Alterations of mitochondrial dynamics, inflammation and mineralization potential of lipopolysaccharide‐induced human dental pulp cells after exposure to N‐acetyl cysteine, Biodentine or ProRoot MTA

Abstract

Aim To investigate the effects of N‐acetyl cysteine (NAC), Biodentine, ProRoot MTA and their combinations, on cell viability, mitochondrial reactive oxygen species (mtROS) production, mineralization and on the expression of genes related to inflammatory cytokine production, mitochondrial dynamics and cell apoptosis of lipopolysaccharide (LPS)‐induced human dental pulp cells (hDPCs). Methodology Isolated hDPCs were exposed to 20 μg mL−1 of Escherichia coli (E. coli) LPS for 24 h, before the experiment, except for the control group. Eight experimental groups were assigned: (i) control (hDPCs cultured in regular medium), (ii) +LPS (hDPCs cultured in LPS medium throughout the experiment), (iii) −LPS/Media, (iv) −LPS/BD, (v) −LPS/MTA, (vi) −LPS/NAC, (vii) −LPS/BD + NAC and (viii) −LPS/MTA + NAC. Cell viability was measured using Alamar blue assay at 24 and 48 h. Production of mtROS was evaluated at 6 and 24 h by MitoSOX Red and MitoTracker Green. The expressions of IL‐6, TNF‐α, Bcl‐2, Bax, Mfn‐2 and Drp‐1 genes were investigated at 6 h using reverse transcriptase‐polymerase chain reaction (RT‐PCR). For differentiation potential, cells were cultured in the osteogenic differentiation media and stained using Alizarin red assay at 14 and 21 days. The Kruskal–Wallis test, Mann–Whitney U test and one‐way anova were performed for statistical analysis. Results NAC was associated with significantly greater LPS‐induced hDPC viability (P < 0.05). Both Biodentine and MTA extracts promoted cell survival, whereas the combination of NAC to these material extracts significantly increased the number of viable cells at 24 h (P < 0.05). Biodentine, MTA or NAC did not alter the mtROS level (P > 0.05). NAC supplementation to the MTA extract significantly reduced the level of IL‐6 and TNF‐α expression (P < 0.05). Regarding mitochondrial dynamics, the use of NAC alone promoted significant Mfn‐2/Drp‐1 expression (P < 0.05). Most of the groups exhibited a level of Bcl‐2/Bax gene expression similar to that of the control group. The increases in mineralization productions were observed in most of the groups, except the LPS group (P < 0.05). Conclusions The antioxidant effect of NAC was not evident under the LPS‐induced condition in DPC in vitro. NAC combined either with Biodentine or MTA improved LPS‐induced hDPCs survival at 24 h. The combination of NAC with MTA promoted mineralization.

Role of Lipopolysaccharide, Derived from Various Bacterial Species, in Pulpitis—A Systematic Review

“…Teneligliptin also reduces expression of TLR4 [ 26 ]. Another study showed that hDPCs treated with NAC (N-acetylcysteine), combined with Biodentine or Mineral trioxide aggregate (MTA) (cement compounds used in pulpitis treatment) [ 27 ], showed better viability. The group with MTA and NAC treatment also promoted mineralization [ 27 ].…”

In vitro biocompatibility and bioactivity of calcium silicate‑based bioceramics in endodontics (Review)

“…There have been numerous studies on the in vitro biocompatibility and bioactivity of calcium silicate-based bioceramics in endodontics ( 16 , 79 , 147 , 192 ). MTA has been investigated most thoroughly and is considered the gold standard.…”

“…Increased Alp activity and dentin matrix protein expression levels have been observed in human DPSCs stimulated with Biodentine ( 75 - 78 ). Furthermore, Biodentine significantly increases calcium deposition ( 79 ) and enhances the production of Ocn and Runx2 in human DPSCs stimulated with lipopolysaccharide (LPS) although there is no change in ALP expression levels ( 53 ). In terms of the inflammatory response, Biodentine does not affect high expression of IL-6 and IL-8 in DPSCs induced by LPS stimulation but decreases levels of the anti-inflammatory cytokine TGF-β1 ( 53 ).…”

Lipopolysaccharides and hydrogen peroxide induce contrasting pathological conditions in dental pulpal cells

“…Isolation of HDPCs was done according to the method described by Weekate and colleagues (Weekate et al, 2021). In brief, the harvested pulp tissues were digested using Collagenase I (Gibco/Invitrogen) and Dispase II (Sigma‐Aldrich) and cultured in α‐MEM (Sigma‐Aldrich) supplemented with 10% fetal bovine serum (Gibco/Invitrogen), 100 U/ml of penicillin and 100 μg/ml of streptomycin (Sigma‐Aldrich), and 100 μmol/L of L‐ascorbic acid (Sigma‐Aldrich) at 37°C with 95% air and 5% CO 2 .…”

“…Each group was treated with either vehicle, 20 μg/ml LPS, 400 μM H 2 O 2 , and combined LPS and H 2 O 2 or the two combined, for 24 h. LPS (Ultra‐Pure LPS‐EB from Escherichia coli , 0111:B4 strain; #0000110081) were purchased from Sigma‐Aldrich. The 20 μg/ml LPS and 400 μM H 2 O 2 were used in this study because previous studies demonstrated that 20 μg/ml LPS (Weekate et al, 2021; Yang et al, 2019) and 400 μM H 2 O 2 (Kim et al, 2017; Lee et al, 2015) induced the pathological condition in HDPCs and osteoblasts. The details of each group are as follows: (1) vehicle group: cells were incubated in ⍺‐MEM for 24 h; (2) LPS group: cells were incubated in ⍺‐MEM containing LPS 20 μg/ml for 24 h; (3) H 2 O 2 group: cells were incubated in ⍺‐MEM containing 400 μM of H 2 O 2 for 24 h; and (4) the two combined group: cells were incubated in ⍺‐MEM containing 400 μM of H 2 O 2 and 20 μg/ml of LPS for 24 h. After 24 h, cells in each group were subdivided into three subgroups.…”