Cell culture
Human PDL fibroblasts and the bone marrow-derived MSC line UE7T-13 were obtained from Lonza (Basel, Switzerland) and the Riken BioResource Research Center (Tsukuba, Japan), respectively. The cells were cultured in α-minimum essential medium (α-MEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 15% fetal bovine serum (FBS, HyClone, Logan, UT, USA), GlutaMax and antibiotic-antimycotic solution (Thermo Fisher Scientific). The UE7T-13 line is an immortalized clone of bone marrow-derived MSCs generated by transduction with the papillomavirus type 16 protein E7 and human telomerase reverse transcriptase.21 We selected this cell line because of its greater potential for differentiation into osteoblasts, adipocytes, and chondrocytes than two other clonal cell lines (UBE6T-7 and UE6E7T-12) in our pilot study. For adipocyte differentiation, hMSC adipogenic differentiation medium (Lonza) was used according to the manufacturer’s instructions, and adipocytes were stained with Oil Red O solution (Sigma-Aldrich, St. Louis, MO, USA) after 4 weeks of differentiation. For osteoblast differentiation, UE7T-13 cells were cultured in α-MEM containing L-ascorbic acid 2-phosphate (50 µg/mL, Sigma-Aldrich), dexamethasone (10− 8 M, Sigma-Aldrich), and β-glycerophosphate (10 mM, Sigma-Aldrich) for 3 weeks and von Kossa staining was performed to visualize the mineral deposits using a commercially available kit (Polysciences, Warrington, PA, USA). For chondrocyte differentiation, pellet culture of UE7T-13 cells was performed using chondrogenic induction medium (Lonza) containing TGF-β3 for 4 weeks. Frozen sections of UE7T-13 spheroids were prepared and stained with Alcian blue (Nacalai Tesque, Kyoto, Japan).
Flow cytometry
UE7T-13 cells were harvested by trypsinization, fixed in 4% paraformaldehyde (PFA) (Nacalai Tesque, Kyoto, Japan) for 15 min, and then blocked with 5× Blocking One solution (Nacalai Tesque) for 15 min on ice. Thereafter, the cells (2 × 105) were incubated with the following antibodies on ice for 30 min: PE-conjugated anti-human CD29, CD44, CD45, and CD105 (1:20; BioLegend, San Diego, CA, USA); PE-conjugated normal mouse IgG1k (1:20; BioLegend); PE-conjugated anti-human CD19 (1:10; BD Bioscience, Franklin Lakes, NJ, USA); FITC-conjugated anti-human CD73 (1:20; Invitrogen); FITC-conjugated anti-human CD34 (1:10; BD Bioscience); FITC-conjugated anti-human CD90 (1:25; Abcam, Cambridge, UK); FITC-conjugated normal mouse IgG1k (1:20; BioLegend); APC-conjugated human CD14 (1:20; BioLegend); and APC-conjugated normal mouse IgG2a (1:20; ExBio, Vestec, Czech Republic). After the cells were washed with phosphate-buffered saline (PBS) containing 2% FBS, flow cytometric analysis was performed using a FACS Verse flow cytometer (BD Bioscience). All flow cytometry data were analyzed using Flow Jo software (BD Bioscience).
MSC-extract and protein collection
A schematic diagram of the MSC-extract and MSC-protein isolation process is shown in Fig. 2A. Briefly, UE7T-13 cells were harvested using 0.05% trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA; Thermo Fisher Scientific), washed with α-MEM, and then suspended in PBS at a density of 106 cells/100 µL of PBS. The cell suspension was alternately placed in a liquid nitrogen and water bath at 37°C to disrupt the cell membranes through three cycles of freezing and thawing. Next, the cells were centrifuged at 15000 rpm at 4°C for 30 min, after which the cell membranes and other debris were deposited at the bottom of the tube. Cellular extracts were then obtained by filtration through a 0.22-µm pore size filter (Kurabo, Osaka, Japan). For MSC-protein isolation, the MSC-extract samples were mixed with four volumes of cold acetone (Nacalai Tesque). After vigorous vortex mixing, the mixture was stored at -20°C for 1 h to precipitate the protein. Following centrifugation at 15000 rpm for 15 min, the supernatant was removed, and the protein pellet was dissolved in PBS or saline using an ultrasonic shaker. The protein concentrations of the samples were measured using a bicinchoninic acid (BCA) protein assay kit (Toyobo). The MSC-extract and protein were stored at -20°C until further use.
Electrophoresis of MSC-extract and protein
To examine the protein contents of MSC-extract and MSC-protein, 7.5 µg of total protein was mixed with an equal volume of sample buffer (ExApply, ATTO Corporation, Tokyo, Japan) plus dithiothreitol (DTT) and boiled for 5 min. Thereafter, the samples were subjected to 10% acrylamide gel electrophoresis, and the proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After electrophoresis, the acrylamide gels were stained with Coomassie Brilliant Blue (Nacalai Tesque), and gel images were obtained using a gel documentation system (ChemiDoc XRS Plus, Bio-Rad, Hercules, CA, USA).
WST-8 proliferation assay
PDL cells were seeded in 96-well plates (3000 cells/well) in α-MEM supplemented with different concentrations of MSC-extract, MSC-protein, recombinant human basic fibroblast growth factor (bFGF) (Pepro-Tech, Cranbury, NJ, USA), or recombinant human hepatocyte growth factor (HGF) (BioLegend, San Diego, CA, USA). After 3 days, the proliferation of PDL cells was examined using WST-8 assay reagent (Dojindo, Kumamoto, Japan) according to the manufacturer’s protocol. The cell proliferation rate was expressed as the absorbance at 450 nm.
Ki67 immunostaining
Ki67 immunocytochemistry was used to examine proliferating cells. PDL cells were fixed with 4% PFA (Nacalai Tesque) for 20 min at room temperature, and the cells were permeabilized with 0.25% Triton X-100 in PBS for 15 min. Blocking of the cells was performed in 10% goat serum-PBS for 1 h. Primary antibody (mouse anti-human Ki67 antibody (DAKO, Tokyo, Japan)) and secondary antibody (goat anti-mouse IgG Alexa 488 (Thermo Fisher Scientific)) were applied to the PDL cells. After nuclear staining with Hoechst 33342 (Dojindo), the cells were observed under a fluorescence microscope (BZ-9000, Keyence, Osaka, Japan). The percentage of Ki67-positive PDL cells was calculated from the total nuclear counts and the number of Ki67-positive cells in 15 randomly selected microscopic field images.
Cell migration assay using a Boyden chamber
PDL cell migration was examined using a Boyden chamber. Chambers (Kurabo) with 8 µm pore membranes were placed in 24 well plates. PDL cells (105 cells in α-MEM without serum) were added to the upper chamber, and α-MEM containing MSC-extract, MSC-protein, bFGF, or HGF was added to the lower well. After 8 h of incubation, the chambers were washed, and the membranes were fixed in 4% PFA. The migrated cells were stained with 0.05% crystal violet (Nacalai Tesque), and images of the migrated cells were captured using a BZ-9000 microscope (Keyence).
Growth factor array and enzyme-linked immunosorbent assay (ELISA)
To investigate the wide range of growth factors in MSC-extract and MSC-protein, a dot-blot-based antibody array kit (Hunan growth factor array C1) (Ray Biotech, Norcross, GA, USA) was used, following the manufacturer’s protocol. A total of 500 µg of MSC-extract or MSC-protein sample was applied to the dot-blot membrane. After the membrane was exposed to the chemiluminescent solution, dots representing the concentration of each growth factor in the sample were photographed using a ChemiDoc XRS Plus (Bio-Rad). The intensity of each dot was measured using ImageJ software and calculated as a percentage of the intensity of the positive control. To assess the amounts of bFGF and HGF in the MSC-extract and MSC-protein, the Human Fibroblast Growth Factor Basic ELISA MAX Deluxe Set (BioLegend) and Human Hepatocyte Growth Factor Sandwich ELISA Kit (Proteintech, Rosemont, IL, USA) were used according to the protocols provided.
Liquid chromatography-mass spectrometry (LC-MS/MS)
The protein content of the MSC-extract was analyzed using LC-MS/MS at Kazusa Genome Technologies Inc. (Chiba, Japan). Proteins were isolated from the MSC-extract through acetone precipitation and reduced with 10 mM DTT at 50°C for 30 min. The reduced protein samples were digested using Lys-C and trypsin at 37°C overnight. After desalinization and extraction, the peptides were dissolved in 3% acetonitrile-0.1% formic acid. Nano LC-MS/MS analysis was performed using a linear acetonitrile gradient (3 to 65%) on an UltiMate 3000 RSLC nano LC system (Thermo Fisher Scientific) coupled to a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific) in ESI positive mode for 40 min. The obtained MS data were further analyzed for the identification and quantification of peptides and proteins using Scaffold DIA (Matrix Science, Tokyo, Japan). Peptides and proteins with a false discovery rate (FDR) less than 1% were identified and quantified.
RNA-sequencing (RNA-seq) and Gene Ontology (GO) analysis
Twenty-four hours after MSC-protein treatment, total RNA was extracted from PDL cells using NucleoSpin RNA (Macherey-Nagel, Düren, Germany). Following DNase treatment of the RNA samples, the RNA concentration and OD260/280 values were measured using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific) and were 0.142 and 0.15 µg/µL and 2.12 and 2.09 for RNA from PDL cells and MSC-protein treated PDL cells, respectively. Thirty microliters of each RNA sample was sent to Veritas International (Madrid, Spain) for RNA-seq analysis. After poly(A) enrichment of the RNA samples, libraries were constructed using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) and sequenced using a NovaSeq 6000 system (Illumina Inc., San Diego, CA, USA). Quality testing of the sequencing data was performed using FastQC (version 0.11.8), and low-quality reads were depleted using Trim Galore (version 0.6.4). After filtering the sequencing data, each read was mapped to the reference genome, GTCh38, using STAR software (version 2.7.4a). FeatureCounts (version 1.6.4) was used to count the reads mapped to each gene symbol, and gene expression comparisons were performed on TMM-normalized count data using edgeR (version 3.22.3). Genes with fold changes greater than 3 and less than 0.3333333 were defined as upregulated and downregulated genes, respectively. GO analysis of the upregulated genes was performed using gprofiler2 (version 0.2.1). Fisher's exact probability test was performed for terms registered in GO (biological process [BP], molecular function [MF], and cellular component [CC]) and Reactome, and a statistical analysis was performed to determine which terms were related to genes with variable expression. The results of GO analysis are depicted as a Manhattan plot with terms on the horizontal axis and log p-values on the vertical axis.
Rat periodontal defect models and MSC-protein transplantation
The animal experiments in this study were approved by the Osaka Dental University Institutional Animal Care and Use Committee (approved #21-07004 and 22-02025) before the initiation of the experiments. All animal experiments were conducted in accordance with the approved guidelines of the Science Council of Japan for proper animal experiments and the ARRIVE guidelines (https://arriveguidelines.org/). The ARRIVE checklist is included in Additional File 6. All animal experiments were performed between March and April 2022 at the Osaka Dental University Animal Facility. The experimental animals were housed in a specific pathogen-free (SPF) environment with free access to food and water. The periodontal defect model was created according to the procedure described in a previous study, with certain modifications.22 Twelve male Sprague-Dawley (SD) rats (8–10 weeks of age) (Shimizu Laboratory Supplies, Kyoto, Japan) were anesthetized through intraperitoneal injection of medetomidine (Zenoaq, Fukushima, Japan), midazolam (Astellas Pharma Inc.,Tokyo, Japan), and butorphanol (Meiji Seika Pharma Co., Ltd., Tokyo, Japan). The gingival flap on the mesial side of the maxillary first molar was elevated and periodontal defects were created with a dental bur using a stereoscopic microscope. Thereafter, a hydrogel scaffold (2×2×2 mm) (MedGel, Nitta Gelatin Inc., Osaka, Japan) with MSC-protein (780 µg/mL) (experimental group) or saline (control group) was transplanted, and the gingival flap was closed using a 7 − 0 silk suture (Johnson & Johnson, New Brunswick, NJ, USA). One periodontal tissue defect was created in each animal. The gingival incision was minimized to reduce invasiveness toward the animal, and careful attention was given to invasion of the surrounding tissues. Fifty microliters of MSC-protein (approximately 400 µg/mL) or saline was injected into the gingival tissues around the maxillary first molar 5 days after surgery. The sample size was determined from similar periodontal tissue defect models conducted previously to minimize the number of rats used in the experiment.23 When postoperative observation revealed symptoms of anguish or rapid weight loss (> 20% of body weight), the animal was excluded from the experiment. In such instances, humane endpoints were applied, and the experiment was terminated by euthanasia with an intraperitoneal overdose of barbiturate. These criteria were established prior to the experiment; however, no animals were excluded from the experiment. Six rats were assigned to the experimental and control groups, all of which were used in subsequent analyses (12 rats in total). The assignment of experimental groups was made by a blinded third individual to minimize bias in the experiments and analyses and K.I. was aware of the group allocation in this study.
Histological analysis
Four weeks after the transplantation surgery, the rats were sacrificed with an overdose of barbiturate (Kyoritsu Seiyaku, Tokyo, Japan), and tissue blocks of the maxillae were collected. Following fixation of the blocks in 4% PFA (Nacalai Tesque) for 2 d, decalcification was performed in 2× K-CX solution (FALMA, Tokyo, Japan) for 3 d at 4°C. Paraffin-embedded sections were prepared and stained with hematoxylin and eosin (H&E) for histological analysis. For picrosirius red staining, after deparaffinization, the sections were stained with 0.1% picrosirius red in saturated picric acid (Muto Pure Chemicals, Tokyo, Japan) for 10 min at 37°C. Images were captured using a BZ9000 microscope (Keyence). The primary outcome measure in this study was the extent of periodontal regeneration. For this purpose, the lengths between the cementoenamel junction (CEJ) and the bottom of the periodontal defect and between the bone crest and the bottom of the defect were measured. Using these two length measurements, the bone height ratio was calculated and compared.
Microcomputed tomography (micro-CT) analysis
Periodontal tissue healing was evaluated using micro-CT (Skyscan 1275, Burka, Antwerp, Belgium). Rat maxillary samples were scanned after PFA fixation (1 mm copper filter, 360° rotation, 0.2° rotation step, high-resolution mode, voltage 100 kV, and current 60 µA). The scanned images were reconstructed using InstaRecon software (Burka). To compare periodontal tissue regeneration, ImageJ software was used to measure the exposed root surface area perpendicular to the maxillary proximal root on the reconstructed images.
Images
For cells and histological sections, images were obtained using X20 Plan Fluor ELWD DM and X10 Plan Apo objective lenses (Nikon, Tokyo, Japan) in a BX-9000 system (Keyence). The fluorescent samples were observed using GFP-B and DAPI-B filters (Keyence). Images of the migration assay filters were captured using a digital camera (EOS40D, Canon, Tokyo, Japan). All images were first placed in PowerPoint for Windows (Microsoft Corporation, Redmond, WA, USA), cropped as needed, and positioned for the layout. Each figure was then exported as a TIFF file, and the resolution was changed to 350 dpi or higher using image analysis software (GIMP for Windows, https://www.gimp.org/) and saved as a TIFF or PDF file. The resolution of the acquired micrographs is shown in the figure legends. Adjustments of each color channel, threshold manipulation, signal range expansion/contraction, alteration of high-signal intensity, and averaging were not performed. The original gel and membrane images are included in Supplementary Figs. 6 and 7 in Additional File 5.
Statistical analysis
The data are expressed as the mean ± standard deviation (SD). To confirm the reproducibility of the experiment, at least three independent similar experiments were conducted, and representative data from the confirmed reproducible data are shown in the figures. All the data are plotted on a bar graph to show their variability. For the comparison of two groups, the variance between the two groups was tested and Student’s t test was used for comparisons between two groups. For comparison of more than three experimental groups, one-way analysis of variance (ANOVA) followed by Tukey’s test was performed. All the statistical analyses were performed using GraphPad Prism 9.0.0 for Windows (San Diego, CA, USA). Differences with p < 0.05 were considered significant in this study.