Spontaneously immortalized human GECs line was established in the authors’ laboratory64.Normal human gingival fibroblasts (HGFs) were isolated from healthy attached gingiva of a healthy 18-year-old female65.Both cells were maintained in basic medium (BM) containing Dulbecco’s modified Eagle’s medium (Gibco, Life Technologies, Grand Island, NY, USA) supplemented with 23 mM sodium bicarbonate, 20 mM HEPES, 1% antibiotics (50 μg/mL of streptomycin sulfate, 100 U/mL of penicillin; Gibco), and 10% heat-inactivated fetal bovine serum (FBS; Gibco).
MV collection was performed as previously described28. Briefly, confluent GECs were rinsed with phosphate-buffered saline (PBS) and cultured in FBS-free medium for 48 h. Conditioned medium from the cell cultures was collected and centrifuged (Sorvall® RC 5B Plus, Mandel Scientific; manufactured by DuPont, Newtown, CT, USA) at 4℃, first at 3,000 g for 15 min to remove cellular debris and then at 25,000 g for 30 min to collect MVs. MV pellets were rinsed once with PBS, homogeneously re-suspended in FBS-free medium and kept at 4℃ until used. Total MV protein content was measured with Bio-Rad Protein Assay reagent (Bio-Rad Laboratories, Hercules, CA, USA) and spectrophotometry at 570 nm and used for standardizing the vesicle amounts in the experiments.
HGF stimulation by epithelial MVs in osteogenic conditions
HGFs were seeded in 24-well plates (2 × 105 cells per well) for 48 h. Then, the medium was changed to osteogenic differentiation medium [OM; BM supplemented with 50 µg/ml of ascorbic acid (Sigma-Aldrich, St Louis, MI, USA), 100 nM dexamethasone (Sigma- Aldrich), 100 nM vitamin D3 (Sigma-Aldrich), and 10 mM ß-glycerophosphate (Sigma- Aldrich)] and MVs were added to the final concentration of 30 μg protein/ml. Medium was changed and fresh MVs were added every 72 h.
Staining of mineralized nodules by von Kossa
Von Kossa staining was performed as previously reported66. In brief, the cells were fixed in 4% formaldehyde (Fisher Scientific, Fair Lawn, NJ, USA) after 7- and 28-days culture periods and incubated with 2% silver nitrate (Fisher Scientific) in dark for 10 min and then exposed to bright light for 15 min. The plates were then washed with distilled water and dehydrated in 100% ethanol. The samples were then examined by light microscopy67.
Calcification of cultures measured by IncuCyte real-time imaging
For real-time imaging of calcification, 10 mM calcein green (Sigma-Aldrich) solution, which fluoresces when bound to calcium crystals, was prepared in 0.1 M NaOH, and the solution was further diluted to 1 mM and sterile-filtered. Calcein solution was added directly to the differentiation medium in final concentration of 1 μM68. The culture medium was changed every three days. The green fluorescence mask of GCU 270 was set in order to exclude background noise to avoid artifacts68. The images were taken in real-time, using an automated IncuCytetm S3 live-cell imaging system (Sartorius Corporation, Edgewood, NY, USA) at a series of time points up to 17 days.
Sample preparation for SEM
HGFs were cultured on 10 mm tissue culture plastic coverslips (Sarstedt, Newton, NC, USA) in 24-well plates for four weeks, fixed with 2.5% glutaraldehyde (EM grade, Electron Microscopy Sciences (EMS), Hatfield, PA, USA), postfixed with 1% osmium tetroxide (EMS) and dehydrated in a graded series of ethanol followed by critical point drying (Samdri-795; Tousimis Research Corporation, Rockville, MD, USA). The coverslips were mounted on stubs and coated with 20-nm carbon (Med020, Leica Microsystems Inc, Concord, ON, Canada). Cells were visualized by SEM (Helios 650 Nanolab dual beam, FEI, Hillsboro, OR, USA) using the backscattered electrons (BSE) imaging mode (atomic number contrast) and electron energy sectioning69. Once a suitable area was identified, a cross section was created with the Focused Ion Beam (FIB), exposing the interior of the cells. The BSE imaging mode was used to distinguish the cell and intracellular calcified vesicles based on atomic number contrast. To confirm the presence of Ca (L alpha of 3.9 keV) and P (K alpha 2.013) areas of high density were analyzed using EDX point analysis (Octane Pro Detector, EDAX, Mahwah, NJ, USA).
Double staining of MVs and mineral deposits
To analyze direct relationship between MVs and calcification, the GECs were labeled using Vybrant® DiI cell labeling solution (1:200; Invitrogen, Eugene, OR, USA) for 1 h. Labeled MVs were collected as above. HGFs were seeded on gelatin (0.2%)-coated glass coverslips in BM. After 48 h, the medium was changed to OM supplemented with 1 μM calcein green, and Dil-labeled MVs were added. MVs were added only once at day 0. Medium was changed at day 4. At each time point, the cells were fixed with 4% formaldehyde at room temperature for 20 min and the nuclei then stained with 300 nM DAPI (Molecular Probes Inc., Eugene, OR, USA) in PBS for 15 min. Samples were mounted with Immuno-mount solution (Thermo Scientific, Pittsburgh, PA, USA) and examined using an Axioplan II Fluorescent microscope (Carl Zeiss Inc., Jena, Germany). The images were captured using Northern Eclipse software (Empix Imaging, Mississauga, ON, Canada).
Gene expression profiling
HGFs were seeded in 6-well plates (2 × 105 cells per well) in BM for 48 h, after which the medium was changed to OM and MVs were added as above. After a 1- or 3-day culture, total RNA was extracted using Purelink RNA Mini kit (Thermo Scientific) according to the manufacturer’s protocol and assessed for purity by the RNA/DNA Calculator (GeneQuant Pro; Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). The quality of RNA samples was assessed using Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and samples with RNA Integrity Number (RIN) ³8 were used for further analysis70. Qualifying samples were then prepped following the standard protocol for the NEBnext Ultra ii Stranded mRNA (New England Biolabs, Ipswich, MA, USA). Sequencing was performed on the Illumina NextSeq 500 (Illumina Inc. San Diego, CA, USA) with Paired End 42bp × 42bp reads. De-multiplexed read sequences were then aligned to the reference sequence using STAR aligners (https://www.ncbi.nlm.nih.gov/pubmed/23104886). Assembly and differential expression were estimated using Cufflinks (http://cole-trapnell-lab.github.io/cufflinks/) through bioinformatics apps available on Illumina Sequence Hub. Gene abundances were normalized by calculating Fragments Per Kilobase of Exon Per Million Fragments Mapped (FPKM). Differentially expressed genes (DEGs) were identified with a Benjamini-Hochberg adjusted q-value of less than 5% and absolute fold-change of ≥ 1.5. The data were analyzed by the BaseSpace Correlation Engine (https://basespace.illumina.com, Illumina, Cupertino, CA) and The Database for Annotation, Visualization, and Integrated Discovery (DAVID) Resources 6.7 (at http://david.abcc.ncifcrf.gov/) using default setting. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was used to evaluate the function of DEGs in two groups.
Quantitative reverse transcription PCR (RT-qPCR)
RT-qPCR was performed as previously described71. Briefly, total RNA was obtained as above. Total RNA (1μg) was reverse-transcribed with high-capacity cDNA reverse transcription kit (Applied Biosystems, Life Technologies, Grand Island, NY, USA), according to the manufacturer’s instructions, and Mastercycler Gradient 5331 Reverse-Transcriptase PCR Instrument (Eppendorf AG, Hamburg, Germany). The cDNA was diluted to a concentration at which the threshold-cycle value was well within the range of its standard curve. cDNA (5 μl) was mixed with 10 μl of 2x iQ SYBR Green I Supermix (Bio-Rad) and 5 pmol of primers in 96-well plate wells. Asparagine-linked glycosylation 9 (ALG9) and glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) were used as reference genes. Real-time qPCR amplification was performed using the CFX96 system (Bio-Rad Laboratories). The data were analyzed and are presented according to the comparative Ct method (CFX Manager Software, version 2.1; Bio-Rad Laboratories). PCR primers used are listed in the supplementary Table S1.
All data are expressed as mean ± SEM from at least three independent experiments. Statistical analysis was performed using SPSS 24.0 software. Statistical differences were evaluated by Student’s t-test for paired comparisons or by one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. Statistical analysis for RT-qPCR data was done using log2-transformed data. P values <0.05 were considered as statistically significant.