Trans-dentinal cytotoxicity assessment of adhesive systems on sound and caries-affected dentin using a microphysiological artificial pulp chamber system

This study evaluated, in vitro, the trans-dentinal cytotoxicity of a universal adhesive system (Single Bond Universal, SBU) applied onto sound and caries-affected dentin (CAD), using a microphysiological artificial pulp chamber (APC) on-a-chip system. Human dentin discs (0.35 mm) from sound (SD) or microbiologically demineralized teeth (CAD) were used in a co-culture model under simulated pulpal pressure into APC-on-a-chip, with MDPC-23 cells seeded on the pulpal surface and human dental pulp cells (HDPCs) cultured in 3D collagen gels beneath the dentin. SBU was applied on the occlusal surface in self-etch (SE) or etch-and-rinse (ER) mode and LED-cured (continuous mode). After 24 h, cell metabolism (Alamar Blue), viability (Live/Dead), dentin permeability (Lp), morphology (SEM), and HEMA diffusion (UV–vis) were evaluated. Data were analyzed using two-way ANOVA/Tukey or Student’s t-tests (p < 0.05). SBU application reduced MDPC-23 metabolism by 71.2% (SE) and 85.7% (ER) on SD, and 46.8% (SE) and 62.2% (ER) on CAD. HDPCs showed similar trends: decreases of 50.2% and 70.1% on SD, and 38.1% and 61.9% on CAD, respectively. Live/Dead staining confirmed increased cell death, especially in SBU-ER groups. CAD discs had ∼82% lower permeability than SD; ER etching increased permeability in both. SEM showed open tubules in SD and demineralized surfaces in CAD. HEMA diffusion was reduced by 36.5% and 54.7% in CAD for SE and ER, respectively. SBU featured cytotoxicity regardless on dentin condition (SD or CAD), highlighting biological risks of using universal adhesives in deep cavities. • A microphysiological APC-on-a-chip model simulated the dentin–pulp interface in vitro. • Universal adhesive cytotoxicity was tested on sound and caries-affected dentin. • Both self-etch and etch-and-rinse modes reduced odontoblast-like cell metabolism. • Caries-affected dentin attenuated but did not prevent trans-dentinal cytotoxicity. • Findings support the cautious use of universal adhesives on very deep dentin.