Introduction

Periodontitis disrupts periodontal supporting structures, including cementum, alveolar bone, and periodontal ligament, potentially leading to tooth loss [1, 2]. The ultimate goal of current periodontal therapy is to anatomically and functionally restore lost periodontal tissues [3, 4]. Some clinical periodontal regenerative techniques, including guided tissue regeneration (GTR) combined with bone grafting and growth factors, have been established to restore the damaged periodontal supporting structures [5, 6]. However, these techniques tremendously rely on the patient’s own regenerative capacity, and their therapeutic efficacy are changeable and unpredictable [7,8,9].

The emergence of nanobiomaterials has made periodontal tissue regeneration a domain with breakthrough potential [10, 11]. 2D nanomaterials such as graphene and MXene had been reported to regulate osteogenic differentiation of PDLCs and accelerate periodontal regeneration [12, 13]. Laponite (LAP) is a two-dimensional (2D) nanosilicate with a diameter of less than 50 nm and a thickness of 1–2 nm created synthetically [14, 15]. LAP has been widely utilized in regenerative biomedical applications due to its unique characteristics, superior biocompatibility and biological activity [16]. Several studies indicated that LAP demonstrated superior cytocompatibility and could stimulate osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs), human mesenchymal stem cells (MSCs), human dental follicle stem cells (DFSCs) and human adipose derived stem cells (ADSCs) [17,18,19,20]. Our previous research has demonstrated that LAP can biofunctionally modify biomaterials [21, 22]. LAP-embedded polycaprolactone (PCL) improved the mechanical characteristics and facilitated rat BMSCs and osteoblasts to directly stimulate osteogenesis and indirectly control osteoclastogenesis and angiogenesis, eventually promoting bone formation in vivo [22]. It indicated that LAP had great potential in manipulating periodontal regeneration.

Periodontal ligament cells (PDLCs), with stem-cell-like properties, are extracted from the periodontal ligament and are considered the most promising seed cells for periodontal regeneration [23]. PDLCs can differentiate into osteoblasts and cementoblasts, regulate vascular endothelial cells to promote the formation of blood vessels, ultimately facilitating the regeneration of periodontal tissue [23,24,25,26]. However, the biological effect and particular mechanisms of LAP on PDLCs and periodontal regeneration is currently unclear. Therefore, the present study aimed to explore the intrinsic regulatory role of LAP on PDLCs and its effect on periodontal tissue regeneration. LAP was firstly co-cultured with PDLCs to evaluate its effects on osteogenesis, cementogenesis and angiogenesis, which exerted substantial effects for periodontal regeneration process. Then we evaluated the processes by which LAP acts on PDLCs and its regulatory effects on intracellular cascade reactions. Subsequently, LAP was implanted into rat experimental periodontal defect. Both microcomputed tomography (micro-CT) and histological assessment were performed to evaluate its effect on periodontal regeneration. This investigation will provide novel insights into the potential application of LAP in periodontal therapy.

Experimental section

Preparation of materials

LAPONITE-XLG (Nanocor Inc, China) was dissolved in 2 mL sterile water, shocked and ultrasonic stirring until complete dissolution to prepare an initial liquid with a concentration of 5 mg·mL-1.

Cell culture and identification

All experiments were approved by the Ethics of the School and Hospital of Stomatology, Fujian Medical University (2021 Ethics Review No. 104). PDLCs were isolated and cultured from mirror 1/3 of the human teeth root as previously described and cultured in α-MEM with 10% FBS [13]. The α-MEM medium was changed to osteogenic induction solutions (Cyagen, China), adipogenic induction medium (Cyagen, China), and chondroblast induction (Cyagen, China) to confirm the ability of differentiation. Mineralized nodules, lipogenic nodules and blue chondroblast cells were stained with Alizarin Red S, oil red O and Alysin blue respectively (Sigma Aldrich, USA). The stained cells were imaged using fluorescence microscopy (Zeiss, Germany). The surface markers of PDLCs were analyzed by flow cytometry. PDLCs were digested with 0.25% trypsin and collected suspension to incubate with FITC-labeled CD34, FITC-labeled CD45, FITC-labeled CD73 and FITC-labeled CD90, respectively, then analyzed by flow cytometer (BD Biosciences, USA).

Cell counting kit-8 (CCK-8) assay

A CCK-8 assay (Dojindo, Japan) was performed to measure cell proliferation at 1, 3, 5 and 7 days following the instructions. Cell viability at each time point was determined by measuring the optical density (OD) value at 450 nm using an iMark microplate reader (iMark, Bio-Rad Laboratories, USA).

Alkaline phosphatase (ALP) staining and mineral nodule formation assays

BCIP/NBT alkaline phosphatase kit (Beyotime, China) and ALP assay kit (Jiancheng Inc, China) were used to evaluate ALP staining and activity following osteogenic induction for 7 days. Alizarin Red S (pH 4.2) (Sigma, USA) and 10% cetylpyridinium chloride in 10 mM sodium phosphate (pH 7.0) were applied to observe and eluted the mineral nodule following osteogenic induction for 21 days.

Quantitative real-time polymerase chain reaction (PCR) analysis

A universal RNA extraction kit, PrimeScript RT Master Mix, and TB Green Premix ExTaq II (Takara, Japan) were used to extract total RNA from each sample, synthesize cDNA and perform qPCR respectively (Takara, Japan). The primers were displayed in Table S1 (Supporting Information) and created by Sangon Biotech (Shanghai, China).

Western blot

Collecting the protein of cells stimulated with different concentrations of LAP for 7 days by using RIPA lysis buffer, proteins were separated by protein gel electrophoresis, and then transferred to polyvinylidene difluoride (PVDF) membranes. After 3 h closure using 5% skim milk powder, PVDF membranes were incubated with the primary antibodies ALPL (1:1000), COL1A1 (1:1000), RUNX2 (1:500), CAP (1:5000), CEMP1 (1:1000), P-AKT(1:1000), AKT(1:1000), PI3K(1:1000), P-mTOR (1:1000), mTOR (1:1000), GAPDH (1:1000) and Actin (1:5000). Then, using a secondary antibody (1:20000) to incubate the membranes. Enhanced chemiluminescence reagents were used to measure the levels of protein expression, and ImageJ was used to evaluate the results.

Intracellular uptake activity of LAP

PDLCs treated by 100 µg·mL− 1 rhodamine-marked LAP were fixed, permeated, and blocked. Then, F-actin staining (FITC-phalloidin) and DAPI solution (Beyotime, China) were incubated in the cells gradually. Confocal laser scanning microscopy was used to visualize the labeled cells.

HUVECs cultured with conditioned medium of PDLCs

PDLCs were cultured by 0 µg·mL− 1 and 100 µg·mL− 1 LAP for 3 days with 1 mL medium. The supernatants of PDLCs without LAP were used as controls (0 µg·mL− 1 group). Human umbilical vein endothelial cells (HUVECs) were obtained from the Cell Bank of the Chinese Academy of Sciences and grown in DMEM with 10% FBS supplemented with the acquired supernatants at a 1:1 ratio. Six-well plates containing HUVECs were scratched using a pipette tip. After that, supernatants were added in the manner previously stated. At each instant, the pictures were taken respectively. The wound regions were approximated by using Image J software. Matrigel matrix (Corning, USA) was put in the bottom of a 96-well plate. Then, HUVECs were cultured on the surface of the fixed matrix with different supernatants. The lengths of the tube-like structures were calculated by Image J software. Gene and protein expression levels of HUVECs were evaluated by qPCR and WB as described before on Day 3.

RNA sequencing and bioinformatics analysis

Transcriptome sequencing was performed to explore the mechanism of osteogenesis and cementogenic promotion by LAP. Total RNA was isolated from PDLCs stimulated by LAP using Trizol reagent (Takara, Japan) after 7 Days of cultivation, according to the manufacturer’s instructions. Cells without LAP were used as the controls. RNA sequencing was performed at Wekemo Bioincloud to determine the expression profiles of the mRNAs in the groups of 0 µg·mL− 1 and 100 µg·mL− 1 LAP. mRNAs with a fold change of log2 > 1 or < -1 and p < 0.05 were considered differentially expressed. Gene ontology (GO) analysis was used for functional annotation of genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to analyze the signaling pathways in which differentially expressed genes were enriched. Bioinformatic analyses were performed using online Wekemo Bioincloud tools (https://bioincloud.tech/pipelines).

Inhibition of Pi3K-AKT signaling pathway

LY294002 (LY, MCE, USA) was dissolved in dimethyl sulfoxide (DMSO, MCE, USA) to prepare an initial liquid with a concentration of 5 mM. LY was diluted with medium to 10 μM, while the control and LAP group was added with the same amount of DMSO. The cells were randomly split into three groups: the Control + DMSO group, the LAP + DMSO group, and the LAP + LY group. After 6 h, the protein of cells was collected, and Western blot was performed as described in Section "Western blot". ALP staining and mineral nodule formation assays were performed as described in Section "Alkaline phosphatase (ALP) staining and mineral nodule formation assays". qPCR was performed as described in Section "Quantitative real-time polymerase chain reaction (PCR) analysis".

Rat periodontal defect surgical procedure

The study protocol was approved by the Animal Care and Use Committee of Fujian Medical University (IACUC FJMU 2023-0036). All rats were anesthetized via intraperitoneal injection of 40 mg/kg ketamine. The periodontal defect model was prepared in 4-week-old rat mandibles as our previously described [27]. A round bur was used to prepare periodontal defects with 2 mm height × 3 mm width × 1 mm depth around the first molar. The periodontal ligament was removed to expose the root surface. 10 rats were randomly split into two groups: the Control group with Collagen Sponge (CS, kejibang, China), the LAP group with LAP infiltrated in CS. 5 rats were included in each group. 24 Days later, all rats were sacrificed to collect mandibles.

Microcomputed tomography (micro-CT) analysis

The fixed mandibles samples were scanned by a micro-CT scanner (SCANCO µCT50, Switzerland). Three-dimensional images were obtained and analyzed using Mimics software (Mimics 20.0, Materialise, Leuven, Belgium). Cross-sections of micro-CT reconstructed images were near apical third area. Bone volume per total volume ratio (BV/TV), bone mineral density (BMD) and trabecular number (Tb.N) were evaluated.

Histologic staining and analysis

The mandibles were decalcified in 10% ethylenediaminetetraacetic acid (EDTA), dehydrated and embedded. Consecutive sections were obtained from the defect area and stained using H&E and Masson’s trichrome staining. For the periodontal defect model, the sections were near apical third area. The new bone areas and new attachment formation rate were assessed using ImageJ software.

Immunofluorescence staining and analysis

Consecutive sections were obtained from the defect area and immersed in antigen retrieval solution. Then, sections blocked with goat serum and incubated with primary antibodies against rat CD31 (1:100; ABclonal, China) as a vascular endothelial cell surface marker. Alexa Fluor 488-conjugated secondary antibody (1:500; Beyotime, China) was incubated for 1 h. Nuclei were stained with DAPI. The CD31-positive stained area was assessed using Image J software.

Statistical analysis

The mean and standard deviation are used to present data. To assess the variations across groups, the t test or one-factor analysis of variance was performed, followed by the Tukey’s HSD post hoc test. p < 0.05 was considered significant for all tests.

Results

LAP promoted osteogenic differentiation of PDLCs

PDLCs were successfully isolated and cultured from human extracted tooth. The multipotent differentiation capacities of PDLCs were confirmed after osteogenic, adipogenic and chondrogenic differentiation induction. Flow cytometry analysis indicated that PDLCs were negative for hematopoietic cell marker CD34 and CD45 (Fig. 1c-d), and were positive for mesenchymal stem cell markers (CD73 and CD90) (Fig. 1e-f). These results suggest PDLCs were successfully cultured.

PDLCs were treated by different concentrations of LAP. LAP showed cytocompatibility in the range of 0–100 µg·mL− 1 (Fig. 2b). Figure 2c and e show the ALPL staining and ALPL activity of PDLCs treated by LAP after 7 days. Dark blue staining indicated an increase in ALP activity as elevated LAP concentrations. There are more cell staining and ALP activity in 50 µg·mL− 1 and 100 µg·mL− 1 groups, particularly evident at 100 µg·mL− 1. Alizarin red S staining showed most pronounced red nodule formed in 100 µg·mL− 1 group (Fig. 2d and f), which indicated that LAP enhanced mineralized matrix formation. The expression levels of osteogenic differentiation genes and proteins in PDLCs were estimated after treated by LAP for 7 days. LAP significantly upregulated the expression of COL1A1, RUNX2 and ALPL, with maximum expression observed at 100 µg·mL− 1 (Fig. 2g-i). Also, increased ALPL and COL1A1 protein levels of PDLCs could be observed in a dose-dependent manner (Fig. 2j-l). The results collectively demonstrated that LAP could promote osteogenic differentiation and mineralization of PDLCs.

Fig. 1
figure 1

Isolated and identification of PDLCs. (a-b) PDLCs cultured from human PDL tissue. (c-f) Flow cytometry analysis of CD34, CD45, CD73 and CD90 in PDLCs. (g) Red nodules of mineralization by Alizarin Red S. Black arrows: Red nodules of mineralization. (h) Positive lipid accumulation by oil red O. Black arrows: Oil Red O-positive lipid accumulation. (i) PDLCs with chondrogenic induction solution, blue chondroid cells can be seen by Alcian blue staining. Black arrows: Blue chondroid cells. Scale bar = 100 μm

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Fig. 2
figure 2

LAP promoted PDLCs osteogenic differentiation. (a) Schematic diagram of LAP promoting osteogenic differentiation of PDLCs. (b) The metabolic activity of PDLCs stimulated by different concentrations of LAP. (c, e) ALPL staining and activity of PDLCs stimulated by LAP for 7 days. (d, f) Alizarin red staining and quantification of PDLCs cultured by LAP for 21 days. (g-i) qPCR results of osteogenic-related genes (COL1A1, RUNX2 and ALPL) of PDLCs stimulated with LAP for 7 days. (j-l) Western blot analysis of osteogenic-related proteins (ALPL and COL1A1) of PDLCs stimulated with LAP for 7 days. Scale bar = 1mm and 100 μm. *p < 0.05; **p < 0.01; ***p < 0.001 compared with the control (LAP at 0 µg·mL− 1)

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LAP induced PDLCs cementogenic differentiation

To further evaluate the capacity of LAP on cementogenic differentiation of PDLCs, typical cementogenic differentiation genes (CAP and CEMP1) were selected to estimate the expression levels following PDLCs were stimulated with LAP for 7 days. As depicted in Fig. 3b and c, the relative expression levels were progressively upregulated and subsequently increased in a dose-dependent manner, with the highest expression observed at a LAP concentration of 100 µg·mL− 1. Accordingly, CAP and CEMP1 protein levels of PDLCs treated by LAP showed the same trend as genes expression. The maximum expression amount is 100 µg·mL− 1 of LAP (Fig. 3d-f). These results suggested that LAP could accelerate cementogenic differentiation of PDLCs.

Fig. 3
figure 3

LAP-induced PDLCs cementogenic differentiation. (a) Schematic diagram of LAP promoting cementogenic differentiation of PDLCs. (b, c) The expression levels of cementogenic differentiation genes (CAP and CEMP1) of PDLCs after 7 days of incubation with various doses of LAP. (d-f) WB analysis of cementogenic differentiation proteins (CAP and CEMP1) of PDLCs stimulated with various doses of LAP for 7 days. *p < 0.05; **p < 0.01; ***p < 0.001 compared with the control (LAP at 0 µg·mL− 1)

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PDLCs stimulated by LAP indirectly enhanced angiogenesis

LAP with concentration of 100 µg·mL− 1 was chosen for further experiments based on the above results. To further elucidate the potential effects of LAP on PDLCs-induced regeneration processes, a series experiments were carried out to evaluate whether LAP modulates angiogenesis by PDLCs in a paracrine manner. PDLCs were cultured by 0 µg·mL− 1 and 100 µg·mL− 1 LAP for 3 days, and the supernatants were then collected and added to HUVECs. As shown in Fig. 4b and c, cell migration was clearly observed in the LAP group compared to the control group. Moreover, more tube-like structures were observed in the LAP group, the tube length and the number of tube junctions were significantly higher than the control group (Fig. 4d-f). Furthermore, qPCR analysis was used to assess the expression levels of different cytokines associated with angiogenesis in HUVECs. The heatmap of mRNA expression levels of angiogenesis-related gene is shown in Fig. 4g. Notably, the expression of angiogenesis-related gene and protein (HIF1A, VEGFA and eNOS) in HUVECs were significantly enhanced in the LAP group compared to control group (Fig. 4g-n). Taken together, these results showed PDLCs stimulated with LAP promoted angiogenesis of HUVECs in vitro.

Fig. 4
figure 4

PDLCs stimulated by LAP indirectly enhanced angiogenesis. (a) Schematic diagram of PDLCs stimulated by LAP enhanced angiogenesis of HUVECs. (b-c) Scratch wound healing assays. (d-f) Tube formation assays. (g). Angiogenesis-related gene expression magnitude heatmap. (h-j) mRNA expression levels of angiogenesis-related genes (VEGFA, HIF1A and eNOS) of HUVECs. (k-n) Western blot analysis of angiogenesis proteins (VEGFA, HIF1A, and eNOS) of HUVECs. Scale bar = 100 μm. *p < 0.05; **p < 0.01; ***p < 0.001 compared with the control (LAP at 0 µg·mL− 1)

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The uptake of LAP by PDLCs

In order to track the pathway and extent of LAP uptake by PDLCs, confocal laser scanning microscopy was used to position rhodamine-marked LAP, which was uptook by PDLCs. Figure 5c illustrates a small amount of LAP could be observed on the cell membrane of PDLCs within 2 h. Over time, LAP cellular uptake exhibited a time dependent increase. The ingested LAP aggregated enlarging in size and number and distributed throughout the cell membrane to the nucleus. At day 7, the internalized LAP was predominantly localized around cell nucleus (Fig. 5g). It demonstrated that LAP could be easily taken up by PDLCs to further regulate cellular activity.

Fig. 5
figure 5

The uptake of LAP by PDLCs. (a) Schematic diagram of cellular uptake of LAP. Cellular uptake of LAP at 0 h (b), 2 h (c), 6 h (d), 24 h (e), 3d (f) and 7d (g). Scale bar = 20 μm. Red (RB-labelled Laponite particles), blue (nucleus), green (actin). Scale bar = 20 μm

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RNA sequencing analysis of PDLCs treated with LAP

To further explore the potential mechanism of LAP on PDLCs, RNA sequencing analysis of PDLCs treated by LAP was carried out. KEGG pathway analysis was performed to investigate the potential signaling pathways and the enriched pathways are presented in Fig. 6b. The PI3K-AKT pathway attracted our attention. GO analysis of differentially expressed genes showed that the PI3K-AKT pathway components were upregulated in the LAP group (Fig. 6c). To verify the influence of PI3K-AKT signaling pathway on PDLCs treated with LAP, the protein expression level of PI3K, P-AKT, AKT, P-mTOR and mTOR, key components of the PI3K-AKT signaling pathway, were evaluated. As shown in Fig. 6d-g, the expression of PI3K and the phosphorylation levels of AKT and mTOR in PDLCs stimulated with LAP were significantly higher than the control group, which is consistent with transcriptome sequencing results.

Fig. 6
figure 6

RNA sequencing analysis of PDLCs treated with LAP. (a) Volcano plots showing mRNA expression profiles of PDLCs cultured in the two groups. (b)KEGG pathway gene enrichment analysis between the PDLCs treated with LAP. (c) The heat map showing expression levels of PI3K-AKT signaling pathways associated genes identified by GO enrichment analysis. (d-g) PI3K, AKT, P-AKT, mTOR and P-mTOR protein expression were measured by Western blot analysis. *p < 0.05 compared with the control (LAP at 0 µg·mL− 1)

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LAP promoted osteogenesis and cementogenesis of PDLCs via PI3K-AKT signaling pathway activation

To verify the effects of PI3K-AKT signaling pathway on LAP-mediated osteogenesis and osteogenic differentiation of PDLCs, LY294002 (LY), an inhibitor of PI3K, was added to the LAP group to inhibit PI3K-AKT pathway activation. The expression of key marker proteins (P-AKT, AKT, P-mTOR, mTOR and PI3K) in the PI3K-AKT signalling pathway was significantly decreased after LY treatment (Fig. 7a-d). Accordingly, the increased ALPL expression and mineralized nodule formation levels also suppressed in the LAP + LY group (Fig. 7e-f). The expression of osteogenic and cementogenic differentiation genes and proteins (RUNX2, COL1A1, CAP and CEMP1) were significantly decreased after LY treatment in LAP + LY group (Fig. 7g-o). The above results indicated that suppressing PI3K-AKT signaling pathway would abort LAP-induced osteogenic and cementogenic differentiation of PDLCs, which suggested LAP promoted osteogenesis and cementogenesis of PDLCs via PI3K-AKT signaling pathway activation.

Fig. 7
figure 7

LAP promoted osteogenesis and cementogenesis of PDLCs. via PI3K-AKT signaling pathway activation. (a-d) PI3K, AKT, P-AKT, mTOR, and P-mTOR protein expression were measured by western blot analysis. (e) ALPL staining. (f) Alizarin Red S staining. (g-j) The expression levels of genes associated to osteogenic and cementogenic differentiation (RUNX2, COL1A1, CAP and CEMP1). (k-o) The expression levels of proteins associated to osteogenic and cementogenic differentiation (RUNX2, COL1A1, CAP and CEMP1) were measured by western blot analysis. Scale bar = 1mm and 100 μm. *p < 0.05; **p < 0.01; ***p < 0.001

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LAP improved rat periodontal defect regeneration in vivo

As shown in Fig. 8a, more bone formation outside mandibular bone contours of periodontal defects. The bone mineral density (BMD) and volume ratios of new bone to total tissue (BV/TV) analysis showed a higher volume and density of new bone in the periodontal defect in LAP group than control group (Fig. 8b-c). Additionally, the Tb.N levels in the LAP group was higher than that in the control group (Fig. 8d). Histological staining and analysis showed only limited amount of bone formed in the control groups, and more new bone formation at the edge of rat periodontal defects in LAP group (Fig. 8e-f). Few attached fibers attached to root surface in control group. But in LAP group, more dense mature bone formed around root surface, and homogeneous collagen fibers and mature bone could also be observed on the adjacent root surfaces, indicating new attachment formation in the periodontal defect. Quantitative analysis also indicated new bone and new attachment formation rate were significantly higher in LAP group than control group (Fig. 8g-h). Additionally, CD31 was used as a marker for vessels in immunofluorescent staining to evaluate new vessel formation in periodontal defects (Fig. 8i). Quantitative analysis indicated a significantly higher area of CD31-positive cells in the LAP group compared to the control group, suggesting that LAP can accelerate blood vessel formation during the periodontal regeneration process (Fig. 8j). These results confirmed the regulatory effects of LAP on periodontal regeneration.

Fig. 8
figure 8

LAP improved rat periodontal defect regeneration. (a) Representative micro-CT reconstructed images of rat periodontal defects. (b-d) BMD, BV/TV ratio and Tb.N of Micro-CT data analysis. (e-f) Histological HE and Masson’s trichome-stained of rat periodontal defects. (g-h) Histological quantitative analysis of new bone formation rate and new attachment formation rate. (i) Immunofluorescent staining of CD31 in periodontal defects areas. (j) Quantitative analysis of CD31-positive cells. NC, new cementum; NP, new periodontal ligaments; NB, new bone. Scale bar = 100 and 200 μm. *p < 0.05; **p < 0.01

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Discussion

Bioactive materials have been widely used in periodontal tissue regeneration, which generate special biological or chemical reactions on the surface or interface of materials [28]. These reactions can impact the interaction between tissues and materials, stimulate cell activity and induce tissue regeneration. In this study, for the first time, we found that LAP could improve osteogenic and cementogenic differentiation of PDLCs, and this regulatory effects of LAP on PDLCs differentiation were closely correlated with activation of PI3K-AKT signaling pathway. Additionally, LAP enhanced angiogenesis indirectly via manipulating paracrine of PDLCs. Furthermore, LAP could enhance periodontal defect regeneration in vivo. Our data refined the underlying mechanisms of LAP on periodontal regeneration and highlighting it could serve as a promising therapeutic option in periodontal therapy.

As contributors to new bone and cementum formation, PDLCs aid in the restoration of damaged tooth-supporting tissue by periodontitis [29]. To promote osteogenesis and cementogenesis of PDLCs, a variety of periodontal regeneration techniques have been used [30, 31]. Several studies have shown that nanomaterials, such as iron oxide nanoparticles, AuNPs, could promote the osteogenic differentiation of PDLCs and further promote periodontal tissue regeneration [32, 33]. Our results indicated that stimulation of PDLCs with LAP can promote the formation of ALP and mineralized nodules, along with activation of the key transcription factor (RUNX2) during osteogenic differentiation. Additionally, the downstream osteogenic related expression factors (COL1A1) was significantly increased by the addition of LAP, which showed dose dependence. These results were consistent with the conclusions of Carrowand Xavier et al., which indicated that LAP could promote human mesenchymal stem cells osteogenic differentiation [18]. Interestingly, we found that the cementum-specific proteins CAP and CEMP1 in PDLCs were also significantly upregulated after being treated with LAP. CEMP1 and CAP are known cementogenic-related proteins capable of stimulating cementogenic differentiation [34, 35]. These results represented the inaugural demonstration that two-dimensional nanomaterial LAP can enhance the cementogenic differentiation of PDLCs and might play regulatory role in cementogenesis.

Vascularization acts as a crucial role in the network of regenerative tissue formation, providing oxygen, nutrients, and growth factors to the regeneration sites [36, 37]. Given the deep link between periodontal tissue regeneration and angiogenesis, the ideal periodontal regeneration therapy must consider the vascularization regulation [38, 39]. Some biomaterials, such as hierarchical porous biomimetic sponges and extrusion-printed scaffolds, markedly enhance endogenous vascularized bone tissue regeneration by augmenting the paracrine effects of MSCs [40, 41]. HUVECs possess the typical functions of vascular endothelial cells and are widely used in angiogenesis research [42, 43]. Iwasaki et al. found that the conditioned medium from PDLCs improved the viability of HUVECs and modulated angiogenesis in a paracrine manner, presenting a promising strategy for periodontal tissue regeneration [44]. In our previously study, we also found that PCL/LAP scaffold improved angiogenesis of HUVECs through regulating the paracrine of osteoblasts [22]. The current study indicated that PDLCs treated with LAP indirectly enhanced the migration capacity of HUVECs, promoted vascularization, and significantly upregulated the expression of angiogenic genes and proteins (VEGFA, HIF1A, and eNOS). It might be attributed to the paracrine effects mediated by PDLCs following stimulation with LAP.

Due to their nanoscale size, nanomaterials have the potential to effectively interact with cell membranes and intracellular fluids via receptor mediated endocytosis [45]. LAP (Na+ 0.7[(Si8Mg5.5Li0.3)O20(OH)4]−0.7) belongs to artificially synthesized nanoclay, comprised of bioactive silicate nanoplatelets. It can degrade into Mg2+, Si(OH)4, and Li+, which are cytocompatible and facilitate normal cellular metabolism and proliferation [18, 46, 47]. Mousa and Gahawar et al. previously found that LAP could be internalized by BMSCs in a time dependent manner and undergone degradation in lysosomes [18, 48]. To further explore the interaction between LAP and PDLCs, LAP was labeled with rhodamine red B using its non-specific adsorption property and then co-cultured with PDLCs. The results revealed that LAP could be rapidly aggregated outside the cell membrane, uptaked by PDLCs via endocytosis, and with prolonged exposure, gradually accumulated around the cell nucleus. Therefore, we speculated that LAP entered the PDLCs through entosis to form phagosomes, and gradually fused with lysosomes to form vesicles. With lysosomal enzymes, LAP gradually degraded into ionic products and released into the cytoplasm to promote the transformation of cells from proliferation to differentiation state, thus playing a role.

To further explore the regulatory mechanism of LAP on PDLCs, transcriptome sequencing of PDLCs was conducted. The results demonstrated a close correlation between the regulation of LAP and the PI3K-AKT signaling pathway. PI3K-AKT signaling pathway has been confirmed plays a crucial role in regulating various cellular processes, including cell growth, survival, and proliferation [49, 50]. It also found to be intimately involved in orchestrating osteogenic differentiation [51,52,53]. Zhu et al. developed a nanozyme to regulate osteogenic differentiation through activating PI3K-AKT pathway and ultimately promote periodontal regeneration [54].In the current study, the expression of P-AKT, P-mTOR, and PI3K in the PI3K-AKT signaling pathway was significantly increased when PDLCs stimulated with LAP. These key marker proteins were significantly decreased when the LY294002, an inhibitor of PI3K, was added to PDLCs co-cultured with LAP. Accordingly, the osteogenic and cementogenic differentiation of PDLCs stimulated with LAP were counteracted by inhibiting the PI3K-AKT signaling pathway. These data demonstrated that LAP degradation may occur subsequent to endocytosis, with the resultant ionic products activating the PI3K-AKT signaling pathway in PDLCs, leading to cementogenesis as well as osteogenesis. This activation subsequently promotes the transcription of RUNX2, COL1A1, CAP and CEMP1, initiating new bone and cementum formation, ultimately facilitating periodontal regeneration. As far as we know, this is the first study to reveal that LAP promoted cell differentiation via PI3K-AKT signaling pathway activation. However, the exact mechanisms involved still need to be further explored.

The goal of periodontal therapy is to achieve periodontal regeneration. Currently, rat mandibular periodontal defect model has been widely used to explore the effectiveness of distinct regenerative approaches [55]. It provides a stable local environment for bone, periodontal ligament and cementum regeneration. In the present study, LAP was implanted into rat mandibular periodontal defects. The results demonstrated that LAP enhanced more vascular bone formation during periodontal regeneration. Furthermore, the formation of new attachments was markedly enhanced. We speculated that the released LAP might mobilize PDLCs within periodontal defect sites, directly enhancing osteogenic and cementogenic differentiation, ultimately leading to the formation of new alveolar bone and cementum. Furthermore, LAP may indirectly modulate the paracrine of PDLCs, thereby promoting vascularization to facilitate periodontal tissue regeneration.

Periodontal tissue regeneration involves a complex interplay of multiple cells and biological processes, including an early macrophage or neutrophil mediated inflammatory response, antibacterial defense, and multi-tissue regeneration of the periodontium [56,57,58]. Although the current study found that LAP promoted periodontal regeneration, only the effect on PDLCs were evaluated, thus limiting the elucidation of the intricate process of periodontal regeneration. Moreover, the mechanisms by which LAP binds to cells for exocytosis remain unclear, as do the precise degradation processes upon cellular entry and the specific regulatory effects on the PI3K-AKT signaling pathway, and the immune effect between LAP and immune cells is also uncertain. These uncertainties need to be further explored to offer new insights into the impact of LAP in periodontal treatment. This encompasses utilizing LAP-functionalized biomaterials for clinical applications and leveraging LAP as a drug carrier, in combination with antibiotics, growth factors, or peptide factors, to enhance its efficacy in periodontal regeneration.

Conclusion

In general, the current study indicated that LAP facilitated osteogenic and cementogenic differentiation of PDLCs via PI3K-AKT signaling pathway. Furthermore, LAP indirectly boosted angiogenesis by modulating the paracrine activity of PDLCs. Also, rat periodontal defect treated with LAP exhibited more periodontal tissue regeneration. To our current knowledge, this study represents the first comprehensive investigation that LAP could regulate PDLCs by activating PI3K-AKT signaling pathway and has the ability to promote periodontal defect regeneration in vivo (Fig. 9). Our results hold that LAP presents a promising candidate for periodontal therapy.

Fig. 9
figure 9

Schematic illustration of the role of LAP on PDLCs for periodontal regeneration

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