The present study was approved by the Ethics Committee of The Affiliated Stomatology Hospital, Southwest Medical University (Luzhou, China; approval no. 20221107003). Premolar teeth were extracted for orthodontic reasons, and written consent was provided by the guardian and patient for the use of these teeth in the present research project. In total, 34 premolars were collected from patients (12-14 years old) from November to December, 2022 at The Affiliated Stomatology Hospital, Southwest Medical University. PBS containing 5, 2 and 1% streptomycin/penicillin were prepared and used for washing roots, while the periodontal membrane was delicately removed from the middle 1/3 of the roots. The periodontal ligament tissue was seeded in a culture flask with 2 ml complete medium, which contained 89% a MEM medium (Gibco; Thermo Fisher Scientific, Inc.), 10% fetal bovine serum (Procell Life Science & Technology Co., Ltd.) and 1% streptomycin/penicillin (Beyotime Biotechnology Co., Ltd.). The flask was placed inverted in an incubator at 37°C (5% CO₂). After 2-3 h, the cell culture flask was inverted. Third-generation cells were collected for adipogenic and osteogenic induction. The medium was changed to either osteogenic medium [complete medium with 50 μg/ml ascorbic acid (Beyotime Biotechnology Co., Ltd.), 100 nmol/l dexamethasone (Beijing Solarbio Science & Technology Co., Ltd.) and 10 mmol/l β-glycerophosphate (Beijing Solarbio Science & Technology Co., Ltd.)] or adipogenic induction medium [complete medium with 100 μmol/l indomethacin (Beijing Solarbio Science & Technology Co., Ltd.), 0.5 mmol/l 3-isobutyl-1-methyl xanthine (Beijing Solarbio Science & Technology Co., Ltd.), 10 μg/ml insulin (Beyotime Biotechnology Co., Ltd.) and 1 μmol/l dexamethasone (Beijing Solarbio Science & Technology Co., Ltd.)] and refreshed every 3 days. Alizarin red S staining (2%) for calcified nodules (37°C, 1 h) was performed after 28 days, while oil red O staining (0.3%) for fat droplet (37°C, 30 min) was conducted after 21 days.

Surface markers for the third-generation hPDLSCs were identified by flow cytometry. Cells were resuspended in 1-2 ml PBS and placed into assay tubes. Flow cytometry assay-associated surface marker antibodies (BD Biosciences), including CD31 (cat. no. 560984; FITC), CD44 (cat. no. 561858; PE), CD45 (cat. no. 560976; FITC), CD105 (cat. no. 560839; PE) and CD90 (cat. no. 555596; PE), were pre-diluted for use at the recommended volume per test. These antibodies were added to the cells and incubated at 4°C for 30 min in the dark. Subsequently, the cells were washed with PBS thrice, resuspended in PBS and subjected to flow cytometry using a BD FACS Calibur (BD Biosciences). Evaluation and analysis of stem cell characteristics were conducted by detecting the specific stem cell surface markers CD31, CD44, CD45, CD90 and CD105. The results were analyzed by BD CellQuest Pro software 5.1 (BD Biosciences).

Cells treated with NAR (10 μmol/l) were also treated with NG-nitro-L-arginine methyl ester (L-NAME; 70 μM) to inhibit endothelial nitric oxide synthase (eNOS), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 20 μM) to inhibit soluble guanylate cyclase (sGC) and AKT inhibitor VIII (58 nM) to inhibit AKT, as recommended by the manufacturer (MedChemExpress for all).

The third-generation hPDLSCs were seeded at a density of 5×10³ cells per well in 96-well plates, and the impact of 10 μmol/l NAR on the viability of hPDLSCs was assessed. After 7 days, the absorbance at 450 nm was measured with a plate reader (Thermo Fisher Scientific, Inc.) following the instructions provided by the manufacturer of Cell Counting Kit-8 (37°C, 1 h; APeXBIO Technology LLC).

After 7 days of culture, cells subjected to different treatments were washed with PBS and lysed by RIPA lysis buffer (cat. no. PC101; Shanghai Epizyme Biotech Co., Ltd.) containing protease (cat. no. GRF101; Shanghai Epizyme Biotech Co., Ltd.) and phosphatase inhibitors (cat. no. GRF102; Shanghai Epizyme Biotech Co., Ltd.; 1:100). The BCA protein assay kit was used to determine the concentration of proteins. After the addition of 5X SDS-PAGE protein loading buffer, the samples underwent a 10-min incubation in a metal water bath at 100°C. Subsequently, 20 μg protein per lane was subjected to 6, 7.5 or 10% SDS-PAGE, then transferred onto PVDF membranes. After blocking (room temperature, 10 min) by Protein Free Rapid Sealing Solution (cat. no. PS108P; Shanghai Epizyme Biotech Co., Ltd.), the membranes were incubated with 1:2,000 diluted primary antibodies (ImmunoWay Biotechnology Company) against Runt-related transcription factor 2 (RUNX2; cat. no. YT5356), osteopontin (OPN; cat. no. YT3467), sGC (cat. no. YN5360), protein kinase G (PKG; cat. no. YN1879), transient receptor potential cation channel, subfamily C, member 6 (TRPC6; cat. no. YN4084), AKT (cat. no. YT0185), phosphorylated (p)-AKT (cat. no. YP0006), eNOS (cat. no. YT3174), p-eNOS (cat. no. YP0514) and GAPDH (cat. no. YN5585) for 24 h at 4°C, followed by a 1-h incubation with the Goat Anti-Rabbit IgG H&L antibody (Beijing Biosynthesis Biotechnology Co., Ltd.; cat. no. YP0514; 1:3,000) at room temperature. Protein bands were developed by Omni-ECL™ Femto Light Chemiluminescence kit (cat. no. SQ201; Shanghai Epizyme Biotech Co., Ltd.) after immersion of the membrane in the developing solution. GAPDH served as an internal reference to calculate the relative expression of other target proteins by ImageJ (version 1.52; National Institutes of Health).

After 7 days of incubation, total RNA extraction was performed with Rapid RNA Extraction kit (Hunan Accurate Bio-Medical Technology Co., Ltd.), and RNA was reverse transcribed using Reverse Transcription Premix Kit according to the manufacturer's protocol (Hunan Accurate Bio-Medical Technology Co., Ltd.). The primer sequences for qPCR are listed in Table I. The primers (Sangon Biotech Co., Ltd.) SYBR (Hunan Accurate Bio-Medical Technology Co., Ltd.) and cDNA were added in octuplet tubes and qPCR was performed following a preset program (step 1: 95°C, 30 sec, 1 cycle; step 2: 95°C, 5 sec, 40 cycles; step 3: 60°C, 30 sec, 40 cycles) (Bio-Rad Laboratories, Inc.). Gene expression was normalized to GAPDH, which served as an internal reference. The 2^−ΔΔCq method was used for data analysis (17).

After 7 days of incubation at 37°C, cells from different treatment groups were washed with PBS thrice then fixed with 4% paraformaldehyde for 30 min at room temperature. The cells were then incubated with freshly prepared ALP staining reagent (Sangon Biotech Co., Ltd.) at 37°C for 30 min. Subsequently, ALP staining was observed and recorded by light microscopes and cameras.

Following 28 days of incubation, cells from various treatment groups were washed with PBS thrice and subsequently fixed with 4% paraformaldehyde for 30 min at room temperature. Next, cells underwent 2-3 washes with 2-3 ml double-distilled water. Finally, 1 ml alizarin red dye (Beijing Solarbio Science & Technology Co., Ltd.) was added to each well, and the cells were incubated for 30 min at 37°C in an incubator. The wells were then washed thoroughly with double-distilled water, and the mineralized nodules were observed and recorded by light microscopes and cameras.

The three-dimensional (3D) structure of the AKT receptor protein (ID: 1UNQ) was downloaded from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (PDB) database (https://www.rcsb.org/). Additionally, the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) was queried for NAR (compound CID: 932), and the molecular ligand 3D structure was retrieved and downloaded. Subsequently, both the AKT protein and NAR molecular files were converted to a PDBQT format, which involved the elimination of all water molecules and the addition of polar hydrogen atoms. Molecular docking was then conducted by using Autodock 4.2 (18) and Autodock Vina 1.2.2 (19), and these tools were employed to calculate the binding energy between AKT and NAR. The visualization of the final docking results was performed using Ligplot v.2.2.4 (EMBL-EBI) and Pymol 2.4.0 (Schrödinger, Inc.).

The high-throughput sequencing service and subsequent bioinformatics analysis were provided by Novogene Co. Ltd. (Beijing, China). Briefly, after 7 days of culture, total RNA was extracted by TRIzol (Thermo Fisher Scientific, Inc.) from the cells of various treatment groups. Subsequently, the gene expression of both the control and experimental groups was assessed, the differentially expressed mRNAs (GEO ID: GSE266150) were identified by Novogene Co. Ltd., with the value of |log2FoldChange|>1 and Padj <0.01. This was followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, which characterized the functional pathways associated with the differentially expressed genes.

The key genes involved in the cGMP-PKG signaling pathway were identified using the KEGG database online tool (https://www.genome.jp/kegg/). The NAR target gene set was obtained through the Encyclopedia of Traditional Chinese (20) database (http://www.tcmip.cn/ETCM/index.php/Home/Index/index.html). These two gene sets were entered into the STRING database (https://cn.string-db.org/) to generate the PPI network. The network was analyzed using Cytoscape 3.9.1 (https://cyto-scape.org/) and the genes were ranked after calculating the score of each gene. The top ten ranked genes were selected to construct the core sub-network.

The cGMP ELISA was conducted using the human cyclic guanosine monophosphate ELISA kit (cat. no. F2502-A; Beijing Huabodeyi Biotechnology Co., Ltd.). After a 2-day incubation, the medium from hPDLSCs from various treatment groups was collected. The cGMP standard solutions were diluted to 0.1, 0.2, 0.4, 0.8 and 1.6 nmol/l according to the provided instructions Subsequently, the samples and the standard solutions were added to a 96-well plate and incubated at 37°C for 30 min. After completion of the reaction between the samples or the standard solutions with the Enzyme Coating Plates, the samples and the standards were discarded. Next, the washing solution was added to the 96-well plate five times, then 50 μl enzyme labeling reaction solution was added to all wells except the blank wells. The reaction was then incubated at 37°C for an additional 30 min. The enzyme labeling reaction solution was then discarded and the 96-well plate was washed with washing solution five times. Color rendering solution A and B were added to the 96-well plate successively, then the 96-well plate was incubated at 37°C for 10 min without light. Subsequently, terminating solution was added to terminate the reaction. Finally, the cGMP level was quantified according to the optical density (OD) at 405 nm by comparison with the standard curves.

NO detection was conducted using the Griess Reagent kit (cat. no. S0021S; Beyotime Biotechnology Co., Ltd.). hPDLSCs were seeded in 6-well plates at a density of 1.0×10⁶ cells per well, subjected to the various aforementioned treatments for 24 h, then the supernatant was collected. The NaNO₂ standard solutions with concentrations ranging from 0 to 100 μM were prepared with complete medium. Next, 50 μl standards and 50 μl samples were added separately to each well of a 96-well plate. Subsequently, NO detection reagents I and II were added (50 μl each; 37°C, 10 min). After completion of the reaction, Finally, the resulting solutions were quantified according to the OD at 540 nm using a microplate reader (Thermo Fisher Scientific, Inc.).

A total of 12 Sprague-Dawley (SD) male rats (180-220 g, 6 weeks old) purchased from Southwest Medical University Laboratory Animal Center (Luzhou, China), were divided equally into the control and NAR groups. Animals were housed in a controlled environment, including a temperature of 23±2°C, a relative humidity of 45±5%, a 12 h light-dark cycle and access to food and water ad libitum. Rats were examined daily for body weight, health and behavior. Anesthesia was induced with 50 mg/kg sodium pentobarbital by intraperitoneal injection. Hair removal was achieved using depilatory cream and sequential incisions were made in the left cheek tissues. Next, a 1×1×2-mm defect was abraded underneath the molar teeth of the rats using a high-speed handpiece. After surgery, subcutaneous injection of carprofen (5 mg/kg) was used for pain relief. The NAR group received a daily gavage of 50 mg/kg NAR (Shanghai Macklin Biochemical Co., Ltd.) (21,22), while the control group received an equal volume of saline. The following conditions were set as the humane endpoints: Initial body weight loss of >20%, inability to eat and drink and animals without anesthesia or sedation showing depression or low body temperatures <37°C. No SD rats exhibited these humane endpoints. After 28 days of continuous treatment, the SD rats were euthanized at a 50% vol/min CO₂ displacement rate (according to the AVMA guidelines) (23). After determining that the rats were not moving, not breathing and had dilated pupils, the CO₂ was switched off and the animals were observed for a further 2 min to determine that they were dead. Next, lower jaw samples were collected and then immersed in 4% paraformaldehyde (room temperature, 24 h). The animal manipulation procedures were approved by The Ethics Committee of the Laboratory Animal Center of Southwest Medical University (approval no. 20221107-016).

After 2-month decalcification using ethylenediaminetetraacetic acid (cat. no. ST069-500 ml; Beyotime Biotechnology Co., Ltd.), the alveolar bone tissues were embedded in paraffin and sectioned (4-μm sections), followed by Masson staining (room temperature, 2 h) (Beijing Solarbio Science & Technology Co., Ltd.) and light microscopy to observe osteogenesis at the bone defects.

For immunofluorescence staining, the tissue sections were deparaffinized with xylene (twice for 15 min each). After deparaffinization, the sections were rehydrated with different concentrations of alcohol (100, 95, 80 and 70%) and purified water. The excess water was discarded, and antigen repair was conducted by adding an appropriate amount of antigen repair solution (cat. no. P0088; Beyotime Biotechnology Co., Ltd.) to the tissues, then washing with water after 10 min. Next, the sections were blocked using 5% goat serum (Beijing Solarbio Science & Technology Co., Ltd.) for 30 min at 37°C and then incubated overnight with anti-RUNX2 (1:100; cat. no. YT5356) and anti-OPN (1:100; cat. no. YT3467) antibodies (ImmunoWay Biotechnology Company) at 4°C. The next day, the sections were incubated with a Multi-rAb CoraLite^® Plus 594-Goat Anti-Rabbit Recombinant Secondary Antibody (1:200; cat. no. RGAR004; Proteintech Group, Inc.) at 37°C for 1 h. Next, the sections were washed in PBS, treated with DAPI (37°C, 10 min) and sealed with glycerol. The sections were then observed and imaged under a fluorescence microscope.

A micro-CT system (SkySCAN 1176 Micro-CT; Bruker Corporation) was employed for imaging. The imaging parameters were as follows: Pixel size=35 mm, angular rotation step=0.8 and voltage/current=50 kV/498 mA. The CT results were reconstructed, and the region of the bone corresponding to the bone defect was specifically selected for subsequent analysis. The regions of interest were analyzed using CT-Analyser (version 1.20.3.0; Bruker Corporation). The total volume of the tissue was calculated by the software by setting uniform parameters. Then, the bone mineral density (BMD), bone volume/tissue volume (BV/TV) ratio and trabecular thickness (TbTh) were assessed.

NAR probes labeled with fluorescein were synthesized through an azide-alkyne cycloaddition reaction. hPDLSCs were treated with NAR probes (10 μmol/l) and fluorescence detection was conducted after a 2-day incubation period. The treated cells were fixed using 4% paraformaldehyde (37°C, 30 min), washed with PBS and blocked with 10% goat serum for 1 h (room temperature). The blocked cells were then incubated with anti-AKT antibody (1:100) at 4°C overnight. The next day, the samples were incubated with a Multi-rAb CoraLite^® Plus 594-Goat Anti-Rabbit Recombinant Secondary antibody (1:200; Proteintech Group, Inc.) at 37°C for 1 h, followed by incubation with DAPI (37°C, 10 min). The hPDLSCs were then washed with PBS thrice, sealed with glycerol and observed and imaged under a laser confocal microscope.

SPSS version 21.0 (IBM Corp.) was used to analyze the data. All data are presented as the mean ± standard deviation. Statistical significance was evaluated using the Student's t-test (unpaired) for independent samples to compare the differences in the data between groups. For ≥3 groups, one-way ANOVA and Dunnett's post hoc test were employed. P<0.05 was considered to indicate a statistically significant difference.

The established third-generation hPDLSCs exhibited robust osteogenic and adipogenic differentiation capabilities (Fig. 1C and D). Flow cytometry analysis confirmed the identity of hPDLSCs, which revealed positive expression of CD44 (99.84%), CD90 (99.95%) and CD105 (98.95%), and negative expression of CD31 (0.46%) and CD45 (0.17%) (Fig. 1E). Compared with the control group, hPDLSCs treated with 10 μM NAR for 7 days demonstrated enhanced osteogenic potential, as indicated by ALP staining, formation of mineralized nodules and elevated gene and protein expression of RUNX2 and OPN (Fig. 1F, G and I-K).

The transcriptome sequencing results revealed differential expression of 104 genes between the control and NAR groups (Fig. 2A and B). KEGG analysis of these differentially expressed genes revealed the involvement of the cGMP-PKG signaling pathway (Fig. 2C). The ELISA result revealed an elevated cGMP protein expression in the NAR group compared with the control group (Fig. 2D). During the assessment of pathway factors associated with the cGMP-PKG signaling axis, the NAR group was observed to exhibit elevated expression levels of sGC, PKG and TRPC6 proteins and genes compared with the control group (Fig. 2E-G).

Strong ALP and alizarin red staining was noted in the NAR group compared with the control group (Fig. 3A and B), indicating that NAR promoted the osteogenic differentiation of hPDLSCs. Additionally, the gene and protein expression levels of the osteogenesis-related factors, RUNX2 and OPN, were higher in the NAR group than in the control group (Fig. 3D-F). This elevation was accompanied by increased expression of p-eNOS, NO and sGC (Fig. 3C-E and G). Upon the addition of L-NAME (an eNOS inhibitor), the NAR + L-NAME group exhibited decreased expression of p-eNOS, NO and sGC compared with the NAR group, in addition to a decreased expression of the osteogenic factors, RUNX2 and OPN. Furthermore, the staining intensity of ALP and alizarin red was lower in the NAR + L-NAME group compared with the NAR group (Fig. 3A and B).

Further experiments were conducted to explore the role of the NO downstream factor, sGC. The results revealed an increased expression of sGC, PKG, cGMP and TRPC6 in the NAR group compared with the control group (Fig. 4C, E, F and H). Upon addition of ODQ (an sGC inhibitor), the NAR + ODQ group exhibited decreased expression of the osteogenesis-related factors, RUNX2 and OPN, at both the gene and protein levels (Fig. 4D, F and G). Additionally, the intensity of both ALP and alizarin red staining in the NAR + ODQ group was lower compared with the NAR group (Fig. 4A and B). Furthermore, the expression of sGC, PKG, cGMP and TRPC6 was reduced in the NAR + ODQ group compared with the NAR group (Fig. 4C-H).

NO-cGMP-PKG signaling pathway-related factors and NAR targeting factors were input into the STRING database for analysis. A PPI network was generated, and the top 10 genes were used to construct the core sub-network by Cytoscape. AKT emerged as the top-ranking gene in this network, which revealed an association between AKT and eNOS (also termed NOS3; Fig. 5A). Subsequently, AKT, eNOS, NO, sGC, cGMP, PKG and TRPC6 were integrated into the cGMP-PKG signaling pathway, which revealed that AKT was situated upstream of this pathway and could directly regulate eNOS (Fig. 5B). Fig. 5C illustrates the docking results of NAR (structure downloaded from PubChem) with AKT molecules (structure downloaded from the PDB), while the 2D representation is shown in Fig. 5D. In addition, a NAR probe labeled with fluorescein was co-cultured with hPDLSCs (Fig. 5E), which revealed co-localization of the fluorescence emission of the NAR probe with that of AKT in the cytoplasm (Fig. 5F).

The results revealed increased expression of p-AKT and p-eNOS in the NAR group compared with the control group (Fig. 6D and F). Upon addition of AKT inhibitor VIII, the NAR + AKT inhibitor VIII group exhibited decreased expression of the osteogenesis-related factors, RUNX2 and OPN, at both the gene and protein levels compared with the NAR group. Additionally, ALP and alizarin red staining exhibited a lower intensity, and the expression of p-AKT and p-eNOS was reduced in the NAR + AKT inhibitor VIII group compared with the NAR group (Fig. 6A-F).

Due to the enhanced osteogenic differentiation observed in hPDLSCs under the influence of NAR during in vitro experiments, it was hypothesized that NAR may promote alveolar bone regeneration. To investigate this, an alveolar bone defect model was established in SD rats, and a daily gavage of 50 mg/kg NAR was administered to the treatment group. Micro-CT analysis of bone morphology revealed that, after 28 days of treatment, new bone, BMD, BV/TV and TbTh at the bone defects were higher in the NAR group compared with the saline-treated (control) group (Fig. 7A-D). Masson staining also indicated increased formation of new bone in the NAR group (Fig. 7E). Immunofluorescence further demonstrated higher expression of RUNX2 (Fig. 7F) and OPN (Fig. 7G) in the periodontal ligament and new bone at the alveolar bone defects in the NAR group. These findings suggested that NAR has the potential to promote the healing of alveolar bone defects.