A quantitative three-dimensional bone mineralisation assay using human dental pulp stem cell spheroids

Abstract

Matrix mineralisation in three-dimensional (3D) human cell culture is still assessed predominantly by terminal histochemical staining. Alizarin Red S and Von Kossa are destructive, semi-quantitative, and require fixation and dye extraction before a number can be obtained, which precludes longitudinal measurement and makes miniaturised multiwell formats impractical. We describe a scaffold-free assay that renders 3D human bone mineralisation as a continuous, well-level quantity. Primary human dental pulp stem cells (DPSCs) were dispensed by automated liquid handling into 384-well plates, where they formed a single, centrally positioned spheroid per well. Eleven plate geometry and surface-coating combinations were compared, and cell-repellent coating rather than well geometry alone was identified as the determinant of single-aggregate formation. Mineral deposition was measured non-destructively by adding the tetracycline-derived near-infrared probe BoneTag680 directly to the existing culture volume and reading the plate from below, with no spheroid transfer, embedding, sectioning or dye extraction at any point. Spheroids maintained in 10% fetal calf serum (FCS) with osteogenic cocktail were separated from a 1% FCS low-mineralisation condition with an assay window of 13.74 plus-or-minus sign 3.29-fold and a Z-factor of 0.627 plus-or-minus sign 0.119 across four independent plates, with a well-level coefficient of variation of 10.88% compared with 55.81% in a commercial alternative. A DNA content measurement acquired from the same wells after near-infrared acquisition showed no increase in cell number under osteogenic conditions, establishing that the mineral signal is not biomass-driven and providing an internal counter-screen against proliferative and cytotoxic artefacts. All data derive from a single donor; the assay is therefore characterised for technical and within-donor reproducibility, and is presented as a defined quantitative measurement intended as a foundation for bone-anabolic compound screening rather than as a screening-validated platform.