin tissue for odontogenic induction. Using the limiting-dilution technique, colony-forming cell population was isolated and characterized by proliferative activity and multilineage differentiation potential. By in vitro exposure to conditioned medium of embryonic and neonatal tooth germ cells in culture, the proliferation and mineralization activity of DMSCs was elevated while the embryonic tooth germ cell conditioned medium (ETGC-CM) produced more significant effects. Meanwhile, TGC-CM treated DMSCs phenocopied the odontoblasts as indicated by specific lineage markers. To further examine the in vivo development potential of induced DMSCs, TGC-CM pretreated scaffolds were loaded with treated DMSCs. Following in vivo subcutaneous transplantation, ETGC-CM treated DMSCs were capable of producing blocks of dentin-like matrix as proved by positive staining of BSP and DSP. While NTGC-CM treated DMSCs formed fewer mineral nodules inside the scaffolds which were morphologically and immunochemically identified as bone-like tissues.These observations suggest that under the sufficiently potent inductive microenvironment, DMSCs can be directed into odontoblast-like lineage cells. Our results further posit that the extrinsic niche where cells reside in is continuously required to maintain odontogenic differentiation. More suitable and optimal odontogenic microenvironment need to be further exploited and will be of substantial utility in the context of choosing non-dental adult stem cells for tooth regeneration. Our work highlights the potential utility of DMSCs as an alternative candidate cell source in hopes of developing more practical strategy of tooth regeneration research and offering promising opportunities for therapeutic approach.Part 3. Differentiation of tooth germ microenvironment induced gingival epithelial stem cells In this study, we isolated and characterized gingival epithelial cell population, from which existed a subpopulation that have stem cell properties, such as colony forming ability, slow cell cycle, capacity for self-renewal, and undifferentiated state as indicated by positive staining ofβ1-Integrin and CK14. To evaluate their odontogenic differentiation potential, cap stage tooth germ development niche was chosen to establish different patterns of cell interaction including TGC-CM, ordered and mixed contact co-culture. The data accumulated here showed that the ordered contact co-culture systems can effectively induce GESCs toward the ameloblasts phenotype, while the TGC-CM and mixed contact co-culture could not effectively initiate the odontogenic differentiation of GESCs. Following in vivo transplantation as cell pellets, there was no sufficient experimental evidence showed that induced GESCs could form enamel-like tissues.Taken together, it confirms the critical role of molecular signals in the microenvironment of cap stage tooth germ. Furthermore, the results also indicate that in order to accomplish tooth regeneration, it will be best if the natural process of epithelial–mesenchymal interactions of teeth embryonic development can be reiterated in vitro or in vivo. Accordingly, growth factors and their reservation and transmission through a substrate to anchor the candidate cells could be achieved as natural tooth initiation and morphogenesis.