Abstract
BackgroundAlthough adult human tissue-derived epidermal stem cells are capable of differentiating into enamel-secreting ameloblasts and forming teeth with regenerated enamel when recombined with mouse dental mesenchyme that possesses odontogenic potential, the induction rate is relatively low. In addition, whether the regenerated enamel retains a running pattern of prism identical to and acquires mechanical properties comparable with human enamel indeed warrants further study.MethodsCultured human keratinocyte stem cells (hKSCs) were treated with fibroblast growth factor 8 (FGF8) and Sonic hedgehog (SHH) for 18 h or 36 h prior to being recombined with E13.5 mouse dental mesenchyme with implantation of FGF8 and SHH-soaked agarose beads into reconstructed chimeric tooth germs. Recombinant tooth germs were subjected to kidney capsule culture in nude mice. Harvested samples at various time points were processed for histological, immunohistochemical, TUNEL, and western blot analysis. Scanning electronic microscopy and a nanoindentation test were further employed to analyze the prism running pattern and mechanical properties of the regenerated enamel.ResultsTreatment of hKSCs with both FGF8 and SHH prior to tissue recombination greatly enhanced the rate of tooth-like structure formation to about 70%. FGF8 and SHH dramatically enhanced stemness of cultured hKSCs. Scanning electron microscopic analysis revealed the running pattern of intact prisms of regenerated enamel is similar to that of human enamel. The nanoindentation test indicated that, although much softer than human child and adult mouse enamel, mechanical properties of the regenerated enamel improved as the culture time was extended.ConclusionsApplication of FGF8 and SHH proteins in cultured hKSCs improves stemness but does not facilitate odontogenic fate of hKSCs, resulting in an enhanced efficiency of ameloblastic differentiation of hKSCs and tooth formation in human–mouse chimeric tooth germs.
Highlights
Adult human tissue-derived epidermal stem cells are capable of differentiating into enamel-secreting ameloblasts and forming teeth with regenerated enamel when recombined with mouse dental mesenchyme that possesses odontogenic potential, the induction rate is relatively low
Enhanced ameloblastic differentiation efficiency of cultured human keratinocyte stem cells (hKSCs) in the presence of fibroblast growth factor 8 (FGF8) and Sonic hedgehog (SHH) our previous studies manifested that hKSCs, when recombined with E13.5 mouse dental mesenchyme, were induced to differentiate into enamelsecreting ameloblasts in the presence of FGF8-asorbed beads, the efficiency was quite low with hKSCs differentiating to ameloblasts in 13 out of 41 formed teeth from 146 tissue recombinants [22]
This prompted us to investigate whether application of FGF8/SHH-absorbed beads, instead of FGF8-absorbed beads alone, in the human–mouse chimeric tooth germ could increase the efficiency of tooth formation and ameloblastic differentiation
Summary
Adult human tissue-derived epidermal stem cells are capable of differentiating into enamel-secreting ameloblasts and forming teeth with regenerated enamel when recombined with mouse dental mesenchyme that possesses odontogenic potential, the induction rate is relatively low. Dental pulp stem cells (DPSCs), stem cells from exfoliated deciduous teeth (SHED), and stem cells from the apical papilla (SCAP) could generate dentin/pulp-like complexes in ex-vivo culture [17,18,19] These adult dental stem cells do not possess either odontogenic inducing capability or competence to support tooth formation when confronted with embryonic dental epithelia [20], they remain promising stem cell sources for regeneration of tooth mesenchymal components. Less than 30% and 10% of these recombinant explants in subrenal culture formed teeth and produced enamel, respectively [22] Such low efficiency of ameloblastic differentiation prevents use of these human stem cells as realistic cell sources for tooth replacement therapy. Whether hKSC-derived dental epithelia exhibit an unusual life cycle and whether the regenerated enamel acquires the unique physicochemical characteristics remain elusive and warrant further exploration
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