Rapid generation of dental pulp stem cell-derived mineralized extracellular matrix for quantitative osteoclast resorption assays

Osteoclasts are large, multinucleated cells responsible for bone resorption, playing a central role in bone development, remodeling, and the pathogenesis of metabolic bone diseases through the degradation of inorganic hydroxyapatite and the organic matrix which is primarily composed of collagen. In the study of osteoclast biology, quantitative evaluation of bone resorption capacity is a key indicator of functional maturation. However, currently available assays largely rely on natural bone substrates, such as large animal teeth, or synthetic mineralized materials, both of which present limitations in accessibility, uniformity, and quantitative analysis. Here, we describe a novel resorption assay based on a rapidly mineralized extracellular matrix (ECM) derived from mouse dental pulp stem cells (DPSCs). Primary DPSCs isolated from adult C57BL/6 mouse incisors exhibited robust expansion within 5 days and generated a structurally uniform, well-mineralized ECM after 7 days of osteogenic induction. In addition, immortalization of primary DPSCs via SV40T lentiviral transduction produced stable cell lines with consistent and sustained mineralization capacity. Following decellularization, H&E, Von Kossa, and Masson’s trichrome staining showed that the decellularized ECM formed a continuous mineralized matrix containing collagen-rich organic components. Scanning electron microscopy and mass spectrometry further identified multiple ECM proteins associated with bone-like extracellular matrix. This mineralized ECM supported the differentiation of multiple osteoclast precursors—including mouse and rat bone marrow mononuclear cells as well as human THP-1 cells—into tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts with characteristic F-actin ring formation and measurable resorption activity. Moreover, the combination of Von Kossa staining enabled convenient and quantitative assessment of resorption areas across entire culture wells. The platform sensitively detected dose-dependent modulation of osteoclast activity by RANKL and IFN-γ, confirming its functional responsiveness. Collectively, the DPSC-derived mineralized ECM provides a natural, economical, rapid, and scalable platform for quantitative osteoclast resorption assays, with broad applicability in mechanistic studies and high-throughput drug screening.