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: Carbon dots, Extracellular matrix, Dentin-pulp complex regeneration, Autophagy, PI3K/Akt/mTOR signaling pathway Graphical abstract [41]Image 1 [42]Open in a new tab 1. Introduction The damage of dentin-pulp complex caused by caries, pulpitis, pulp necrosis, periapical lesions, and tooth trauma, is a common disease in daily life, which gives rise to unbearable pain, functional loss of the tooth, and eventually loss of quality life [[43]1]. Root canal therapy as a standard of care is utilized in clinical management of this disease, during which all pulp tissues are removed, and then the root canal is enlarged and obturated by the filling materials [[44]2,[45]3]. Nevertheless, this clinical treatment modality is only a cavity filling procedure without the regeneration of the dentin-pulp complex, thus resulting in a devitalized and brittle tooth. Tissue engineering based on stem cells has emerged as an exciting platform for developing therapeutic strategies aimed at the replacement of the damaged tissues and thereby the repair of the physiological function [[46][4], [47][5], [48][6]]. Recent studies have demonstrated that human dental pulp stem cells (DPSCs) have been successfully engineered to regenerate the dentin-pulp complex, where a scaffold is usually required to load the DPSCs to decrease the loss of DPSCs compared to isolated stem cell suspensions [[49][7], [50][8], [51][9]]. Yet most scaffolds are exogenous and suffer from potential immune rejection [[52]10]. As a natural, multifunctional and endogenous scaffold, extracellular matrix (ECM) secreted by DPSCs can store growth factors, promote cellular differentiation, regulate intercellular communication, and particularly provide structural and biochemical support to surrounding cells [[53]11]. Further, ECM could promote cell adhesion on ECM and makes it possible to develop DPSCs-based cell sheet engineering for the dentin-pulp complex regeneration without immune rejection [[54]12,[55]13]. However, owing to the limited production of ECM, the DPSCs show a low viability and the resultant cell sheet is usually fragile or broken, leading to an inefficient regeneration of the dentin-pulp complex [[56]14]. Consequently, it is significant to explore an effective way to enhance the ECM secretion of DPSCs for improving the structural integrity of the cell sheet and further promoting the dentin-pulp complex regeneration. Currently, one strategy to promote the ECM secretion is to utilize growth factors [[57][15], [58][16], [59][17]], while the extraction processes are quite complex and the products are hard to be preserved for a long time at room temperature, thus resulting in a high cost and a limited application [[60]18]. Besides, ascorbic acid as a reductant can induce the synthesis of collagen (one of the major components of ECM) through reducing Fe^3+ to Fe^2+ and ensuring the hydroxylase reaction cycle, but the easy oxidizability of ascorbic acid causes concern about their long-term stability in storage [[61]19]. Nanomaterials have gained increasing attention in biology owing to their distinct advantages, including facile preparation [[62]20], low cost [[63]21], long-term preservation [[64]22], abundant surface groups [[65]23], and so forth [[66][24], [67][25], [68][26]]. Several kinds of nanomaterials, such as Ag nanoparticle [[69]27], TiO[2] nanotube [[70]28], and carbon nanotube [[71][29], [72][30], [73][31]], have been demonstrated to be capable of increasing the collagen secretion in non-stem cells, whereas there are few studies about their applications in stem cells. More importantly, although collagen plays a key role in cell-ECM adhesion, another two components of ECM (namely, fibronectin and integrin) are needed as well, since the establishment of the cell-ECM connections is via the binding of fibronectin to collagen and cell-surface integrin [[74]11]. With excellent biocompatibility, strong fluorescence properties, and nanoscale size, carbon dots (CDots) as an emerging class of carbon-based nanomaterials have great potential in various biomedical applications, including anti-inflammation [[75]32], bacteriostasis [[76]33], bioimaging [[77]34] and so on [[78]35,[79]36]. A majority of CDots are usually prepared from the organic precursors with carbonyl and amine groups through amidation reaction similar to the formation of peptide bond in protein. Thus some CDots share similar properties as biomacromolecule, such as enzyme [[80]37,[81]38]. Therefore, these accomplishments inspire us to develop a novel CDots-based medicine to address the challenges of promoting the ECM secretion of DPSCs and the dentin-pulp complex regeneration thereof. In this study, we reported the synthesis of a blue photoluminescent CDots with excellent biocompatibility through one-step hydrothermal treatment of ascorbic acid and polyethyleneimine (PEI), and offered an approach to regenerate the dentin-pulp complex, which is hardly achieved by other CDots that have been reported. The as-prepared CDots were characterized by abundant functional groups along with a large π-conjugated structure, and could induce a reasonable activation of the autophagy of DPSCs through phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway, which was confirmed by transcriptome analysis, Western blot assay and molecular dynamics simulation. As a result, DPSCs treated by CDots could secret more ECM-related proteins, including fibronectin, integrin β1 and collagen type Ι (COL1), leading to the cell-ECM adhesion reinforcement, and a stronger osteogenic/odontogenic differentiation capacity of DPSCs, all of which promoted in vivo the efficient regeneration of dentin-pulp complex as well as blood vessels ([82]Scheme 1). Scheme 1. [83]Scheme 1 [84]Open in a new tab Schematic illustration of the synthetic procedure of CDots and the working mechanism for promoting dentin-pulp complex regeneration. 2. Materials and methods 2.1. Materials Ascorbic acid (99.99%) and polyethyleneimine (PEI, MWCO ​= ​1800 D, 99%) were purchased from Macklin (Shanghai, China) and Aladdin Ltd (Shanghai, China), respectively. Dulbecco's modified Eagle medium (DMEM) with high glucose and penicillin-streptomycin were purchased from Hyclone (Logan, UT, USA). Fetal bovine serum was obtained from Biological Industries (Cromwell, CT, USA). Cell counting kit-8 (CCK-8) was purchased from Apexbio Technology LLC (Houston, TX, USA). Cell cycle and Annexin V-APC apoptosis analysis kit were obtained from Sungene Biotech (Tianjin, China). RNeasy mini purification kit was purchased from Qiagen (Valencia, CA, USA), PrimeScript RT Master Mix and SYBR Premix Ex Taq were from TaKaRa (Dalian, China). Protease/phosphatase inhibitor cocktail was obtained from MedChenExpress LLC (Monmouth Junction, NJ, USA). Radioimmunoprecipitation assay (RIPA) buffer, 4,6-diamidino-2-phenylindole (DAPI), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and bovine serum albumin (BSA) were purchased from Beyotime (Shanghai, China). Secondary antibodies and Cy3-labeled IgG antibody were from Proteintech (Rosement, IL, USA). Ascorbic acid, β-glycerophosphate disodium salt hydrate, alizarin red S (ARS), alkaline phosphatase (ALP) staining kit, dexamethasone, indomethacin, insulin, 3-isobutyl-1-methylxanthine and Oil Red O were purchased from Sigma Aldrich Co. (St Louis, MO, USA). Spautin-1 was purchased from Selleckchem (Houston, TX, USA). SC79 and 740Y–P were obtained from MCE (Monmouth Junction, NJ, USA). 2.2. Synthesis and characterization of carbon dots (CDots) In this study, CDots were synthesized according to the following procedures. First, 1 ​g of ascorbic acid and 0.5 ​g of PEI were dissolved in 15 ​mL of deionized water. The clear solution was transferred into a 20 ​mL Teflon-lined stainless-steel autoclave and placed into an oven heated at 200 ​°C. After 8 ​h, the autoclave was cooled to room temperature and the CDots aqueous solution was obtained. Afterwards, to purify the CDots, the as-prepared CDots solution was dialyzed in deionized water using a dialysis bag (MWCO of 1000) for 48 ​h to remove large particles including unreactive PEI and other agglomerated particles. Then, the vacuum rotary evaporator was used to concentrate the dialysate outside dialysis bag by the evaporation of water. The concentrated sample was further dialyzed in deionized water using a dialysis bag (MWCO of 100–500) for 24 ​h to remove small molecular reagents. Next, the purified CDots solution was condensed and free-dried. Finally, The CDots were redissolved in deionized water for the following experiments. For the characterization of the CDots, ultraviolet–visible (UV–vis) absorption spectra were collected using a Lambda 800 UV–vis spectrophotometer. Photoluminescence (PL) spectroscopy was acquired using a Shimadzu RF-5301 PC spectrophotometer. To obtain the images of high-resolution transmission electron microscopy, the CDots solution was dropped on the micro grid copper mesh, dired in the oven, and the as-prepared sample was then observed with a Hitachi H-800 electron microscope at an acceleration voltage of 200 ​kV with a CCD camera. Zeta potential of the CDots was analyzed by dynamic light scattering (DLS) (Zetasizer NanoZS, Malvern Instruments, Britain) at a neutral pH and room temperature. Energy-dispersive spectra (EDS) was performed with the help of Inca X-Max instrument (Oxford Instruments). For detecting functional groups in the CDots, Fourier transform infrared (FTIR) spectra were implemented by a Nicolet AVATAR 360 FTIR instrument. For measuring the elemental composition of the CDots, the CDots solution was dropped on the silicon wafer, and then the sample was dried at room temperature. X-ray photoelectron spectroscopy (XPS) was conducted using a VG ESCALAB MKII spectrometer with a Mg KR excitation (1253.6 ​eV). Binding energy calibration was based on C 1s at 284.6 ​eV. 2.3. In vitro cell culture, cytotoxicity assays and cell imaging All procedures were approved by the Medical Ethics Committee of Stomatological Hospital of Jilin University (approval number: 202045), and all samples were collected with informed consents. Human dental pulp stem cells (DPSCs) were isolated from adult third molars and incubated in DMEM with high glucose containing 10% FBS, 100 U/mL of penicillin and 100 ​μg/mL of streptomycin. DPSCs were cultured in a humidified environment at 37 ​°C under 5% CO[2] and culture medium was replaced every 2–3 days. DPSCs at passages 3–4 were used for this study. The multiple differentiation capacity of DPSCs was identified by alizarin red S (ARS) and Oil Red O staining. DPSCs were seeded into 6-well plates at a density of 1 ​× ​10^5 ​cells/well and cultured overnight. Culture medium was replaced with osteogenic induction medium, containing 50 ​μg/mL of ascorbic acid and 10 ​mmol/L of β-glycerophosphate for 21 days. The cells were fixed with 4% paraformaldehyde for 30 ​min and then stained with ARS for 15 ​min. Then the general photograph of ARS staining was obtained by a scanner and the red nodules were observed under an inverted microscope. For adipogenic induction, culture medium was replaced with adipogenic induction solution, containing 2 ​μM of dexamethasone, 0.2 ​mM of indomethacin, 0.01 ​mg/mL of insulin and 0.5 ​mM of 3-isobutyl-1-methylxanthine. The medium was changed every 3 days, after which the induction and fixation steps were performed, and 0.3% Oil Red O (Cyagen Biosciences) staining was used for 30 ​min at room temperature. The adipogenic droplets were then observed under an inverted microscope. The phenotype of DPSCs was identified by flow cytometry (FCM, BD Biosciences, San Jose, CA, USA) using CD44 (Biolegend, San Diego, CA, USA), CD90 (Biolegend) and CD105 (Biolegend) as positive surface markers, CD3 (Biolegend), CD45 (Biolegend), CD14 (Proteintech) and CD 34 (Proteintech) as negative surface markers. In vitro cytotoxicity of CDots was assessed by CCK-8, cell cycle and apoptosis assays. DPSCs were seeded at 5 ​× ​10^3 ​cells/well in a 96-well plate and cultured overnight. Culture medium was replaced with 200 ​μL of medium containing different concentrations of CDots at 0, 20, 50, 100, 200, or 300 ​μg/mL, respectively. After 24 ​h of incubation, the medium was removed, and 100 ​μL of medium containing 10 ​μL of CCK-8 reagent was added into each well. After 30 ​min of incubation, the absorbance was measured at 450 ​nm using a microplate reader (RT-6000; Rayto Life and Analytical Science Co, Shenzhen, China). Percentage of cell viability was calculated compared to the control group. DPSCs were seeded in a 6-well plate and cultured overnight. Culture medium was replaced with 2 ​mL of medium containing CDots at 0, 20, 50, 100, or 200 ​μg/mL, respectively. After 24 ​h of incubation, cells were collected, washed twice with cold PBS. Subsequently, for cell cycle assay, cells were stained by PI/RNase staining solution. For cell apoptosis assay, cells were resuspended by 100 ​μL of binding buffer and stained using 5 ​μL of Annexin-V-FITC and 5 ​μL of 7-AAD working solution according to the manufacturer's instructions. Finally, these stained cells were analyzed with flow cytometry in 1 ​h. DPSCs were seeded at 1 ​× ​10^5 ​cells/well in a 6-well plate and cultured overnight. Then, culture medium was replaced with 2 ​mL of medium containing CDots at 200 ​μg/mL. After 4 ​h of incubation, the cells were fixed in 4% paraformaldehyde for 10 ​min at room temperature, washed twice with PBS and observed by confocal laser scanning microscopy (CLSM, Olympus, Japan). 2.4. Western blot analysis DPSCs were seeded at 1 ​× ​10^6 ​cells/dish in 10 ​cm cell culture dishes and cultured overnight. Then the culture medium was replaced with 10 ​mL of medium containing CDots at 0, 50, 100 and 200 ​μg/mL. Cells were washed twice with PBS and lysed in RIPA buffer containing protease and phosphatase inhibitor. Then proteins were quantified using a BCA protein assay. Subsequently, 30 ​μg of protein from each group was separated by 10% SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was first incubated in 5% BSA for 1 ​h at room temperature for blocking nonspecific binding sites and then incubated with the following primary antibodies overnight at 4 ​°C: anti-GAPDH (10494-1-AP, 1:2000 (Proteintech)), anti-fibronectin (15613-1-AP, 1:1000 (Proteintech)), anti-integrin β1 (26918-1-AP, 1:1000 (Proteintech)), anti-collagen type Ι (14695-1-AP, 1:1000 (Proteintech)), anti-E-cadherin (20874-1-AP, 1:1000 (Proteintech)), anti-DSPP (SC-73632, 1:100 (Santa Cruz)), anti-DMP1 (SC-73633, 1:100 (Santa Cruz)), anti-BSP (SC-73630, 1:100 (Santa Cruz)), anti-LC3B (18725-1-AP, 1:1000 (Proteintech)), anti-P62 (18420-1-AP, 1:1000 (Proteintech)), anti-Beclin1 (11306-1-AP, 1:1000 (Proteintech)), anti-p-Akt (9271S, 1:1000 (CST)), anti-Akt (9272S, 1:1000 (CST)), anti-p-mTOR (67778-1-Ig, 1:1000 (Proteintech)) and anti-mTOR (66888-1-Ig, 1:1000 (Proteintech)). The following day, the membrane was incubated with the secondary antibodies at room temperature for 1 ​h. Finally, enhanced chemiluminescence reagent was used to visualize the protein bands and ImageJ software was used to analyze the band density. To further evaluate the role of autophagy, Akt and PI3K in CDots-enhanced secretion of ECM, spautin-1, SC79 and 740Y–P were added and the same procedure was used. 2.5. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) DPSCs were cultured in 6-well plates with 2 ​mL of medium containing CDots at 0 and 200 ​μg/mL for 3, 7, 10 and 14 days. Total RNA was extracted using an RNeasy mini purification kit, and cDNAs were synthesized using iScript cDNA synthesis kit. The RT-qPCR assays were conducted using SYBR Premix Ex Taq and MxPro Mx3005P real-time PCR detection system (Agilent Technologies, Santa Clara, CA). Primer sequences are listed in [85]Table S1. ACTB was used as an internal control. 2.6. Immunofluorescence staining DPSCs were seeded on coverslips at 1 ​× ​10^4 ​cells/well in a 6-well plate and cultured overnight. Then the medium was replaced with 2 ​mL medium containing CDots at 0 and 200 ​μg/mL. After 24 ​h of incubation, the cells were fixed by 4% paraformaldehyde for 20 ​min and permeabilized with 0.2% Triton X-100 for 5 ​min at room temperature. 5% BSA was used to block nonspecific binding sites for 1 ​h at room temperature. Then, the cells were incubated with fibronectin antibody (15613-1-AP, 1:500 (Proteintech)), collagen type Ι antibody (14695-1-AP, 1:500 (Proteintech)) and integrin β1 antibody (26918-1-AP, 1:500 (Proteintech)) at 4 ​°C overnight. The following day, the cells were incubated with Cy3-labeled secondary antibody at room temperature for 1 ​h in the dark. Finally, the cells were treated with DAPI for 5 ​min and captured by an inverted fluorescence microscope. 2.7. Alkaline phosphatase staining DPSCs were seeded into 6-well plates at a density of 1 ​× ​10^5 ​cells/well and cultured overnight. Culture medium was replaced with osteogenic induction medium, containing 50 ​μg/mL of ascorbic acid and 10 ​mmol/L of β-glycerophosphate for 7 days and 14 days. Then, cells were fixed in 4% paraformaldehyde for 15 ​min at room temperature and incubated with the ALP staining kit following the manufacturer's instruction. The stained cells were observed and imaged using an inverted microscope. 2.8. Fabrication and observation of dental pulp stem cell sheets DPSCs were seeded at 1 ​× ​10^6 ​cells/dish in 10 ​cm cell culture dishes and cultured overnight in an incubator that is dark environment. Then the culture medium was replaced with 10 ​mL of medium containing CDots at 200 ​μg/mL. After an additional 10 days of culture, dental pulp stem cell sheet was harvested. Then, the cell sheet of each group was observed by an inverted fluorescence microscope to confirm the CDots imaging in the cell sheet. Finally, the cell sheet was fixed by 4% paraformaldehyde and underwent dehydration, paraffin-embedding, sectioning, and hematoxylin and eosin (H&E) staining. The structure of the cell sheet was observed by microscope. 2.9. Transmission electron microscope (TEM) DPSCs were seeded at 1 ​× ​10^6 ​cells/dish in 10 ​cm cell culture dishes and cultured overnight. Then the culture medium was replaced with 10 ​mL of medium containing CDots at 0 and 200 ​μg/mL. After 24 ​h of incubation, cells were harvested and fixed in 4% glutaraldehyde for 4 ​h, dehydrated with graded ethanol and embedded in epon. Ultrathin sections were stained with citrate and uranyl acetate and observed using TEM (TECNAI SPIRIT, FEI Company, Czech Republic). 2.10. Transcriptome analysis by RNA sequencing Total RNA was extracted using the TRIzol reagent according to the manufacturer's protocol. RNA purity and quantification were measured using the NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Then the libraries were constructed using TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. The transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). mRNA and RNA sequencing were performed to investigate the global expression profile of DPSCs and CDots-treated DPSCs (n ​= ​3), and 150 bp paired-end reads were generated. Raw data (raw reads) of fastq format were firstly processed using Trimmomatic and the low quality reads were trimmed to obtain the clean reads. Then the clean reads were mapped to the human genome (GRCh38) using HISAT2. FPKM of each gene was calculated using Cufflinks, and the read counts of each gene were obtained by HTSeq-count. Differential expression analysis was obtained using the DESeq (2012) R package. P value ​< ​0.05 and foldchange >2 or foldchange <0.5 was set as the threshold for significantly differential expression. Hierarchical cluster analysis of differentially expressed genes (DEGs) was performed to demonstrate the expression pattern of genes in different groups and samples. GO enrichment and KEGG pathway enrichment analysis of DEGs were conducted respectively using R based on the hypergeometric distribution. 2.11. Homology modeling and molecular dynamics (MD) simulations The homology modeling was performed via MODELLER package, where the whole FASTA sequence of PI3K (i.e., p110α and p85α) were retrieved from RCSB Protein Data Bank (PDB; the PDB ID is4ovu), and the structures from PDB entry 4ovu were selected as the overall chain templates. The resultant homology models were evaluated by Ramachandran plot analysis. The simplified model of CDots was proposed based on the HRTEM and XPS analysis and optimized by using density functional theory method as carried out in the Gaussian09 software package. The 6-31G∗ basis set was selected to combine with the functional B3LYP throughout all calculations (B3LYP/6-31G∗). All the structures were visualized by the Visual Molecular Dynamics (VMD) program. The MD simulation was carried out utilizing the GROMACS package version 2018.8 under AMBER99SB-ILDNP force field for all-atom simulations. Transferable intermolecular interaction potential 3 points (TIP3P) water molecules were used to solvate the system in a cubic box with a minimum distance of 10 ​Å between the protein and the edge of the box. The extra charges of the system were neutralized with Na^+ ions. Periodic boundary conditions were used in all directions to mimic the infinite system. Steepest descents and/or conjugate gradient minimization (with a tolerance of up to 1000 ​kJ/mol/nm) were made to eliminate any adverse interaction. Before the production simulation, a two-step equilibration was made. During these two stages, all atoms of the system were restrained in position to prevent any conformational change. The systems were first simulated under a constant volume (NVT) ensemble to achieve 300 ​K by the V-rescale method for 1 ​ns? Then, the equilibrated structures from the NVT ensemble were subjected to constant pressure (NPT) equilibration (1 ​ns) using the Parrinello-Rahman barostat under an isotropic pressure of 1.0 ​bar. Production MD was conducted for 120 ns without any restraints. The trajectories from the simulations were saved for every 10 ps for analysis of root mean square deviation (RMSD), root mean square fluctuation (RMSF) as well as the protein-ligand contacts. The binding energy was calculated by the molecular mechanics Poisson-Boltzmann accessible surface area (MM-PBSA) method. 2.12. Fabrication of human treated dentine matrix fragments (hTDMFs) and dentin-pulp complex regeneration in vivo Briefly, the premolar teeth were collected and periodontal ligament tissues were removed mechanically. Then, outer cementum, inner dental pulp, predentin and partial root dentin were also ground carefully. The hTDMFs were cut into 5–7 ​mm sections and the root space was formed to a diameter of 2–4 ​mm. Next, the hTDMFs were soaked in deionized water for 5 ​h and meanwhile concussed using an ultrasonic cleaner for 20 ​min each hour. The deionized water was changed once every hour. Then, the hTDMFs were first soaked in 17% ethylenediamine tetraacetic acid (EDTA) for 5 ​min, washed in deionized water for 10 ​min, then exposed to 5% EDTA for 10 ​min and washed in deionized water for 10 ​min. The hTDMFs were exposed to sterile PBS with 100 units/mL of penicillin and 100 ​μg/mL of streptomycin for 3 days, then washed in sterile deionized water for 10 ​min. Finally, the hTDMFs were stored in DMEM at 4 ​°C. All animal procedures were performed in accordance with animal care guidelines approved by the Animal Ethics Committee of No. 1 Hospital of Jilin University (approval number: 20200470). Cell sheets in each group were put into the cavity of hTDMFs and then the grafts were transplanted into subcutaneous space at the back of 6 weeks old male BALB/c nude mice. Fifteen weeks later, the mice were euthanized and all samples were harvested for histological analysis. All the animal experiments were conducted in accordance with the committee guidelines of Jilin University for animal experiments. All implants were fixed in 4% paraformaldehyde overnight at 4 ​°C, decalcified with 10% EDTA, embedded in paraffin, and cut into 5 ​μm sections. Finally, deparaffinized sections were stained with H&E. Immunohistochemical antibodies included DSPP antibody (SC-73632, 1:100 (Santa Cruz)), DMP1 antibody (SC-73633, 1:100 (Santa Cruz)) and CD31 antibody ([86]ARG52748, 1:50 (arigobio)). 2.13. Statistical analysis Each experiment was repeated at least three times. All measurements were presented as the mean ​± ​standard deviation. Statistical analyses were performed by one-way ANOVA. A value of P ​< ​0.05 was considered statistically significant. 3. Results and discussion 3.1. Synthesis and characterization of CDots In this study, CDots were synthesized via a one-pot hydrothermal treatment of ascorbic acid and PEI. Afterwards, the as-prepared CDots solution was dialyzed in deionized water using a dialysis bag (MWCO of 1000) for 48 ​h to remove large particles including unreactive PEI and other agglomerated particles. The aqueous solution outside the dialysis membrane, which is mainly composed of CDots and other small molecular reagents, was further dialyzed in deionized water using a dialysis bag (MWCO of 100–500) for 24 ​h to remove small molecular reagents. Thus, these dialysis processes ensure the purity of the CDots solution [[87]33], and the purified CDots solution presented yellow color under sunlight, while under ultraviolet (UV) light a bright blue photoluminescence (PL) emission could be observed. [88]Fig. 1a showed the UV–vis absorption spectrum of the CDots solution, which was obviously different from those of the raw material ([89]Fig. S1), indicating the formation of CDots after the hydrothermal treatment, which was also confirmed by the variations of their PL spectra ([90]Fig. 1a and [91]S1). The PL spectrum of the CDots solution exhibited a peak located at 460 ​nm at an excitation wavelength of 350 ​nm, while both ascorbic acid and PEI had no PL emission under UV light ([92]Fig. S1a and b). Additionally, the excitation-emission map of the CDots solution presented a single emission centered at 460 ​nm ([93]Fig. 1b), suggesting the size uniformity and providing an efficient way to examine the behaviors of CDots in biological systems therewith. To elucidate their morphology, high-resolution transmission electron microscopy (HRTEM) was used to characterize the morphology and structure of CDots. As shown in [94]Fig. 1c, CDots possessed a uniform size with an average diameter of 2.5 ​nm, which was beneficial to enter into the cell and thereby exert the pharmacological function of CDots. The well-resolved lattice spacing of 0.21 ​nm conforms to the (100) crystallographic facets of graphitic carbon [[95]39], indicating the formation of a graphene-like structure. Besides, the zeta potential of CDots in aqueous solution was measured to be 15.7 ​mV ([96]Fig. S2). This positive potential is mainly due to the presence of plentiful amine groups in CDots originated from PEI, which can be confirmed by the following characterizations, and this positive value also facilitates the entry of CDots into cells, since the cell membrane usually possesses a negative potential [[97]40]. Energy dispersive spectrum (EDS) demonstrated the existence of C, N and O elements in CDots ([98]Fig. S3). All of O and a portion of C were from ascorbic acid, whereas PEI provided all of N and the other portion of C. To further account for the chemical structure of CDots, Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS) characterizations were conducted. Fig. 1. [99]Fig. 1 [100]Open in a new tab 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