Dentinogenic Capacity Of Dental Pulp Stem Cells And Their Applications In Making Chimeric Teeth

The inductive microenvironment is usually composed of bioactive scaffolds or single growth factors, which are necessary for the odontogenic differentiation of dental pulp stem cells (DPSCs) during the dental tissue engineering. As we all know, tooth developmental environment is much complicated, including the participation of epithelial-mesenchymal interactions, extracellular matrix, mineral ions and growth factors. Therefore, it is almost impossible to achieve the regular tooth morphogenesis with non-physiological inductions mediated by scaffolds or some growth factors. In this paper, the recombined microenvironment from developmental or adult teeth was used to stimulate the differentiation and morphogenesis of dental pulp stem cells, as well as the formation of chimeric bio-teeth. Our results provide an interesting progress in the tooth engineering and present a new strategy for the ultimate clinical applications.Partâ… . Selection and confirmation of the ectopic environment for the bioengineered tooth regeneration.We investigated the ectopic growth and development of four-day-old postnatal molar germs from Sprague-Dawley rats in different sites of allogenic hosts. The hematoxylin-eosin results demonstrated that tooth germs in renal capsules developed much better than those in other places. To further confirm this phenomenon, we implanted tooth-related tissues in renal capsules and got the same conclusion. Moreover, cell pellets from dental papillae can reexhibit the morphogenesis of dentin-pulp complexes, and bioengineered teeth can be made from primary tooth germ cell pellets in renal capsules. However, tooth germ cells at the secondary passage can not generate enough enamel matrices covering the surface of denin-pulp-like complexes in renal capsules.Together, the environment in renal capsules is favorable for the sustainable development of tooth germs, dental tissues and cells. Postnatal dental papilla cells and tooth germ cells can reperform the developmental patterns of dental mesenchyme and tooth germ respectively, indicating that the genetic information necessary for tooth morphogenesis is held in reserve in every dissociated dental cell.Partâ…¡. Differentiation of dental pulp stem cells induced by tooth germ cells. To evaluate the effects of developmental microenvironment from tooth germ cells on the proliferation and differentiation of DPSCs, we used three different coculture methods including tooth germ cell conditioned medium, stratified and mixed culture systems. The data accumulated here showed that the conditioned medium and stratified coculture systems can effectively initiate the odontogenic differentiation and morphogenesis of DPSCs. Both can bring about the formation of typical dentin-pulp complexes in renal capsules. However, the proliferation and regular dentinogenesis from DPSCs can not be induced in the mixed culture system.Taken together, it seems that soluble factors from tooth germ cells in the non-contact coculture systems may facilitate the differentiation and dentinogenesis of DPSCs, while insoluble cell surface molecules and extracellular matrix geneated by tooth germ cells in the mixed coculture system may inhibit the cell proliferation and subsequently affect the sustainable differentiation of DPSCs into odontoblast lineages.Partâ…¢. Effects of adult dental pulp cells on the proliferation and differentiation of dental pulp stem cells.To study the effects of adult microenvironment created by dental pulp cells on the proliferation and differentiation of DPSCs, STRO-1+ DPSCs were cocultured with dental pulp cells by means of three different culture systems as we have mentioned in Partâ…¡. Our observations showed that the adult microenvironment can effectively stimulate the proliferation activity of DPSCs which may due to the existence of growth factors, extracellular matrix and cell surface molecules generated by dental pulp cells. However, this adult microenvironment can not stimulate the odontoblast differentiation and dentinogenesis of DPSCs, which may attribute to a lack of dental epithelial signals.Partâ…£. Making chimeric teeth from dental pulp stem cells.To further test whether the inductive actions of developmental microenvironment are from epithelial components or mesenchymal parts in tooth germ cells, we recombined STRO-1+ DPSCs with apcal bud cells (ABCs) and dental papilla mesenchymal cells (DPMCs) respectively which were isolated from several postnatal incisor germs. After 14-day incubation in vivo, DPSC-ABC cytochimeras developed into typical tooth crown-like structures, while DPSC-DPMC chimeras formed osteodentin-like tissues and dentin-pulp complexes in which PKH26-labelled DPSCs failed to participate in the odontoblast differentiation and dentinogenesis. This suggests that the dental epithelial components may play a predominant role in the odontogenic differentiation of DPSCs induced by tooth germ cells.Although apical bud cells can initiate the odontoblast differentiation of bone marrow stromal stem cells (BMSSCs) in vitro, the BMSSC-ABC chimeras in vivo can only generate dentin-pulp-like tissues with no enamel formation. This may have general meanings for the selection of candidate cells between dental and non-dental stem cells in future tooth engineering and clinical applications. When DPSCs were recombined with apical bud cells at different proportions, typical crown-like tissues only appeared in those cytochimeras at 1:1 ratio, indicating that epithelial-mesenchymal cell proportions can affect the regular morphogenesis of chimeric bio-teeth.In summary, the effects of developmental and adult microenvironment on the differentiation and morphogenesis of DPSCs are different at all, in which the regular dentinogenesis of DPSCs is mostly controlled by dental epithelial signals, more accurately, by the epithelial-mesenchymal interactions. Based on these findings, we successfully reconstructed the chimeric tooth germs using DPSCs.