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

Version 1.0
Chronic exposure to lipopolysaccharides as an in vitro model to simulate the impaired odontogenic potential of dental pulp cells under pulpitis conditions
Soares, Igor Paulino Mendes; Anselmi, Caroline; Pires, Maria Luiza Barucci Araújo; Ribeiro, Rafael Antonio de Oliveira; Leite, Maria Luísa; Soares, Diana Gabriela; Costa, Carlos Alberto de Souza; Hebling, Josimeri, 2023, "Chronic exposure to lipopolysaccharides as an in vitro model to simulate the impaired odontogenic potential of dental pulp cells under pulpitis conditions", https://doi.org/10.48331/scielodata.96UYNU, SciELO Data, V1, UNF:6:IjHO+b7kj6Myf0Mc0pVuhg== [fileUNF]
Learn about Data Citation Standards.
Make Data Count (MDC) Metrics
since
0 Views
0 Downloads
0 Citations
Description
Background: Simulating a bacterial-induced pulpitis environment in vitro may contribute to exploring mechanisms and bioactive molecules to counteract these adverse effects. Aim: 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 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 (Alizarin Red) at 14 and 21 days. Data were analyzed with ANOVA and post-hocs (α=5%). Results: After one and seven days of exposure to LPS, NF-κB was activated in a dose-dependent fashion. One and 10 µg/mL LPS down-regulated the gene expression of odontogenic markers and up-regulated cytokines. Ten µg/mL LPS increased both the production of reactive nitrogen and oxygen species. LPS decreased cell viability seven days after the end of exposure. One and 10 µg/mL LPS decreased hDPCs mineralization in a dose-dependent fashion. 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, simulating a pulpitis-like condition. (2023-06-19)
Subject Medicine, Health and Life Sciences
Keyword Lipopolysaccharides, Cell Culture Techniques, Dental Pulp, Pulpitis, Biomineralization
Related Publication Soares IP, Anselmi C, Pires ML, Ribeiro RA, Leite ML, Soares DG, et al. Chronic exposure to lipopolysaccharides as an in vitro model to simulate the impaired odontogenic potential of dental pulp cells under pulpitis conditions. J Appl Oral Sci. 2023;31:e20230032. doi: 10.1590/1678-7757-2023-0032 doi: 10.1590/1678-7757-2023-0032
License/Data Use Agreement

CC BY 4.0

Filter by
1 to 10 of 12 Files
Alizarin_red(mineralization).tab
Tabular Data - 3.8 KB
Published Jun 21, 2023
0 Downloads
17 Variables, 26 Observations UNF:6:LSNwFGn+dwi2ez+SuniLog==
Preview "Alizarin_red(mineralization).tab"
Cell_viability.tab
Tabular Data - 7.5 KB
Published Jun 21, 2023
0 Downloads
23 Variables, 26 Observations UNF:6:EOXFvVKC/SHxlFnGE/tSBw==
Preview "Cell_viability.tab"
Figure_1.jpg
Figure_1.jpg
JPEG Image - 1.7 MB
Published Jun 21, 2023
2 Downloads
MD5: ea22cae28745d876524ad54498e02320
A) Schematic representation of the experimental design to access the effects of different concentrations of lipopolysaccharides (LPS) on human dental pulp cells (HDPCs). Cells were tested under two different experimental conditions: #1) LPS concentrations diluted in FBS-free α-MEM (basal medium); or #2) LPS concentrations diluted in α-MEM + 10% FBS (growth medium). After 24 hours 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 hours 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. The formation of a mineralized matrix was evaluated after 14 and 21 days.
Preview "Figure_1.jpg"
Figure_2.jpg
Figure_2.jpg
JPEG Image - 726.4 KB
Published Jun 21, 2023
2 Downloads
MD5: 6966e2b3847eda0c6dab4236c0585fbe
Flow cytometry histograms of the mesenchymal stem cell antigens expressed in the established primary culture of human dental pulp cells (HDPCs). Sections A) and B) show representative negativity of FITC and PE channels of fluorescence controls. Representative histograms and percentage of expression are given for C) CD45, D) CD34 (both negative markers), E) CD73, F) CD90, G) CD 105, and H) CD146 (all positive markers).
Preview "Figure_2.jpg"
Figure_3.jpg
Figure_3.jpg
JPEG Image - 804.3 KB
Published Jun 21, 2023
2 Downloads
MD5: 8b74bb2e0f6823e2cd90148e3b424ff3
A) Cell viability after seven days (% of control FBS-free medium) for both experimental conditions: #1) LPS concentrations diluted in FBS-free α-MEM (basal medium); or #2) LPS concentrations diluted in α-MEM + 10% FBS (growth medium). Columns are means and error bars are standard deviations (n=8). Uppercase letters compare the different conditions and lowercase letters compare the LPS concentrations within the same experimental condition. Distinct letters are statistically different (two-way ANOVA/Sidak, α=5%). B) Cell viability after 21 days (% of control FBS-free medium) for both experimental conditions. Columns are means and error bars are standard deviations (n=8). Distinct letters compare each column, showing statistical differences (two-way ANOVA/Sidak, α=5%). C) Mineralized matrix formation (% of control FBS-free medium) for both experimental conditions. Columns are means and error bars are standard deviations (n=8). Distinct letters compare each column, showing statistical differences (two-way ANOVA/Sidak, α=5%).
Preview "Figure_3.jpg"
Figure_4.jpg
Figure_4.jpg
JPEG Image - 1.7 MB
Published Jun 21, 2023
2 Downloads
MD5: 14ee549758209488a35b53ea7d23f2b3
A) Cell viability (% of 1-day control) of human dental pulp cells (HDPCs) cultured with the LPS concentrations (0 - control, 0.1, 1, and 10 µg/mL) diluted in α-MEM + 10% FBS assessed weekly (on days 1, 7, 14, and 21). Columns are means and error bars are standard deviations (n=8). Uppercase letters compare each LPS concentration at different time points and lowercase letters compare LPS concentrations within the same time point. Distinct letters are statistically different (repeated-measures ANOVA/Sidak, α=5%). B) Mineralized matrix formation (% of control) by HDPCs cultured with the LPS concentrations after 14 days. Columns are means and error bars are standard deviations (n=8). Distinct letters indicate statistically different groups (one-way ANOVA/Tukey, α=5%). C) Mineralized matrix formation (% of control) by HDPCs cultured with 0 or 10 µg/mL LPS after 21 days. Columns are means and error bars are standard deviations (n=8). Groups indicated with distinct letters are statistically different (t-test, α=5%). D) Representative images of mineral deposits in an extracellular matrix formed by HDPCs cultured with 0 or 10 µg/mL LPS after 21 days (in the row: an overview of a 96-well plate compartment stained with Alizarin Red, detailed view of each well, and inverted light microscopy image).
Preview "Figure_4.jpg"
Figure_5.jpg
Figure_5.jpg
JPEG Image - 1.1 MB
Published Jun 21, 2023
2 Downloads
MD5: cbf541822fc13cf8c6f0001b8922f6f3
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.
Preview "Figure_5.jpg"
Figure_6.jpg
JPEG Image - 3.3 MB
Published Jun 21, 2023
0 Downloads
MD5: 2501b3cad13af8db16588738eb8a9c4c
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).
Preview "Figure_6.jpg"
Nitrite.tab
Tabular Data - 471 B
Published Jun 21, 2023
0 Downloads
5 Variables, 11 Observations UNF:6:nqrJ20BOWltA0ue6ynMMpw==
Preview "Nitrite.tab"
qPCR.tab
Tabular Data - 6.1 KB
Published Jun 21, 2023
0 Downloads
23 Variables, 20 Observations UNF:6:SQsOkDJkWIMYP3PsjPfRCg==
Preview "qPCR.tab"
Edit File

This file has already been deleted (or replaced) in the current version. It may not be edited.

Restrict Access

Restricting limits access to published files. People who want to use the restricted files can request access by default. If you disable request access, you must add information about access to the Terms of Access field.

Learn about restricting files and dataset access in the User Guide.

Enable access request
You must enable request access or add terms of access to restrict file access.
Save Changes
Edit Embargo

The selected file or files have already been published. Contact an administrator to change the embargo date or reason of the file or files.
Delete Files

The file will be deleted after you click on the Delete button.

Files will not be removed from previously published versions of the dataset.

Select File(s)

Please select one or more files.

Share Dataset

Share this dataset on your favorite social media networks.

Dataset Citations

Citations for this dataset are retrieved from Crossref via DataCite using Make Data Count standards. For more information about dataset metrics, please refer to the User Guide.

Sorry, no citations were found.
Restricted Files Selected

The selected file(s) may not be downloaded because you have not been granted access.

You may request access to the restricted file(s) by clicking the Request Access button.

Download Options

The files selected are too large to download as a ZIP.

You can select individual files that are below the 100.0 MB download limit from the files table, or use the Data Access API for programmatic access to the files.

Select File(s)

Please select a file or files to be downloaded.

Restricted Files Selected

The restricted file(s) selected may not be downloaded because you have not been granted access.

Click Continue to download the files you have access to download.

Delete Dataset

Are you sure you want to delete this dataset and all of its files? You cannot undelete this dataset.

Delete Draft Version

Are you sure you want to delete this draft version? Files will be reverted to the most recently published version. You cannot undelete this draft.

Unpublished Dataset Private URL

Private URL can only be used with unpublished versions of datasets.

Unpublished Dataset Private URL

Are you sure you want to disable the Private URL? If you have shared the Private URL with others they will no longer be able to use it to access your unpublished dataset.

Delete Files

The file(s) will be deleted after you click on the Delete button.

Files will not be removed from previously published versions of the dataset.

Compute

This dataset contains restricted files you may not compute on because you have not been granted access.

Deaccession Dataset

Are you sure you want to deaccession? This is permanent and the selected version(s) will no longer be viewable by the public.

Deaccession Dataset

Are you sure you want to deaccession this dataset? This is permanent an it will no longer be viewable by the public.

Version Differences Details

Please select two versions to view the differences.

Version Differences Details
 
Version:
Last Updated:
Version:
Last Updated:
Select File(s)

Please select a file or files for access request.

Select File(s)

Embargoed files cannot be accessed. Please select an unembargoed file or files for your access request.

Edit Tags

Select existing file tags or create new tags to describe your files. Each file can have more than one tag.

Request Access

  You need to Log In to request access.

Dataset Terms

This dataset is made available under the following terms. Please confirm and/or complete the information needed below in order to continue.

Our Community Norms as well as good scientific practices expect that proper credit is given via citation. Please use the data citation shown on the dataset page.

CC BY 4.0

Preview Guestbook

Upon downloading files the guestbook asks for the following information.

Account Information

Package File Download

Use the Download URL in a Wget command or a download manager to download this package file. Download via web browser is not recommended. User Guide - Downloading a Dataverse Package via URL

https://data.scielo.org/api/access/datafile/

Request Access

Please confirm and/or complete the information needed below in order to request access to files in this dataset.

Compute Batch
Clear Batch
Dataset Persistent Identifier Change Compute Batch
Compute Batch
Submit for Review

You will not be able to make changes to this dataset while it is in review.

Publish Dataset

Are you sure you want to republish this dataset?

Select if this is a minor or major version update.

Publish Dataset

This dataset cannot be published until Journal of Applied Oral Science is published by its administrator.

Publish Dataset

This dataset cannot be published until Journal of Applied Oral Science and SciELO Data are published.

Return to Author

Return this dataset to contributor for modification. The reason for return entered below will be sent by email to the author.