We confirm that all methods have been carried out in accordance with ARRIVE guidelines and relevant guidelines and regulations.
Informed consent was obtained from all subjects and ethics committee approval from the Ethics Committee Review Board of the University of São Paulo School of Dentistry (protocol number FO-USP 029/2018) was obtained prior to collection of the patient’s teeth at the hospital. clinic of the School of Dentistry of the University of São Paulo (FO-USP). The use of Balb/c nude mice in this research has also been approved by the Board of Directors of the Ethics Committee on the Use of Animals (CEUA) of the Institute of Chemistry of the University of Sao Paulo ( IQ-USP) under protocol number 98/2018.
Periodontal ligament stem cells were harvested from third molars of 3 systemically healthy individuals (I1, I2, and I3—18-30 years old). The inclusion criteria were: partially or totally erupting third molars and absence of periodontal disease.
Periodontal ligament tissue samples were obtained by scaling the middle third of the root and PDLSCs were isolated using the explant technique12.48 in alpha-modified Eagle’s medium (α-MEM) (Thermo Fisher Scientific, Waltham, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, USA), 100 μg/mL penicillin , 100 μg/mL streptomycin and 0.5 mg/mL amphotericin B (Gibco, Invitrogen, Carlsbad, USA) at 37°C in an atmosphere of 5% CO2 and 95% humidityseven. After 14 days, cells from the explants reached 70% confluence and PDLSC populations were used at passage 4 for all experiments.
Characterization of pluripotency
In order to identify the mesenchymal stem cell phenotype, approximately 5 × 105 PDLSCs were incubated in 5% bovine serum albumin (BSA)/phosphate buffered saline (PBS) (Gibco, Invitrogen, Carlsbad, USA) 1 × at 4°C in the dark for 1 h with the following monoclonal antibodies: PAR1-FITC (Abcam, Cambridge, UK), OCT4-FITC (Abcam, Cambridge, UK), SOX2-FITC (Abcam, Cambridge, UK), STRO-1-FITC (Abcam, Cambridge, UK Uni), CD14-FITC (eBioscience, San Diego, USA), CD90-FITC (eBioscience, San Diego, USA), CD31-PE (eBioscience, San Diego, USA), CD-44-PE (eBioscience, San Diego, USA), CD34-FITC (Biolegend, San Diego, USA) and CD146-PE (Biolegend, San Diego, USA) for 30 min at 4°C. An uncolored witness was used to define the gates. A total of 10-50,000 events were recorded and the data analyzed via FlowJo (Becton Dickinson, Oregon, USA).
Cell sheet culture and experimental design
PDLSC at 1×106 cells/cm2 were seeded in 100 mm plates for 24 h with α-MEM (Thermo Fisher Scientific, Waltham, USA) supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, USA) and 50 μg/mL vitamin C to induce cell sheet formation, as previously describedseven. Subsequently, cell sheets were assigned to one of the following experimental groups: (1) control medium (CTRL) composed of α-MEM (Thermo Fisher Scientific, Waltham, USA) supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, USA) and 50 μg/mL vitamin C (Sigma-Aldrich, St Louis, USA); (2) osteogenic medium (OST) (CTRL + 100 nM dexamethasone, 5 mM b-glycerophosphate and 50 µg/ml ascorbic acid (Sigma-Aldrich, St Louis, USA) and (3) osteogenic medium with the addition of BY1 agonist peptide (100 nM TFLLR-NH2) (Tocris Bioscience Inc., Bristol, UK) (OST + PAR1). The culture medium was changed every 3 days for 14 days.
Alizarin red staining
I2 PDLSCs were seeded in 6-well plates (5 × 104 cells/cm2), in triplicate. After processing with CTRL, OST and OST + PAR1 groups for 14 days, cell sheets were washed with 1 × PBS (Sigma-Aldrich, St Louis, USA), fixed with 4% paraformaldehyde (Sigma-Aldrich, St Louis, USA) for 15 min at room temperature ( TA), washed with 1 × PBS (Sigma-Aldrich, St Louis, USA) and incubated with a stirred solution of 2% Alizarin Red S (Sigma-Aldrich, St Louis, USA) in PBS (Sigma-Aldrich, St Louis , USA) (pH 4.2) (A5533) for 30 min at RT. For qualitative and macroscopic analysis, images were acquired using a CDD microscope camera (D7000, Nikon, Minato, Japan). For quantitative analysis, 10% ammonium hydroxide solution (Sigma-Aldrich, St Louis, USA) was used to dilute the calcium deposits and a spectrophotometer (Biotek, Winooski, USA) was used to measure the absorbance at 405 nm. The quantification of ARS was calculated using a standard curve.
Characterization of the cell sheet
Scanning electron microscopy
Cell sheets at 14 days of treatment were fixed with Karnovsky’s solution (Thermo Fisher Scientific, Waltham, USA) for 24 h. After this period, post-fixation was performed with osmium tetroxide (Sigma-Aldrich, St Louis, USA) for 2 h and dehydration, passing the material through an increasing series of alcohol (Sigma-Aldrich, St. Louis, USA) from 70 to 100%. The samples were then dried under critical point conditions and sputtered gold coated. Observations were then made using a scanning electron microscope (S-4800; Hitachi, Tokyo, Japan).
Hematoxylin and eosin (Sigma-Aldrich, St Louis, USA) staining of cell sheets was also performed. Briefly, after detachment and fixation in 4% paraformaldehyde (Sigma-Aldrich, St Louis, USA) for 24 h, specimens were embedded in paraffin and sectioned at 4 µm thickness. After the sectioning process, the histological specimens were placed in a 50°C water bath and captured on a histological slide and stored in a 65°C oven for 12 h to allow removal of paraffin remnants. Sequentially, the paraffin removal dehydration protocol was completed using a sequence of solutions of xylol (Sigma-Aldrich, St Louis, USA) and alcohol (Sigma-Aldrich, St Louis, USA) and specimens underwent routine H&E staining according to manufacturer’s guidelines.
Ectopic bone formation assays
Bone formation in vivo was assessed using transplantation of cell sheets pre-incubated with the experimental groups described above. At 14 days of incubation, the cell sheets were detached using a cell scraper (Corning, New York, USA), folded 4 times to acquire a cylindrical shape and each Balb/c nude mouse aged 6 weeks received 2 back grafts from the same experiment. group, bilaterally. A total of 25 mice were divided into the groups as follows: 11 (OST + PAR1), 11 (OST) and 3 (CTRL). After 60 days, mice were euthanized and subcutaneous samples were collected and processed for analysis described below.
After fixation, only the subcutaneous sample from the left side of each animal was scanned using a micro-CT system (Skyscan 1176, Bruker Biospin, Billerica, USA) with the following acquisition parameters: 45 kV, 8.71 μm resolution and 550 μA. The data obtained were processed, reconstructed into three-dimensional images using reconstruction software (NRecon, Micro Photonics, Allentown, USA) and analyzed using system software (CTAn, Bruker Biospin, Billerica, USA).
Immunohistochemistry and histological analysis
Histological procedures were performed as previously described. H&E, Trichrome de Masson, Van Gieson and Von Kossa stains (Sigma-Aldrich, St Louis, USA) were performed according to the manufacturer’s protocol. For immunohistochemical analysis, after fixation in a 4% paraformaldehyde solution in PBS for 30 min, samples were embedded in paraffin and sectioned at 20 µm. Sections were subjected to deparaffinization, rehydration and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, St Louis, USA) for 10 min. Next, sections were blocked in 3% hydrogen peroxide solution (Sigma-Aldrich, St Louis, USA) for 10 min, followed by antigen retrieval with 10% citrate buffer ( Sigma-Aldrich, St Louis, USA) for 15 min and blocking in 5% BSA (Sigma-Aldrich, St Louis, USA) for another 30 min. Then, exposure to specific primary antibodies was performed for: integrin β1 – AB52971 (Abcam, Cambridge, UK), type I collagen – AB34710 (Abcam, Cambridge, UK) and BSP – AB8448 (Abcam, Cambridge, UK). Slides were then incubated with secondary conjugated anti-rabbit IgG antibody – AB205718 (Abcam, Cambridge, UK) followed by incubation of avidin-biotin complex (Abcam, Cambridge, UK) for 30 min. . Diaminobenzidine (DAB) solution (Abcam, Cambridge, UK) was applied every 2 min and the reaction observed under the microscope. Specimens were dehydrated, cleaned and mounted. Images of the specimens were obtained with an optical microscope (E600, Nikon, Tokyo, Japan) and three images of each sample were acquired and quantified using the J-frame (NIH, Bethesda, USA).
Experiments were performed in triplicate and data were expressed as mean ± standard deviation (SD). Data were analyzed based on the comparison between the experimental group (OST + PAR1) with osteogenic control group (OST) and control group (CTRL). A one-way ANOVA followed by Tukey’s post-hoc test was used. A meaning P-value of 0.05 was established for all tests and data analysis was performed (GraphPad Prism™ Version 6.0c, La Jolla, USA).