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. 2024 Jun 24:35:e245529.
doi: 10.1590/0103-6440202405529. eCollection 2024.

Cytotoxic effects on human dental pulp stem Cells after exposure to adhesive bonding agents

Antonella Castellanos Caroprese  1 , Alexa Nicolle Chinchilla Navarrete  1 , Sandra Janeth Gutiérrez Prieto  1 , Jean Carlos Villamil  1 , Octavio Alejandro Castañeda Uribe  2 , Juan Carlos Salcedo Reyes  3 , And Henry Alberto Méndez Pinzón  3
Affiliations

Cytotoxic effects on human dental pulp stem Cells after exposure to adhesive bonding agents

Antonella Castellanos Caroprese et al. Braz Dent J. .

Abstract
in English, Portuguese

Studies regarding cytotoxic effects attributed to the use of adhesive bonding agents on pulp tissue are not conclusive. To point out whether these materials are safe for clinical use, in vivo exposure of dental pulp to adhesive bonding agents was simulated using an experimental setup in which Human Dental Pulp Stem Cells (hDPSC) are exposed to the action of two kinds of adhesives: self-etching adhesives and two-step bonding agents through a dentine barrier. Cytotoxic effects on these cells were evaluated by MTT assay protocol and fluorescence microscopy, and their results were contrasted to those obtained through Raman spectra taken on single hDPSCs. Overall, no significant cytotoxic effects were observed by combining all the techniques, and cell viability close to 90% was achieved for a dentine barrier of at least 1 mm thick. Moreover, Raman spectroscopy was able to detect structural DNA damage in some dental pulp cells when exposed to two-step bonding agents, suggesting that this technique could be considered a complementary tool with the potential to evaluate cell toxicity beyond cell viability.

Os estudos sobre os efeitos citotóxicos atribuídos ao uso de agentes de união adesivo no tecido pulpar não são conclusivos. Para determinar se esses materiais são seguros para uso clínico, a exposição in vivo da polpa dentária a agentes de união adesiva foi simulada por meio de uma configuração experimental na qual as células-tronco da polpa dentária humana (hDPSC) são expostas à ação de dois tipos de adesivos: adesivos autocondicionantes e agentes de união de duas etapas por meio de uma barreira de dentina. Os efeitos citotóxicos nessas células foram avaliados pelo protocolo de ensaio MTT e microscopia de fluorescência, e seus resultados foram contrastados com os obtidos por meio de espectros Raman obtidos em hDPSCs individuais. De modo geral, não foram observados efeitos citotóxicos significativos com a combinação de todas as técnicas, e a viabilidade celular próxima a 90% foi obtida para uma barreira de dentina de pelo menos 1 mm de espessura. Além disso, a espectroscopia Raman foi capaz de detectar danos estruturais ao DNA em algumas células da polpa dentária quando expostas a agentes de colagem de duas etapas, sugerindo que essa técnica poderia ser considerada uma ferramenta complementar com potencial para avaliar a toxicidade celular além da viabilidade celular.

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Figures

Figure 1
Figure 1. Experimental design for simulating exposure of dental pulp to adhesive agents in clinical practice: a) Diagram of cutting area of dentin discs in molars. b) a Real dentine disc image and their placement as a barrier between adhesive agents and cell culture and c) a Picture showing a 12.0 mm diameter dentin disc fixed to the transwell.
Figure 2
Figure 2. Scanning Electron Microscopy (SEM) images of a dentine disc: a) Smear layer on the occlusal surface (1000X) and b) Under 10.0 kX. The structure and morphology of dentine debris within the smear layer are observed.
Figure 3
Figure 3. Monolayer of hDPSC under an inverted microscope (40x).
Figure 4
Figure 4. SERS substrates elaborated by deposition of a metallic layer on colloidal crystals (CC). a) SEM image of FCC colloidal crystal grown by stacked 250 nm Silica spheres. b) Structure of the SERS substrate with a 50 nm thickness gold layer on top (Ref. 24 ).
Box 1
Box 1. Protocol established by the manufacturers for the application of adhesives on dentine discs
Figure 5
Figure 5. SEM images at the dentine/adhesive interface. a) Self-etching adhesive Single Bond Universal 3M™. b) Two-step adhesive Adper single bond 3M™.
Figure 6
Figure 6. Representative normalized Raman Spectrum of hDPSC
Figure 7
Figure 7. Raman spectra of hDPSC a) with no adhesive applied (control) and under exposure to the adhesives: b) Single bond universal, c) Optibond all in one, and d) Adper single bond.
Figure.8. Renormalized
Figure.8. Renormalized. spectra of the two-step adhesive Adper single bond 3M™ and the control spectrum taking the Adenine, Guanine band as reference.
Figure 9
Figure 9. Images of Fluorescence emitted by hDPSC after exposure to adhesives: Single bond universal (a,b), Optibond all in one (c,d), and Adper single bond (e,f).
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