SAL was purchased from Chengdu Must Bio-Technology Co., Ltd. (cat. no. MUST-19021504) and was dissolved in dimethyl sulfoxide (stock concentration of 200 mM). α-modification of Eagle's minimum essential medium (α-MEM) and penicillin/streptomycin were purchased from Gibco; Thermo Fisher Scientific, Inc. Alizarin Red S (ARS), Triton X-100 and DAPI were obtained from Sigma-Aldrich; Merck KGaA. Primary antibodies against GAPDH (Cell Signaling Technology, Inc., cat. no. 5174T), Smad 1 (Cell Signaling Technology, Inc., cat. no. 6944T), phosphorylated (p-)-Smad1/5/8 (Cell Signaling Technology, Inc., cat. no. 13820T), runt-related transcription factor 2 (RUNX2) (Cell Signaling Technology, Inc., cat. no. 12556S), bone sialoprotein (BSP) (Cell Signaling Technology, Inc., cat. no. 5468S), and osteocalcin (OCN) (Cell Signaling Technology, Inc., cat. no. 12888) were purchased from Cell Signaling Technology, Inc. Osterix (OSX) (Abcam, cat. no. ab209484) were purchased from Abcam. Alkaline Phosphatase (ALP) assay kit (cat. no. P0321S) was obtained from Beyotime Institute of Biotechnology.

The present study was approved by The Shanghai Stomatological Hospital Ethics Association [approval no. SSDC-(2020)0006] and all patients or their parents provided written informed consent. The hDPSCs were isolated from completely healthy premolars, without caries or periodontitis, which were extracted during the course of orthodontic treatment. The donors were aged between 14 and 18 years (n=10, five male, five female, aged 15.6±1.26 years) and they had their teeth extracted in Shanghai Stomatological Hospital between August and September 2019. The pulp was gently separated from the tooth and cultured with a modified tissue piece method in maintenance medium containing Dulbecco Modified Eagle Medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 20% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 100 units ml-1 penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C and 5% CO₂ in an incubator. The medium was replaced every 3 days after cells grew out from the tissue pieces until cells reached 80-90% confluence. hDPSCs were digested by 0.05% trypsin-EDTA and then seeded into a new dish at 1:1. Flow cytometric analysis was used to determine the cell surface markers present on hDPSCs. The hDPSCs of each donor were isolated and cultured alone.

The effects of SAL on hDPSC proliferation were measured using a Cell Counting Kit-8 kit (CCK-8; Dojindo Molecular Technologies, Inc.) according to the manufacturer's protocols (17). In brief, hDPSCs were plated into 96-well plates at a density of 5x10³ cells/well and were incubated for 48 or 96 h at 37˚C with serial dilutions of SAL (0, 10, 20, 50 and 100 µM). Subsequently, 10 µl CCK-8 solution was mixed with 90 µl complete α-MEM and added to each well. Following incubation for 2 h at 37˚C, the optical density was measured using an ELX800 absorbance microplate reader (Bio-Tek Instruments, Inc.) at 450 nm (650 nm reference).

hDPSCs were first cultured in 12-well plates (4x10⁴ cells per well). The wells were then divided into eight groups as follows at 37˚C and 5% CO₂ in an incubator for 21 days: i) Growth medium group (GM); ii) osteo/odontogenic induction medium (OM) group; iii) OM + 1 µM SAL group (1 µM); iv) OM + 5 µM SAL group (5 µM); v) OM + 10 µM SAL group (10 µM); vi) OM + 20 µM SAL group (20 µM); vii) OM + 50 µM SAL group (50 µM); viii) and OM + 100 µM SAL group (100 µM).

The osteo/odontogenic medium was comprised of 100 nM dexamethasone, 50 µM ascorbic acid and 10 mM β-glycerophosphate dissolved in α-MEM (all from Sigma-Aldrich; Merck KGaA) (18). The growth medium was 10% FBS and 1% Penicillin-Streptomycin in α-MEM. To assess osteogenic differentiation, the cells were stained with ARS (18). Briefly, hDPSCs were collected on day 21, fixed in 4% paraformaldehyde for 15 min and stained with 40 mM ARS (pH 4.9; Sigma-Aldrich; Merck KGaA) for 15 min, both at 25˚C. The plates were observed under an inverted microscope (magnification, x200; Nikon Corporation). The darker the intensity of the red dots, the higher the number of calcium nodules and therefore the higher degree of differentiation.

hDPSCs were cultured in 12-well plates (4x10⁴ cells per well). The wells were divided into three groups as follows: i) GM; ii) OM; and iii) 10 µM SAL group. hDPSCs were then treated for 7 days at 37˚C. Subsequently, they were fixed in 4% paraformaldehyde for 10 min at room temperature. The cells were stained for ALP using the ALP assay kit (cat. no. P0321S; Beyotime Institute of Biotechnology) according to the manufacturer's protocols (19,20). The cells were subsequently washed three times with distilled water. ALP staining was observed under an inverted microscope (magnification, x200; Nikon Corporation). Increases in the concentration of the insoluble nitroblue tetrazolium formazan was associated with a change in color from dark blue to blue/purple, which is in turn associated with higher ALP activity.

hDPSCs were cultured in six-well plates (8x10⁴ cells per well). The wells were divided into three groups as follows: i) GM; ii) OM; and iii) 10 µM SAL. hDPSCs were in turn treated for 3 and 7 days at 37˚C. Total mRNA was extracted using the acid guanidinium thiocyanate-phenol-chloroform method (21). Complementary DNA synthesis was performed using a FastQuant RT kit (Tiangen Biotech Co., Ltd. cat. no. KR106-03). qPCR was performed using the SYBR Green Premix (Tiangen Biotech Co., Ltd.; cat. no. FP207) according to the manufacturer's protocols under the following conditions: 15 min of denaturation at 42˚C and 1 cycle of 1 min of denaturation at 95˚C and 40 cycles each of 5 sec of denaturation at 95˚C and 15 sec of amplification at 60˚C. The following primer sequences were used as previously described: human actin (22) forward, 5'-CATGTACGTTGCTATCCAGGC-3' and reverse, 5'-CTCCTTAATGTCACGCACGAT-3'; human ALP (22) forward, 5'-CCTCCTCGGAAGACACTCTG-3' and reverse, 5'-GCAGTGAAGGGCTTCTTGTC-3'; human RUNX2(22) forward, 5'-CCACTGAACCAAAAAGAAATCCC-3' and reverse, 5'-GAAAACAACACATAGCCAAACGC-3'; human OCN (23) forward, 5'-GCGCTACCTGTATCAATGGC-3' and reverse, 5'-AACTCTCACAGTCCGGATT-3'; human dentin sialophosphoprotein (DSPP) (7) forward, 5'-CGACATAGGTCACAATGAGGATGTCG-3' and reverse, 5'-TTGCTTCCAGCTACTTGAGGTC-3'; human BSP (7) forward, 5'-CAGGCCACGATATTATCTTTACA-3'and reverse, 5'-CTCCTCTTCTTCCTCCTCCTC-3' and human OSX (24) forward, 5'-CTGTGAAACCTCAAGTCCTATGGA-3' and reverse, 5'-GCTCTGCAGTCAAGGGAGATG-3'. The relative gene expression was calculated according to the comparative 2^-ΔΔCq method and normalized to that of actin (25).

hDPSCs were cultured in 24-well plates (4x10⁴ cells per well). The wells were divided into three groups as follows: i) GM; ii) OM; and iii) 10 µM SAL. hDPSCs were treated for 3 days at 37˚C. The cell samples were then fixed in 4% paraformaldehyde in PBS for 20 min at room temperature. Subsequently, the cells were permeabilized in the presence of PBS with 0.25% Triton X-100 for 15 min (26) and blocked with 5% non-fat milk diluted in TBS at room temperature for 1 h. The cells were incubated overnight at 4˚C with anti-DSPP (1:200; cat. no. SC-73632; Santa Cruz Biotechnology, Inc.) in the presence of 2.5% BSA. The cells were subsequently washed three times in PBS with 0.1% Tween-20 (PBST) and stained for 1 h at room temperature with a goat anti-mouse FITC-conjugated secondary antibody (1:1,000; cat. no. F-2761; Thermo Fisher Scientific, Inc.) in 2.5% BSA. The samples were washed five times with PBST and the cells were counterstained with DAPI (1:10,000) at room temperature for 10 min. The cells were imaged using a fluorescence microscope (magnifications, x200; Leica Microsystems, Inc.).

hDPSCs were cultured in six-well plates (8x10⁴ cells per well). The wells were divided into three groups as follows: i) GM; ii) OM; and iii) 10 µM SAL group. hDPSCs were treated for 3 days at 37˚C. The cells were collected in RIPA lysis buffer (Thermo Fisher Scientific, Inc.). The protein concentration was determined by BCA method (Thermo Fisher Scientific, Inc., cat. no 23225). The lysate proteins (30 µg) were separated using 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. The PVDF membranes were blocked with 5% non-fat milk diluted in TBS at room temperature for 1 h and were incubated overnight at 4˚C with a series of primary antibodies targeted at GAPDH, Smad 1, p-Smad1/5/8, RUNX2, BSP, OSX, and OCN (1:1,000 dilution). The following day, the PVDF membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, Inc. cat. nos. 7074 and 7076; 1:10,000 dilution) for 1-2 h at room temperature. A Western Chemiluminescent HRP Substrate system (EMD Millipore; cat. no. WBKLS0100) was used for visualizing the bands. The signals were captured with an Amersham Imager 600 monitoring system (GE Healthcare). ImageJ 1.8.0 (National Institutes of Health) was used to quantify the bands.

Data analysis was performed using the SPSS software (ver. 23.0; IBM Corp.). Experiments were repeated independently at least three times. The results were expressed as mean ± standard deviation. Statistical analysis of the data was performed with one-way analysis of variance followed by the Scheffé's post hoc test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effects of SAL on the osteo/odontogenic differentiation of hDPSCs, CCK-8 assay was first used. Treatment with SAL for 48 or 96 h at concentrations <100 µM did not induce cytotoxicity. SAL (10 or 20 µM) significantly increased cell viability at 96 h time points of treatment compared with that in the control group (Fig. 1A and B).

ARS was subsequently used to detect the effect of SAL on the osteogenic differentiation of hDPSCs. SAL promoted the osteo/odontogenic differentiation of hDPSCs at 100 µM; Fig. 1C showed that differentiation showed by the calcium nodules was increased in all treatment groups vs. GM. SAL (10 µM) markedly increased the osteo/odontogenic differentiation of hDPSCs (Fig. 1C). Based on this observation, 10 µM SAL was selected for the following experiments.

ALP staining indicated that OM increased ALP activity compared with GM and SAL (10 µM) markedly increased ALP activity in the hDPSCs compared with OM (Fig. 2A). Additionally, SAL further promoted the expression levels of ALP mRNA following incubation with the cells for 3 and 7 days compared with OM (Fig. 2B). The expression levels of OSX were also significantly increased in hDPSCs following treatment of the cells with 10 µM SAL for 3 and 7 days compared with those in the OM group (Fig. 2C). Although SAL promoted the expression levels of RUNX2, OCN, DSPP and BSP mRNA following incubation with the cells for 3 and 7 days to some extent, there was no significant difference (Fig. 2).

DSPP is a key protein involved in the process of odontogenic differentiation (23). In the OM group, the expression of DSPP was higher compared with the GM group. SAL (10 µM) effectively promoted the expression of DSPP in hDPSCs as detected by immunofluorescence analysis compared with the OM group (Fig. 3).

The effects of SAL on the expression of the osteo/odontogenic differentiation-associated proteins were detected in hDPSCs by western blot analysis. Western blot analysis indicated that SAL markedly increased the expression levels of the RUNX2 and BSP proteins compared with the OM group (Fig. 4A). To explore the specific mechanism underlying the involvement of SAL in the osteo/odontogenic differentiation of hDPSCs, the effects of SAL on the p38, JNK, ERK and Smad1/5/8 signaling pathways were examined. The results indicated that whilst SAL could significantly activate Smad1/5/8 signaling compared with that in the OM group, it had no effect on the p38, ERK or the JNK MAPK signaling pathways (Fig. 4B-C).