Isolation and characterization of SCAPs

The study was approved by the Ethics Committee of the Universidad Antonio Nariño (code: 06252018), and all the participants responded voluntarily to an informed consent form, according to the Helsinki Declaration. Blood and teeth were taken from healthy volunteers, no smokers or drug consumption, and blood analysis with normal ranges results. The teeth were collected from patients who required pre-orthodontic surgery for their dental treatments, and the radiography analysis was used to determine the Nola classification.

The apical papilla tissues were obtained from four third molars, extracted for pre-orthodontics treatment, without complete radicular development (Nolla Stage 8 and 9) of three volunteer participants, with a mean age of 17.5 ± 0.6 years. As previously reported, SCAPs were isolated and cultured by the explant culture method [17]. Cells were maintained in Dulbecco's modified Eagle medium (DMEM) (Lonza® High glucose), supplemented with penicillin/streptomycin (10,000 IU penicillin, 10,000 μg/ml streptomycin), l-glutamine (0.1 μM), and fetal bovine serum (10%) (Gibco®) until their ~ 85% confluence. In this study, we used cells between the 2 and 5 passages. For the SCAPs characterization, the cell adhesion and morphology were evaluated using optical microscope observations. A colony formation unit (UFC) assay was performed in 6-well tissue culture plates, and the colonies (≥ 50 cell aggregates) were scored.

Additionally, for chondrogenic and osteogenic differentiation, cells were incubated with chondrogenic [25], and osteogenic culture media [26]. Chondrogenic differentiation was evaluated by Alcian blue staining and the osteogenic differentiation with Alizarin red staining after 14 days. The immunophenotype was determined by immunostaining using specific monoclonal antibodies for CD90 (eBioscience # 12-0909-42), CD105 (Sigma-Aldrich # SAB4700259-100), CD34 (LifeSpan Bioscience #LS C20 4500), and CD 45 (eBioscience # 11-9459-42), cell acquisition was performed in a flow cytometry (BD Accuri ™ C6 flow cytometer). Samples were analyzed using FlowJo™ Software. Data from three independent experiments were acquired.

PRF preparation

Peripheral blood of a healthy participant (25 years old) was collected in tubes without anticoagulant (Vacutainer® system, Becton Dickinson) that immediately were centrifuged (SCILogex® DMO 412) at 3200 rpm for 12 min, as previously we reported [27]. The PRF membrane [28] was sectioned in two parts (4 × 12 mm), and was placed into transwell inserts (pore of 0.4 μm Costar®) to avoid cell adhesion.

Stem cell exposure to LLLT stimulation and PRF

Stem cells were seeded into 12-well plates and cultured for 24 h to allow cell adhesion. They were then exposed once to LLLT as previously described [17]. Briefly, we applied a Diode laser using a therapeutic laser lamp (DMC Therapy-XT®), emitting red radiation at a wavelength of 660 nm, spot size 15 mm with circular shape of the laser beam, energy density of 6 J/cm2, pulse frequency of 60Hz, in a continuous mode. The equipment was fixed to a base and placed perpendicularly in contact with the plate’s lid (angle of 90°) to warranty the irradiation conditions. In the PRF + LLLT group, we irradiated the cells and then put the transwell inserts with the PRF sections inside.

Evaluation of osteogenic potential

The cultures were divided into four experimental groups. The first group refers to SCAPs stimulated with PRF. The second group refers to SCAPs stimulated with LLLT. The third group refers to SCAPs stimulated with PRF + LLLT. Finally, the fourth group refers to SCAPs without any stimuli (control group). In each group, the stem cells (1 × 104 SCAPs/mL) were seeded into 12-well plates in DMEM supplemented with 10% fetal bovine serum and cultured for 24 h to allow cell adhesion. At the end of this culture period, the medium was changed in all experimental groups to osteogenic medium [26]. The osteogenic medium was replaced every 3 days. The osteogenic potential of SCAPs was assessed at different periods of time (7, 14, and 21 days).

Evaluate bone nodule formation

The Alizarin red staining was performed at baseline, 7, 14, and 21 days after the exposure to PRF, LLLT, or PRF + LLLT. For this, the cultures were fixed with 70% ethanol, then overlaid with Alizarin red solution for 30 min. The cultures were then washed extensively, observed under an optical microscope, and photographed (Leica DM300). The nodules of mineralization in each microscope field (> 0.04 mm2) were counted.

Secretion of BMP-2, BMP-4 proteins

The supernatant medium of each experimental condition was collected at 24 h, 7, 14, and 21 days. According to the manufacturer's instructions, enzyme-linked immunosorbent assays (ELISA) were used to quantify BMP-2 and BMP-4 levels (Quantikine® ELISA R&D Systems—DBP200 and DBP400, respectively).

Gene expression of osteoblastic markersRNA-seq analysis of the differentiated SCAPs transcriptomic profile

Total RNA was extracted (RNeasy Mini kit, Qiagen) 21 days after cells were cultured in osteogenic medium in the presence or absence of PRF, LLLT, or PRF + LLLT. The RNA quality and yield were determined by NanoDrop spectrophotometer. Samples presenting RNA integrity number ≥ 7 and a 28S/18 ≥ 1.0 ratio were considered suitable for transcriptome sequencing. cDNA libraries were prepared with 1 μg of starting total RNA and sequenced using a DNBseq system with 100 bp paired-end reads length (Beijing Genomic Institute, BGI).

Quantitative RT-PCR (qRT-PCR) analyses

The gene expressions of relevant osteoblastic markers, including osteocalcin (OCN), osteopontin (OPN), and type 1 collagen (COL 1), were analyzed at 7, 14, and 21 days of incubation in an osteogenic medium in the presence or absence of PRF, LLLT, or PRF + LLLT by qRT-PCR. Briefly, total RNA from all experimental groups was extracted (RNeasy Mini kit, Qiagen) and reverse transcribed with MultiScribeTM RTranscripase (Thermofisher®), according to the manufacturer’s instruction. The amplicons were generated using primers listed in Table 1. Expression levels were estimated in duplicate, and phosphoglycerate kinase (PGK) was used as a normalization gene. ΔΔCt method was used to quantify the relative expression of each target gene.

Table 1 List of primers used for expression analysis by qRT-PCRStatistical analysis

The normality of the data was determined with Shapiro–Wilk test. Data were presented as means ± standard deviations of at least three independent experiments. A one-way ANOVA test with Tukey and Dunnet post hoc test was performed for comparisons. Paired student t test was used for intragroup comparison. p values < 0.05 (*) and p < 0.01 (**) were considered significant. The statistical analyses were performed by the statistical package SPSS V21 (IBM, Armonk, NY, USA).