Chronic exposure to lipopolysaccharides as an in vitro model to simulate the impaired odontogenic potential of dental pulp cells under pulpitis conditions

Abstract Simulating a bacterial-induced pulpitis environment in vitro may contribute to exploring mechanisms and bioactive molecules to counteract these adverse effects. Objective To investigate the chronic exposure of human dental pulp cells (HDPCs) to lipopolysaccharides (LPS) aiming to establish a cell culture protocol to simulate the impaired odontogenic potential under pulpitis conditions. Methodology HDPCs were isolated from four healthy molars of different donors and seeded in culture plates in a growth medium. After 24 h, the medium was changed to an odontogenic differentiation medium (DM) supplemented or not with E. coli LPS (0 - control, 0.1, 1, or 10 µg/mL) (n=8). The medium was renewed every two days for up to seven days, then replaced with LPS-free DM for up to 21 days. The activation of NF-κB and F-actin expression were assessed (immunofluorescence) after one and seven days. On day 7, cells were evaluated for both the gene expression (RT-qPCR) of odontogenic markers (COL1A1, ALPL, DSPP, and DMP1) and cytokines (TNF, IL1B, IL8, and IL6) and the production of reactive nitrogen (Griess) and oxygen species (Carboxy-H2DCFDA). Cell viability (alamarBlue) was evaluated weekly, and mineralization was assessed (Alizarin Red) at 14 and 21 days. Data were analyzed with ANOVA and post-hoc tests (α=5%). Results After one and seven days of exposure to LPS, NF-κB was activated in a dose-dependent fashion. LPS at 1 and 10 µg/mL concentrations down-regulated the gene expression of odontogenic markers and up-regulated cytokines. LPS at 10 µg/mL increased both the production of reactive nitrogen and oxygen species. LPS decreased cell viability seven days after the end of exposure. LPS at 1 and 10 µg/mL decreased hDPCs mineralization in a dose-dependent fashion. Conclusion The exposure to 10 µg/mL LPS for seven days creates an inflammatory environment that is able to impair by more than half the odontogenic potential of HDPCs in vitro, simulating a pulpitis-like condition.


Introduction
Mesenchymal stem cells in dental pulp play a vital role in the defense and regenerative potential of the dentin-pulp complex against lesions by producing reparative dentinogenesis. 1,2 However, the ability of dental pulp to recover from persistent infectious and inflammatory conditions is challenging. Clinically, inflamed pulp tissues are mostly characterized by spontaneous or long-lasting pain, and self-healing is not expected. Considering the anatomical restriction and limited circulation of the dental pulp, the inflammation process becomes self-destructive and irreversible. 2,3 Histologically, teeth with irreversible pulpitis often present anaerobic gram-negative bacteria, which was not observed in normal/reversibly-inflamed pulps. 3 Gram-negative bacteria outer membrane contains endotoxins known as lipopolysaccharides (LPS). 4 LPS present potent biological effects, being able to stimulate pulp cells to produce reactive nitrogen/ oxygen species, 5-8 proteolytic enzymes (e.g., matrix metalloproteinases, especially MMP-9), 9-10 and inflammatory cytokines (e.g., tumor necrosis factors -TNFs, and interleukins, especially IL-8, IL-6, and IL-1). 4,[10][11][12][13][14] The immunomodulatory effects of LPS on pulp cells are mainly mediated by cell receptors that activate the nuclear factor kappa B (NF-κB) signaling pathway. 4,8,14 LPS are found in carious lesions of symptomatic and asymptomatic teeth at increasing numbers as the lesions get deeper and more painful. [15][16][17] Therefore, the presence of LPS has been directly associated with pulpitis symptoms and its concentration seems to be positively correlated to the irreversibility of an inflammatory environment in dental pulp. [15][16][17][18] Clinically, relatively low doses of stimuli in the early or resolving stages of bacterial invasion and caries development stimulate regenerative responses, inducing reactionary dentinogenesis. 2, 19 However, intense bacterial stimuli in active and chronic caries potentially impair regenerative processes. 19 Therefore, many studies have investigated in vitro different LPSchallenged dental pulp cell populations, including dental pulp fibroblasts, odontoblasts, and dental pulp stem cells (DPSCs). [4][5][6][7][8][9][10][20][21][22] Despite several studies on LPS effects in different pulp cells, 4 there is not an established protocol to properly simulate the behavior of dental pulp cells under a bacterialmediated degenerative pulpitis environment in vitro, i.e., able to impair biomineralization induced by pulp cells metabolism. There is a substantial divergence in the relationships between concentration and time of exposure to LPS with the inflammatory response and odontogenic potential of pulp cells in vitro. [4][5][6][7][8][9][10][20][21][22] Therefore, depending on cell culture design, the regenerative potential of mesenchymal pulp cells may be stimulated or impaired, impacting the gene expression and protein translation of odontogenic markers, including type I collagen (partially encoded by COL1A1), alkaline phosphatase (encoded by ALPL), dentin sialophosphoprotein (encoded by DSPP), and dentin matrix acid phosphoprotein 1 (encoded by DMP1); all these events directly affect the ability of pulp cells to produce a mineralized matrix. 4,10,13,[20][21][22] Simulating an LPS-induced pulpitis environment in vitro may contribute exploring the mechanisms involved in pulp cell regeneration and screening bioactive molecules to counter these adverse effects. 23 Thus, this study aimed to establish a cell culture protocol to simulate the impaired odontogenic potential of human dental pulp cells (HDPCs) under pulpitis conditions. The null hypotheses tested were that 1)  After 10 min of incubation in the dark, the absorbance of the resulting reaction was determined at 540 nm (Synergy H1). 5,6 A cell-free culture medium mixed with Griess reagent (1:1) was used as a blank and the data were presented in fold increase of control (LPS-free medium).

Reactive oxygen species production (n=8)
Furthermore, 3 h after the last exposure to LPS After 24 h of cell seeding (Day 0), the culture media were supplemented or not with LPS (0 -control, 0.1, 1, and 10 µg/mL) for seven days. After this time interval, cell viability was evaluated, and cells were cultured in an odontogenic differentiation medium without LPS for further 21 days, when cell viability and mineralized matrix formation were evaluated. B) Schematic representation of the second experimental design. After 24 h of cell seeding (Day 0), the culture medium was supplemented or not with LPS (0 -control, 0.1, 1, and 10 µg/mL) for seven days. After this time interval, cells were cultured in an odontogenic differentiation medium without LPS for up to 21 days. Cell viability was assessed weekly (from Day 1). On day 7, cells were also evaluated for NF-κB activation, gene expression, oxidative stress, and nitrite production.   regarding cell viability (p=0.62), and only 10 μg/mL LPS increased cell viability without FBS compared to control (p=0.02, Figure 3A). Conversely, there were no differences in cell viability for neither LPS concentrations diluted in a medium containing FBS (p≥0.75, Figure 3A). Overall, cell viability was lower when cells were cultured without FBS after seven days (p<0.0001, Figure 3A). At 21 days, there was an interaction effect for cell viability and formation of a mineralized matrix under the two conditions and LPS concentrations (p<0.0001). The absence of LPS (control) did not show significant differences in cell viability whether FBS was added to the culture medium or not. However, a detrimental effect of LPS on HDPCs metabolism was detected only when FBS was added to the culture medium ( Figure 3A). In that experimental condition, all LPS concentrations significantly reduced cell viability compared to the control (p<0.0001, Figure   3B). The same phenomenon was seen in the formation of a mineralized matrix. The different concentrations of Figure 5-A) Gene expression modulation (fold change of control) of odontogenic markers (ALPL and DSPP) and cytokines (TNF, IL1B, and IL8) by human dental pulp cells (HDPCs) cultured with LPS concentrations (0 -control, 0.1, 1, and 10 µg/mL) on day 7. Columns are means and error bars are standard deviations (n=6). Distinct letters indicate that groups are statistically different (one-way ANOVA/ Tukey or Welch's one-way ANOVA/Games-Howell, α=5%). B) Reactive nitrogen species (nitrite, fold increase of control) production by human dental pulp cells (HDPCs) cultured with LPS concentrations (0 -control, 0.1, 1, and 10 µg/mL) on day 7. Distinct letters are statistically different (Welch's one-way ANOVA/Games-Howell, α=5%). C) Reactive oxygen species (oxidative stress, fold increase of control) production by human dental pulp cells (HDPCs) cultured with LPS concentrations (0 -control, 0.1, 1, and 10 µg/mL) on day 7. Distinct letters are statistically different (one-way ANOVA/Tukey, α=5%). On the right, there are representative images (10×) of intracellular oxidative stress (green fluorescence -Carboxy-H2DCFDA probe) in human dental pulp cells (HDPCs) cultured with LPS concentrations (0 -control, 0,1, 1, and 10 µg/mL) on day 7. In the bottom row of the images, the same green fluorescence images (with higher brightness and contrast) merge with the bright field, showing cell confluency  Figure   3C). However, no LPS concentrations affected the formation of a mineralized matrix when cells were cultured under a basal medium (p≥0.99, Figure 3C).   Figure 5A) and down-regulated by 10 µg/mL LPS by half (p=0.009, Figure 5A). No concentration modulated DMP1 gene expression (p=0.052, Figure 5A). Overall, 1 and 10 µg/mL LPS significantly up-regulated inflammatory-related gene expression. TNF, IL1B, IL8, and IL6 genes were significantly up-regulated around 8-fold, 4-fold, 13fold, and 8-fold by 10 µg/mL LPS, respectively (p≤0.03, Figure 5A). After seven days of exposure to LPS, only 10 µg/mL LPS significantly increased nitrite production (p<0.0001, Figure 5B). However, all LPS concentrations increased oxidative stress in a dose-dependent manner (p<0.0001, Figure 5C), increasing around 1.5× for 10 µg/mL LPS.

Considering that the dose-dependent pattern of LPS
After one and seven days, the nuclear translocation of NF-κB subunit p65 increased in a dose-dependent fashion for the LPS concentrations, showing the activation of the NF-κB transcription factor ( Figure   6). Additionally, after seven days, the exposure to 10 µg/mL LPS demonstrated more intense effects on NF-κB, also affecting the F-actin distribution, aggregated around the nucleus (Figure 6). Figure 6-Immunofluorescence of NF-κB subunit p65 (green fluorescence -FITC) and actin filaments (F-actin, red fluorescence -ActinRed555) expressed in human dental pulp cells (HDPCs) after one and seven days of exposure to 0 (control) or 10 µg/mL LPS. The cell nuclei (blue fluorescence -Hoechst) were counterstained and the channels were merged (40×). After one and seven days, cell cultures without LPS (control) showed an expression of p65 mostly in the cytoplasm. Conversely, the exposure to 10 µg/mL LPS increased the nuclear translocation of p65 (pointers), demonstrating the activation of the NF-κB transcription factor. Chronic LPS exposure also abnormally affected the F-actin distribution around the nuclei after seven days (arrows) 1 and 10 µL/mL LPS exerted significant effects, using the higher concentration (10 µL/mL) shall provide a higher challenge, thus providing safer responses.
Since regenerative endodontics is an emerging field, researchers should consider balancing inflammation and the degenerative effects of chronic LPS stimuli in bacteria-affected environments to properly stimulate an osteo/odontoblastic phenotype to induce mineralization by resident stem cells, thus resulting in tissue regeneration. This should help predict better clinical outcomes of minimally invasive vital pulp therapies under pulpitis conditions.

Conclusion
The exposure to 10 µg/mL LPS for seven days creates an inflammatory environment able to impair by more than half the odontogenic potential of HDPCs in vitro. This protocol may be suitable to simulate the regenerative potential of pulp cells under pulpitis conditions in vitro.