Carbon dots enhance extracellular matrix secretion for dentin-pulp complex regeneration through PI3K/Akt/mTOR pathway-mediated activation of autophagy

Abstract

Pulp injury is one of the most common clinical diseases, and severe cases are usually associated with the functional loss of the tooth, while the current clinical treatment modality is only a cavity filling procedure without the regeneration of the dentin-pulp complex, thus leading to a devitalized and brittle tooth. In this study, carbon dots (CDots) with excellent biocompatibility are prepared from ascorbic acid and polyethyleneimine via a hydrothermal method. The as-prepared CDots can enhance extracellular matrix (ECM) secretion of human dental pulp stem cells (DPSCs), giving rise to increased cell adhesion on ECM and a stronger osteogenic/odontogenic differentiation capacity of DPSCs. Further, the mechanism underlying CDots-enhanced ECM secretion is revealed by the transcriptome analysis, Western blot assay and molecular dynamics simulation, identifying that the pharmacological activities of CDots are originated from a reasonable activation of the autophagy, which is mediated by regulating phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway. Based on the abundant CDots-induced ECM and thereby the reinforcement of the cell-ECM adhesion, an intact dental pulp stem cell sheet can be achieved, which in return promote in vivo the efficient regeneration of dentin-pulp complex as well as blood vessels.

Keywords: Autophagy; Carbon dots; Dentin-pulp complex regeneration; Extracellular matrix; PI3K/Akt/mTOR signaling pathway.

Graphical abstract

Scheme 1

Schematic illustration of the synthetic procedure of CDots and the working mechanism for promoting dentin-pulp complex regeneration.

Fig. 1

Characterizations and cytotoxicity assays of CDots. (a) UV–vis absorption (black) and PL emission spectra (red) of CDots. Insets: photographs of CDots solution taken under sunlight (left) and UV light (right), respectively. (b) Excitation-emission map of CDots. (c) HRTEM image of CDots. (d) FTIR spectra of ascorbic acid (black), PEI (blue), and CDots (red). (e–f) High-resolution XPS spectra of CDots: (e) C 1s and (f) N 1s. (g) CCK-8 assay of DPSCs treated with CDots at different concentrations of 20, 50, 100, 200, and 300 ​μg/mL on day 1 and 7, respectively. Data are presented as mean ​± ​SD from three independent experiments. ∗ indicates P ​< ​0.05 vs. vehicle. (h) Cell cycle assay and (i) apoptosis assay of DPSCs treated with CDots at different concentrations of 20, 50, 100, and 200 ​μg/mL, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Cell imaging of CDots-treated DPSCs and ECM secretion of DPSCs enhanced by CDots. (a) Cell imaging of CDots-treated DPSCs. (a1) CLSM image, (a2) optical image and (a3) merged image of optical and CLSM images. (b) Western blot analysis of ECM-related proteins: fibronectin, integrin β1 and COL1. (c) Corresponding quantitative data from Western blot. The relative expressions are fibronectin/GAPDH, integrin β1/GAPDH and COL1/GAPDH, respectively. (d) Detection of mRNA levels of fibronectin, integrin β1 and COL1 by RT-qPCR on day 3, 7 and 10. (e) Immunofluorescence staining for ECM-related proteins by fluorescence microscopy. Data are presented as mean ​± ​SD from three independent experiments. ∗ indicates P ​< ​0.05, ∗∗ indicates P ​< ​0.01.

Fig. 3

Osteogenesis/odontogenesis of DPSCs and cell sheet formation induced by CDots. (a and b) ALP staining on day 7 and 14. (c) Corresponding quantitative data of ALP. (d and e) Validation of osteogenic/odontogenic genes expression by RT-qPCR on day 7 and 14. (f) Western blot analysis of osteogenic/odontogenic-related proteins: BSP, DSPP and DMP1. (g) Corresponding quantitative data from Western blot. The relative expressions are BSP/GAPDH, DSPP/GAPDH and DMP1/GAPDH, respectively. (h) Photographs of cell sheet, where the red and blue arrows represent the scraps and holes of the cell sheet in the control group, respectively. (i) Morphology of cell sheet observed by fluorescence microscope. (j and k) H&E staining of the cell sheet. (l and m) IHC staining of ECM-related proteins in the cell sheet. Data are presented as mean ​± ​SD from three independent experiments. ∗ indicates P ​< ​0.05, ∗∗ indicates P ​< ​0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

CDots-promoted ECM secretion by activating autophagy via regulating PI3K/Akt/mTOR pathway. (a) Volcano diagram and (b) heat map of differentially expressed genes. (c) Signaling pathway where FOXA2 is enriched. (d) Western blot results of autophagy-related proteins (Beclin1 and LC3B) and autophagy substrate protein (P62) in DPSCs treated with CDots (200 ​μg/mL) from 0 to 24 ​h. (e) TEM images. Blue arrow indicates lysome, green arrow indicates mitochonsria, yellow arrow indicates autophagosome, and red arrow indicates autolysosomes. The two images in the left panel are taken under low magnification, and the images in the middle and right panels, which are taken under high magnification, are the enlargement of several specific areas of the corresponding images in the left panel. (f) Western blot results of Beclin1, P62, LC3B and ECM-related proteins (fibronectin, integrin β1, and COL1) in DPSCs treated without or with CDots (200 ​μg/mL) and spautin-1, respectively. (g) Western blot results of p-Akt, Akt, p-mTOR, and mTOR. DPSCs were treated without or with CDots (200 ​μg/mL) from 0 to 24 ​h. (h) Western blot results of Beclin1, P62, LC3B in DPSCs treated without or with CDots (200 ​μg/mL) and 740Y–P, respectively. (i) The structures of CDots binding to p110α/p85α complex, which are extracted from the molecular dynamics simulation trajectory at 0, 60, and 120 ​ns, respectively. Secondary structural elements are depicted as ribbons. Color is based on secondary structures (α-helix, purple; 3–10 helix, blue; β-sheets, yellow; turn, cyan; coil, white). (j) The numbers of residues in diverse secondary structures of (j1) p110α and (j2) p85α. (k) RMSD of the Cα atoms of p110α/p85α in p110α/p85α/CDots complex against time. (l) RMSF values of p110α in the p110α/p85α complex and p110α/p85α/CDots complex, respectively. (m) 3D diagram of interaction between CDots and p110α. Amino acid residues involved in the binding of CDots are drawn as sticks, and CDots are shown as ball-and-stick model. Carbon, hydrogen, oxygen and nitrogen atoms are colored in green, gray, red and blue, respectively. (n) 2D diagram of interaction between CDots and p110α. Interactions involved in the binding of CDots to amino acid residues are shown as dashed lines in different corresponding colors. Data are presented as mean ​± ​SD from three independent experiments. ∗ indicates P ​< ​0.05, ∗∗ indicates P ​< ​0.01. “−” and “+” symbols represent “without” and “with,” respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

CDots-induced cell sheet enabled regeneration of dentin-pulp complex tissue in vivo. (a) Schematic diagram of hTDMF transplantation in nude mice. Cell sheet was inserted into hTDMF before subcutaneously transplanted into nude mice. (b–d) H&E staining for histological analysis of tissues regenerated in hTDMF after subcutaneous transplantation: (b) control group, (c) untreated DPSCs group and (d) CDots-treated DPSCs group. Blue arrows show the newly formed dentin. Green arrows show odontoblast-like cells that aligned the newly formed dentin surface. Red arrows present the newly formed blood vessel. nd, newly formed dentin; np, new formed pulp; bv, blood vessel. (e–g) Masson's Trichrome staining: (e) control group, (f) untreated DPSCs group and (g) CDots-treated DPSCs group. Green arrows show odontoblast-like cells that aligned the newly formed dentin surface. Red arrows present the newly formed blood vessel. (h–m) IHC staining of DSPP, DMP1 and CD31 and the quantitative analysis. Blue arrows show DSPP. Green arrows show DMP1. Red arrows show CD31-positive cells. Data are presented as mean ​± ​SD from three independent experiments. ∗∗ indicates P ​< ​0.01. N.D., not detected. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)