Applicability of Whole Blood Monocyte Activation Test for Endotoxin Activity Assessment in Hydroxyapatite-Based Ceramics

Hydroxyapatite-based ceramics are widely used in dental bioengineering, yet the reliable assessment of endotoxin activity in solid porous materials remains challenging. This study evaluated the applicability of a whole blood Monocyte Activation Test (MAT) to a hydroxyapatite ceramic relevant to dental applications. Two endotoxin association strategies (immersion and dropwise) were compared, followed by nonlinear modelling of cytokine dose-response curves using four-parameter logistic (4PL) regression and spike-recovery analysis to assess potential material interference. Immersion-based spiking produced reproducible, concentration-dependent cytokine responses, whereas dropwise application resulted in minimal functional recovery. IL-1β, IL-6, and TNF-α displayed sigmoidal dose-response profiles with high goodness-of-fit values (R² ≥ 0.93). Spike recovery remained within the 50-200% acceptance range for most concentrations, with IL-6 showing the most consistent analytical performance. TNF-α exhibited signal saturation at higher endotoxin levels, limiting its dynamic range. Multiplex cytokine profiling confirmed that classical MAT readouts were among the most strongly induced mediators and that hydroxyapatite did not trigger baseline inflammatory activation. These findings demonstrate that whole blood MAT can be reliably applied to hydroxyapatite-based dental ceramics when immersion-based endotoxin association and material-specific methodological optimization are employed.