PDLSCs were isolated from the periodontal membrane of healthy first premolars reserved for extraction due to orthodontic needs of patients with no prior history of periodontal disease. The subjects, aged between 18 and 25 years, received treatment at the Oral and Maxillofacial Surgery Department of Qingdao Stomatological Hospital and showed no underlying systemic conditions. Primary PDLSCs were cultured in medium supplemented with 20% fetal bovine serum (FBS, Pricella, China), and all other cultures were maintained with a 10% concentration. This study was approved by the Ethics Committee of Qingdao University (QDU-HEC-2024266).

When PDLSCs reached their third passage, they were plated at a density of 1 × 10⁵ cells per well in a six-well plate and subjected to adipogenic differentiation using specific medium (Pricella, China), with medium refreshed every 3 days for 21 days. Following this period, the cells were fixed in 4% paraformaldehyde (Solarbio, China), rinsed with PBS, and stained with oil red O (Pricella, China) for subsequent microscopic examination.

After the third passage, PDLSCs were similarly plated at a density of 1 × 10⁵ cells per well in a six-well plate and induced with osteogenic differentiation medium (Pricella, China). The cells were then fixed with 4% paraformaldehyde, rinsed with PBS, and subjected to alizarin red (Pricella, China) and alkaline phosphatase (ALP) staining (Pricella, China) for microscopic examination.

The cell density at the third-generation of PDLSCs was adjusted to 1 × 10⁶ cells/mL. The cellular suspension was aliquoted into centrifuge tubes at 100 µL per tube, and antibodies generated against CD34, CD45, CD73, CD90, and CD105 (Pricella, China) (at 1:50 dilutions) were added separately, while the control group received an equivalent volume of PBS. All cells were then incubated at room temperature in the dark for 20 min. After centrifugation, the supernatant was discarded, and the pellet was washed twice with PBS. The cells were resuspended in 500 µL of PBS for analysis on a flow cytometer (CytoFLEX; Beckman Coulter, Fullerton, California, United States).

PDLSCs were seeded at a density of 2 × 10⁴ cells per well in a 96-well plate and cultured with a gradient of lipopolysaccharide (LPS) concentrations at 0 μg/mL, 20 ng/mL, 50 ng/mL, 100 ng/mL, 150 ng/mL,and 200 ng/mL to screen the concentration of LPS in the simulated periodontitis microenvironment. On this basis, the effective concentration of Pso was screened at concentrations of 2 μg/mL, 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL, and 25 μg/mL. We evaluated cellular proliferation using a microplate reader (Bio-Tek, United States).

PDLSCs were co-cultured with Pso after LPS-induction and the LPS-induced stem cells served as the control group. We followed the same experimental protocol as in section 2.1.2, with alizarin red staining and ALP staining performed.

Network pharmacology analysis was employed to screen the potential targets of Pso in relation to chronic periodontitis (Beijing Allwegene Tech.). We implemented this approach to determine the specific targets that Pso may affect with respect to chronic periodontitis. Pathways with a significant enrichment of target genes were then analyzed to construct a drug-target-pathway network.

Cells were allocated to four groups: PDLSCs, PDLSCs induced by LPS, PDLSCs cultured with Pso, and cells cultured with both LPS/PSO. Total RNA was extracted using TRIzol reagent (Takara, Japan), and high-throughput sequencing was conducted by Beijing Allwegene Tech., with P < 0.05 designating differentially expressed genes (DEGs). We performed gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses to identify the pathways associated with the DEGs.

Total cell protein from all groups noted above were extracted using RIPA lysis solution (Elabscience, China) on ice, and a BCA kit (Elabscience, China) was employed to determine protein concentration. With GADPH as the internal reference, ALP (1: 1000; (Abcam, United Kingdom), OCN (1: 5000; Abcam, United Kingdom), Runt-related transcription factor 2 (RUNX2; 1: 1000; Abcam, United Kingdom), and mTOR (1: 1000; Boster, United States) were added sequentially and proteins incubated at 4°C overnight. After incubation with second antibody, ECL luminescent reagent (Elabscience, China) was added to observe HRP-labeled protein bands.

Total RNA from all groups noted above was extracted using TRIzol reagent, and the expression levels of PI3K, LC3, Deptor, PI3K, and WIPI 1 (Sangon Biotech, China) were determined by real-time q-PCR. GADPH (Sangon Biotech, China) was used as the internal reference, and relative expression levels were calculated using the 2^−ΔΔCT method. All analyses were repeated three times. The primers that we used were PI3K, 101 CTT TGC GAC AAG ACT GCC GAG AG (forward) and 101 CGC CTG AAG CTG AGC AAC ATC C (reverse); WIPI 1, 114 TGC TTG GCT CAG GAA CAA CAG AAG (forward) and 114 GCA CCG TGG AGG CTG AAG ATG (reverse); LC3, 89 TCT GAG GGC GAG AAG ATC CGA AAG (forward) and 89 TCC AGG TCT CCT ATC CGA GCT TTG (reverse); RagC, 139 CAG CGG CAA GTC CTC CAT CC (forward) and 139 CAT GTA GTC ATC CTG TGC GTC AAT G (reverse); and Deptor, 118 TTG TGG TGC GAG GAA GTA AGC C (forward) and 118 AGG ACA TTG AGC CCG TTG ACA G (reverse).

Rapamycin is a potent and specific mTOR inhibitor and an autophagic activator (Rangaraju et al., 2010; Edwards and Wandless, 2007), and can be used to effectively inhibit the expression of mTOR. PDLSCs were cultured with rapamycin (MCE, United States) and dissolved in DMSO (Solarbio, China), and PCR was conducted to verify mTOR gene expression.

We added Pso to the control group to form the experimental group, extracted RNA, and implemented PCR to evaluate the changes in gene-expression levels. Cells were then allocated to the control group or mTOR-inhibitor group. The gene and protein expression levels for Rag C and for the osteogenic genes ALP and RUNX2 were ultimately ascertained.

Lentiviruses for Rag C knockdown were constructed by Jikai Gene (Shanghai, China), and PDLSCs in optimal growth conditions were selected for lentiviral transfection. After 16 h of PDLSC transfection with lentivirus, normal medium was replaced, cultures were continued for an additional 72 h, and cells were observed under a fluorescence microscope. Following successful transfection, puromycin was employed to select for cell lines with stable expression, and PCR was utilized to assess expression changes in ALP and RUNX2 genes.

After mixing absolute ethanol and purified water, F127 and TMB were added and the mixture stirred for 30 min at room temperature. Dopamine hydrochloride (Aladdin, China) and Tris were then added, and the mixture was stirred for 24 h at room temperature. The mixture was centrifuged at 12,000 rpm for 10 min, and MPDA was obtained by washing the mixture of absolute ethanol/acetone (v/v = 2:1) three times by centrifugal precipitation.

MPDA and Pso were dispersed in absolute ethanol by ultrasonic shock. The evenly distributed MPDA and Pso were subsequently mixed and stirred in a magnetic stirrer for 24 h at room temperature.

The surface morphology and diameter of MPDA and MPDA-Pso composites were determined by transmission electron microscopy (TEM). The zeta potential was used to assess the stability of MPDA and MPDA-Pso, and the peaks for MPDA and MPDA-Pso were characterized using a Fourier transform infrared spectrometer (NICOLET iS50 FT-IR) and ultraviolet spectrophotometer.

PDLSCs were added to six-well plates at 2 × 10⁵ cells per well and divided into four groups: an MPDA group, Pso group, MPDA-Pso group, and blank control group. The medium was changed every 3 days for 21 days, and the cells were then fixed with 4% paraformaldehyde, washed with PBS, and stained with alizarin red.

PDLSCs were added to six-well plates at a concentration of 2 × 10⁵ cells per well and grouped as 2.5.1 cells, and we changed the solution every 3 days and co-cultured them for 14 days. The cells were then fixed in 4% paraformaldehyde (Solarbio, China) for 30 min, washed with PBS, and stained with an ALP staining kit.

After 14 days, the existing medium was removed, and the cells were fixed with 4% paraformaldehyde and rinsed with PBS. The cells in each group were then lysed on ice. Protein concentration was assessed with a BCA kit, and absorbance was measured at 520 nm after treatment with an ALP kit, and ALP activity was calculated according to the formula.

PDLSCs were seeded in six-well plates and grouped as in 2.5.1 above. After 7 days of culture, the RNA was extracted and its concentration was analyzed. The expression of ALP was determined by q-PCR using QuantiNova PCR Kits (QIAGEN, Germany). We adopted GAPDH as an internal reference for all samples, and the results were quantified as relative expression using the 2^−ΔΔCt method. The primers that we used for ALP were 92 ACT CTC CGA GAT GGT GGTGGT G (forward) and 92 CGT GGT CAA TTC TGC CTC CTT CC (reverse). Three replicates of the experiment were performed for each group.

To verify the osteogenic effects of MPDA-Pso nanoparticles in vivo, we anesthetized eight-week-old Sprague-Dawley rats using sodium pentobarbital, extracted their maxillary first molar, and established an alveolar bone-defect model. Following tooth extraction we applied a gelatin sponge, gelatin sponge combined with MPDA, gelatin sponge combined with MPDA-Pso, or a control group with no material to the alveolar socket. The maxilla (which included the alveolar bone-defect site) was dissected from the sacrificed rats on days seven and 28. Micro-computed tomography (micro-CT) was employed to reconstruct 3D images of alveolar bone defects and to conduct a quantitative analysis. We also performed hematoxylin and eosin (H&E) staining to evaluate the degree of alveolar bone healing. Our experimental procedures complied with the NIH Guide for the Care and Use of Laboratory Animals (United States); and the present study was approved by the Ethics Committee of Qingdao University (QDU-AEC-2024475).

PDLSCs have the potential of self-renewal and multi-lineage differentiation, and are vital to the repair of periodontal tissue damage and the regenerative treatment of periodontal disease (Calabrese, 2021). These cells are also the primary source of newly attaching cells following periodontitis treatment (Yu et al., 2017). We first cultured PDLSCs and identified them, and observed that the primary stem cells grew radially around the tissue block and were arranged in a vortex shape, exhibiting a typically long spindle shape (S1 A). After osteogenic and adipogenic induction, mineralized red nodules (S1 C) and pink lipid droplets (S1 D) were observed microscopically. Flow-cytometric analysis showed that the primary stem cells exhibited characteristics of mesenchymal stem cells (MSCS)—i.e., the cells were positive for CD73, CD90, and CD105 (S1 F), while they were negative for CD34 and CD45 (S1 E). These results indicated that the cells we extracted were, in fact, PDLSCs, and that PDLSCs manifested the basic characteristics of BMSCs.

LPS is known to stimulate the production of pro-inflammatory cytokines in vitro, and has been employed to simulate periodontal inflammatory disease (Stemmler et al., 2021). In order to emulate the periodontal microenvironment, PDLSCs were co-cultured with LPS, according to the references and our CCK-8 assay showed that 100 ng/mL LPS can simulate periodontitis environment and did not inhibit the proliferation of PDLSCs (Figure 1A). On this basis, we found that 10 μg/mL Pso significantly promoted the activity of PDLSCs (Figure 1B). Additionally,We noted that as LPS concentration increased, the enhancement in cellular vitality became less pronounced. Consequently, we selected 10 μg/mL Pso and 100 ng/mL LPS for subsequent experiments. ALP staining revealed a distinct blue-black coloration in the Pso group relative to the control group (Figure 1C), denoting a higher production of ALP in the former group. Additionally, alizarin red staining demonstrated an increased presence of calcium nodules in the Pso group (Figure 1D), further supporting the conclusion that Pso promoted the osteogenic differentiation of PDLSCs.

To elucidate the mechanism underlying Pso-induced osteogenic differentiation of PDLSCs, we initially conducted a network pharmacology analysis to predict potential targets of Pso. After deduplication through database queries, we obtained 24 active compound targets and 1,800 chronic periodontitis-related therapeutic targets (Figure 2A). Eight genes were ultimately identified that served as both target genes of active ingredients and genes associated with chronic periodontitis. Enrichment analysis of the target proteins revealed that Pso targeted a variety of immune- and inflammation-related signaling pathways, including the mTOR signaling pathway, Toll-like receptor signaling pathway, and MAPK signaling pathway (Figures 2B–D); and of these, the mTOR signaling pathway was specifically related to osteogenesis (Laplante and Sabatini, 2012).

In order to explore the mechanism(s) underlying Pso influences on the osteogenic differentiation of PDLSCs, we sequenced RNA extracted from the cultured cells using an Illumina second-generation high-throughput sequencing platform. This approach allowed us to assess the impact of Pso on PDLSCs, particularly in the context of LPS-induced differential gene expression. In comparison to the control group, we identified 2,377 DEGs in the cells cultured with both LPS/PSO, of which 1,606 were upregulated and 771 were downregulated (all P < 0.05) (Figure 3A). When Pso was administered to LPS-induced PDSLCs, the DEGs were predominantly enriched in biological processes, including developmental processes, cell differentiation, and cell regulation (Figure 3B).

In order to further identify the targeting pathway for Pso, we performed GO and KEGG functional-enrichment analyses of the DEGs, and noted that the most significantly enriched differences were observed in the pathways related to autophagy and mitophagy (Figure 3D). Among these factors, the enriched difference in the mTOR pathway was particularly significant, implying that this pathway was critical to the osteogenic process with respect to inflammatory PDLSCs. We therefore speculate that mTOR signaling may constitute a key pathway involved in the osteogenic differentiation of PDLSCs.

When we evaluated the expression of related proteins by Western blot analysis, we observed that the expression levels of the osteogenesis-related genes OCN, RUNX2, and ALP were significantly downregulated in the LPS group, while they were elevated following the addition of Pso. Relative to the control group, mTOR expression was notably downregulated in the PSO group. These results suggested that Pso promoted the osteogenic differentiation of PDLSCs, a process that may be associated with mTOR (Figure 4A).

In order to further clarify the mechanism by which mTOR regulated osteogenesis, we evaluated the differential expression of genes related to the mTOR pathway based on the results of transcriptomic sequencing. As can be viewed from the figure, after adding LPS and compared to the control group, the expression levels of LC3 and PI3K increased, while the expression of Deptor diminished; and again compared to the control group, the genes with augmented expression included Deptor, Rag C, and WIPI 1 in the Pso group (Figures 4B–F). These findings were thus consistent with our sequencing results.

In order to examine the relationship between the mTOR-signaling pathway and osteogenesis of PDLSCs, we added rapamycin as an mTOR inhibitor to cultured PDLSCs. Results indicated that the addition of the mTOR inhibitor significantly attenuated the gene-expression level of mTOR (Figure 5A) while simultaneously upregulating Rag C (Figure 5B). We also ascertained that after adding Pso to PDLSCs incubated with the mTOR inhibitor, the expression of Rag C protein was upregulated; and that the gene- and protein-expression levels for the osteogenic markers ALP and RUNX2 tended to increase, but not to a significant degree (Figures 5C–F). This indicated that Pso regulated osteogenic differentiation via mTOR.

In order to further assess the underlying mechanism of action for Pso in the Rag C/mTOR-signaling axis and to investigate the role of mTOR in the osteogenic differentiation of PDLSCs, we constructed lentiviral vectors to establish PDLSCs with Rag C knockdown. The presence of green fluorescence in the cells validated the success of the lentiviral transfection (Figure 5G), and we observed that the osteogenic genes ALP and RUNX 2 were upregulated after lentiviral knockdown compared to the controls (Figures 5H,I). This result confirmed that Rag C functioned as an upstream regulator of mTOR and that Pso limited mTOR signaling via Rag C, thus modulating the osteogenic differentiation of PDLSCs.

In order to improve the physical properties of Pso, we prepared MPDA nanoparticles loaded with Pso. The optimal drug loading rate was approximately 80.30% when the Pso:MPDA ratio was 5:1 (Figure 6E). Under electron microscopy, the prepared MPDA nanoparticles were spherical and showed a pore structure (Figures 6A,C). The mean size of the MPDA nanoparticles was 196.60 ± 21.90 nm, and the zeta potential was −23 mv. Pso was encapsulated in MPDA mesopores, and the MPDA-Pso nanoparticles were spherical in shape (Figure 6B) and of various particle sizes with a mean ± SD of 220.95 ± 28.20 nm (Figure 6D). After drug loading, the particle size and shape did not change significantly, and the zeta potential was −22.7 mv.

Fourier infrared spectra (Figure 6F) showed a series of changes in the MPDA-Pso nanoparticles compared to MPDA alone. The characteristic peak change at 1395 cm-1 represented the bending and tensile vibration of the C-O-H bond, the peak change at 910 cm-1 represented the vibration of the C-O bond of the oxygen-containing functional group in MPDA, and the peak change at 1510 cm-1 represented the shear vibration of the N-H bond. In conclusion, these characteristic peak changes further proved that Pso was successfully loaded and that the MPDA-Pso nanomaterials were prepared successfully.

To verify an effect of the prepared MPDA-Pso nanomaterials on osteogenic differentiation of PDLSCs, MPDA-Pso nanoparticles were co-cultured with PDLSCs, with the control group and the MPDA group displaying no significant blue coloration. In contrast, the cells in both the Pso and MPDA-Pso groups were stained intensely bluish; however, the staining area of the Pso group was notably smaller than that of the MPDA-Pso group (Figure 7A). The results of alizarin red staining mirrored those of ALP staining, with the most prominent red calcium nodules observed in the MPDA-Pso group, while such nodules were infrequently observed in the control and MPDA groups (Figure 7B). After 14 days of cell culture, ALP activity and ALP gene expression were assessed, and we noted significant differences among the Pso group, the MPDA-Pso group, and the control group. Both the Pso group and the MPDA-Pso group exhibited significantly enhanced ALP activity and ALP gene expression in the cells (Figures 7C,D). These results indicated that MPDA-Pso nanomaterials and Pso promoted the osteogenic differentiation of PDLCs.

To evaluate the osteogenic effects of MPDA-Pso nanomaterials in vivo, we constructed a rat model of maxillary first molar defect, conducted micro-CT analysis 7 days and 28 days after the application of the materials, and noted that the amount of new bone in the MPDA-Pso group increased compared to the control group. Alveolar bone healing was also better at 28 days than at 7 days (Figures 8A,B).

To assess the extent of alveolar bone healing, we conducted histologic analyses to evaluate alveolar inflammation and cellular proliferation at seven and 28 days post-reaction, and observed that all groups manifested an inflammatory response at 7 days. Although the proportion of new bone within the alveolar socket had increased significantly in the MPDA-Pso group by 28 days, the remaining three groups demonstrated more pronounced inflammatory responses in alveolar bone compared to the MPDA-Pso group (Figure 8C). We attribute the diminished inflammatory response observed in the MPDA-Pso group to the material’s beneficial effect on the osteogenic differentiation of BMSCs, as it facilitated the transformation of granulation tissue into woven bone.

Our results also revealed that the bone volume fraction (BV/TV) in the MPDA-Pso group was significantly higher than that in the control group (P < 0.01) (Figure 8D). However, there was no significant difference in BV/TV between the gelfoam group or the MPDA group compared to the control group. Additionally, trabecular separation (Tb.Sp) in the MPDA-Pso group was significantly lower than in the control group (P < 0.001) (Figure 8E). These findings suggested that MPDA-Pso nanomaterials effectively promoted osteogenesis in rat alveolar sockets.