Cytotoxicity of released monomers, additives, and ions from bioactive dental liners in human dental pulp stem cells

The biological performance of dental liner materials is commonly attributed to mineralizing ion release and presumed bioactivity. However, these materials represent complex chemical systems that may also release organic constituents with potential biological effects. While ion release has been characterized previously, quantitative data on co-released organic constituents and their cytotoxic thresholds in human dental pulp stem cells (hDPSCs) remain limited. This study aimed to identify and quantify organic substances released from six dental liner materials and to assess the cytotoxicity of selected organic and inorganic components in hDPSCs.

Specimens (n = 4 per material and elution medium) were prepared as standardized discs and incubated for 72 h in methanol or water. Organic substances released from six dental liner materials (an experimental liner/base (EXP), TheraCal LC® (TC), Dycal® (DY), Lime-Lite Enhanced™ (LL), Fuji Lining LC Paste Pak (FL), and Medcem MTA (MC)) were identified and quantified using gas chromatography-mass spectrometry (GC/MS). Based on the analytical findings and previously published ion release data, individual organic compounds and representative inorganic salts were subsequently tested separately in hDPSCs. Cytotoxicity after 24 h exposure was assessed using WST-1 assays, and EC₅₀ values were derived from four-parameter logistic (4PL) nonlinear regression with variable slope.

GC/MS detected nine organic substances released into methanol, whereas no organic substances were detected in water eluates. FL showed the highest release of 2-hydroxyethyl methacrylate (HEMA, ≈0.30 mg/mL) and camphorquinone (CQ, ≈0.05 mg/mL). TC released the highest amounts of butylated hydroxytoluene (BHT, ≈0.009 mg/mL) and 4-(dimethylamino) benzoic acid ethyl ester (DMABEE, ≈0.10 mg/mL). In hDPSCs, sodium fluoride (NaF) exhibited the highest cytotoxicity (EC₅₀ ≈0.056 mg/mL), followed by CQ (≈0.068 mg/mL) and triethylene glycol dimethacrylate (TEGDMA, ≈0.475 mg/mL).

The biological performance of dental liner materials cannot be inferred from ion release alone. The integrated evaluation of organic substance release, inorganic ion elution, and substance-specific cytotoxicity provides a more comprehensive framework for the biological evaluation of so-called "bioactive" liner materials and supports substance-specific hazard characterization under standardized experimental conditions.