- Email: [email protected]
Odontogenic Differentiation of Stem Cells From Apical Papilla by Inducing Autophagy
BASIC RESEARCH – BIOLOGY Shuang Lei, MS, DDS, Xue-Mei Liu, MS, DDS, Yao Liu, DDS, PhD, Jing Bi, MS, DDS, Shu Zhu, MS, DDS, and Xu Chen, DDS, PhD
Lipopolysaccharide Downregulates the Osteo-/ Odontogenic Differentiation of Stem Cells From Apical Papilla by Inducing Autophagy
ABSTRACT Introduction: The dentinogenesis potential of stem cells during dentin-pulp tissue regeneration may be compromised by microorganism components. Here we aimed to investigate the cell viability and osteo-/odontogenic differentiation of stem cells from apical papilla (SCAP) exposed to bacterial lipopolysaccharide (LPS) and to evaluate the molecular mechanism in vitro. Methods: CCK8 assay was used to assess the SCAP proliferation rate on exposure to different concentrations of LPS in medium. Dentin matrix protein-1 (DMP-1), runt-related transcription factor-2 (Runx-2), and alkaline phosphatase (ALP) expression and mineralized nodule formation were detected by Western blotting and alizarin red S staining to evaluate SCAP osteo-/odontogenic differentiation. Autophagosomes in SCAP and the autophagyrelated markers Beclin 1, autophagy-related gene 5 (Atg5), and microtubule-associated proteins light chain 3 (LC3) were detected by transmission electron microscopy and Western blotting, respectively. Effects of the autophagy inhibitor 3-methyladenine on LPS-treated SCAP osteo-/odontogenic differentiation were also examined. Results: SCAP cell viability was decreased by 5 mg/mL LPS treatment on day 3. LPS (5 mg/mL) inhibited SCAP osteo-/ odontogenic differentiation and decreased DMP-1, Runx-2, and ALP expression. Moreover, LC3, Atg5, and Beclin 1 expression and autophagosome number were elevated. Conversely, autophagy inhibition rescued the number of mineralized nodules and DMP-1, Runx-2, and ALP expression in the LPS-treated SCAP. Conclusions: Our findings indicated that autophagy was involved in the suppression of SCAP osteo-/odontogenic differentiation in an LPS-induced inflammatory environment. This study discloses autophagy modulation as a potential new strategy to improve SCAP osteo-/odontogenic differentiation in an inflammatory environment. (J Endod 2020;-:1–7.)
SIGNIFICANCE This study disclosed the role of autophagy in regulating the biological effects of lipopolysaccharide on stem cells from apical papilla, which contributes to the damage and repair mechanisms of immature permanent teeth affected with periapical periodontitis.
KEY WORDS Autophagy; lipopolysaccharide; osteo-/odontogenic differentiation; proliferation; stem cells from apical papilla Pulp and periapical disease in immature permanent teeth comprises a group of inflammatory diseases caused by microorganisms (mainly bacteria) infecting the developing dental root1. Stem cells from apical papilla (SCAP), which reside at the root apex of incompletely developed teeth, have been identified as essential tissue in tooth root development and apexogenesis. SCAP are presumed to constitute a prospective stem cell source of the primary odontoblasts that are responsible for the formation of root dentin and regeneration of the pulp-dentin complex2. However, the dentinogenic capacity of SCAP may be impaired by the presence of microorganisms and their components. For example, it has been reported that the cell number and viability of SCAP were reduced during prolonged infection periods, although SCAP could survive in the infectious environment of immature teeth3. Bacteria involved in the pathogenesis of apical periodontitis may have participated in the early stages of pulp inflammation and necrosis. Bacteria virulence factors comprise structural-cellular components and released products that are involved in every step of the infectious process including
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From the Department of Pediatric Dentistry, School of Stomatology, China Medical University, Shenyang, China; and Liaoning Provincial Key Laboratory of Oral Diseases, Shenyang, China Address requests for reprints to Dr Xu Chen, Department of Pediatric Dentistry, School of Stomatology, China Medical University, 117 Nanjing North Street, Shenyang, 110002, China. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright © 2020 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2020.01.009
Autophagy and Differentiation of SCAP Exposed to LPS
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attachment, invasion, survival, and damage4. Lipopolysaccharide (LPS), a major component of gram-negative bacteria, acts as an endotoxin that elicits a variety of immune responses in odontoblasts, fibroblasts, dental pulp stem cells (DPSCs), and SCAP5–7. Studies have reported that 1 mg/mL LPS protects mesenchymal stem cells (MSCs) against apoptosis and enhances their proliferation, whereas 5 mg/mL LPS increases MSCs apoptosis in vitro8,9. However, the exact mechanism by which LPS influences the biological behaviors of SCAP remains unclear. Dental stem cell differentiation and tooth development are influenced by macroautophagy (hereafter referred to as autophagy)10,11, a ubiquitous, evolutionarily conserved cellular process in which characteristic double- or multi-membrane autophagosomes form and include components of the cytoplasm and parts of intracellular organelles12. The outer membrane of the autophagosome then fuses with a lysosome or endosome, generating a singlemembrane autolysosome13. Accumulating evidence has shown that autophagy is a normal part of the cell life cycle and is involved in cell growth, differentiation, cell survival or death, melanosome degradation, and inflammatory response14,15. Generally, autophagy can be induced by starvation, chemical drugs, or inflammatory environment. It can be inhibited by 3-methyladenine (3-MA) through inhibiting class III phosphatidylinositol 3-kinase. In addition, chloroquine, which inhibits lysosomal enzymes and prevents the fusion of autophagosome and lysosome, can also inhibit autophagy in stem cells16. However, the potential role of autophagy in periapical periodontitis of immature permanent teeth has not yet been elucidated. To address these issues, in this study we aimed to explore the effect of LPS on the committed osteo-/ odontogenic differentiation of SCAP and whether this effect is modulated by autophagy.
MATERIALS AND METHODS SCAP Isolation and Culture SCAP were collected from intact, caries-free impacted third molars with immature roots from healthy patients (12–15 years of age) at the dental clinic of the School of Stomatology affiliated with China Medical University. This study was approved by the Ethics Committee of the School of Stomatology, China Medical University (201508). Both the patients and their guardians provided written informed consent for use of the collected cells. SCAP were freshly isolated and cultured as previously reported17. The apical papilla was gently separated from the tooth and then minced and digested with 4 mg/
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mL dispase II (Boehringer Ingelheim, Mannheim, Germany) and 2 mg/mL collagenase type I (Worthington Biochemical Co, Lakewood, CO) at 37 C. Single-cell suspensions were seeded and cultured in alpha minimum essential medium (a-MEM) (HyClone, Logan, UT) supplemented with 15% fetal bovine serum (Excell Bio, Shanghai, China), 100 U/mL penicillin-streptomycin (HyClone), and 0.1 mmol/L L-ascorbic acid (Sigma-Aldrich, St Louis, MO) in a humidified atmosphere at 37 C with 5% CO218. SCAP from passages 4–5 were used for all experiments in the present study.
Cell Proliferation Assay SCAP were seeded in 96-well plates (3000 cells/well) in serum-free culture medium containing 0, 0.05, 0.5, and 5 mg/mL Porphyromonas gingivalis LPS (InvivoGen, San Diego, CA). The cells were starved for 12 hours before the stimulus was added. Medium without LPS served as the control. The experiment was conducted according to the manufacturer’s instructions. Briefly, after incubation for 1, 2, and 3 days, 10 mL CCK8 solution (Dojindo, Kumamoto, Japan) with 90 mL a-MEM was added to each well, and the plates were incubated for another 2 hours in the dark. The optical density was detected by using a microplate reader (Tecan, Salzburg, Austria) at a wavelength of 450 nm.
Western Blot Analysis SCAP were treated with different concentrations of LPS (0.05, 0.5, and 5 mg/ mL) or LPS plus 5 mmol/L of the autophagic inhibitor 3-MA (Selleckchem, Shanghai, China). 3-MA was diluted in phosphate buffered saline (PBS) and used to pretreat cells for 24 hours before application of LPS to inhibit autophagy. Cells were washed 3 times with ice-cold PBS and incubated with RIPA lysis buffer (Beyotime Biotech Co, Shanghai, China) containing 1 mmol/L phenylmethanesulfonyl fluoride on ice for 30 minutes. Protein concentrations were measured by using a BCA protein assay kit (Beyotime Biotech Co) according to the manufacturer’s instructions. After the samples were boiled with sample loading buffer (Beyotime), equal amounts of protein extracts (20 mg protein per sample) were subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA). The membranes were blocked with 4% bovine serum albumin for 1 hour at room temperature and then incubated with an anti– dentin matrix protein 1 (DMP-1) antibody (1:1000; Abcam, Cambridge, UK), anti–runtrelated transcription factor-2 (Runx-2) antibody
(1:250; Proteintech, Rosemont, IL), anti– alkaline phosphatase (ALP) antibody (1:200; Santa Cruz Biotechnology, Dallas, TX), anti– microtubule-associated proteins light chain 3 (LC3) antibody (1:1000; Abcam), anti-Beclin 1 antibody (1:1000; Abcam), anti–autophagy related gene 5 (Atg5) antibody (1:1000; Abcam), and antibody against glyceraldehyde3-phosphate dehydrogenase (GAPDH) (1:1000; Proteintech) at 4 C overnight. The membranes were washed with Tris-buffered saline Tween-20 and exposed to goat antirabbit/anti-mouse immunoglobulin G IRDyel 800cw secondary antibody (1:1000; Abbkine, Inc, Redlands, CA) at room temperature for 1 hour. The protein bands were detected by using an Odyssey CLx instrument (LI-COR, Lincoln, NE), and grey scale analysis was performed with ImageJ software (1.50i; National Institutes of Health, Bethesda, MD).
Alizarin Red S Staining SCAP were seeded into 6-well plates (1 ! 105 cells/well) containing the conditional medium with LPS at different concentrations (0, 0.05, 0.5, and 5 mg/mL) and LPS plus 3-MA for 3 days. Then the culture medium was exchanged with osteo-/odontogenic inductive medium containing 1.8 mmol/L monopotassium phosphate (Sigma-Aldrich) and 10 nmol/L dexamethasone (SigmaAldrich). After 4 weeks, all specimens were fixed with 60% isopropanol for 1 minute at room temperature. Cells were stained with 1% alizarin red S dissolved in distilled water for 3 minutes and then washed with distilled water to remove the unbound alizarin red S. The stained cells were photographed by using an inverted phase-contrast microscope (Olympus IX41; Shinjuku-ku, Tokyo, Japan). ImageJ software was used for the semiquantitative analysis of mineralized nodule formation.
Transmission Electron Microscopy Untreated and LPS-treated (5 mg/mL LPS for 3 days) SCAP released from the culture dish by trypsinization were centrifuged for 10 minutes at 12,000 rpm and collected. Cells were washed with PBS twice, fixed in 2% glutaraldehyde, and dehydrated by using an alcohol gradient series before being embedded in Epon812 resin (SPI, West Chester, PA). Subsequently, tissues were cut into 1-mm sections and stained with uranyl acetate and lead citrate. Autophagosomes were observed and photographed by using transmission electron microscopy (TEM) (Hitachi 7650, Tokyo, Japan).
Statistical Analysis Each experiment was conducted in triplicate. All values are reported as the mean
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values 6 standard deviation. The quantitative data were tested for normal distribution. The data were analyzed by using one-way analysis of variance with SPSS 17.0 software (SPSS Inc, Chicago, IL). A post hoc multiple comparison test was conducted to compare the differences between each of the 2 groups. A P value less than .05 was considered statistically significant.
RESULTS LPS at High Dose Inhibits SCAP Cell Viability After continuous LPS exposure for 3 days, the cell morphology of SCAP was observed by microscope. As shown in Figure 1A, control cells exhibited a normal fibroblastic morphology typical of MSCs. Compared with the control group, the cell morphology of SCAP exhibited no significant changes after 0.05 mg/mL LPS exposure, whereas after stimulation with 0.5 mg/mL and 5 mg/mL LPS,
SCAP were morphologically characterized by increased cell debris, enlarged cell shape, and more dead cells. To further clarify the effect of LPS on SCAP cell viability, the CCK8 assay was conducted to measure the SCAP proliferation after the cells were exposed to LPS for different time periods (1, 2, and 3 days) and at different doses (0.05, 0.5, and 5 mg/mL). As shown in Figure 1B, compared with the control group, 0.05 mg/mL LPS had no effect on SCAP proliferation for days 1, 2, and 3, respectively. On days 1 and 2, 0.5 mg/mL LPS increased the SCAP proliferation rate. However, LPS at 5 mg/mL suppressed SCAP proliferation rate on day 3, whereas it had no effect on the proliferation rate on days 1 and 2.
High Dose of LPS Inhibits SCAP Osteo-/Odontogenic Differentiation The dentinogenesis potential of SCAP is critical to pulp-dentin tissue regeneration in the infectious environment of immature teeth, so
we evaluated the osteo-/odontogenic differentiation capacity of SCAP after continuous LPS exposure for 3 days. Alizarin red S staining showed that the number of mineralized nodules clearly decreased with the addition of 5 mg/mL LPS (Fig. 2A). DMP-1, Runx-2, and ALP protein expression in the 5 mg/mL LPS-challenged group decreased compared with that in the control group (Fig. 2B).
LPS Triggers SCAP Autophagy We further explored whether LPS induced autophagy in SCAP. On the basis of the LPS effects on SCAP cell viability and differentiation, we used 5 mg/mL LPS exposure in further experiments. As shown in Figure 3A, TEM confirmed the presence of autophagy, as evidenced by the large number of double- or multiple-layered autophagic vacuoles (arrows) containing cytoplasmic components in the LPSchallenged SCAP group compared with the
FIGURE 1 – Cell proliferation of SCAP treated with different concentrations of LPS. (A ) Morphology of SCAP after exposure to control medium and medium with different concentrations of LPS (0.05, 0.5, and 5 mg/mL). Scale bars 5 200 mm. (B ) Cell proliferation rate of SCAP after treatment by different doses of LPS. *P , .05. Error bars: mean 6 standard deviation.
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FIGURE 2 – Osteo-/odontogenic differentiation capacity of SCAP treated with different concentrations of LPS. (A ) Alizarin red S staining showed the capacity of mineralized nodule formation. (B ) DMP-1, Runx-2, and ALP protein expression in SCAP exposed to LPS detected by Western blot. *P , .05. Error bars: mean 6 standard deviation. Scale bar 5 50 mm.
control SCAP group. LC3 plays a key role in initiating autophagosome biogenesis, and during the process of autophagy, the ratio of LC3II/LC3I is a standard for autophagy level of cell. Western blot analysis demonstrated that exposure of SCAP to LPS resulted in
elevated LC3II/LC3I ratio, Atg5, and Beclin 1 protein levels compared with the control (Fig. 3B), further supporting that autophagy was involved in the downregulation of SCAP osteo-/odontogenic differentiation capacity after LPS stimulation.
Autophagy Suppression by 3-MA Reverses the Osteo-/Odontogenic Differentiation Potentials of SCAP Exposed to LPS To further research the role of autophagy in LPS-mediated alterations in SCAP
FIGURE 3 – LPS at 5 mg/mL induces autophagy in SCAP. (A ) TEM of SCAP after exposure to LPS; arrow indicates autophagosome. (B ) Western blot results shows the expression level of Beclin 1, Atg5, and the ratio of LC3II/LC3I in SCAP treated with LPS. **P , .01. Error bars: mean 6 standard deviation.
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FIGURE 4 – Autophagy is involved in the regulation of osteo-/odontogenic differentiation. (A ) Western blot analysis shows that expression of Beclin 1 and Atg5 is upregulated, and the ratio of LC3II/LC3I is increased after stimulation with LPS, and the autophagy inhibition after cells were treated with 3-MA. (B ) Increased capacity for mineralized nodule formation for SCAP treated with LPS plus 3-MA by alizarin red S staining. (C ) Protein expression of DMP-1, Runx-2, and ALP in 3-MA plus LPS group is increased by Western blot detection. *P , .05. Error bars: mean 6 standard deviation. Scale bar 5 50 mm. differentiation, the autophagic process was inhibited by using the autophagy inhibitor 3MA. Western blot analysis revealed that Beclin 1, Atg5 expression, and LC3II/LC3I ratio in the LPS plus 3-MA group were markedly decreased compared with those in the LPS group (Fig. 4A). Alizarin red S staining showed the clearly increased number of mineralized nodules in the LPS plus 3-MA group (Fig. 4B). Meanwhile, 3-MA partially upregulated the DMP-1, Runx-2, and ALP protein levels as shown by Western blot analysis, albeit not to the level of the control group, indicating that autophagy inhibition partially reversed the osteo-/odontogenesis differentiation of SCAP exposed to LPS (Fig. 4C).
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DISCUSSION Periapical periodontitis in immature permanent teeth is mainly caused by bacterial infection of dental pulp, which usually leads to damage to the apical papilla and Hertwig’s sheath, resulting in the cessation of dental root development. It has been reported that the released endotoxin and cytokines that passed through the root canal induced periapical bone destruction and inflammatory response from a distance rather than from the direct effects of bacteria on the periradicular tissues19. In this study, we used LPS, a major component of gram-negative bacteria, to elucidate the role of inflammatory response. Endotoxin contents
ranged from 17–228 EU/mL (equate to 0.17– 0.228 mg/mL) in asymptomatic teeth. Higher levels of endotoxin were found in teeth that had clinical symptomatology, ranging from 270– 696 EU/mL (equate to 0.27–0.696 mg/mL)20. However, sampling from the root canal might obtain only some parts of LPS molecules, and the exact LPS concentration in the root canal might be higher than what has been reported. Five mg/mL LPS has been reported to induce the biological responses of many MSCs including SCAP in vitro7,21. In the CCK-8 assay, this concentration significantly suppressed SCAP proliferation rate. Therefore, we chose this appropriate concentration for the mechanism research.
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Odontogenic differentiation potential of SCAP is essential for the development of dental root and tissue regeneration in immature permanent teeth. DMP-1 was the main marker for odontoblast differentiation. ALP is an early marker for both osteogenesis and odontogenesis, and Runx-2 is a master transcription factor and is also essential for the later stage of tooth formation. A favorable microenvironment is required for stem cells to proliferate and differentiate in the periapical area of young permanent teeth. Various tendencies focused on the regulation of SCAP properties in bacterial environment revealed that an appropriate concentration of LPS (0.1 mg/mL) could promote the proliferation and osteo-/odontogenic differentiation of SCAP22, whereas another study did not detect any biological change on cell proliferation, mineralization capacity, and DSPP gene expression of SCAP after pretreatment with lower doses of LPS (0.1 mg/mL and 1 mg/ mL)23. Apoptosis, autophagy, and ferroptosis have been reported to be involved in the regulation of MSCs properties in inflammatory environment24. Autophagy participates in the renewal, pluripotency, differentiation, proliferation, and aging of stem cells. As common markers of autophagy, LC3 plays a key role in initiating autophagosome biogenesis, whereas Beclin 1 governs the autophagic process by regulating the Ptdlns3KC3-dependent generation of phosphatidylinositol 3-phosphate and the
recruitment of additional Atg proteins that activate autophagosome formation. Atg5 is also critical for autophagosome formation12. During the process of autophagy, Atg5 often forms a complex with Atg12, which is necessary for the transformation of LC3I into LC3II. Our results showed that LPS treatment of SCAP activated autophagy, as indicated by the increased expression of autophagy-related proteins (Beclin 1, LC3II, and Atg5) and the accumulation of autophagosomes. Moreover, we inhibited autophagy in SCAP by chemical inhibitor (3-MA), showing that 3-MA treatment also partially rescued LPS-induced damage of SCAP differentiation. It has been demonstrated that autophagy plays a dual role on SCAP properties in an inflammation environment. These results were similar to the research that autophagy was shown to be involved in controlling the osteogenic differentiation of human DPSCs, odontoblasts, and MSCs25–28. Some researchers found that autophagy plays a protective role against the tumor necrosis factor a–induced apoptosis of bone marrow MSCs29. However, Wang et al30 reported that resveratrol could suppress the tumor necrosis factor a–induced inflammatory cytokines produced by DPSCs by regulating the inhibitory autophagy-JNK signaling cascade, suggesting that resveratrol might be beneficial to ameliorate pulpal damage in the acute phase of pulp inflammation. Together with our results, these findings support that autophagy may constitute a new and
promising target for improving the osteo-/ odontogenic differentiation of SCAP in an inflammatory environment. In turn, this induced differentiation is expected to inform potential strategies for dentin-pulp complex regeneration. However, the detailed mechanisms of autophagy in the regulation of SCAP in inflammatory environment in vivo are topics of further investigation.
CONCLUSION In summary, we showed that LPS treatment could inhibit the osteo-/ odontogenic differentiation of SCAP via autophagy. This study helps to explain the role of autophagy in the regulation of the osteo-/odontogenic differentiation of SCAP in an inflammatory environment and clarifies the damage and repair mechanisms of immature permanent teeth affected with periapical periodontitis.
ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grant no. 81771059), Liaoning Provincial Science and Technology Project (grant no. 2018225061), and Scientific Research Fund of Liaoning Provincial Education Department (grant no. LQNK201723). The authors deny any conflicts of interest related to this study.
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Akiyama K, Chen C, Gronthos S, Shi S. Lineage differentiation of mesenchymal stem cells from dental pulp, apical papilla, and periodontal ligament. Methods Mol Biol 2012;887:111–21.
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Tsukiboshi M, Ricucci D, Siqueira JF Jr. Mandibular premolars with immature roots and apical periodontitis lesions treated with pulpotomy: report of 3 cases. J Endod 2017;43:S65–74.
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Liu J, Du J, Chen X, et al. The effects of mitogen-activated protein kinase signaling pathways on lipopolysaccharide-mediated osteo/odontogenic differentiation of stem cells from the apical papilla. J Endod 2019;45:161–7.
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Lipopolysaccharide Downregulates the Osteo-/ Odontogenic Differentiation of Stem Cells From Apical Papilla by Inducing Autophagy
ABSTRACT Introduction: The dentinogenesis potential of stem cells during dentin-pulp tissue regeneration may be compromised by microorganism components. Here we aimed to investigate the cell viability and osteo-/odontogenic differentiation of stem cells from apical papilla (SCAP) exposed to bacterial lipopolysaccharide (LPS) and to evaluate the molecular mechanism in vitro. Methods: CCK8 assay was used to assess the SCAP proliferation rate on exposure to different concentrations of LPS in medium. Dentin matrix protein-1 (DMP-1), runt-related transcription factor-2 (Runx-2), and alkaline phosphatase (ALP) expression and mineralized nodule formation were detected by Western blotting and alizarin red S staining to evaluate SCAP osteo-/odontogenic differentiation. Autophagosomes in SCAP and the autophagyrelated markers Beclin 1, autophagy-related gene 5 (Atg5), and microtubule-associated proteins light chain 3 (LC3) were detected by transmission electron microscopy and Western blotting, respectively. Effects of the autophagy inhibitor 3-methyladenine on LPS-treated SCAP osteo-/odontogenic differentiation were also examined. Results: SCAP cell viability was decreased by 5 mg/mL LPS treatment on day 3. LPS (5 mg/mL) inhibited SCAP osteo-/ odontogenic differentiation and decreased DMP-1, Runx-2, and ALP expression. Moreover, LC3, Atg5, and Beclin 1 expression and autophagosome number were elevated. Conversely, autophagy inhibition rescued the number of mineralized nodules and DMP-1, Runx-2, and ALP expression in the LPS-treated SCAP. Conclusions: Our findings indicated that autophagy was involved in the suppression of SCAP osteo-/odontogenic differentiation in an LPS-induced inflammatory environment. This study discloses autophagy modulation as a potential new strategy to improve SCAP osteo-/odontogenic differentiation in an inflammatory environment. (J Endod 2020;-:1–7.)
SIGNIFICANCE This study disclosed the role of autophagy in regulating the biological effects of lipopolysaccharide on stem cells from apical papilla, which contributes to the damage and repair mechanisms of immature permanent teeth affected with periapical periodontitis.
KEY WORDS Autophagy; lipopolysaccharide; osteo-/odontogenic differentiation; proliferation; stem cells from apical papilla Pulp and periapical disease in immature permanent teeth comprises a group of inflammatory diseases caused by microorganisms (mainly bacteria) infecting the developing dental root1. Stem cells from apical papilla (SCAP), which reside at the root apex of incompletely developed teeth, have been identified as essential tissue in tooth root development and apexogenesis. SCAP are presumed to constitute a prospective stem cell source of the primary odontoblasts that are responsible for the formation of root dentin and regeneration of the pulp-dentin complex2. However, the dentinogenic capacity of SCAP may be impaired by the presence of microorganisms and their components. For example, it has been reported that the cell number and viability of SCAP were reduced during prolonged infection periods, although SCAP could survive in the infectious environment of immature teeth3. Bacteria involved in the pathogenesis of apical periodontitis may have participated in the early stages of pulp inflammation and necrosis. Bacteria virulence factors comprise structural-cellular components and released products that are involved in every step of the infectious process including
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From the Department of Pediatric Dentistry, School of Stomatology, China Medical University, Shenyang, China; and Liaoning Provincial Key Laboratory of Oral Diseases, Shenyang, China Address requests for reprints to Dr Xu Chen, Department of Pediatric Dentistry, School of Stomatology, China Medical University, 117 Nanjing North Street, Shenyang, 110002, China. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright © 2020 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2020.01.009
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attachment, invasion, survival, and damage4. Lipopolysaccharide (LPS), a major component of gram-negative bacteria, acts as an endotoxin that elicits a variety of immune responses in odontoblasts, fibroblasts, dental pulp stem cells (DPSCs), and SCAP5–7. Studies have reported that 1 mg/mL LPS protects mesenchymal stem cells (MSCs) against apoptosis and enhances their proliferation, whereas 5 mg/mL LPS increases MSCs apoptosis in vitro8,9. However, the exact mechanism by which LPS influences the biological behaviors of SCAP remains unclear. Dental stem cell differentiation and tooth development are influenced by macroautophagy (hereafter referred to as autophagy)10,11, a ubiquitous, evolutionarily conserved cellular process in which characteristic double- or multi-membrane autophagosomes form and include components of the cytoplasm and parts of intracellular organelles12. The outer membrane of the autophagosome then fuses with a lysosome or endosome, generating a singlemembrane autolysosome13. Accumulating evidence has shown that autophagy is a normal part of the cell life cycle and is involved in cell growth, differentiation, cell survival or death, melanosome degradation, and inflammatory response14,15. Generally, autophagy can be induced by starvation, chemical drugs, or inflammatory environment. It can be inhibited by 3-methyladenine (3-MA) through inhibiting class III phosphatidylinositol 3-kinase. In addition, chloroquine, which inhibits lysosomal enzymes and prevents the fusion of autophagosome and lysosome, can also inhibit autophagy in stem cells16. However, the potential role of autophagy in periapical periodontitis of immature permanent teeth has not yet been elucidated. To address these issues, in this study we aimed to explore the effect of LPS on the committed osteo-/ odontogenic differentiation of SCAP and whether this effect is modulated by autophagy.
MATERIALS AND METHODS SCAP Isolation and Culture SCAP were collected from intact, caries-free impacted third molars with immature roots from healthy patients (12–15 years of age) at the dental clinic of the School of Stomatology affiliated with China Medical University. This study was approved by the Ethics Committee of the School of Stomatology, China Medical University (201508). Both the patients and their guardians provided written informed consent for use of the collected cells. SCAP were freshly isolated and cultured as previously reported17. The apical papilla was gently separated from the tooth and then minced and digested with 4 mg/
2
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mL dispase II (Boehringer Ingelheim, Mannheim, Germany) and 2 mg/mL collagenase type I (Worthington Biochemical Co, Lakewood, CO) at 37 C. Single-cell suspensions were seeded and cultured in alpha minimum essential medium (a-MEM) (HyClone, Logan, UT) supplemented with 15% fetal bovine serum (Excell Bio, Shanghai, China), 100 U/mL penicillin-streptomycin (HyClone), and 0.1 mmol/L L-ascorbic acid (Sigma-Aldrich, St Louis, MO) in a humidified atmosphere at 37 C with 5% CO218. SCAP from passages 4–5 were used for all experiments in the present study.
Cell Proliferation Assay SCAP were seeded in 96-well plates (3000 cells/well) in serum-free culture medium containing 0, 0.05, 0.5, and 5 mg/mL Porphyromonas gingivalis LPS (InvivoGen, San Diego, CA). The cells were starved for 12 hours before the stimulus was added. Medium without LPS served as the control. The experiment was conducted according to the manufacturer’s instructions. Briefly, after incubation for 1, 2, and 3 days, 10 mL CCK8 solution (Dojindo, Kumamoto, Japan) with 90 mL a-MEM was added to each well, and the plates were incubated for another 2 hours in the dark. The optical density was detected by using a microplate reader (Tecan, Salzburg, Austria) at a wavelength of 450 nm.
Western Blot Analysis SCAP were treated with different concentrations of LPS (0.05, 0.5, and 5 mg/ mL) or LPS plus 5 mmol/L of the autophagic inhibitor 3-MA (Selleckchem, Shanghai, China). 3-MA was diluted in phosphate buffered saline (PBS) and used to pretreat cells for 24 hours before application of LPS to inhibit autophagy. Cells were washed 3 times with ice-cold PBS and incubated with RIPA lysis buffer (Beyotime Biotech Co, Shanghai, China) containing 1 mmol/L phenylmethanesulfonyl fluoride on ice for 30 minutes. Protein concentrations were measured by using a BCA protein assay kit (Beyotime Biotech Co) according to the manufacturer’s instructions. After the samples were boiled with sample loading buffer (Beyotime), equal amounts of protein extracts (20 mg protein per sample) were subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA). The membranes were blocked with 4% bovine serum albumin for 1 hour at room temperature and then incubated with an anti– dentin matrix protein 1 (DMP-1) antibody (1:1000; Abcam, Cambridge, UK), anti–runtrelated transcription factor-2 (Runx-2) antibody
(1:250; Proteintech, Rosemont, IL), anti– alkaline phosphatase (ALP) antibody (1:200; Santa Cruz Biotechnology, Dallas, TX), anti– microtubule-associated proteins light chain 3 (LC3) antibody (1:1000; Abcam), anti-Beclin 1 antibody (1:1000; Abcam), anti–autophagy related gene 5 (Atg5) antibody (1:1000; Abcam), and antibody against glyceraldehyde3-phosphate dehydrogenase (GAPDH) (1:1000; Proteintech) at 4 C overnight. The membranes were washed with Tris-buffered saline Tween-20 and exposed to goat antirabbit/anti-mouse immunoglobulin G IRDyel 800cw secondary antibody (1:1000; Abbkine, Inc, Redlands, CA) at room temperature for 1 hour. The protein bands were detected by using an Odyssey CLx instrument (LI-COR, Lincoln, NE), and grey scale analysis was performed with ImageJ software (1.50i; National Institutes of Health, Bethesda, MD).
Alizarin Red S Staining SCAP were seeded into 6-well plates (1 ! 105 cells/well) containing the conditional medium with LPS at different concentrations (0, 0.05, 0.5, and 5 mg/mL) and LPS plus 3-MA for 3 days. Then the culture medium was exchanged with osteo-/odontogenic inductive medium containing 1.8 mmol/L monopotassium phosphate (Sigma-Aldrich) and 10 nmol/L dexamethasone (SigmaAldrich). After 4 weeks, all specimens were fixed with 60% isopropanol for 1 minute at room temperature. Cells were stained with 1% alizarin red S dissolved in distilled water for 3 minutes and then washed with distilled water to remove the unbound alizarin red S. The stained cells were photographed by using an inverted phase-contrast microscope (Olympus IX41; Shinjuku-ku, Tokyo, Japan). ImageJ software was used for the semiquantitative analysis of mineralized nodule formation.
Transmission Electron Microscopy Untreated and LPS-treated (5 mg/mL LPS for 3 days) SCAP released from the culture dish by trypsinization were centrifuged for 10 minutes at 12,000 rpm and collected. Cells were washed with PBS twice, fixed in 2% glutaraldehyde, and dehydrated by using an alcohol gradient series before being embedded in Epon812 resin (SPI, West Chester, PA). Subsequently, tissues were cut into 1-mm sections and stained with uranyl acetate and lead citrate. Autophagosomes were observed and photographed by using transmission electron microscopy (TEM) (Hitachi 7650, Tokyo, Japan).
Statistical Analysis Each experiment was conducted in triplicate. All values are reported as the mean
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values 6 standard deviation. The quantitative data were tested for normal distribution. The data were analyzed by using one-way analysis of variance with SPSS 17.0 software (SPSS Inc, Chicago, IL). A post hoc multiple comparison test was conducted to compare the differences between each of the 2 groups. A P value less than .05 was considered statistically significant.
RESULTS LPS at High Dose Inhibits SCAP Cell Viability After continuous LPS exposure for 3 days, the cell morphology of SCAP was observed by microscope. As shown in Figure 1A, control cells exhibited a normal fibroblastic morphology typical of MSCs. Compared with the control group, the cell morphology of SCAP exhibited no significant changes after 0.05 mg/mL LPS exposure, whereas after stimulation with 0.5 mg/mL and 5 mg/mL LPS,
SCAP were morphologically characterized by increased cell debris, enlarged cell shape, and more dead cells. To further clarify the effect of LPS on SCAP cell viability, the CCK8 assay was conducted to measure the SCAP proliferation after the cells were exposed to LPS for different time periods (1, 2, and 3 days) and at different doses (0.05, 0.5, and 5 mg/mL). As shown in Figure 1B, compared with the control group, 0.05 mg/mL LPS had no effect on SCAP proliferation for days 1, 2, and 3, respectively. On days 1 and 2, 0.5 mg/mL LPS increased the SCAP proliferation rate. However, LPS at 5 mg/mL suppressed SCAP proliferation rate on day 3, whereas it had no effect on the proliferation rate on days 1 and 2.
High Dose of LPS Inhibits SCAP Osteo-/Odontogenic Differentiation The dentinogenesis potential of SCAP is critical to pulp-dentin tissue regeneration in the infectious environment of immature teeth, so
we evaluated the osteo-/odontogenic differentiation capacity of SCAP after continuous LPS exposure for 3 days. Alizarin red S staining showed that the number of mineralized nodules clearly decreased with the addition of 5 mg/mL LPS (Fig. 2A). DMP-1, Runx-2, and ALP protein expression in the 5 mg/mL LPS-challenged group decreased compared with that in the control group (Fig. 2B).
LPS Triggers SCAP Autophagy We further explored whether LPS induced autophagy in SCAP. On the basis of the LPS effects on SCAP cell viability and differentiation, we used 5 mg/mL LPS exposure in further experiments. As shown in Figure 3A, TEM confirmed the presence of autophagy, as evidenced by the large number of double- or multiple-layered autophagic vacuoles (arrows) containing cytoplasmic components in the LPSchallenged SCAP group compared with the
FIGURE 1 – Cell proliferation of SCAP treated with different concentrations of LPS. (A ) Morphology of SCAP after exposure to control medium and medium with different concentrations of LPS (0.05, 0.5, and 5 mg/mL). Scale bars 5 200 mm. (B ) Cell proliferation rate of SCAP after treatment by different doses of LPS. *P , .05. Error bars: mean 6 standard deviation.
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FIGURE 2 – Osteo-/odontogenic differentiation capacity of SCAP treated with different concentrations of LPS. (A ) Alizarin red S staining showed the capacity of mineralized nodule formation. (B ) DMP-1, Runx-2, and ALP protein expression in SCAP exposed to LPS detected by Western blot. *P , .05. Error bars: mean 6 standard deviation. Scale bar 5 50 mm.
control SCAP group. LC3 plays a key role in initiating autophagosome biogenesis, and during the process of autophagy, the ratio of LC3II/LC3I is a standard for autophagy level of cell. Western blot analysis demonstrated that exposure of SCAP to LPS resulted in
elevated LC3II/LC3I ratio, Atg5, and Beclin 1 protein levels compared with the control (Fig. 3B), further supporting that autophagy was involved in the downregulation of SCAP osteo-/odontogenic differentiation capacity after LPS stimulation.
Autophagy Suppression by 3-MA Reverses the Osteo-/Odontogenic Differentiation Potentials of SCAP Exposed to LPS To further research the role of autophagy in LPS-mediated alterations in SCAP
FIGURE 3 – LPS at 5 mg/mL induces autophagy in SCAP. (A ) TEM of SCAP after exposure to LPS; arrow indicates autophagosome. (B ) Western blot results shows the expression level of Beclin 1, Atg5, and the ratio of LC3II/LC3I in SCAP treated with LPS. **P , .01. Error bars: mean 6 standard deviation.
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FIGURE 4 – Autophagy is involved in the regulation of osteo-/odontogenic differentiation. (A ) Western blot analysis shows that expression of Beclin 1 and Atg5 is upregulated, and the ratio of LC3II/LC3I is increased after stimulation with LPS, and the autophagy inhibition after cells were treated with 3-MA. (B ) Increased capacity for mineralized nodule formation for SCAP treated with LPS plus 3-MA by alizarin red S staining. (C ) Protein expression of DMP-1, Runx-2, and ALP in 3-MA plus LPS group is increased by Western blot detection. *P , .05. Error bars: mean 6 standard deviation. Scale bar 5 50 mm. differentiation, the autophagic process was inhibited by using the autophagy inhibitor 3MA. Western blot analysis revealed that Beclin 1, Atg5 expression, and LC3II/LC3I ratio in the LPS plus 3-MA group were markedly decreased compared with those in the LPS group (Fig. 4A). Alizarin red S staining showed the clearly increased number of mineralized nodules in the LPS plus 3-MA group (Fig. 4B). Meanwhile, 3-MA partially upregulated the DMP-1, Runx-2, and ALP protein levels as shown by Western blot analysis, albeit not to the level of the control group, indicating that autophagy inhibition partially reversed the osteo-/odontogenesis differentiation of SCAP exposed to LPS (Fig. 4C).
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DISCUSSION Periapical periodontitis in immature permanent teeth is mainly caused by bacterial infection of dental pulp, which usually leads to damage to the apical papilla and Hertwig’s sheath, resulting in the cessation of dental root development. It has been reported that the released endotoxin and cytokines that passed through the root canal induced periapical bone destruction and inflammatory response from a distance rather than from the direct effects of bacteria on the periradicular tissues19. In this study, we used LPS, a major component of gram-negative bacteria, to elucidate the role of inflammatory response. Endotoxin contents
ranged from 17–228 EU/mL (equate to 0.17– 0.228 mg/mL) in asymptomatic teeth. Higher levels of endotoxin were found in teeth that had clinical symptomatology, ranging from 270– 696 EU/mL (equate to 0.27–0.696 mg/mL)20. However, sampling from the root canal might obtain only some parts of LPS molecules, and the exact LPS concentration in the root canal might be higher than what has been reported. Five mg/mL LPS has been reported to induce the biological responses of many MSCs including SCAP in vitro7,21. In the CCK-8 assay, this concentration significantly suppressed SCAP proliferation rate. Therefore, we chose this appropriate concentration for the mechanism research.
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Odontogenic differentiation potential of SCAP is essential for the development of dental root and tissue regeneration in immature permanent teeth. DMP-1 was the main marker for odontoblast differentiation. ALP is an early marker for both osteogenesis and odontogenesis, and Runx-2 is a master transcription factor and is also essential for the later stage of tooth formation. A favorable microenvironment is required for stem cells to proliferate and differentiate in the periapical area of young permanent teeth. Various tendencies focused on the regulation of SCAP properties in bacterial environment revealed that an appropriate concentration of LPS (0.1 mg/mL) could promote the proliferation and osteo-/odontogenic differentiation of SCAP22, whereas another study did not detect any biological change on cell proliferation, mineralization capacity, and DSPP gene expression of SCAP after pretreatment with lower doses of LPS (0.1 mg/mL and 1 mg/ mL)23. Apoptosis, autophagy, and ferroptosis have been reported to be involved in the regulation of MSCs properties in inflammatory environment24. Autophagy participates in the renewal, pluripotency, differentiation, proliferation, and aging of stem cells. As common markers of autophagy, LC3 plays a key role in initiating autophagosome biogenesis, whereas Beclin 1 governs the autophagic process by regulating the Ptdlns3KC3-dependent generation of phosphatidylinositol 3-phosphate and the
recruitment of additional Atg proteins that activate autophagosome formation. Atg5 is also critical for autophagosome formation12. During the process of autophagy, Atg5 often forms a complex with Atg12, which is necessary for the transformation of LC3I into LC3II. Our results showed that LPS treatment of SCAP activated autophagy, as indicated by the increased expression of autophagy-related proteins (Beclin 1, LC3II, and Atg5) and the accumulation of autophagosomes. Moreover, we inhibited autophagy in SCAP by chemical inhibitor (3-MA), showing that 3-MA treatment also partially rescued LPS-induced damage of SCAP differentiation. It has been demonstrated that autophagy plays a dual role on SCAP properties in an inflammation environment. These results were similar to the research that autophagy was shown to be involved in controlling the osteogenic differentiation of human DPSCs, odontoblasts, and MSCs25–28. Some researchers found that autophagy plays a protective role against the tumor necrosis factor a–induced apoptosis of bone marrow MSCs29. However, Wang et al30 reported that resveratrol could suppress the tumor necrosis factor a–induced inflammatory cytokines produced by DPSCs by regulating the inhibitory autophagy-JNK signaling cascade, suggesting that resveratrol might be beneficial to ameliorate pulpal damage in the acute phase of pulp inflammation. Together with our results, these findings support that autophagy may constitute a new and
promising target for improving the osteo-/ odontogenic differentiation of SCAP in an inflammatory environment. In turn, this induced differentiation is expected to inform potential strategies for dentin-pulp complex regeneration. However, the detailed mechanisms of autophagy in the regulation of SCAP in inflammatory environment in vivo are topics of further investigation.
CONCLUSION In summary, we showed that LPS treatment could inhibit the osteo-/ odontogenic differentiation of SCAP via autophagy. This study helps to explain the role of autophagy in the regulation of the osteo-/odontogenic differentiation of SCAP in an inflammatory environment and clarifies the damage and repair mechanisms of immature permanent teeth affected with periapical periodontitis.
ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grant no. 81771059), Liaoning Provincial Science and Technology Project (grant no. 2018225061), and Scientific Research Fund of Liaoning Provincial Education Department (grant no. LQNK201723). The authors deny any conflicts of interest related to this study.
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