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Thesis
Interaction of dental pulp stem cells with bacterial products modulates the protein expression of Cathepsin B
University of Iowa
Master of Science (MS), University of Iowa
Summer 2022
DOI: 10.25820/etd.006575
Abstract
Title: Interaction of Dental Pulp Stem Cells with Bacterial Products modulates the Protein Expression of Cathepsin B.Objective: Endogenous enzymes, such as cysteine cathepsins, have been showed to be the key proteases driving dentin degradation in caries progression. While acid-activation is the currently accepted mechanism for the activation of endogenous proteases from the dentin, odontoblasts-derived enzymes might be released by pulp in response to bacterial infiltration. The aim of this study was to characterize the effect of stimulation of human dental pulp stem cells (DPSC) with bacterial products on the in vitro protein expression of cathepsin B. More specifically, DPSC response to purified bacterial products as well as spent-culture filtrates of Streptococcus mutans (SM) and Lactobacillus rhamnosus (LR) was investigated. The specific hypotheses were: 1. purified molecules of lipopolysaccharide (LPS) and/or lipoteichoic acid (LTA) would modulate the protein expression of cathepsin B by human DPSC; and 2. the spent-culture filtrates from pure colonies of SM and/or LR would modulate the protein expression of cathepsin B by human DPSC.
Methods: The first part of the in vitro study addressed the first hypothesis and has three experimental groups: 1) ⍺-MEM (control); 2) ⍺-MEM + LPS; and 3) ⍺-MEM + LTA. The second part addressed the second hypothesis and has three experimental groups: 1) ⍺-MEM + D-MEM (control); 2) ⍺-MEM + D-MEM + SM; and 3) ⍺-MEM + D-MEM + LR. The study was conducted using immortalized human DPSC, LPS from Escherichia coli O111:B4, LTA from Bacillus subtilis, Streptococcus mutans (ATCC 25175), and Lactobacillus rhamnosus (ATCC 53103). Initially, cell viability assays were performed to determine the ideal concentrations and incubation time for DPSC stimulation with the bacterial products. Then, DPSC supernatants and lysates were collected from DPSC following the stimulation of the DPSC with LPS, LTA, SM, or LR filtrates for 24 h. DPSC supernatants and lysates were analyzed for the quantitative detection of total protein and expression of cysteine cathepsin B. Data analysis was performed using Shapiro-Wilk’s test, two-way ANOVA and Tukey HSD post hoc tests (α=0.05).
Results: The cell viability results for different concentrations of LTA and LPS revealed that DPSC incubated with 10 µg/mL LPS showed the highest viability amongst the LPS groups, while 2.5 µg/mL LTA showed the highest viability amongst the LTA groups. The cell viability results for the LTA, LPS, SM and LR groups showed an overall decrease in DPSC viability from 24 h to the 48 h of stimulation, with statistically significant difference for LPS (p = 0.037) and LTA (p < 0.001) groups. The cell viability results for DPSC stimulated with bacterial filtrates showed an increase in the viability of the DPSC from the 24 h to the 48 h incubation, however, there was no statistically significant differences among the groups (p > 0.05). The DPSC stimulation with LTA resulted in the highest total protein for the supernatants, but the lowest for lysates when compared to LPS and control. However, the ANOVA tests revealed no significant difference in the total protein concentration among the groups for the supernatants and lysates for both LPS, LTA, and culture filtrates (p > 0.05). The results for cathepsin B expression for the DPSC supernatants after 24 h of stimulation with purified products and culture filtrates showed a significant reduction in the cathepsin B expression by the LTA group (p = 0.036) and the LR (p = 0.008) group compared to their controls.
Conclusion: The purified molecules of LTA and spent-culture filtrates from pure colonies of Lactobacillus rhamnosus modulated the protein expression of cathepsin B by human DPSC and resulted in a significant decrease in the levels of this enzyme.
Details
- Title: Subtitle
- Interaction of dental pulp stem cells with bacterial products modulates the protein expression of Cathepsin B
- Creators
- Olajide Obe
- Contributors
- Cristina Vidal (Advisor)Steven Armstrong (Committee Member)Carolina Cucco (Committee Member)Jeffrey Banas (Committee Member)Erliang Zeng (Committee Member)
- Resource Type
- Thesis
- Degree Awarded
- Master of Science (MS), University of Iowa
- Degree in
- Oral Science
- Date degree season
- Summer 2022
- Publisher
- University of Iowa
- DOI
- 10.25820/etd.006575
- Number of pages
- xvi, 94 pages
- Copyright
- Copyright 2022 Olajide Obe
- Language
- English
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 81-94).
- Public Abstract (ETD)
Tooth decay occurs when there are disruptions of the bacterial communities in the mouth, which then affects the balance of demineralization and remineralization. The early stages of tooth decay affect the enamel, and in its advanced stages, it affects the dentin, pulp and can lead to loss of the tooth. The composition and profile of the bacteria involved in tooth decay is dependent on the tooth tissue involved in the decay. Furthermore, these bacteria change with progression of the decay, depth of the decay and changes in the pH level. When the tooth decay reaches the dentin, the organic component is broken down following the breakdown of the minerals and causes the decay and cavity to further progress. In addition to bacterial organisms that produce acid from the breakdown of sugars, the breakdown of the organic component of dentin is driven by endogenous enzymes, such as cysteine cathepsins. The cysteine cathepsins are activated by the low pH of the acid and cleave dentinal collagen to promote the breakdown of dentin’s organic component. The composition of bacteria involved in tooth decay that is dependent on the tissue involved, the changes in the types of bacteria as the decay advances, and the breakdown of dentinal collagen by endogenous proteases such as cysteine cathepsin can be put together to suggest interactions between the host and the bacteria for tooth decay in dentin. Therefore, this study investigated the interaction of odontoblasts with bacterial products and its influence on the expression of cysteine cathepsin B. More specifically, the aim of our study was to characterize the effect of stimulation of human dental pulp stem cells with bacterial products on the expression of cathepsin B. For that, dental pulp stem cells were stimulated with different bacterial products and the levels of cathepsin B expressed by these cells were quantified. Our results demonstrated that specific bacterial products were able to reduce the levels of cathepsin B expressed by the dental pulp stem cells, supporting the hypothesis that the expression of this enzymes can be modulated in response to the stimulation with bacterial products.
- Academic Unit
- Oral Pathology, Radiology and Medicine
- Record Identifier
- 9984285051502771
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