Extracellular IL-37 Enhances Osteogenic and Odontogenic Differentiation of Human Dental Pulp Stem Cells via Autophagy Pathway

Background The osteogenic and odontogenic differentiation of dental pulp stem cells (DPSCs) contributes to the restoration and regeneration of dental tissues. Previous study indicated that IL-37 has often been identied as an anti-inammatory factor that affects other pro-inammatory signals. It is known to be a factor capable of inducing in vitro osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs). The aims of this study were to explore the effects of IL-37 on the differentiation of DPSCs. Methods DPSCs were cultured in growth medium with different concentration of IL-37, ALP activity was done to detect the optimal concentration for the following experiments. CCK-8 were conducted to assess the effect of IL-37 on proliferation of DPSCs. To assess differentiation, alkaline phosphatase activity, ALP staining, alizarin red S staining and real ‐ time RT ‐ PCR of DSPP, Runx2, ALP, and OSX were measured. Western blot was conducted to examine the levels of autophagy related markers (Beclin1, P62, LC3).


Introduction
Dental pulp is the soft tissue inside tooth, which plays an indispensable role in the homeostasis of vital teeth [1]. The function of dental pulp is to support dentin formation and regeneration [2]. A range of injuries or diseases including caries, pulpitis, periapical periodontitis, tooth trauma, etc. can result in pulp necrosis, further teeth losing [3]. For therapeutic strategies for the kind of teeth above, vital pulp therapy (VPT) and regenerative endodontic treatment (RET) attracted recent attention to remain functional of teeth. The aims of VPT are to preserve and save dental pulp vitality, to induce dental pulp stem cells (DPSCs) to differentiate into osteoblasts and odontoblasts and ultimately form the hard tissue such as the tertiary dentin. RET known as revascularization aims to promote normal physiological development in teeth with necrotic pulp and act as a substitute for injured dental structures. DPSCs have been regarded as an important candidate for such treatment. Their capability of differentiation into odontogenic and osteogenic stem cells associated with biomaterials and growth factors is critical for dental pulp regeneration [4,5]. Mesenchymal stem cells (MSCs) have a signi cant role in pulp regeneration therapy for the reconstruction of tissues. Due to the easily available source of dental tissues, dental stem cells are considered good candidates for tissue engineering applications.
Human MSCs are a serviceable therapeutic tool for tissue engineering. DPSCs are identi ed as a type of MSCs that were originally isolated from human dental pulp tissue [6]. DPSCs have a lot of merits of easy access with the least invasive procedures and without any ethical controversy, and retain capacities of clonogenic formation, high proliferation, excellent regeneration, multilineage differentiation potential, and little inherent immunogenicity. As has been widely acknowledged, DPSCs can be induced to differentiate into a good many of directions, including osteogenic, dentinogenic, adipogenic, chondrogenic, myogenic, and neurogenic differentiation [7]. Compared with other MSCs from tissues as follows: bone marrow [8], peripheral blood [9], adipose tissue [10], and umbilical cord blood [11,12], DPSCs demonstrate higher clonogenic and proliferative potential. Therefore, DPSCs become an engaging tool cell source for tissue engineering and regenerative medicine.
During the developmental process of MSCs, extracellular cues tend to exert its function in determining the fate of MSCs. Previous studies have revealed that in chronic in ammatory bone diseases, bone regeneration can be inhibited, and the osteogenic differentiation of DPSCs can be in uenced by in ammatory microenvironments [13]. Interleukin 1 family member 7 (IL-1F7), a novel anti-in ammatory cytokine was recently proposed to be renamed Interleukin-37 (IL-37) [14]. IL-37 functions as a natural inhibitor of in ammatory and immune responses [15]. IL-37 is an anti-in ammatory factor that affects other pro-in ammatory signals, such as those mediated by tumor necrosis factor α (TNF-α), IL-1β, and IL-18 [16]. It was reported that IL-37 can promoted BMMSCs to differentiate into osteogenic lineage cells in vitro [17]. Recent studies have shown that abnormal expression of IL-37 in several autoimmune orthopedic diseases, such as ankylosing spondylitis and rheumatoid arthritis [18]. Most recently, studies demonstrated a connection between IL-37 and several bone metabolism-related in ammatory cytokines and reported that recombinant IL-37 (rhIL-37) inhibited the expression of pro-in ammatory cytokines, such as IL-6, TNF-α, IL-17, and IL-23 in patients with ankylosing spondylitis [19]. The new study has indicated that IL-37 suppresses osteoclast formation and bone resorption in vivo [20].
A better understanding of the molecular mechanisms that govern odontogenesis and osteogenesis might provide us with new perspectives on treatment of many oral diseases. Recent reports have provided evidence that macromolecular degradation in stem cells in a process of differentiation occurs through autophagy [21]. Autophagy or "self-eating", is a conservative cellular degradation pathway that recycles cellular content [22]. Upon activation of autophagy, degradation of intracellular protein and organelles via a process that involves the delivery of cytoplasmic cargo to lysosomes and release of metabolites required for anabolic processes, such as cell growth, proliferation and differentiation [23]. When cells are subjected to external stress, such as nutrition de ciency, oxidative stress, hypoxia, tumor formation, aging or infection, autophagy plays an important role as a cell survival mechanism [24,25]. Recent studies have indicated that autophagy is an essential part in the functioning and maintenance of stem cells, acting to maintain their stemness, regulate their self-renewal, and mediate their differentiation capacity [26,27] [28]. In addition, accumulating evidence has demonstrated that autophagy is also involved in osteogenesis and bone development [29,30]. Autophagy is also an essential process that maintains mineralizing capacity, and balance the population of osteoblasts and osteoclasts [31]. The members in our team have demonstrated that autophagy is involved in the odontogenic and osteogenic differentiation in stem cells of apical papilla and rBMMSCs [32,33].
We hypothesis that autophagy might be involved in IL-37-mediated DPSCs osteogenic and odontogenic differentiation. Therefore, the aim of the present study was to verify whether IL-37 is able to promote the osteogenic and odontogenic differentiation of DPSCs and whether autophagy was involved in the regulation of osteo/odontogenic differentiation of DPSCs in vitro.

Isolation and Cell culture
Fresh extracted third molars were collected from patients (age:17-23 years old) with the patient's consent. Tooth was cleaned with PBS (Gibco, USA). The pulp tissue was minced into smaller fragments and then immersed into a solution (3 mg/mL collagenase type I (Sigma, USA) and 4 mg/mL dispase (Gibco, USA)) for 1 h. Cell suspensions were seeded into culture dishes containing complete medium.

Immuno uorescence staining
Single cell suspensions were seeded on top of coverslips. After removing the medium, cells were xed and washed. Brie y, after permeabilization with Triton X-100 for 12 min, cells were blocked with BSA (Boster, China) overnight, and then incubated with primary antibodies (anti-STRO-1, 1:100) overnight at 4℃. Cells were incubated with Cy3-conjugated secondary antibodies (Abcolonal, China). Following counterstained with DAPI, cells were photographed using a uorescence microscope (Leica, Germany).

Colony-forming assay
After cultured for 10 days later, cells were stained with toluidine blue (Beyotime, China). Photographs were taken under the microscope (Leica, Germany).

Multiple lineage differentiation
Cells were cultured in osteoinductive medium (OM) of 50 mg/ml ascorbic acid, 10 mM sodium βglycerophosphate, 10 nM dexamethasone (Sigma, USA). After 2 weeks, cells were stained with Alizarin Red S (Sigma, USA) to detect mineral deposits.
Cells were incubated in adipogenic medium (Cyagen Biosciences Inc, USA) for 4 weeks. Then, cells were stained with Oil Red O reagent of the kit to reveal lipid droplets.
Cell pellets were prepared for a three-dimensional culture system. Cells were cultured in chondrogenic differentiation medium (Cyagen Biosciences Inc, USA) for 28 days. Alcian blue staining was utilized to examine the cartilage nodules.

ALP activity assay
Cells were incubated with different concentrations of IL-37 (0, 0.1, 1, 10, and 100 ng/mL) for 5 days. ALP activity of DPSCs was detected according to manufacturer's instruction.

ALP staining
Cells were seeded on 12-well plates and incubated with different cultured medium in different groups for 5 days. ALP staining was performed using an ALP staining kit (Beyotime Biotechnology, China) according to the manufacturer's instruction.

Alizarin red staining
DPSCs were cultured in OM for 14 days in the presence or absence of IL-37. The formation of mineralized nodules of DPSCs was evaluated by alizarin red S staining. For quantitative analysis, 10% cetylpyridinium chloride (CPC) was added and the absorbance was measured at 562 nm.

EdU assay
Cell proliferation ratio was measured using EdU Cell Proliferation Assay Kit according to the protocol of manufacturer.

RNA extraction and PCR analysis
Total RNA was isolated and reversed to cDNA by the RT Reagent Kit (Vazyme, China). Real-time qPCR reactions were carried out SYBR Green Mix (Vazyme, China). The gene speci c primers are listed in Table 1.

TEM
Cells were collected after treated with or without 1 ng/mL IL-37 for 12 h. After xation, dehydration, embedding, sectioning and staining, samples were viewed with a Hitachi Model H-7500 TEM (Hitachi, Japan).

Immuno uorescence staining
After cell attachment, the culture solid was replaced by complete medium and IL-37 (1 ng/mL) for 12 h. Cells were stained with primary antibody including DSPP, RUNX2, ALP, LC3 (1:100) according to the steps above.

Statistical analysis
Data are shown as mean ± SEM and were tested for statistical signi cance using ANOVA.

Characterization of SCAPs and screening for the optimal IL-37 concentration
DPSCs were isolated and displayed a typical cobblestone-like morphology. Primary DPSCs were observed as shown in Fig. 1A. PDSCs in passage 3 were typical broblast-like or spindle-like in Fig. 1B. Immuno uorescence staining indicated that isolated DPSCs were positive for the established MSCsspeci c surface marker STRO-1 (Fig. 1C). Flow cytometry analysis showed a pronounced expression of mesenchymal stem cell markers including CD73, CD90, and CD105, while they also showed the negativity for hematopoietic cell marker CD34 and leucocyte maker CD45. Cell surface markers: CD34 (0.07%); CD45 (0.30%); CD73 (99.6%); CD90 (93.3%); CD105 (87.5%) (Fig. 1D). The result of colony-forming assay showed that DPSCs could form single cell colonies. A single colony stained with crystal violet was observed under the microscope (Fig. 1E). Furthermore, results of oil red O staining, alizarin red S (ARS) staining and Alcian blue staining showed that DPSCs could differentiate into adipocytes, osteoblasts, and chondrocyte (Fig. 1F, G, H).

Effect of IL-37 on the proliferation of DPSCs
At day 5, ALP activity assay showed that ALP activity was the highest in the 1 ng/mL IL-37 group ( Fig. 2A; P < 0.01). As compared with other groups, the 1 ng/mL IL-37 group also presented the highest ALP protein and gene expression after being induced for 5 days (Fig. 2B, C, D; P < 0.01). Therefore, 1ng/mL IL-37 was selected to be the optimal concentration for our further study. To evaluate the impact of IL-37 on the proliferation of DPSCs, CCK-8 analysis and EdU assay were performed. As indicated by the results of EdU assay (Fig. 2E, F; p > 0.05), and CCK-8 ( Fig. 2G; p > 0.05), 1ng/mL IL-37 exerted almost no signi cant effect on the proliferation of SCAPs as compared with the control group.

IL-37 induced the osteo/odontogenic differentiation of SCAPs
To analyze the in uence of IL-37 on the osteo/odontogenic differentiation and mineralization of DPSCs, cells were treated with 1 ng/mL IL-37. The protein expression of osteogenic and odontogenic markers in hDPSCs were examined cultured with or without 1 ng/mL IL-37 for 0, 3 and 7 days. Cells cultured with 1 ng/mL IL-37 expressed markedly higher levels of DSPP, RUNX2, ALP and OSX than those control groups for 3, 7 days, while, there is no difference between control group and IL-37 group for 0 day (Fig. 3A and B). Consistently, qRT-PCR showed that the osteogenic and odontogenic markers DSPP, RUNX2, ALP and OSX were also increased in the IL-37 treatment groups for 3, 7 days ( Fig. 3C; p < 0.05 or 0.01). To further con rm whether IL-37 mediate osteogenic and odontogenic differentiation of DPSCs, cells were then cultured in OM with and without IL-37. Compared with control group, ALP staining revealed that ALP activity was signi cantly higher with administration of 1 ng/mL IL-37 (Fig. 3D). ALP activity assay con rmed the results of staining (Fig. 3E, p < 0.01). A similar trend in Alizarin Red S staining was detected after induction for 14 days. DPSCs treated with 1 ng/mL IL-37 and IL-37 + MM presented more mineralized nodules than control group or MM group (Fig. 3F). CPC assay revealed that extracellular calcium deposition was signi cantly increased in IL-37 group at day 14 as compared with the control group ( Fig. 3G; p < 0.01). In addition, immuno uorescence assay showed that the osteoblastic and odontoblastic markers DSPP and RUNX2 were upregulated in 1 ng/mL IL-37-treated group (Fig. 2H, I). Above all, these results indicated that 1 ng/mL IL-37 enhanced the osteo/odontogenic differentiation of DPSCs.

1 ng/mL IL-37 triggered autophagy of DPSCs
To explored the mechanism of IL-37-enhanced odontoblastic and osteoblastic differentiation potential of DPSCs, western blot was conducted. DPSCs were treated with 1 ng/mL IL-37. Exposure of DPSCs to complete medium containing 1 ng/mL IL-37 resulted in elevated protein expression of autophagy-related protein Beclin1 and the expression ratio of LC3-II/I in a time-dependent manner while the expression of p62 was decreased (Fig. 4C, D). The images of immunostaining with LC3 showed that endogenous LC3 dot form in IL-37-treated DPSCs was signi cantly higher than control group (Fig. 4E). Meanwhile, to further con rm the induction of autophagy in IL-37-treated DPSCs, the formation of autophagosomes was observed by means of TEM. The photos of TEM showed more autophagosomes in 1 ng/mL IL-37treated DPSCs group at 7 days than those in the control group ( Figure F). These observations strongly suggested IL-37 activated autophagy in IL-37-treated DPSCs.

The autophagy inhibitor 3-MA suppresses IL-37mediated enhancement osteoblastic and odontoblastic differentiation of DPSCs
To further validate our ndings, autophagy was inhibited with speci c inhibitor 3-MA, respectively. Western blot analysis indicated that the ratio of LC3-II to LC3-I, Beclin1 expression levels in IL-37 + 3-MA group were signi cantly lower than the IL-37 group, while 3-MA signi cantly up-regulated P62 protein levels, suggesting that autophagy was suppressed when autophagy inhibitor3-MA treatment was applied ( Fig. 5A, B). The result indicated that 3-MA showed obviously autophagy inhibition. RT-PCR and western blot were conducted to investigate the effects of IL-37 on osteoblastic and odontoblastic differentiation of DPSCs following autophagy inhibitor treatment. Western blot analysis showed that the inhibitor 3-MA dramatically suppressed IL-37-mediated enhancement of DSPP, RUNX2, ALP and OSX expression in DPSCs at protein levels (Fig. 5C, D). Similarly, the inhibitor 3-MA suppressed IL-37-enhanced the expression of DSPP, OCN, RUNX2 and OSX in DPSCs at gene level (Fig. 5E). To investigate the role of autophagy in IL-37-induced mineralization, ALP staining, ALP activity assay, ARS staining and CPC assay were performed. The results of ALP staining and ALP assay showed that ALP activity of IL-37-treated DPSCs was inhibited with the addition of autophagy inhibitor 3-MA on day 5 (Fig. 5F, G). After induction for 2 weeks, the amount of minerals was signi cantly decreased in the presence of autophagy inhibitor 3-MA compared with IL-37 treatment alone (Fig. 5H, I). As shown by immunostaining analysis, the expression of DSPP and RUNX2 increased remarkably in IL-37-treated DPSCs compared with IL-37 + 3-MA (Fig. 5J, K). These results indicated that IL-37-enhanced osteoblastic and odontoblastic differentiation of DPSCs were repressed by autophagy inhibition.

Upregulation of autophagy by rapamycin further promoted odontogenic and osteogenic differentiation of DPSCs
To further examine the effect of autophagy on osteogenic and odontogenic differentiation of DPSCs, cells were cultured in medium in the presence of rapamycin. The result also indicated that rapamycin treatment signi cantly up-regulated Beclin1 protein levels, increased the ratio of LC3-II to LC3-I and downregulated P62 protein level compared with the IL-37 group (Fig. 5A, B). Besides, hDPSCs exposed to rapamycin signi cantly increased autophagy activity. Western blot analysis and qRT-PCR were conducted to detect the inducer of autophagy rapamycin on the osteogenic and odontogenic differentiation of DPSCs at the RNA and protein levels. The results of western blot showed that the expression of osteogenic and odontogenic markers DSPP, RUNX2, ALP and OSX were all up-regulated in IL-37 + rapamycin group comparing with IL-37 (Fig. 6C, D). PCR assay for mRNA expression of odontogenesis/osteogenesis-related molecules showed that autophagy activation by rapamycin enhanced IL-37-induces differentiation (Fig. 6E). ALP staining and ALP activity assay showed that rapamycin upregulated ALP activity compared with IL-37 groups (Fig. 6F, G). ARS staining indicated that the number of mineralized nodules was observed more in the rapamycin group (Fig. 6H). CPC assay showed that the group treated with IL-37 + rapamycin presented higher calcium contents as compared with IL-37 group (Fig. 6I). Besides, immuno uorescence assay showed that osteogenic and odontogenic markers DSPP, RUNX2 were upregulated in IL-37 + rapamycin-treated group (Fig. 6J, K). All these data indicated that autophagy plays a catalytic role in IL-37-induced osteogenic and odontogenetic differentiation of hDPSCs.

Discussion
Osteogenic differentiation and odontogenic differentiation are crucial characteristics of the oral stem cells pluripotency. They play key roles in the maintenance, tissue regeneration and engineering. In this study, we demonstrated that IL-37 and the related target autophagy pathway play an important role in the osteo/odontogenic differentiation of DPSCs. DPSCs consist of heterogeneous multipotent cell populations, which have the potential to differentiate into osteoblasts and odontoblasts. Osteogenic and odontogenic differentiation result in the expression of related genes and proteins such as DSPP, RUNX2, ALP, OSX. This study emphasized a novel and promising role of IL-37 in regulating the odontogenic and osteogenic differentiation of DPSCs.
IL-37 is recently identi ed as an anti-in ammatory cytokine [34]. A Chinese study reported that patients carrying a mutation in the coding region of IL-37 have lower disease activity scores and less pain, which indicated signi cant function for the anti-in ammatory effects of IL-37 [35]. Previous studies demonstrated that cytokines were associated with autophagy. For example, in addition to controlling the migration of macrophages into degenerative tissues, IL-33, one of cytokines, also regulated autophagy in these tissues [36]. The level of IL-37 is normally low, but this signi cantly increases under severe in ammatory conditions. It has been previously found that IL-37 plays an important regulatory role in the development of several in ammatory and autoimmune diseases [37,38]. Recent studies investigated that IL-37 mediates a variety of anti-cancer effects in multiple types of cancer [39]. Recently, it was reported that IL-37 promoted the osteogenic differentiation of BMSCs [17]. In this study, we found that extracellular IL-37 accelerated the osteogenic and odontogenic differentiation of DPSCs via autophagy when at 1 ng/mL, while there is no signi cant effect on the proliferation.
The autophagy pathway is normally inhibited by 3-MA and activated by rapamycin [40]. Autophagy is a major intracellular mechanism to protect cells from stress stimulation and maintain the properties of stem cells [30,41]. Autophagy plays an important role in the osteogenic and odontogenic differentiation of hDPSCs under speci c condition [1]. During cellular differentiation, autophagy occurs in order to meet metabolic needs associated with morphological and functional changes. Several proteins, including LC3, p62, and Beclin1 have been involved in autophagy and are used as markers for the activation of the process. LC3 is the most widely used autophagic marker. LC3 is usually produced by ubiquitin, the residues of which are exposed on the vesicular membrane surface after ATG4 homolog catalysis, and forms LC3-I in the cytoplasm [41]. LC3-I can speci cally bind phosphatidylethanolamine on the vesicle membrane surface and eventually forms LC3-II, which is an integral membrane protein present in autophagosomes [42]. Accumulation of LC3 II is thus used as a marker for activation of autophagy, which can directly reveal autophagy. Beclin-1 is an autophagy-initiated protein. P62 possesses LC3interacting region and serves as a signaling hub of autophagy. P62 is degraded through the process of autophagy, therefore the degradation of P62 expression level can serve as a marker of autophagic clearance [30]. The increases of Beclin1, an autophagy initiation protein, is also accompanied by activation of the process. In this study, IL-37 promoted LC3 I/II conversion, increased the protein levels of Beclin1 and decreased the protein expression of P62.
ALP has a vital function during mineralization of osteoblasts and odontoblast, and its activity is upregulated at an early stage of calci cation [43]. In this study, 1 ng/ml was the optimal concentration of IL-37 to promote differentiation of DPSCs by detecting the ALP activity at day 5, as well as the mRNA level and protein level of ALP at day 5. Our results showed that the expression levels of osteogenic and odontogenic markers, including DSPP, RUNX2, ALP, and OSX were upregulated at 3, 7 days of differentiation, indicating that 1 ng/mL IL-37 can enhance the osteo/odontogenic differentiation of DPSCs. The autophagy pathway was shown to positively regulate differentiation of MSCs. We proceeded to investigate whether autophagy was activated by IL-37, the key proteins in autophagy signaling were evaluated. Consistent with pathway analysis, the protein expressions of Beclin 1, the ratio of LC3 II/I were signi cantly increased while the expression of P62 was reduced, when DPSCs were treated with IL-37 for 6, 12, 24 h. To further con rm whether autophagy signaling was implicated in the process of IL-37 enhanced osteogenic and odontogenic differentiation of DPSCs, we evaluated the expression of related markers above with or without autophagy inhibitor 3-MA. While 3-MA was used, we found that all the markers of osteogenesis and odontogenesis were dramatically reduced. These in vitro results demonstrated that autophagy is an important mechanism in IL-37 enhanced osteo/odontogenic differentiation of hDPSCs. Rapamycin, as autophagy activator was used to further detect the relationship between autophagy and osteo/odontogenic differentiation of IL-37 treated DPSCs. Osteogenic and odontogenic differentiation of IL-37 enhanced DPSCs further increased with rapamycin treatment.
In summary, the present study revealed that IL-37 could induce osteogenic and odontogenic differentiation in DPSCs, which involved the upregulation of autophagy. The results expanded our knowledge on the role of IL-37 in osteogenesis and odontogenesis of DPSCs, and also suggested that IL-37 may be a potential drug for pulp treatment in the future and provided insight into the molecular mechanism of vital pulp therapy. Also, kinetic in vivo measurements should be carried out in future studies.

Conclusion
Overall, IL--37 enhances differentiation of DPSCs into odontoblasts and osteoblasts via autophagy signaling pathways.

Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate Studies were carried out in accordance with the Declaration of Helsinki and got the approval of the Ethical Committee of Nanjing Medical University.

Consent for publication
Not applicable.