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LL-37 regulates odontogenic differentiation of dental pulp stem cells in an inflammatory microenvironment

Stem Cell Research & Therapy volume 15, Article number: 469 (2024) Cite this article

Abstract

Background

Inflammation often causes irreversible damage to dental pulp tissue. Dental pulp stem cells (DPSCs), which have multidirectional differentiation ability, play critical roles in the repair and regeneration of pulp tissue. However, the presence of proinflammatory factors can affect DPSCs proliferation, differentiation, migration, and other functions. LL-37 is a natural cationic polypeptide that inhibits lipopolysaccharide (LPS) activity, enhances cytokine production, and promotes the migration of stem cells. However, the potential of LL-37 in regenerative endodontics remains unknown. This study aimed to investigate the regulatory role of LL-37 in promoting the migration and odontogenic differentiation of DPSCs within an inflammatory microenvironment. These findings establish an experimental foundation for the regenerative treatment of pulpitis and provide a scientific basis for its clinical application.

Materials and methods

DPSCs were isolated via enzyme digestion combined with the tissue block adhesion method and identified via flow cytometry. The impact of LL-37 on the proliferation of DPSCs was evaluated via a CCK-8 assay. The recruitment of DPSCs was assessed through a transwell assay. The mRNA expression levels of inflammatory and aging-related genes were assessed via reverse transcription‒polymerase chain reaction (RT‒PCR), western blotting, and enzyme‒linked immunosorbent assay (ELISA). The odontogenic differentiation of DPSCs was assessed through alkaline phosphatase (ALP) staining, alizarin red staining, and RT‒PCR analysis.

Results

LL-37 has the potential to enhance the migration of DPSCs. In an inflammatory microenvironment, LL-37 can suppress the expression of genes associated with inflammation and aging, such as TNF-α, IL-1β, IL-6, P21, P38 and P53. Moreover, it promotes odontogenic differentiation in DPSCs by increasing ALP activity, increasing calcium nodule formation, and increasing the expression of dentin-related genes such as DMP1, DSPP and BSP.

Conclusion

These findings suggest that the polypeptide LL-37 facilitates the migration of DPSCs and plays a crucial role in resolving inflammation and promoting cell differentiation within an inflammatory microenvironment. Consequently, LL-37 has promising potential as an innovative therapeutic approach for managing inflammatory dental pulp conditions.

Background

Dental pulp is the only soft tissue in dental tissue and is a loose connective tissue located in a cavity composed of dentin. It plays essential roles in nutrition, sensory perception, and defense mechanisms [1]. Dental pulp possesses the inherent capacity for repair and regeneration. Following odontoblast damage, undifferentiated mesenchymal stem cells located in the corresponding region of the pulp can undergo differentiation into odontoblasts, thereby forming odontoblastic bridges [2, 3]. The most prevalent cause of pulp infection is deep caries, whereby bacteria can infiltrate the pulp cavity through dentin tubules before their toxic byproducts induce inflammation in the pulp, particularly when caries affects the deeper layers of dentin [4,5,6]. An excessive or prolonged inflammatory response ultimately results in complete tissue necrosis. The most commonly employed approach for managing irreversible pulpitis in clinical practice involves root canal treatment subsequent to pulp removal. Nevertheless, this treatment modality is associated with several limitations. Following root canal treatment, teeth lacking pulp become more fragile and susceptible to fracture. Additionally, tooth color may change, and there is a potential risk of reinfection [7, 8]. Therefore, in the early stages of pulpal inflammation, addressing pulp inflammation and augmenting the odontogenic differentiation of undifferentiated mesenchymal stem cells are imperative, as these processes hold paramount importance for the effective management of pulpitis [9, 10].

Investigating the role of individual cells in the development of inflammation and reparative dentin within dental pulp tissue poses significant challenges, thus necessitating the utilization of in vitro cell culture as an effective approach. In 2000, Gronthos et al. [11] successfully isolated and characterized dental pulp stem cells from adult dental pulp tissue. DPSCs originate from neural ridges and are a type of mesenchymal stem cell that is crucial for differentiation in dental pulp tissue [11, 12]. In certain inflammatory responses, proinflammatory factors such as tumor necrosis factor and interleukins can exert diverse effects on various functions of DPSCs, including cell proliferation, differentiation, and calcium deposition [13, 14]. The inflammatory process impairs the capacity of DPSCs to undergo differentiation [15, 16]. Given the irreversible damage inflicted by pulpitis, investigating the impact of pulp inflammation on the differentiation potential of DPSCs and exploring strategies for safeguarding DPSCs against inflammation-induced damage, aging, and apoptosis while preserving their multilineage differentiation capacity are imperative [17].

LL-37 is the only antimicrobial polypeptide found in the human body and belongs to the cathelicidin family [18]. It is expressed and distributed in many cells, tissues, and body fluids in the human body [19]. Initially, research focused on the antibacterial activity of LL-37, which has good antibacterial activity against pathogenic microorganisms such as bacteria and fungi [20, 21]. In recent years, extensive research on LL-37 has revealed its potent immunomodulatory effects. The local application of LL-37 significantly attenuates the expression of inflammatory mediators, exerting a profound effect on the regulation and eradication of periodontitis [22]. LL-37 has also been demonstrated to interact with mesenchymal stem cells (MSCs), thereby modulating their proliferation, migration, and regeneration [23]. A previous study revealed that LL-37 significantly enhanced the differentiation, migration, and proliferation of MSCs in mouse models of osteolytic bone defects induced by LPS [24]. Studies on the toxicity of LL-37 toward DPSCs have shown that LL-37 at a concentration of 10 μg/mL or less does not affect the survival of DPSCs [25]. However, the regulatory role of LL-37 in pulp inflammation and its potential to induce the differentiation of DPSCs within an inflammatory microenvironment remain uncertain.

Therefore, we constructed an inflammatory microenvironment with LPS to investigate the potential of LL-37 in enhancing the migration and differentiation of DPSCs, as well as controlling inflammation. These findings offer valuable insights for the clinical implementation of LL-37 in the management of dental pulp inflammation.

Materials and methods

Cell culture and characterization

Cell isolation and culture

Primary human dental pulp cells were isolated according to the procedure reported by Gronthos et al. [11]. Pulp tissues were obtained from normal impacted mandibular third molars or normal premolars extracted for orthodontic treatment in healthy patients aged 18–25 years from the Affiliated Stomatologic Hospital of Anhui Medical University, Hefei, Anhui (approval no. LLSC20240974). The pulp tissue was washed twice with PBS, chopped into pieces, and digested with 500 μL of type I collagenase (Sigma‒Aldrich) for 30 min. After centrifugation for 5 min at 1000 rpm, the separated cells were cultured in α-MEM (Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, USA), 100 U/mL penicillin (Beyotime Shanghai, China) and 100 U/mL streptomycin (Beyotime Shanghai, China) and incubated at 37 °C in 5% CO2. The state of the cells was observed under an inverted microscope, and when the cells reached 80% to 90% confluence, they were digested with trypsin (Servicebio, Wuhan, China) and passaged at a ratio of 1:3. Cells at passages 3 to 5 were used in the following experiments.

Characterization of DPSCs

The characterization of DPSCs was analyzed by flow cytometry as previously described [26].The cell suspension was divided into 1.5 mL EP tubes. After the cells were washed twice with PBS, 100 μL of PBS was added to EP tubes to resuspend the cell pellet. Five microlitres of anti-CD34-PE (BD Biosciences, USA), anti-CD45-PE (R&D Systems), anti-CD105-APC (BD Biosciences, USA), and anti-CD73-PE (R&D Systems) antibodies were added to each tube, and the samples were incubated at room temperature for 30 min. The EP tubes were centrifuged at 300–400 rpm for 5 min at room temperature, and the supernatant was discarded. The stained cells were resuspended in 400 μL of PBS and analyzed by flow cytometry (CytoFLEX, BECKMAN).

Differentiation potential of DPSCs

The multilineage differentiation of DPSCs assays as previously described [27, 28]. For the induction of osteogenesis and lipogenesis, 2 × 105 cells/well were seeded in 6-well plates and incubated at 37 °C in 5% CO2. The osteogenic induction medium containing 2 mM β-glycerophosphate (Sigma‒Aldrich), 50 mg/mL ascorbic acid (Sigma‒Aldrich), and 10–7 M dexamethasone (Sigma‒Aldrich) was changed every 3 days, and the adipogenic induction medium (OriCell, Guangzhou, China) was changed according to A medium induction for 2 days and B medium for 1 day. Osteogenesis induction lasted for 21 days, and adipogenesis induction lasted for 28 days. Alizarin red staining solution (Beyotime Shanghai, China) was used to detect calcium nodule deposition, and oil red O staining (Servicebio, Wuhan, China) was used to detect lipid droplets in the cells.

Formation of cell colonies

To assess the ability of DPSCs to form colonies, 200 cells were seeded in each well of a 6-well plate. After 14 days, the cells were fixed with 4% PFA and stained with 0.5% crystal violet, and the aggregation of the cells was observed under a stereomicroscope (Leica, Germany).

Biocompatibility of LL-37

In a previous study, 10 μg/mL LL-37 inhibited the proliferation of stem cells from the apical papilla at day 3 and whereas low concentrations (1.25 and 2.5 μg/mL) markedly stimulated cell viability [29]. To verify the effect of low concentrations LL-37 (< 10 μg/mL) and 10μg/mL LL-37 on the biocompatibility of DPSCs, the cells were exposed to 1.25, 2.5, 5 and 10 μg/mL LL-37 after incubation for 24 h. DPSCs were seeded in a 96-well plate at a density of 2 × 103 cells/well and cultured with α-MEM (Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin (Beyotime Shanghai, China), and 100 U/mL streptomycin (Beyotime Shanghai, China) at 37 °C in 5% CO2. After 24 h of incubation, the α-MEM was replaced with 100 μL α-MEM containing different concentrations of LL-37 (1.25, 2.5, 5 and 10μg/mL) or control (PBS) for 1, 3, 5, and 7 days. After incubation for 1, 3, 5, and 7 days, CCK-8 reagent (Biosharp, Beijing, China) was added to each well, and the plates were incubated for an additional two hours. The OD value was subsequently measured via a microplate reader at an absorbance of 450 nm (Axio Observer 3, Germany). The polypeptides were made into a solution in phosphate buffer saline (PBS).

Effects of LL-37 on the migration of DPSCs

DPSCs migration was evaluated by using a Boyden chamber assay [30]. To verify the effect of low concentrations LL-37(1.25, 2.5 and 5 μg/mL) on the migration of DPSCs, a previous protocol was modified and performed [31]. The cells were resuspended in serum-free culture medium. Then, 200 μL of each cell suspension was added to the upper chamber of a transwell plate at a density of 2 × 104 cells/well. In the lower chamber, 750 μL of α-MEM (Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, USA) was added. The treatment groups were further exposed to 1.25, 2.5, or 5 μg/mL LL-37 after incubation for 24 h and 48 h. After 24 or 48 h, the transwell chambers were fixed with 4% paraformaldehyde at room temperature for 15 min and stained with 0.1% crystal violet for 10 min. The chamber was washed with PBS, and any nonmigrating cells in the upper chamber were removed with a moist cotton ball. The cells that had passed through the insert membrane were observed under a microscope (Leica, Germany). Positively stained cells were measured with ImageJ (National Institute of Health, Bethesda, MD, USA). After comprehensive analysis of the results of biocompatibility and cell migration experiments, we selected LL-37 with a concentration of 5 μg/mL for further study.

Gene expression of DPSCs by RT‒PCR

DPSCs (1 × 106) were seeded in a 6-well plate. After 24 h, the DPSCs were treated with 1 μg/mL LPS (LPS group), 5 μg/mL LL-37 + LPS (LL-37 group) or PBS (control group). The cells were cultured for 12 h to determine the mRNA levels of TNF-α, IL-1β, and IL-6; 48 h to determine the mRNA levels of P21, P38, and P53; and 7, 14, and 21 days to determine the mRNA levels of dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), and bone sialoprotein (BSP), respectively. Total RNA was extracted with an RNA-Quick Purification Kit (EScience Biotech, Shanghai, China). Complementary DNA (cDNA) was reverse transcribed via a PrimeScript RT kit (Takara, Kusatsu, Japan) according to the manufacturer’s instructions. The prepared reverse transcription system was put into the reverse transcription system, and the reverse transcription was finished after the reaction at 37 °C for 15 min and 85 °C for 5 s. Subsequently, the RT‒PCR procedure was performed by using a SYBR green PCR mix kit (Takara) on a real-time PCR system (Mx3000P, Agilent). After ensuring that there was no air in the EP tube, the sample was put into the PCR instrument, and the corresponding program was set. After the program was completed, the expression of each cytokine was analyzed according to the CT value. The primers (Tsingke Biotech, Beijing, China) used for PCR are listed in Table S1.

Western blot

DPSCs were harvested and lysed with radioimmunoprecipitation assay (RIPA) buffer (Beyotime Shanghai, China) for 30 min to extract total protein. The protein concentration was determined via a BCA protein assay kit (Beyotime Shanghai, China). Each protein sample (20 μg) was electrophoresed via a 10% PAGE Fast Gel Preparation Kit (EpiZyme Biotechnology, Shanghai, China) and then transferred onto a polyvinylidene fluoride membrane (Biosharp, China). After the transfer was completed, the PVDF membranes were immersed in 5% skim milk for 2 h and incubated overnight at 4 °C with primary antibodies against TNF-α (1:1000; Abcam) and β-actin (1:10,000; Zhongshan, Beijing, China). The membranes were washed three times with TBST buffer for 10 min each time and then incubated with horseradish peroxidase-conjugated IgG antibodies (Huabio, Hangzhou, China) for 1 h at 37 °C. The IgG antibodies were washed off three times with TBST buffer for 10 min each time. A chemiluminescent HRP substrate (Millipore, USA) was used for chemiluminescence. Band intensity was measured via a chemiluminescence western blot detection system. At least three samples from each group were assayed, and the blots were analyzed via ImageJ software (National Institute of Health, Bethesda, MD, USA).

ELISA

The IL-6 and IL-1β protein contents of the cell supernatants were determined via human-specific enzyme-linked immunosorbent assay (ELISA) kits (DAKEWE, Beijing, China) following the instructions provided with the kits.

Alkaline phosphatase (ALP) activity assay and alizarin red staining

The ALP activity and the mineralization nodules of the DPSCs assays as previously described [32,33,34].The cells were seeded onto 6-well plates at a density of 5 × 105 cells/well and cultured until they reached 75–85% confluence. The medium was subsequently changed to osteogenic-induced medium (OM) containing 2 mM β-glycerophosphate (Sigma‒Aldrich), 50 mg/mL ascorbic acid (Sigma‒Aldrich), and 10–7 M dexamethasone (Sigma‒Aldrich). The medium was supplemented with either 1 μg/mL LPS (LPS group), 1 μg/mL LPS + 5 μg/mL LL-37 (LL-37 group), or PBS (control group). The medium was changed every three days. After 4 and 7 days, the cells were fixed with 4% paraformaldehyde for 30 min, and ALP staining was carried out via an ALP chromogenic kit (Beyotime Shanghai, China). For quantitative analysis, the cells were lysed with noninhibitor cell lysate. The ALP activity was quantitatively analyzed via an ALP quantitative assay kit (Beyotime Shanghai, China). The plates were further incubated at 37 °C for 10 min, after which 100 μL of reaction stop solution was added to each well to terminate the reaction. The absorbance at a wavelength of 405 nm was measured. After 14 and 21 days, the cells were fixed with 4% paraformaldehyde for 30 min. Alizarin red staining was used to stain the induced cells, and calcium nodules were dissolved in a solution of cetylpyridine chloride after staining. OD values at a wavelength of 562 nm were determined to quantitatively analyze the formation of calcium nodules.

Statistical analysis

Statistical analysis was performed with Prism version 9.0 (GraphPad Software, USA). Data analysis was performed via one- or two-way analysis of variance (ANOVA), and the results are expressed as the means ± standard deviations (SDs). All the experiments were repeated three times, and the statistical significance was expressed as P < 0.05 (*), P < 0.01 (**), P < (***), and P < 0.0001 (****).

Results

Morphological observation and identification of DPSCs

After 72 h, the dental pulp tissue fragments had adhered to the bottom wall of the culture flask, and a limited number of cells had migrated outward from the surrounding tissue fragments (Fig. 1A). Flow cytometry analysis revealed positive and high expression of the mesenchymal markers CD73 (99.68%) and CD44 (99.40%) but negative expression of the hematopoietic markers CD45 (0.01%) and CD34 (0.05%), which is consistent with the phenotypic profile of human mesenchymal cells (Fig. 1B). The results of the colony formation assay demonstrated that the DPSCs exhibited robust growth and proliferation after 14 days of isolation and culture. Notably, numerous cell colonies with consistent cellular morphology and elongated shuttle-like shapes were observed under a microscope (Fig. 1C,a). Following a 21-day induction period with an osteogenic induction solution, the cells formed numerous red mineralized nodules of varying sizes (Fig. 1C,b). Furthermore, following 28 days of incubation in lipid-inducing medium, the cells demonstrated proficient formation of appropriately sized and abundant lipid droplets, which exhibited distinct red staining upon Oil Red O application (Fig. 1C,c).

Fig. 1
figure 1

A Morphology of primary dental pulp stem cells. B Identification of surface antigen markers of DPSCs via flow cytometry. C, a Crystal violet staining showing the formation of cell colonies. C, b Alizarin red staining showing the formation of calcium nodules. C, c Oil red O staining showing the formation of lipid droplets

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Biocompatibility of LL-37

The absorbance values of the experimental group and the control group were measured at 1, 3, 5 and 7 days (Fig. 2A). The results revealed no statistically significant difference in the absorbance values (ODs) at 450 nm between the low concentrations LL-37-treated group (< 10 μg/mL) and the control group within the same assay node (P > 0.05). Each group exhibited significant cell proliferation over the course of the culture period, suggesting that the low concentrations polypeptide LL-37 (< 10 μg/mL)did not exert a substantial inhibitory effect on the proliferation of DPSCs.

Fig. 2
figure 2

A Cell viability of DPSCs treated with different concentrations of LL-37 for 1, 3, 5, and 7 days, as determined by a CCK-8 assay. B Quantitative results of cell migration. C Staining results of cell migration. *P < 0.05. The results are presented as the means ± SDs of triplicate measurements from three independent experiments

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LL-37 promotes the migration of DPSCs

The ability of LL-37 to promote the migration of DPSCs was evaluated via a transwell assay and microscopic observation at 24 and 48 h. The results demonstrated that varying concentrations of LL-37 (1.25 μg/mL, 2.5 μg/mL, and 5 μg/mL) elicited different migratory responses in DPSCs. The migratory potential of LL-37 was positively correlated with its concentration (Fig. 2B,C). After ImageJ software was used to quantify cell migration through the chambers in each experimental group, we observed a progressive increase in the migratory cell population over time. Compared with the control group, the group treated with 5 μg/mL LL-37 presented significantly greater numbers of migrating cells at both 24 and 48 h (P < 0.05). Although there was an increase in cell migration in the groups treated with LL-37 at concentrations of 1.25 μg/mL and 2.5 μg/mL, no statistically significant difference was observed compared with that in the control group (P > 0.05). On the basis of the combined results of cell migration and biocompatibility, a concentration of 5 μg/mL LL-37 was selected for subsequent experiments.

Immunomodulatory and antiaging activity

RT‒PCR

To determine the effect of LL-37 on the expression of inflammatory factors secreted by DPSCs, DPSCs were cultured with or without LL-37 in the presence of 1 μg/mL LPS from E. coli for 24 h to simulate the inflammatory microenvironment and were examined by RT‒PCR to detect the release of cytokines (Fig. 3). The results revealed a significant increase in the expression of inflammatory factors secreted by DPSCs, including TNF-α, IL-1β, and IL-6, after stimulation with 1 μg/mL LPS. This observation suggests that the presence of 1 μg/mL LPS is capable of inducing an inflammatory response in DPSCs. Compared with the LPS group, the LL-37 group presented a significant reduction in the secretion of inflammatory factor mRNAs, indicating that LL-37 plays a crucial role in regulating inflammation progression during dental pulp inflammation. Excessive stimulation within an inflammatory microenvironment induces premature aging of DPSCs, thereby significantly impeding tissue regeneration. The expression levels of typical aging-related genes (P21, P38, and P53) were significantly increased in the LPS group, indicating the accelerated aging of DPSCs within an inflammatory microenvironment. In contrast, treatment with LL-37 led to partial downregulation of the expression of these genes. These findings suggest that LL-37 possesses remarkable antiaging properties.

Fig. 3
figure 3

Effects of LL-37 on the expression of inflammatory cytokines and typical aging-related genes in LPS-stimulated DPSCs. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The results are presented as the means ± SDs of triplicate measurements from three independent experiments

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Elisa and western blot

In this study, we assessed the impact of LL-37 on the secretion levels of proinflammatory factor proteins via both ELISA (Fig. 4A,B) and Western blot analysis (Fig. 4C,D). Full-length blots are presented in Supplementary Fig. 1 (Fig. S1). The ELISA results revealed a significant increase in the expression of the inflammatory factors IL-1β and IL-6 following LPS stimulation (P < 0.0001), confirming successful inflammation modeling. Conversely, treatment with the polypeptide LL-37 led to a substantial decrease in the secretion of IL-1β and IL-6 by the cells (P < 0.0001). The results of protein band analysis and quantitative assessment revealed significant upregulation of TNF-α expression in the LPS group compared with the control group (P < 0.05). After 24 h of LL-37 treatment, the expression level of TNF-α was significantly lower than that in the LPS group. The findings of this study suggest that LL-37 has the potential to attenuate the inflammatory response of DPSCs and demonstrates superior therapeutic efficacy in controlling inflammation progression.

Fig. 4
figure 4

Effects of LL-37 on the secretion levels of proinflammatory factor proteins in different groups, as determined by ELISA and Western blotting. A ELISA results of IL-1β. B ELISA results of IL-6. C Western blotting analysis of the protein expression of TNF-α. D Quantification of TNF-α. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The results are presented as the means ± SDs of triplicate measurements from three independent experiments

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LL-37 promotes odontogenic differentiation of DPSCs in an inflammatory microenvironment

ALP activity assay and alizarin red staining

ALP activity levels were assessed on days 4 and 7, revealing that the LL-37 group exhibited significantly deeper staining than the LPS group at both time points. The quantification of ALP was in accordance with the staining results (Fig. 5A). After 14 and 21 days of induction, alizarin red staining revealed significantly greater deposition of calcium nodules in the LL-37 group than in the control and LPS groups. The quantification results of the calcium nodules were in accordance with the staining results (Fig. 5B).

Fig. 5
figure 5

A ALP staining and quantitative analysis. B Alizarin red staining and quantitative analysis. ****P < 0.0001. The results are presented as the means ± SDs of triplicate measurements from three independent experiments

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RT‒PCR

The mRNA expression levels of DMP-1, BMP-2, and DSPP were assessed at 7, 14 and 21 days poststimulation (Fig. 6). Our findings demonstrated that the mRNA levels of DMP-1, BSP, and DSPP were significantly greater in both the control group and the LL-37 group than in the LPS group (P < 0.05).

Fig. 6
figure 6

Relative mRNA expression levels of odontoblast-related genes. * P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The results are presented as the means ± SDs of triplicate measurements from three independent experiments

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Discussion

Considering the regenerative properties of pulp tissue, DPSCs respond to external stimuli during the initial stages of inflammation, leading to their recruitment and subsequent formation of reparative dentin. However, when pulpitis progresses further, the potential for pulp repair and regeneration becomes unattainable. Therefore, timely control of inflammation is imperative to preserve the intrinsic self-repair capacity of pulp tissue.

The ability of the polypeptide LL-37 to interact with MSCs has been demonstrated, thereby modulating their proliferation, migration, and regenerative potential [35]. As a pivotal signaling molecule, LL-37 plays a crucial role in mediating intracellular and extracellular signal transduction, exhibiting interactions with diverse cell types and facilitating tissue repair processes. In recent years, an increasing number of studies have explored the potential of LL-37 in oral tissue regeneration and repair [36]. The application of LL-37 significantly increased the production of basic fibroblast growth factor, hepatocyte growth factor, and keratinocyte growth factor in gingival fibroblasts. By exerting its influence on these cells, damaged gingival tissue can undergo effective repair and reshaping [37]. An investigation of dental pulp tissue repair revealed that LL-37 has the ability to stimulate DPSCs migration [38, 39]. Additionally, LL-37 induces activation of the ERK pathway, thereby facilitating the secretion of vascular endothelial growth factor in DPSCs [40]. The presence of a minimal concentration of LL-37 can facilitate the odontogenic differentiation process in DPSCs [25]. Previous studies on the repair of pulp tissue with LL-37 have demonstrated that LL-37 has promising potential for application in the restoration of pulp tissue damaged by pulpitis.

As the sole identified Cathelidin family antimicrobial polypeptide in humans, LL-37 is expressed predominantly in neutrophils and epithelial cells, with widespread distribution across various tissues, including the intestine, airways, genitalia, and skin [41, 42]. In recent years, LL-37 has received widespread attention; however, its application has been somewhat limited because of its interaction with mammalian cell membranes and resulting cytotoxicity [43, 44]. The interaction between LL-37 and the cell membrane phospholipid bilayer has been demonstrated to be concentration dependent in research studies. Within the range of 0.1–1.0 μM, LL-37 exhibited antibacterial, immunomodulatory, and noncytotoxic properties; however, at concentrations of 2.0–13 μM, it induced hemolysis and certain cytotoxicity. Moreover, higher concentrations (> 13 μM) result in increased cytotoxicity [45]. Therefore, in this study, we adopted the effective concentration of LL-37 from previous studies and investigated its biocompatibility to establish a gradient of low-concentration LL-37 within a range that has been determined to exhibit reduced cytotoxicity toward DPSCs. LL-37 is the only human cathelicidin antibacterial peptide discovered at present and expressed in saliva so the healthy and biocompatibility of LL37 in the environment of oral cavity are assured [46]. Moreover, during the induction of odontogenic differentiation of DPSCs, we changed the induction solution every three days and added LL-37 at the same time. The density of DPSCs in the experimental group and the control group were no significant difference at 4, 7, 14 and 21 days after induction and dosing, which can prove the long-term safety of LL-37 on DPSCs.

The polypeptide LL-37 exerts a direct immunomodulatory effect, regulating the response of immune cells to inflammatory stimulation and attenuating the release of proinflammatory cytokines, including TNF-α, IL-1β and IL-6 [47]. DPSCs may be involved in immune responses during pulpal infection through activating NF- κB and LPS activated the I-κB kinase complex (IKK) in DPSCs to induce the phosphorylation and degradation of IκBα, resulting in the nuclear translocation of NF-κB [48]. LL-37 has been shown to indirectly modulate TLR when it binds to LPS, thereby preventing TLR4 signalling and LPS-induced inflammation [23]. In this study, we induced an inflammatory microenvironment via LPS and demonstrated that LL-37 effectively attenuated the production of proinflammatory cytokines by DPSCs within the inflamed microenvironment, thereby controlling the progression of pulpitis. As a potent inflammatory stimulator, LPS can serve as a ligand for toll-like receptors, triggering the activation of various downstream signaling pathways upon recognition. For example, the interaction between LPS and TLR4 can elicit the activation of the mitogen-activated protein kinase and nuclear factor kappa B (NF-κB) signaling pathways [49]. The NF-κB signaling pathway plays a pivotal role in the pathogenesis and progression of pulpitis. In response to inflammatory stimulation, this pathway is activated, leading to the release of proinflammatory cytokines such as TNF-α, IL-1β and IL-6 [50]. TNF-α orchestrates a diverse array of inflammatory and immunomodulatory activities, including vasodilation, facilitating leukocyte extravasation from blood vessels to infected tissues, and pivotal regulatory involvement in the pathogenesis of pulpitis [32]. The potent proinflammatory cytokine IL-1β triggers a diverse range of innate immune processes and is strongly implicated in both infectious and autoimmune diseases [51]. IL-1β is indispensable for the inflammatory response and host defense against pathogen invasion; however, it can also exacerbate chronic diseases and acute tissue damage. The cytokine IL-6 plays a pivotal role in both antigen-specific immune responses and inflammatory reactions [52].

Previous studies have demonstrated the ability of DPSCs to differentiate into inflammatory microenvironments, whereby the release of proinflammatory factors during the early stages of inflammation can facilitate the odontogenic differentiation of DPSCs [53]. However, repetitive stimulation with LPS can induce aging in DPSCs [54]. The capacity of aging DPSCs to withstand inflammation and undergo differentiation is impacted [55,56,57,58]. Excessive stimulation of oxidative stress in an inflammatory microenvironment induces aging in DPSCs, thereby significantly impeding tissue regeneration. The expression levels of canonical aging-associated genes (P21, P38, and P53) were significantly increased in the LPS group, indicating that aging-related deterioration and concurrent functional decline in DPSCs occurred. The relative expression of these genes in the LL-37 group was moderately downregulated, suggesting that LL-37 had a positive antiaging effect. Under the same conditions, LL-37 eliminated the activation of cell cycle regulators P21, P38 and P53. It is involved in the aging of DPSCs, but its exact function and mechanism are unknown. We found that LL-37 promoted the odontogenic differentiation of DPSCs and prevented their aging, with the most significant changes in the expression of the age-related marker P21 and P53. LL-37 attenuates cellular senescence by regulating the p53-p21(Cip1) pathway and restores differentiation in DPSCs [59, 60].

The LL-37 peptide can facilitate angiogenesis and synergistically collaborate with growth factors to enhance tissue repair and promote growth [61]. These biological effects are intricately linked to the intercellular communication between LL-37 and diverse cell types, particularly mesenchymal stem cells (MSCs). LL-37 specifically influences MSC proliferation, migration, and osteogenic differentiation. By inducing cell migration, LL-37 can effectively enhance the osteogenic differentiation of MSCs and facilitate in vivo bone formation [62]. The differentiation process of DPSCs resembles osteoblast-mediated bone formation and can be categorized into three distinct developmental stages: proliferation, early differentiation involving stromal secretion and maturation, and late differentiation characterized by stromal mineralization [63]. Differentiation at various stages can be identified via conventional histological staining techniques. ALP is a well-established biomarker for early differentiation, and the formation of mineralized nodules can be assessed in vitro through alizarin red staining [64]. In this study, we demonstrated that LL-37 not only enhances the migratory capacity of DPSCs but also stimulates their odontogenic differentiation by upregulating ALP expression and facilitating calcium nodule formation within the inflammatory microenvironment. The verification of odontogenic differentiation-related gene expression at different time points further substantiated these findings. During DPSC differentiation, a diverse array of collagen and noncollagen proteins are expressed, each exhibiting distinct functionalities. Therefore, we postulate that LL-37 facilitates differentiation through the regulation of mineral-related gene expression. The results were validated through RT‒PCR analysis. The dentin matrix primarily consists of type I collagen. COL1A is secreted to facilitate the formation of a collagen matrix during early odontoblast differentiation, whereas BSP and DMP-1 play crucial roles in promoting the initial mineral crystallization process [65, 66]. Therefore, these proteins can be considered early indicators of osteoblast and odontoblast differentiation. The results demonstrated that the expression of both genes increased on day 7, implying that LL-37 stimulated extracellular matrix secretion and maturation. Among noncollagenous proteins, dentin sialophosphoprotein (DSPP) plays a pivotal role in dental tissue [67]. DSPP effectively binds Ca2+ and induces mineral formation, making it a key marker of late differentiation. Induced DPSCs constitutively expressed DSPP, with its expression gradually increasing during the onset of induced differentiation. These findings suggest that LL-37 has promising potential in the regeneration of dental pulp tissue.

This study demonstrated the effective control of pulpitis and promotion of odontogenic differentiation in DPSCs by LL-37, thereby identifying a potential novel therapeutic option for future pulpitis treatment. However, this study focused solely on the short-term effects of LL-37, while its long-term effects remain inadequately assessed. In clinical applications, comprehending the prolonged efficacy and potential adverse reactions of LL-37 is crucial. Furthermore, despite the positive role of LL-37 in pulpitis observed in this study, research on its underlying mechanism of action is scarce. Further elucidation of the precise mechanism underlying the regulatory role of LL-37 in modulating inflammatory factors and facilitating the migration and odontogenic differentiation of DPSCs would contribute to a more accurate assessment of its therapeutic efficacy and potential risks.

Conclusions

The low concentration of LL-37 results in favorable biocompatibility, as evidenced by the fact that 5 μg/mL LL-37 does not impede the proliferation of DPSCs and significantly enhances cell migration. In the inflammatory microenvironment induced by 1 μg/mL LPS, 5 μg/mL LL-37 suppressed the release of proinflammatory factors while promoting odontogenic differentiation in DPSCs. These findings highlight the potential clinical application prospects of LL-37 in treating pulpitis, suggesting its utility as a promising pulp capping material for reversible pulpitis treatment.

Availability of data and materials

All data generated or analyzed in this study are included in this article and its supplementary information files. Other relevant data are available from the corresponding author upon reasonable request.

Abbreviations

ALP:

Alkaline phosphatase

α-MEM:

α-Modified eagle’s medium

BSP:

Bone sialoprotein

CCK-8:

Cell counting kit-8

DMP1:

Dentin matrix protein 1

DSPP:

Dentin sialophosphoprotein

DSP:

Dentin sialoprotein

FBS:

Fetal bovine serum

hDPSCs:

Human dental pulp stem cells

IL:

Interleukin

LPS:

Lipopolysaccharide

LL-37:

Peptide LL-37

OD:

Optical density

PBS:

Phosphate buffer saline

PFA:

Paraformaldehyde

RT-PCR:

Real-time quantitative PCR

RNA:

Ribonucleic acid

TNF-α:

Tumor necrosis factor-α

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Acknowledgements

The authors declare that they have not use AI-generated work in this manuscript.

Funding

This work was supported by the Research Fund of the Anhui Institute of Translational Medicine (2023zhyx-B17), the Anhui University Natural Science Research Project (2023AH050597), and the Health Research Program of Anhui (AHWJ2023A10089).

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Authors and Affiliations

  1. Key Lab. of Oral Diseases Research, College and Hospital of Stomatology, Anhui Medical University, Hefei, 230032, Anhui Province, China

    Yunfeng Ma, Xinyuan Liu & Chris Ying Cao

  2. Department of Stomatology, Longgang Otorhinolaryngology Hospital of Shenzhen, Institute of Oral Science, Shenzhen, 518172, China

    Quanli Li

  3. Department of Comprehensive Care, Tufts University School of Dental Medicine, Boston, MA, 02111, USA

    Ruoxi Dai

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Yunfeng Ma contributed to conception of manuscript, design of experiments, interpretation, and analysis of data, drafting and revision of manuscript. Xinyuan Liu contributed to interpretation, and analysis of data. Ruoxi Dai g contributed to the study concept and design, data analysis and interpretation. Quanli Li contributed to conception of manuscript, interpreted data and revised manuscript. Chris Ying Cao contributed to conception of manuscript, design of experiments, interpretation, and analysis of data, drafting and revision of manuscript.

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Correspondence to Chris Ying Cao.

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The extraction procedures of human DPSCs were conducted in accordance with the Declaration of Helsinki, and informed consent was obtained from the donors and/or their guardians before the tooth collection. The extraction procedures of human DPSCs in this study were approved by the Ethics Committee of Anhui Medical University. (Project title: The key physicochemical factors and mechanisms of collagen intra/extra-fibrillar mineralization mediated by polyelectrolyte-calcium phosphate prenucleation clusters; Approval No: LLSC20240974; Date of approval: 2024–03-01).

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Ma, Y., Liu, X., Dai, R. et al. LL-37 regulates odontogenic differentiation of dental pulp stem cells in an inflammatory microenvironment. Stem Cell Res Ther 15, 469 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-024-04075-7

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-024-04075-7

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Stem Cell Research & Therapy

ISSN: 1757-6512