Experimental design and statistical rationale
All studies included three biological replicates. The two experimental groups were: (1) SHED and (2) SHED + HPL. The SHED group was a hydrogel supplemented with components FBS. The SHED + HPL group was the hydrogel supplemented with HPL. Both FBS (Cat. No. C04001-050) and HPL (Cat. No. PLTGOLD100R) were purchased from Biological Industries (BI, Israel). Significance analysis was performed. Differentially expressed proteins, p < 0.05 while fold change ≥ 1.5, were considered significant.
Hydrogel preparation and stem cell culture
Modified gelatin and oxidized gellan gum were prepared as previously reported [15, 22]. A schematic diagram of the preparation of hydrogel and SHED cultures was shown in Fig. 1. Chitosan (Sigma-Aldrich, MO, USA), gelatin (Sigma-Aldrich, MO, USA) and GG (Sigma-Aldrich, MO, USA) solutions were prepared by dissolving each polymer in dulbecco’s minimum essential medium (DMEM, BI, Israel) containing 10% FBS or HPL, respectively. Before preparing the hydrogel, the chitosan solution and gelatin polymer solution were filtered at 37 °C using Whatman FP 30/0.2 CA-S sterile filters (Thermo Fisher Scientific, MA, USA), and the GG solution was filtered using Sterivex- GP 0.22 μm filtered Millipore Express sterile filter (Merck Millipore, MA, USA) at 60 °C. Keep the solution at 37 °C, then mix an equal volume (1:1) of the solution by pipette for a few seconds. In 3D culture experiments, cell suspensions were simultaneously mixed with chitosan, gelatin, and GG during gelation. The cell culture medium for all groups was DMEM, and the SHED group was supplemented with 10% FBS, while the SHED + HPL group was supplemented with 5% HPL. After approximately 20 min of gelation time, spread cell culture medium over the samples.
Scanning electron microscopy
After vacuum freeze-drying, the samples were adhered to the conductive tape of the scanning electron microscope (SEM) base, and were coated by ion sputtering instrument and observed by scanning electron microscope. A scanning electron microscope (SEM, IdC-8010, Japan) was used to observe the physical morphology of the hydrogel at an acceleration voltage of 3 kV.
Cell isolation and culture
The stem cells from human exfoliated deciduous teeth were separated according to the previous method . This study was approved by the ethical committee of Peking University Third Hospital, and all the participants was obtained informed consent (2021144-02). The study was conducted in accordance with ethical approval granted by the ethical committee of Peking University Third Hospital and followed the Declaration of Helsinki and informed consent was taken from all individual participants. In short, the healthy deciduous teeth from children between the ages of 6 and 8 with informed consent were collected and preserved. The pulp was exposed after the crown was opened, and then the pulp tissue was extracted and cut into pieces. The digestive mixture, containing 0.3% collagenase I and 0.4% dispase (Sigma-Aldrich, MO, USA), was used to prepare single cell suspension by overnight digestion. SHED was seeded in a 7 cm2 petri dish (Eppendorf, USA) and was inoculated in DMEM (BI, Israel) supplemented with 10% FBS, 1% 100 U/mL penicillin and 100 mg/mL streptomycin (BI, Israel), and 100 μmol L-ascorbic acid (BI, Israel). SHED was placed in an incubator with 37 ℃ constant temperature, specific humidity and 5% CO2. SHED was expanded and cryopreserved after 3–4 weeks in culture. Passages 3 to 5 of SHED were used in the experiments.
Cell proliferation test
A total of 1 × 104 cells of SHED were seeded in 96-well plates and covered with DMEM supplemented with 10% FBS or 5% HPL. After 24 h of culture, methylthiazolyldiphenyl-tetrazolium bromide (MTT, Solarbio, China) was added to the culture medium and incubated for 4 h at 37 °C. After discarding the supernatant, Formazan was dissolved in DMSO (Sigma-Aldrich, MO, USA) and the OD value at 490 nm was measured in the microplate reader. Independent 3 repeated experiments were performed.
SHED were harvested using 0.05% Trypsin–EDTA (BI, Israel) and washed twice in PBS (BI, Israel). Cells were filtered through a 70 mm cell strainer. A total of 1 × 105 SHED cells were prepared into a single cell suspension, fixed with 4% paraformaldehyde (Solarbio, China) and washed with PBS three times. The cells were labeled with CD14 (PE, BD Biosciences, USA), CD19 (PE, BD Biosciences, USA), CD34 (PE, BD Biosciences, USA), CD45 (PE, BD Biosciences, USA), CD73 (FITC, BD Biosciences, USA), CD90 (FITC, BD Biosciences, USA), CD105 (FITC, BD Biosciences, USA) and HLA-DR (PE, BD Biosciences, USA). And the intensity of SHED was analyzed by flow cytometry (BD Biosciences, NJ).
Osteogenesis and adipogenesis induce differentiation
The hydrogel coating was seeded on the bottom of the culture plate. Osteogenic and adipogenic differentiation of SHED were performed according to the human dental pulp stem cell osteogenic differentiation medium kit (Cyagen Biosciences, China) and the human dental pulp stem cell adipogenic differentiation medium kit (Cyagen Biosciences, China), respectively. A total of 1 × 106 cells were seeded in 6-well plates and the induction medium was replaced after the cells grew adherently. Following the protocol for osteogenic differentiation, replace the fresh induced differentiation medium every 3 days. Similarly, according to the adipogenic differentiation protocol, change the fresh induction medium A for 3 days, and then change the fresh induction medium B for 1 day. The calcium nodules and lipid droplets of the cells were stained by Alizarin Red (Cyagen Biosciences, China) and Oil Red O (Cyagen Biosciences, China) after 3–5 weeks of induction, respectively.
Differentiation to neural-like cells
A total of 1 × 105 SHED cells were seeded in 12-well plates and were encapsulated in hydrogel. The fresh neural-like cells induction medium, supplemented with EGF (10 ng/mL, Sigma-Aldrich, MO, USA) and bFGF (10 ng/mL, Sigma-Aldrich, MO, USA), was replaced until the cell fusion rate reaches 80–90%. The neural-like cells markers were detected after 2 weeks.
Peptide preparation and labeling
After SHED were encapsulated in hydrogel and cultured for 3 days, the supernatants were collected and concentrated to extract proteins. A total of 300 μg protein was extracted. Then, the protein solution was cleaved into peptides in trypsin (Sigma-Aldrich, MO, USA) at 37 ℃ overnight after alkylation and methylation by DL-Dithiothreitol (DTT, Macklin, China) and Iodoacetamide (IAA, Macklin, China). Desalting of the peptide solution was performed at Sep-Pak C18 1 cc Vac Cartridge (Waters, USA) and labeled by TMTsixplex™ isobaric label reagent set (Thermo Fisher Scientific, USA) and terminated in hydroxylamine solution (Thermo Fisher Scientific, USA) at room temperature.
LC–MS/MS and bioinformatics analysis
The peptide was analyzed and identified by LC–MS/MS. After re-dissolved in 1% formic acid (FA, Rhawn, China), the peptide was redistributed in the C18-reversed phase trap Gemini column (Phenomenex, Torrance, CA) and Orbitrap Fusion MS (Thermo Fisher Scientific) fitted with an online Easy-nLC 1000 system (Thermo Fisher Scientific). The raw file was analyzed in Maxquant (version 18.104.22.168) for TMT6-126, TMT6-127, TMT6-128, TMT6-129, TMT6-130 and TMT6-131 based on the human FASTA database and the false discovery rate (FDR) was limited to 0.01. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed in Database for Annotation, Visualization and Integrated Discovery (DAVID) online tool (https://david.ncifcrf.gov). The protein–protein interaction network was constructed in STRING (https://string-db.org/) and visualized in Cytoscape (version 3.7.2). Weighted correlation network analysis (WGCNA) was performed according to the following procedure. Data files containing protein expression datasets and stem cell characteristics were prepared and organized in a standard format. First, protein abundances were clustered in R software (https://www.r-project.org/) and R studio (https://www.rstudio.com/) to construct weighted gene networks. Second, the correlations and correlation coefficients of protein profiles and groupings were calculated, and important modules were identified and associated with stem cell characteristics.
Cell migration assay
The hydrogel was spread evenly in the upper insert (Eppendorf, USA). The human umbilical vein endothelial cells (HUVEC) were obtained from the American Type Culture Collection (ATCC, USA) and cultured in DMEM medium. A total of 1 × 105 cells of HUVEC were seeded in a 6-well plate and co-cultured with SHED. The 200μL yellow pipette tip was used to scratch the bottom. Then, SHED was seeded in the insert and transferred to a 6-well plate to form a co-culture system with HUVEC. After 12 h of culture, the migration of cells was observed and the migration rate was calculated. Independent 3 repeated experiments were performed.
The tube formation assay in vitro
A total 1 × 105 cells of HUVEC was seeded on a 6-well plate pre-covered with SHED-encapsulated hydrogel supplemented with HPL. A co-cultivation system between SHED and HUVEC was constructed. After culturing for 12 h, the angiogenic differentiation of the HUVEC was observed and photographed by a fluorescence inverted phase contrast microscopy (CNOPTEC, China). Independent 3 repeated experiments were performed.
RNA isolation, reverse transcription and real-time quantitative PCR
Trizol (CWBio, China) was added to lyse the cells for 10 min. Chloroform (Tgreag, China) and isopropanol (Tgreag, China) were used for RNA isolation and precipitation, respectively. The cDNA library was constructed by reverse transcription in a RT-PCR kit (CWBio, China). UltraSYBR Mixture (CWBio, China) was used for fluorescence quantification and signal feedback to detect gene amplification. The relative expression calculation method of mRNA and primer sequence refers to our previous method . The experiment was repeated three times.
The cells were washed with PBS and fixed with 4% paraformaldehyde (Solarbio, China). The cells were blocked with 5% goat serum and incubated with primary antibodies for 1 h including GFAP (Beyotime, China) and Nestin (Beyotime, China). The FITC-linked secondary antibody (Beyotime, China) was used to bind to neural markers and the cells were observed on an inverted fluorescence microscope (Evos D840, CNOPTEC, China).
Visualization and analysis of data were performed in Prism software (version 8.0, GraphPad, San Diego, CA, USA). Student’s t test or one-way ANOVA was used to analyze the significance of the data. Values of p < 0.05 were set statistically significant, and p < 0.05*, p < 0.01**, p < 0.001***.