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Harnessing developmental processes for tissue engineering represents a promising yet challenging approach to regenerative medicine. Tooth avulsion is among the most serious traumatic dental injuries, whereas functional tooth regeneration remains uncertain. Here, we established a strategy using decellularized tooth matrix (DTM) combined with human dental pulp stem cell (hDPSC) aggregates to simulate an odontogenesis-related developmental microenvironment. The bioengineered teeth reconstructed by this strategy regenerated three-dimensional pulp and periodontal tissues equipped with vasculature and innervation in a preclinical pig model after implantation into the alveolar bone. These results prompted us to enroll 15 patients with avulsed teeth after traumatic dental injuries in a pilot clinical trial. At 12 months after implantation, bioengineered teeth led to the regeneration of functional teeth, which supported continued root development, in humans. Mechanistically, exosomes derived from hDPSC aggregates mediated the tooth regeneration process by upregulating the odontogenic and angiogenic ability of hDPSCs. Our findings suggest that odontogenic microenvironment engineering by DTM and stem cell aggregates initiates functional tooth regeneration and serves as an effective treatment for tooth avulsion.

LIM homeobox 8 (Lhx8) is a highly conserved transcriptional factor with recently illustrated roles in cholinergic and GABAergic differentiation, and is expressed in neural crest derived craniofacial tissues during development. However, Lhx8 functions and signaling pathways are largely elusive. Here we showed that Lhx8 regulates dental mesenchyme differentiation and function via Wnt and TGFβ pathways. Lhx8 expression was restricted to dental mesenchyme from E11.5 to a peak at E14.5, and absent in dental epithelium. By reconstituting dental epithelium and mesenchyme in an E16.5 tooth organ, Lhx8 knockdown accelerated dental mesenchyme differentiation; conversely, Lhx8 overexpression attenuated dentin formation. Lhx8 overexpressed adult human dental pulp stem/progenitor cells in β-tricalcium phosphate cubes attenuated mineralized matrix production in vivo. Gene profiling revealed that postnatal dental pulp stem/progenitor cells upon Lhx8 overexpression modified several matrix related gene expression including Dspp, Cola1 and osteocalcin. Lhx8 transcriptionally activates Wnt and TGFβ pathways, and its attenuation upregulates multiple dentinogenesis genes. Together, Lhx8 regulates dentin development and regeneration by fine-turning Wnt and TGFβ signaling.

While it is known that precise dental epithelial-mesenchymal (DE-DM) cell interactions provide critical functions in tooth development, reliable methods to establish proper DE-DM cell interactions for tooth regeneration have yet to be established. To address this challenge, and to generate bioengineered teeth of predetermined size and shape, in this study, we characterize three dimensional (3D) pre-fabricated DE-DM cell constructs. Human dental pulp cell seeded Collagen gel layers were co-cultured with porcine DE cells suspended in Growth Factor Reduced (GFR) Matrigel. The resulting 3D DE-DM cell layers were cultured in vitro, or implanted and grown subcutaneously in vivo in nude rats. Molecular, histological and immunohistochemical (IHC) analyses of harvested implants revealed organized DE-DM cell interactions, the induced expression of dental tissue-specific markers Amelogenin (AM) and Dentin Sialophosphoprotein (DSPP), and basement membrane markers Laminin 5 and collagen IV, and irregular mineralized tissue formation after 4 weeks. We anticipate that these studies will facilitate the eventual establishment of reliable methods to elaborate dental tissues, and full sized teeth of specified sized and shape.

Rodent incisors provide a classic model for studying epithelial-mesenchymal interactions in development. However, postnatal stem/progenitor cells in rodent incisors have not been exploited for tooth regeneration. Here, we characterized postnatal rat incisor epithelium and mesenchyme stem/progenitor cells and found that they formed enamel- and dentin-like tissues in vivo. Epithelium and mesenchyme cells were harvested separately from the apical region of postnatal 4–5 day rat incisors. Epithelial and mesenchymal phenotypes were confirmed by immunocytochemistry, CFU assay and/or multi-lineage differentiation. CK14+, Sox2+ and Lgr5+ epithelium stem cells from the cervical loop significantly enhanced amelogenin and ameloblastin expression upon BMP4 or FGF3 stimulation, signifying their differentiation towards ameloblast-like cells, whereas mesenchyme stem/progenitor cells upon BMP4, BMP7 and Wnt3a treatment robustly expressed Dspp, a hallmark of odontoblastic differentiation. We then control-released microencapsulated BMP4, BMP7 and Wnt3a in transplants of epithelium and mesenchyme stem/progenitor cells in the renal capsule of athymic mice in vivo. Enamel and dentin-like tissues were generated in two integrated layers with specific expression of amelogenin and ameloblastin in the newly formed, de novo enamel-like tissue, and DSP in dentin-like tissue. These findings suggest that postnatal epithelium and mesenchyme stem/progenitor cells can be primed by pivotal signals towards bioengineered tooth regeneration.

Stem cell mediated tissue engineering has been acknowledged as a prospective strategy for repairing and replacing damaged and lost tissues. However, the low survival rate of implanted stem cells proves to be a major challenge in the management of transplantation failures. While previous studies have indicated the effectiveness of tissue engineered cell sheets in improving the survival rate of implanted cells, we have recently demonstrated the use of treated dentin matrix (TDM) as a biological scaffold and dental follicle cells (DFCs) as the seeding cells for dentinogenesis and tooth root construction. This study proposes a strategy utilizing TDM with human dental follicle cell sheets (DFCSs) for root regeneration. The biological characteristics and changes of human DFCSs under the effect of TDM were studied with scanning electron microscopy, transmission electron microscopy, immunofluorescence microscopy, immunohistochemistry and quantitative real-time PCR. DFCSs combined with TDM were implanted subcutaneously into the dorsum of mice. Histological examination of the harvested grafts revealed a whirlpool-like alignment of the DFCs in multiple layers that were positive for COLI, integrinβ1, fibronectin and alkaline phosphatase (ALP), suggestive of the formation of a rich extracellular matrix. DFCSs, under the effect of TDM, highly expressed DMP-1 and bone sialoprotein (BSP), indicating their potential for odontogenesis and osteogenesis. Importantly, in vivo, TDM could induce and support DFCSs to develop new dentin-pulp like tissues and cementum-periodontal complexes that were positive for markers such as DSP, nestin and VIII factors, COLI and cementum attachment protein (CAP), implying successful root regeneration. Therefore, DFCSs combined with TDM may prove to be a better strategy for the construction of tooth root, and is a prospective approach that could be utilized for the treatment of root or tooth defect or loss in future.

In this study, several in vivo and in vitro comparisons were performed to test the possibility of using adipose-derived stem cells (ADSCs), a more convenient cell source than dental pulp stem cells (DPSCs), in tooth regeneration. Using an efficient, non-engineering implantation method, we first demonstrated that both implants of ADSCs and DPSCs were able to grow self-assembled new teeth in adult rabbit extraction sockets with high success rate. The stem cells were necessary because the implants grew no tooth without them. A stepwise comparison showed that the regenerated teeth from these two types of adult stem cells were living with nerves and vascular system and remarkably similar to a normal tooth in many details. Further strictly controlled, side-by-side comparisons between the two types of stem cells also showed that the expression patterns of gene markers and the broad differentiation potentials induced by specific methods in vitro were very similar. Although a few differences were found, they did not affect the tested tooth regeneration in vivo or differentiation in vitro. Furthermore, rabbit ADSCs had a higher growth rate and a better senescence resistance in culture. All these findings suggest that ADSCs, one of the richest adult stem cells in mammals, are very similar and useful as DPSCs for regenerative dentistry.

Progress is being made toward regenerating teeth by seeding dissociated postnatal odontogenic cells onto scaffolds and implanting them in vivo, but tooth morphology remains difficult to control. In this study, we aimed to facilitate tooth regeneration using a novel technique to sequentially seed epithelial cells and mesenchymal cells so that they formed appropriate interactions in the scaffold. Dental epithelium and mesenchyme from porcine third molar teeth were enzymatically separated and dissociated into single cells. Mesenchymal cells were seeded onto the surface of the scaffold and epithelial cells were then plated on top so that the two cell types were in direct contact. The cell-scaffold constructs were evaluated in vitro and also implanted into immunocompromised rats for in vivo analysis. Control groups included constructs where direct contact between the two cell types was prevented. In scaffolds seed using the novel technique, alkaline phosphatase activity was significantly greater than controls, the tooth morphology in vivo was developed in similar to that of natural tooth, and only one tooth structure formed in each scaffold. These results suggest that the novel cell-seeding technique could be useful for regulating the morphology of regenerated teeth.