HEMA Effects on Autophagy Mechanism in Human Dental Pulp Stem Cells

Autophagy is a complex mechanism that permits the degradation of cellular components in order to enhance cell homeostasis, recycling the damaged, dysfunctional, or unnecessary components. In restorative dentistry practice, free resin monomers of 2-hydroxyethyl methacrylate (HEMA) can be released. The aim of this study was to investigate the effect of HEMA on proliferation and autophagy in human dental pulp stem cells (hDPSCs). Human DPSCs were treated with different concentrations of HEMA (3 and 5 mmol L−1). To evaluate the proliferation rate, MTT and trypan blue assays were used. Autophagic markers such as microtubule-associated protein 1 light chain 3 (LC3-I/II) and ubiquitin-binding protein (p62) were analyzed through immunofluorescence observations. Beclin1, LC3-I/II, and p62 were evaluated by means of Western blotting detection. Considering that activity of extracellular signal–regulated kinase (ERK) and its phosphorylated form (pERK) mediates several cellular processes, such as apoptosis, autophagy, and senescence, the involvement of ERK/pERK signaling was also evaluated. Obtained results showed a decreased cell proliferation associated with morphological changes in HEMA-treated cells. The Western blot results showed that the expression levels of Beclin1, LC3-I/II, and ERK were significantly elevated in HEMA-treated cells and in cells co-treated with rapamycin, an autophagic promoter. The expression levels of p62 were significantly reduced compared to the untreated samples. Protein levels to the autophagic process, observed at confocal microscopy confirmed the data obtained from the Western blot. The up-regulation of ERK and pERK levels, associated with nuclear translocation, revealed that ERK pathway signaling could act as a promoter of autophagy in dental pulp stem cells treated with HEMA.


Cell Culture
The Ethics Committee of the University of Chieti approved the research study (n • 266/University of Chieti). To obtain hDPSCs, dental pulp was collected by a procedure previously reported [28]. Dental pulp was collected from human teeth scheduled to be removed for orthodontic therapy. Explants were cultured in petri dishes with mesenchymal stem cells growth medium-chemically defined (MSCGM-CD) (Lonza, Basel, Switzerland) [29]. Three times a week, the medium was replaced with fresh medium. After two weeks in culture, cells spontaneously migrated from explants. All experiments were performed with cells at second passage.
To evaluate their capacity to adhere to the plastic substrate, cells were fixed and the toluidine blue solution was then used to stain hDPSCs in order to evaluate cell morphology using inverted light microscopy [31].
For osteogenic differentiation, the cells were cultured in osteogenic differentiation medium kit (Lonza). The cells were stained with Alizarin red S solution after 21 days of induction [32]. Adipogenic differentiation was induced by culturing the hDPSCs in adipogenic medium kit (Lonza) for 28 days, and the cells were assessed using Adipo Oil red O staining solution [33].

Cell Proliferation and Viability Assay
Cell proliferation was evaluated through MTT assay, as previously reported [36]. A total 2 × 10 3 cells per well were seeded into 96 well plates in 200 µL of medium to test all considered conditions: hDPSCs, hDPSCs treated with HEMA 3 mmol L −1 , and hDPSCs treated with HEMA 5 mmol L −1 for 24 h. At different endpoints-24, 48, and 72 h-20 µL of MTT (Promega, Milan, Italy) solution was added to each well. Absorbance at 490 nm was measured with a reference wavelength of 630 nm [37].
Cell viability was assessed by trypan blue exclusion test. For this purpose, all samples were incubated with trypan blue solution at the same endpoints used for the MTT test (24,48, and 72 h) and subsequently analyzed by light microscopy, using a Burker's chamber as previously described [38].

Immunohistochemistry and Confocal Laser Scanning Microscope (CLSM) Analysis
For immunofluorescence detection, cells grown on eight well chamber slides were fixed using 4% paraformaldehyde, diluted in 0.1M sodium phosphate buffer (PBS, Lonza) [39]. After the fixation step, cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min, followed by blocking with 5% skimmed milk in PBS for 30 min [40]. Primary antibodies used for immunofluorescence were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA). P62  [41]. Samples were observed using a Zeiss LSM800 confocal system (Zeiss, Jena, Germany). The relative fluorescence intensities of p62, ERK, pERK, and LC3 were quantified using NIS-Elements AR imaging software (Nikon). For the counting statistics of immunofluorescence-positive nuclei for ERK and pERK, ten views (100×) were randomly chosen in each experimental group and analyzed using NIS-Elements AR imaging software (Nikon). All the experiments were repeated at least three times. Data are presented as the mean and standard error of the mean (mean ± SEM). The comparison analysis of different groups was done using a one-way analysis of variance followed by a post hoc Bonferroni evaluation using GraphPad Prism5. Differences were termed statistically significant at p < 0.05.

Statistical Analysis
Graph Pad Prism 6.0 (GraphPad Software, La Jolla, CA, USA) was used to perform the statistical evaluation. Student's t-test was used to assess the differences between the groups. Obtained results are reported as mean ± SEM. A p-value < 0.05 was considered statistically significant.

Characterization of hDPSCs
To characterize the hDPSC profiles, cytofluorimetric analysis and mesengenic differentiation ability were evaluated after cell isolation. The phenotype profile of the hDPSCs was determined by cytofluorimetric analysis. Human DPSCs showed a positivity for the following markers: Sox-2, Oct3/4, CD13, CD29, CD73, CD90, and CD105, while they were negative for C14, CD34, and CD45 ( Figure 2A).
Plastic-adherent cells showed a fibroblastic morphology with long cytoplasmic processes and an evident nucleus ( Figure 2B).
The capacity of hDPSCs to differentiate into osteoblasts and adipoblasts has been demonstrated through in vitro experiments. Alizarin Red S solution staining has been used to assess osteogenic ability; for this purpose, cells were stained within 21 days of the induction period. Calcium deposits were detected in red by inverted light microscopy ( Figure 2C). Adipo Oil red O staining was used to evaluate the hDPSCs' capacity to differentiate towards an adipogenic phenotype. Cells were maintained under adipogenic conditions, and after 28 days of culture, lipid vacuoles at the cytoplasmic level were evident by inverted light microscopy observation ( Figure 2D). Gene expression was assessed to confirm the qualitative data obtained at light microscopy. RT-PCR was carried out on undifferentiated and differentiated samples. Differentiated hDPSCs showed an up-regulation in the mRNA expression of RUNX2 and ALP in cells maintained under osteogenic conditions when compared to the undifferentiated cells ( Figure 2E). Human DPSCs cultured under adipogenic conditions showed an up-regulation in the expression of FABP4 and PPAR ( Figure 2F).

HEMA Effects on Cell Proliferation Rate
To evaluate cell proliferation and viability, MTT and Trypan blue assays were performed on all considered samples. Cells treated with HEMA 3 mmol L −1 and HEMA 5 mmol L −1 showed a lower proliferation rate from 24 to 72 h of culture when compared to the untreated cells ( Figure 3A). The same trend has been demonstrated in the Trypan blue line graph ( Figure 3B). undifferentiated and differentiated hDPSCs. (F) RT-PCR of Fatty Acid-Binding Protein 4 (FABP4) and Peroxisome Proliferator-Activated Receptor Gamma (PPARɣ) performed in undifferentiated and differentiated hDPSCs. Scale bar: 10 μm. ** p < 0.01.

HEMA Effects on Cell Proliferation Rate
To evaluate cell proliferation and viability, MTT and Trypan blue assays were performed on all considered samples. Cells treated with HEMA 3 mmol L −1 and HEMA 5 mmol L −1 showed a lower proliferation rate from 24 to 72 h of culture when compared to the untreated cells ( Figure 3A). The same trend has been demonstrated in the Trypan blue line graph ( Figure 3B).

Autophagic Marker Expression
Immunofluorescence staining was performed to evaluate the expression of markers related to the autophagic mechanism. P62 showed a decreased expression in hDPSCs treated with HEMA 3 mmol L −1 and HEMA 5 mmol L −1 compared to the untreated cells, observed by confocal microscopy ( Figure 4A1-C4). LC3 and ERK showed an opposite regulation, untreated cells showed a negative expression of LC3 and a light positivity for ERK. As shown in Figure 5C1,C4, hDPSCs were positive to ERK, demonstrating a nuclear translocation after HEMA 5 mmol L −1 treatment. The phosphorylated form of ERK showed the same trend of ERK expression, as demonstrated by the percentage of nuclear localization of pERK in HEMA 5 mmol L −1 treated cells when compared to

Autophagic Marker Expression
Immunofluorescence staining was performed to evaluate the expression of markers related to the autophagic mechanism. P62 showed a decreased expression in hDPSCs treated with HEMA 3 mmol L −1 and HEMA 5 mmol L −1 compared to the untreated cells, observed by confocal microscopy ( Figure 4A1-C4). LC3 and ERK showed an opposite regulation, untreated cells showed a negative expression of LC3 and a light positivity for ERK. As shown in Figure 5C1,C4, hDPSCs were positive to ERK, demonstrating a nuclear translocation after HEMA 5 mmol L −1 treatment. The phosphorylated form of ERK showed the same trend of ERK expression, as demonstrated by the percentage of nuclear localization of pERK in HEMA 5 mmol L −1 treated cells when compared to untreated ( Figure 6E). In hDPSCs treated with HEMA 3 mmol L −1 and HEMA 5 mmol L −1 , LC3 showed a high expression localized at the cytoplasmic level ( Figure 7A1-C4). untreated ( Figure 6E). In hDPSCs treated with HEMA 3 mmol L −1 and HEMA 5 mmol L −1 , LC3 showed a high expression localized at the cytoplasmic level ( Figure 7A1-C4).

Autophagic Marker Levels
Beclin-1 and P62 are classical autophagic markers. To evaluate their expression, Western blot analyses were performed. The protein levels of p62 decreased in HEMA 3 and 5 mmol L −1 treated

Autophagic Marker Levels
Beclin-1 and P62 are classical autophagic markers. To evaluate their expression, Western blot analyses were performed. The protein levels of p62 decreased in HEMA 3 and 5 mmol L −1 treated samples, while Beclin1-specific protein bands showed an up-regulation in treated cells ( Figure 8A). In particular, the results were enhanced in samples treated with rapamycin, an autophagic inductor ( Figure 8B).The expression of ERK, pERK, and LC3-I/II was increased in HEMA 3 and 5 mmol L −1 treated cells when compared to the control (p < 0.05). All experiments were repeated in triplicate. ( Figure 8C).

Discussion
Methacrylate-based restorative materials are common materials used in clinical practice to restore tooth damage, function, and at the same time ensure the desired aesthetic component. These materials are commercialized in a viscous form to be better manipulated during clinical practice, and they are then subjected to a polymerization process to be converted into their solid form [44]. Frequently, an incomplete polymerization can induce the release of monomers into the oral cavity, which move towards dentin micro-channels to enter the vascular system, inducing an inflammatory response, causing an alteration to the odontoblastic function and subsequent pulp tissue damage.
HEMA has been reported to alter the homeostasis of various cell lines in vitro, inducing DNA damage, apoptosis, and necrosis and autophagy [12].
Autophagy is an intracellular catabolic process that preserves cell homeostasis, involving the degradation of damaged organelles and/or toxic macromolecules. Autophagy machinery plays a direct or indirect role in health and disease by way of lysosomal activity. Mitophagy could be considered a selective autophagy mechanism that induces the degradation of mitochondria in response to damage or stress. Damaged mitochondria produce high levels of reactive oxygen species (ROS) that trigger the mitophagy process [26]. Autophagy has a dual role in tumor cells, acting as a tumor suppressor and, at the same time, enhancing tumor cell growth. Autophagy also plays a key role in the suppression or not of inflammatory processes. Meanwhile, inflammatory processes can induce or block the autophagy pathway.
The main goal of this work was to demonstrate the involvement of autophagy in response to low concentrations 3 and 5 mmol L −1 of HEMA treatment in hDPSCs as an adaptive machinery to ensure cell homeostasis. For this purpose, the capacity of LC3, p62, and ERK signaling to counteract on the control of autophagy induced by HEMA treatment in stem cells from dental pulp was evaluated. Our findings provided evidence that hDPSCs express stemness markers [30], and are competent to differentiate into osteoblast and adipoblast lineages [27].
Colorimetric detection to assess cell metabolic activity (MTT assay) and the Trypan blue exclusion test to determine the number of viable cells provided evidence that the exposure to 3 and 5 mmol L −1 of HEMA induced lower growth relative to untreated cells, from 24 h to 72 h of treatment. The reduction of cell proliferation and the change in classical cell morphology can thus be directly associated with the HEMA treatment [5]. This research paper focused on metabolic changes that occur in the first 24 h of treatment. In particular, the expression at the confocal microscopy level and Western blotting analyses of autophagic markers such as LC3 and p62, in addition to the molecular signaling pathway ERK, were evaluated.
These types of intracellular signal transduction cascades are activated through phosphorylation that induces nuclear translocation [20]. The role of MEK/ERK in autophagy activation has been reported in the literature. MEK/ERK signaling mediates autophagy, involving several mechanisms, including amino acid deprivation [22], aurintricarboxylic acid [23], B-group soyasaponins [24], and curcumin [25]. Beclin-1 has been shown to be regulated by MEK/ERK signaling to stimulate the autophagy process [16]. In this study, we have provided evidence that after 24 h of 3 and 5 mmol L −1 HEMA treatment, an up-regulation of LC3 in parallel with a p62 down-regulation was detected. Moreover, a significant increase of ERK protein level was demonstrated, together with its nuclear translocation in treated samples, demonstrating that HEMA treatment induced an autophagic process through the positive modulation of ERK signaling. Beclin-1 is the first mammalian gene found to mediate autophagy, such as regulating the turnover of proteins controlling the growth and proliferation of cells [45]. Our results demonstrated a decreased level of p62 in HEMA treated cells and in HEMA + rapamycin treated samples and at the same time we highlighted a overexpression of Beclin1 in HEMA treated cells, more pronounced in samples treated with HEMA + rapamycin treated hDPSCs showed a decrease of p62 and an overexpression of Beclin1 when compared to untreated hDPSCs. ERK, pERK, and LC3-II showed an upregulation in 3 and 5 mmol L −1 HEMA when compared to control cells, while LC3-I showed an opposite regulation.
Rapamycin is a special prophylactic for the mammalian target of rapamycin (mTOR), which binds fk506-binding protein 12 kDa (FKBP12) to form a molecular complex that inhibits mTOR activity [46], and is considered an autophagy promoter. We also evaluated the influence of rapamycin on the expression of classical autophagic markers in treated and untreated hDPSCs. In this study, Western blot analysis indicated that rapamycin increased Beclin1 levels, but decreased p62 levels. In response to HEMA injury, dental pulp stem cells activate autophagy as a pro-survival cytoprotective mechanism. Further studies are necessary to consider the strategic and therapeutic applications of this research in tissue repair and regeneration.

Conflicts of Interest:
The authors declare no conflict of interest.