November 5, 2024

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Human dental pulp stem cells derived extracellular matrix promotes mineralization via Hippo and Wnt pathways

Human dental pulp stem cells derived extracellular matrix promotes mineralization via Hippo and Wnt pathways

Cell isolation and culture

The study was approved by the Human Research Ethics Committee of Chulalongkorn University (approval no. 106/2022). Inform consent was obtained from study participants. Methods were carried out in accordance with the Declaration of Helsinki. Pulp and gingival tissues were collected from those tissues surgically removed according to the patient’s treatment plan. Tissues were collected from the patients who met the predefined inclusion criteria in accordance with the scientific protocol at the Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Chulalongkorn University. The inclusion criteria were as follows: healthy donors, permanent dentition, impacted molars, age range of 18–35 years, no gender specificity, and absence of tooth pathology. The explantation method was used to obtain the cells. Briefly, the collected tissues were chopped into small pieces without using enzymatic dissociation. Subsequently, the fragmented tissues were placed in 35 mm culture dishes for cells to migrate out from the tissue. The isolated cells were cultured in a growth medium composed of Dulbecco’s Modified Eagle Medium (DMEM, cat. no. 11960, Gibco, USA) containing 10% fetal bovine serum (FBS, cat. no. 10270, Gibco, USA), 2 mM l-glutamine (GlutaMAX-1, cat. no. 35050, Gibco, USA), 100 unit/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin B (Antibiotic–Antimycotic, cat. no. 15240, Gibco, USA). The cells were incubated at 37 °C in a humidified 5% carbon dioxide atmosphere. The culture medium was changed every 48 h. The cells between passages 3 and 7 were used in the subsequent experiments. The overall experimental scheme is indicated in Fig. 10.

Figure 10
figure 10

A diagrammatic representation of the experimental strategy. DPSCs and GSCs were isolated from pulp and gingival tissues collected from impacted permanent molars of healthy individuals. Cells were cultured in normal growth medium and osteogenic induction medium for 21 days to facilitate ECM production. Subsequently, decellularization was performed to obtain dECMs. A comprehensive proteomic analysis was conducted to assess the matrisome profiling of these dECMs. Subsequent experimentation involved the reseeding of GSCs onto each dECM, followed by culture in normal growth medium and osteogenic induction medium to elucidate the cellular responses and osteogenic differentiation capabilities of the reseeded cells. Furthermore, RNA sequencing techniques were employed to analyze differential gene expression, identify enriched pathways, and characterize GO terms associated with the experimental conditions. Created by biorender.com.

For inhibitory experiments, GSCs were pretreated with inhibitors for 30 min prior to reseeding on the dECM. The cell signaling inhibitors used in this study were as follows: 4 μM SB431542 (TGF-β inhibitor, cat. no. 1614, Sigma–Aldrich, USA), 20 µM dobutamine hydrochloride (DH, YAP inhibitor, cat. no. D0676, Sigma–Aldrich, USA), 25 µM IWP-2 (Wnt inhibitor, cat. No. 3533, Tocris Bioscience, USA), 3.75uM JAK inhibitor I (cat. no. 420099, Calbiochem, USA), 1.5 nM ERK inhibitor (cat. no. 328006, Calbiochem, USA), and 5 nM LY294002 (PI3K inhibitor, cat. no. A0231, Sigma–Aldrich, USA).

Flow cytometry analysis

Surface protein expression was analyzed using flow cytometry. Single-cell suspensions were stained with fluorescence conjugated antibodies (1:50 dilution) as follows: FITC conjugated anti-human CD44 (Cat. No. 555478, BD Bioscience, USA), PE-conjugated anti-human CD105 (Cat. No. 21271054, Immuno Tools, Germany), FITC-conjugated anti-human CD90 (Cat. No. ab124527, Abcam, USA), and PerCP-conjugated anti-CD45 (Cat. No. 21810455, Abcam, USA). Mean fluorescence intensity was calculated using a FACSCalibur flow cytometer (BD Bioscience, San Jose, CA, USA).

Osteogenic differentiation

Cells (50,000 cells/well in a 24-well plate) were cultured in an osteogenic medium consisting of growth medium supplemented with 50 µg/mL ascorbic acid (cat. no. A-4034, Sigma-Aldrich, USA), 250 nM dexamethasone (cat. no. D8893, Sigma-Aldrich, USA), and 5 mM β-glycerophosphate (cat. no. G9422, Sigma-Aldrich, USA). Osteogenic differentiation potential was elucidated using ALP, ARS, and Von Kossa staining.

For ALP staining, the cells were washed with phosphate buffer saline (PBS) and fixed with 4% formaldehyde for 10 min. Then, cells were incubated with BCIP/NBT (Roche, USA) in the dark at room temperature for 30 min. The ALP-positive cells were observed using an inverted microscope (Olympus, USA).

For the ARS staining, the cells were fixed with cold methanol for 10 min and washed with deionized water. The samples were then stained with 2% ARS solution (Sigma-Aldrich Chemical) for 3 min at room temperature with gentle agitation. The mineral deposits were solubilized with 10% cetylpyridinium chloride monohydrate in 10 mM sodium phosphate. The optical density was measured at 570 nm with a microplate reader (ELx800, BIO-TEK®, United States).

For Von Kossa staining, the cells were fixed with 4% formaldehyde in PBS and further incubated with 5% silver nitrate in sterile deionized water. The samples were exposed to ultraviolet light for 5 min at room temperature. The stained cells were examined under an inverted microscope.

Adipogenic differentiation

Cells (12,500 cells/well in a 24-well plate) were cultured in adipogenic medium comprising growth medium containing 0.1 mg/ml insulin (cat. no. 11070738 Sigma-Aldrich, USA), 1 μM dexamethasone (cat. no. D8893, Sigma-Aldrich, USA), 1 mM IBMX (cat. no. PHZ1124, Thermo Fisher Scientific, USA), and 0.2 mM indomethacin (cat. no. 53861, Sigma-Aldrich, USA) for 16 days. The intracellular lipid droplet was stained by Oil red O staining. Briefly, cells were fixed with 4% formaldehyde in PBS for 10 min, followed by incubating with 0.2% Oil Red O solution for 15 min. Lipid accumulation was examined using an inverted microscope.

Extracellular matrix production and decellularization

The culture plate was coated with 0.2% gelatin for 2 h at 37 °C. The cells were seeded on a gelatin-coated surface and divided into two groups: N-ECM and OM-ECM. In N-ECM, cells were maintained in a growth medium for 7 days and subsequently cultured in a growth medium supplemented with 50 μg/ml l-ascorbic acid for 14 days. For OM-ECM, cells were maintained in an osteogenic medium for 21 days.

Decellularization was performed using 0.5% Triton X-100 in 20 mM ammonium hydroxide and washed with a protease inhibitor in PBS. Deoxyribonuclease A at a concentration of 0.0025% in sterile PBS was added to the samples and incubated for 30 min at room temperature for DNA removal.

Protein extraction and digestion

Protein extraction was performed from dECM on day 21 using a Compartment Protein Extraction Kit (MERCKMillipore, USA). The protein pellets’ solubilization and digestion were performed as previously described43. In brief, dECM pellets were solubilized in a solution containing 8 M urea, 100 mM ammonium bicarbonate, and 10 mM dithiothreitol. Cysteines were alkylated by adding iodoacetamide, and samples were deglycosylated with PNGaseF (New England BioLabs, USA, 1:100 units for 1 mg sample) and subsequently digested with trypsin/LysC (Promega, USA), at a ratio of 1:10,000 enzyme: substrate. Final digestions were done using trypsin (Worthington Biochemical Corporation, USA) at a ratio of 1:1000 (enzyme: substrate), followed by a second aliquot of trypsine/LysC (Promega, USA), at a ratio of 1:10,000 (enzyme:substrate).

Mass spectrometry

Mass Spectrometry (LC–MS/MS) was performed as previously described10. In brief, chromatography was performed with an RSLCnano system (Ultimate 3000, Thermo Scientific) coupled online to a Q Exactive HF-X with a Nanospay Flex ion source (Thermo Scientific).

Peptides were trapped on a C18 column (75 μm inner diameter × 2 cm: nanoViper Acclaim PepMapTM 100, Thermo Scientific) at a flow rate of 2.5 μl/min over 4 min and subsequently separated on a 50 cm × 75 μm C18 column (nanoViper Acclaim PepMapTM RSLC, 2 μm, 100 Å, Thermo Scientific) at 50 °C at a flow rate of 300 nl/min over 211 min. MS full scans were performed in the ultra-high-field Orbitrap mass analyzer. Top 20 intense ions were further fragmented via high-energy collision dissociation activation. Ions with a charge range from 2 + to 6 + were selected for screening. Data were searched against the Homo sapiens (UP000005640) SwissProt database using Sequest HT through proteome discoverer (version 2.2). The data were subsequently processed using myProMS v3.9.3 (https://github.com/bioinfo-pf-curie/myproms) FDR calculation used Percolator. The label-free quantification was performed by peptide Extracted Ion Chromatograms (XICs) computed with MassChroQ version 2.2. To correct the XICs, median and scale normalization was applied on the total signal. For statistical analysis, a linear model was performed, and p-values were adjusted using Benjamini–Hochberg FDR procedure. Matrisome proteins database (Human Matrisome (Updated December 2022): http://matrisomeproject.mit.edu/other-resources/human-matrisome/) has been updated and used for selecting the ECM proteins out of the whole proteomics data. The mass spectrometry proteomics raw data have been deposited to the ProteomeXchange Consortium via the PRIDE (PMID: 34,723,319) partner repository with the data identifier “PXD040575” and “PXD018951” ([email protected] & DYdXRjUC).

Matrisome protein–protein interaction and enrichment pathway analysis

The significant ECM proteins detected from matrisome database were analyzed using Metascape (https://metascape.org/gp/index.html#/main/step1). PPI enrichment was determined using minimum network size = 3 and maximum network size = 500. GO enrichment analysis, categorized as a biological process, cellular component, and molecular function, was performed. The PPI figures were created by using Cytoscape version 3.9.1.

ECM seeding experiment

GSCs were seeded at a density of 25,000 cells on dECM_DPSCs or dECM_GSCs and cultured in a growth medium for 30 min, 24 h, or 7 days. Evaluation of cell morphology, attachment, and spreading was done using SEM. Cell viability was assessed using MTT assay. For mineralization assay, GSCs were reseeded and maintained in osteogenic induction medium for 14 days.

Immunofluorescence staining

Samples were fixed with 4% formaldehyde in PBS and incubated with 0.1% Triton-X100 in PBS. Non-specific binding was blocked with horse serum (2% v/v). Samples were stained with mouse monoclonal IgG anti-type I collagen (1:200 dilution, Abcam, UK) or mouse monoclonal IgG anti-fibronectin (1:500 dilution, Invitrogen, United States) at 4 °C overnight. The secondary antibody labeled with AlexaFluor 488 was added at a 1:2000 dilution for 2 h. F-actin organization was examined using AlexaFluor 594 Phalloidin (1:1000 dilution, Invitrogen, United States). DAPI (1:500 dilution, Invitrogen, United States) was used to counterstain the nuclei. Visualization of the target protein was detected using a fluorescent microscope with an ApoTome system (Carl Zeiss, Germany).

Scanning electron microscopy

The samples were fixed with 3% glutaraldehyde in PBS for 30 min and dehydrated with a graded series of ethanol. Hexamethyldisiloxane was added for 5 min and the gold sputter-coat was performed for SEM analysis.

Cell viability test

GSCs (12,500 cells/well) were reseeded on dECM. At day 1, 3, and 7, the cells were incubated with 0.5 mg/mL MTT solution (USB Corporation) for 30 min, allowing formazan crystal formation. The precipitated crystals were solubilized using a dimethyl sulfoxide and glycine buffer. The solution was measured absorbance at 570 nm by a microplate reader (ELx800, BIO-TEK®, United States). The percentage cell number was calculated and normalized with the control.

Quantitative real-time polymerase chain reaction

Total cellular RNA was extracted using TRIzol reagent (RiboEx solution, cat. no. 301-001, GeneAll, South Korea). The cDNA was obtained by converting one microgram of total RNA using ImProm-II Reverse Transcription System (cat. no. A3800, Promega, USA). qPCR was performed using FastStart Essential DNA Green Master (Roche Diagnostic, Germany) in a CFX connect Real-Time PCR machine (Bio-Rad, Singapore). Product specificity was evaluated using melt curve analysis. The targeted mRNA expression levels were normalized to GAPDH gene. The relative expression was calculated using 2−ΔΔCt method44. The primer oligonucleotide sequences are shown in Supplementary Table 2.

High-throughput RNA sequencing

Total RNA was extracted using a RNeasy kit (Qiagen, USA). The RNA quality was examined using an Agilent 2100 BioAnalyzer (Agilent Technologies, USA), NanoDrop (Thermo Fisher Scientific Inc.), and 1% agarose gel. Library preparation was constructed using a NEBNext® UltraTM RNA Library Prep Kit for Illumina®. The constructed library was validated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). Sequencing was performed on the illumine HiSeq platform in a 2X150bp paired-end configuration. Base-calling is performed by Illumina RTA software. Demultiplexing is performed by Illumina bcl2fastq 2.17 software based on index information and the number of reads and quality score (Q30) were counted. Data were aligned to reference genome via software HISAT2 (v2.0.1)45,46. Differential expression analysis used the DESeq2 Bioconductor package. The sequencing data were submitted to the NCBI’s Gene Expression Omnibus (GSE226347).

Statistical analysis

All experiments were repeated using cells derived from at least four different donors (n = 4). The statistical analysis was performed using Prism 8 (GraphPad Software, USA). For a two-group comparison, the Mann–Whitney U test was used. For three or more group comparisons, statistical differences were assessed using the Kruskal–Wallis test, followed by Dunn’s test as a posthoc pairwise comparison. Statistical significance was considered at p < 0.05.