Effects of Adper™ Scotchbond™ 1 XT, Clearﬁl™ SE Bond 2 and Scotchbond™ Universal in Odontoblastic Activity †

: This study aimed to assess in vitro cytotoxicity for Adper™ Scotchbond™ 1 XT (SB1), Clearﬁl™ SE Bond 2 (CSE) and Scotchbond™ Universal (SBU), using MDPC-23 cell cultures. The metabolic activity, protein content, cell death types and cellular morphology were evaluated. All extracts determined a signiﬁcant reduction in cell metabolism and viability. CSE extracts signiﬁcantly reduced cell’s metabolic activity at its higher concentrations (50% and 100%). All adhesives determined a reduction in the number of viable cells. Changes were dependent on the adhesive, concentration and incubation time. CSE was the most cytotoxic and showed a higher degree of reactivity. and investigation, and and data and writing—


Introduction
Adhesive systems allow the adhesion of restorative materials to dental substrate [1,2]. These materials are evolving towards simpler clinical application protocols and better clinical performance [1,3]. With the increasing complexity of adhesive formulations, several substances present in these materials have been identified and studied, which can induce adverse biological reactions [4,5].

Types of Cell Death
The cells were incubated with the adhesive extracts at 25% and 50% concentrations. The types of cell death were determined using double labelling with annexin V (AnV-FITC), and propidium iodide (PI).

Morphology and Qualitative Cytotoxicity Assessment
Cells were stained with May-Grünwald Giemsa for morphology evaluation, followed by optical microscopy analysis. The grading of reactivity described in the ISO 10993-5 [7] was applied.

Statistical Analysis
The statistical analysis was performed using GraphPad Prism 8 ® (GraphPad Software, San Diego, CA, USA). For the metabolic activity and protein content results, the Shapiro-Wilk test followed by the t-test or the Wilcoxon test were used (control cultures were normalized at 100%). Two-factor ANOVA or Kruskal-Wallis tests were used to compare the experimental conditions along the incubation periods. Multiple comparisons and corrections were performed using the Tukey or Dunn corrections. Regarding flow-cytometry results, one factor ANOVA or Kruskal-Wallis were used, and multiple comparisons with the corrections of Bonferroni or Dunn were performed as applicable.

Metabolic Activity and Protein Content
Incubation of the cells with the adhesive's extracts determined a metabolic activity reduction, significantly for the higher concentrations ( Figure 1). culture medium (DMEM, 13.4 g/L-D-5648, Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, F7524, Sigma Aldrich, St. Louis, MO, USA), for 24 h [6]. For all studies, the odontoblast-like cell line MDPC-23 was used.

Types of Cell Death
The cells were incubated with the adhesive extracts at 25% and 50% concentrations. The types of cell death were determined using double labelling with annexin V (AnV-FITC), and propidium iodide (PI).

Morphology and Qualitative Cytotoxicity Assessment
Cells were stained with May-Grünwald Giemsa for morphology evaluation, followed by optical microscopy analysis. The grading of reactivity described in the ISO 10993-5 [7] was applied.

Statistical Analysis
The statistical analysis was performed using GraphPad Prism 8 ® (GraphPad Software, San Diego, CA, USA). For the metabolic activity and protein content results, the Shapiro-Wilk test followed by the t-test or the Wilcoxon test were used (control cultures were normalized at 100%). Two-factor ANOVA or Kruskal-Wallis tests were used to compare the experimental conditions along the incubation periods. Multiple comparisons and corrections were performed using the Tukey or Dunn corrections. Regarding flowcytometry results, one factor ANOVA or Kruskal-Wallis were used, and multiple comparisons with the corrections of Bonferroni or Dunn were performed as applicable.

Metabolic Activity and Protein Content
Incubation of the cells with the adhesive's extracts determined a metabolic activity reduction, significantly for the higher concentrations ( Figure 1).  Protein content was significantly reduced after the incubation of the cultures with the adhesive extracts at 25% and 50% concentrations. CSE extracts significantly reduced cell viability at both concentrations compared to SB1 and SBU extracts (Figure 2).
Protein content was significantly reduced after the incubation of the cultures with the adhesive extracts at 25% and 50% concentrations. CSE extracts significantly reduced cell viability at both concentrations compared to SB1 and SBU extracts (Figure 2).

Types of Cell Death
Cultures exposed to the extracts showed reduced numbers of live cells with a consequent increase of cells in apoptosis, late/apoptosis and in necrosis ( Figure 3).

Morphology and Qualitative Cytotoxicity Assessment
CSE extracts led to the greater inhibition of cell growth with the destruction of the membrane, being classified with a higher degree of reactivity among the adhesives under study.

Conclusions
Adhesive extracts determined changes in the cultures depending on the adhesive and its concentration. CSE extracts were the most cytotoxic. The clinical application of these materials has to be cautious, and the possibility of pulpal-induced cytotoxicity must be taken into account.

Types of Cell Death
Cultures exposed to the extracts showed reduced numbers of live cells with a consequent increase of cells in apoptosis, late/apoptosis and in necrosis (Figure 3).
Biol. Life Sci. Forum 2021, 9, 3 3 of 4 Protein content was significantly reduced after the incubation of the cultures with the adhesive extracts at 25% and 50% concentrations. CSE extracts significantly reduced cell viability at both concentrations compared to SB1 and SBU extracts (Figure 2).

Types of Cell Death
Cultures exposed to the extracts showed reduced numbers of live cells with a consequent increase of cells in apoptosis, late/apoptosis and in necrosis ( Figure 3).

Morphology and Qualitative Cytotoxicity Assessment
CSE extracts led to the greater inhibition of cell growth with the destruction of the membrane, being classified with a higher degree of reactivity among the adhesives under study.

Conclusions
Adhesive extracts determined changes in the cultures depending on the adhesive and its concentration. CSE extracts were the most cytotoxic. The clinical application of these materials has to be cautious, and the possibility of pulpal-induced cytotoxicity must be taken into account.

Morphology and Qualitative Cytotoxicity Assessment
CSE extracts led to the greater inhibition of cell growth with the destruction of the membrane, being classified with a higher degree of reactivity among the adhesives under study.

Conclusions
Adhesive extracts determined changes in the cultures depending on the adhesive and its concentration. CSE extracts were the most cytotoxic. The clinical application of these materials has to be cautious, and the possibility of pulpal-induced cytotoxicity must be taken into account.