Functional extracellular vesicles from SHEDs combined with gelatin methacryloyl promote the odontogenic differentiation of DPSCs for pulp regeneration.

Pulp regeneration is a novel approach for the treatment of immature permanent teeth with pulp necrosis. This technique includes the combination of stem cells, scaffolds, and growth factors. Recently, stem cell-derived extracellular vesicles (EVs) have emerged as a new methodology for pulp regeneration. Emerging evidence has proven that preconditioning is an effective scheme to modify EVs for better therapeutic potency. Meanwhile, proper scaffolding is of great significance to protect EVs from rapid clearance and destruction. This investigation aims to fabricate an injectable hydrogel loaded with EVs from pre-differentiated stem cells from human exfoliated deciduous teeth (SHEDs) and examine their effects on pulp regeneration. We successfully employed the odontogenic induction medium (OM) of SHEDs to generate functional EV (OM-EV). The OM-EV at a concentration of 20 µg/mL was demonstrated to promote the proliferation and migration of dental pulp stem cells (DPSCs). The results revealed that OM-EV has a better potential to promote odontogenic differentiation of DPSCs than common EVs (CM-EV) in vitro through Alizarin red phalloidin, alkaline phosphatase staining, and assessment of the expression of odontogenic-related markers. High-throughput sequencing suggests that the superior effects of OM-EV may be attributed to activation of the AMPK/mTOR pathway. Simultaneously, we prepared a photocrosslinkable gelatin methacryloyl (GelMA) to construct an OM-EV-encapsulated hydrogel. The hydrogel exhibited sustained release of OM-EV and good biocompatibility for DPSCs. The released OM-EV from the hydrogel could be internalized by DPSCs, thereby enhancing their survival and migration. In tooth root slices that were subcutaneously transplanted in nude mice, the OM-EV-encapsulated hydrogel was found to facilitate dentinogenesis. After 8 weeks, there was more formation of mineralized tissue, as well as higher levels of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1). The effects of EV can be substantially enhanced by preconditioning of SHEDs. The functional EVs from SHEDs combined with GelMA are capable of effectively promoting dentinogenesis through upregulating the odontogenic differentiation of DPSCs, which provides a promising therapeutic approach for pulp regeneration.

Lu H ，Mu Q ，Ku W ，Zheng Y ，Yi P ，Lin L ，Li P ，Wang B ，Wu J ，Yu D ，Zhao W ... -  《JOURNAL OF NANOBIOTECHNOLOGY》

Odontogenic Differentiation of Human Dental Pulp Stem Cells on Hydrogel Scaffolds Derived from Decellularized Bone Extracellular Matrix and Collagen Type I.

Paduano F ，Marrelli M ，White LJ ，Shakesheff KM ，Tatullo M ... -  《PLoS One》

Biomimetic pulp scaffolds prepared from extracellular matrix derived from stem cells from human exfoliated deciduous teeth promote pulp-dentine complex regeneration.

To evaluate the role of biomimetic pulp scaffolds derived from the extracellular matrix derived of stem cells from human exfoliated deciduous teeth (SHED-ECM-PS) in promoting pulp-dentine complex regeneration. SHED-ECM-PS was prepared through cell aggregation and decellularization techniques. Histological and immunofluorescence analyses, scanning electron microscopy, and DNA quantification assays were used to characterize the SHED-ECM-PS. Additionally, a tooth slice implantation model was established to evaluate the effects of SHED-ECM-PS on regeneration of the pulp-dentine complex in vivo. Extraction medium for SHED-ECM-PS was prepared, and its effect on bone marrow mesenchymal stem cells (BMMSCs) was assessed in vitro. Cell counting kit-8 and Ki-67 staining assays were performed to determine cell proliferation. The rate of apoptosis was evaluated by flow cytometry. Wound healing and transwell assays were conducted to evaluate cell migration. Alizarin red S staining was performed to examine mineralized nodule formation. Western blotting was used to detect the expression of osteogenic and odontogenic markers. The results were analysed using an independent two-tailed Student's t-test. p < .05 was considered statistically significant. SHED-ECM-PS was successfully constructed, exhibiting a striped dental pulp-like shape devoid of nuclear structures or DNA components, and rich in fibronectin, collagen I, DMP1 and DSPP. Notably, SHED-ECM-PS showed no impact on the proliferation or apoptosis of BMMSCs. Histological analysis revealed that dental pulp fibroblasts formed an interwoven mesh in the root canal, and angiogenesis was observed in the SHED-ECM-PS group. Moreover, a continuous, newly formed tubular dentine layer with polarized odontoblast-like cells was observed along the inner wall of the root canal. SHED-ECM-PS promoted the migration, polar alignment and mineralized nodule formation of BMMSCs and specifically elevated the expression levels of odontogenic markers, but not osteogenic markers, compared with the control group in vitro. SHED-ECM-PS exhibited no cytotoxicity and promoted pulp-dentine complex regeneration in vivo as well as cell migration and odontogenic differentiation of BMMSCs in vitro. These findings provide evidence that SHED-ECM-PS, as a novel biological scaffold, has the potential to improve the outcomes of REPs.

Yang N ，Shen R ，Yang W ，Zhang S ，Gong T ，Liu Y ... -  《-》

Effects of HEMA and TEDGMA on the in vitro odontogenic differentiation potential of human pulp stem/progenitor cells derived from deciduous teeth.

The aim of this study was to investigate the effects of HEMA and TEGDMA on the odontogenic differentiation potential of dental pulp stem/progenitor cells. Dental stem/progenitor cell cultures were established from pulp biopsies of human deciduous teeth of 1-3 year-old children (Deciduous Teeth Stem Cells-DTSCs). Cultures were characterized for stem cell markers, including STRO-1, CD146, CD34, CD45 using flow cytometry. Cytotoxicity was evaluated with the MTT assay. DTSCs were then induced for osteo/odontogenic differentiation by media containing dexamethasone, KH(2)PO(4),β-glycerophosphate and L-ascorbic acid phosphate in the presence of nontoxic concentrations of HEMA (0.05-0.5mM) and TEGDMA (0.05-0.25mM) for 3 weeks. Additionally, the effects of a single exposure (72 h) to higher concentrations of HEMA (2mM) and TEGDMA (1mM) were also evaluated. DTSCs cultures were positive for STRO-1 (7.53±2.5%), CD146 (91.79±5.41%), CD34 (11.87±3.02%) and negative for CD45. In the absence of monomers cell migration, differentiation and production of mineralized dentin-like structures could be observed. Cells also progressively expressed differentiation markers, including dentin sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN and alkaline phosphatase-ALP. On the contrary, long-term exposure to nontoxic concentrations of HEMA and TEGDMA significantly delayed the differentiation and mineralization processes of DTSCs, whereas, one time exposure to higher concentrations of these monomers almost completed inhibited mineral nodule formation. BSP, OCN, ALP and DSPP expression were also significantly down-regulated. These findings suggest that HEMA and TEGDMA can severely disturb the odontogenic differentiation potential of pulp stem/progenitor cells, which might have significant consequences for pulp tissue homeostasis and repair.

Bakopoulou A ，Leyhausen G ，Volk J ，Tsiftsoglou A ，Garefis P ，Koidis P ，Geurtsen W ... -  《-》

Novel metformin-containing resin promotes odontogenic differentiation and mineral synthesis of dental pulp stem cells.

Wang S ，Xia Y ，Ma T ，Weir MD ，Ren K ，Reynolds MA ，Shu Y ，Cheng L ，Schneider A ，Xu HHK ... -  《-》