Investigating Precursor Spatial Organization in Osteoclastogenesis Using DNA-Directed Cell Patterning

Abstract Osteoclasts are large, multinucleated cells in the bone marrow that are of macrophage lineage and are responsible for bone resorption. Disruption of osteoclast differentiation or resorption activity can cause a broad spectrum of skeletal diseases ranging from osteoporosis to cancer-associated bone loss. Despite their key role in homeostasis, studying osteoclasts in vitro remains challenging, largely because no commercially available cell line exists. Consequently, in vitro studies rely on differentiation from precursor cells using osteoclastogenic factors. While prior studies have optimized various culture-based parameters, most in vitro models lack control over the spatial organization of precursors. This highlights the need for a platform offering such control to study inter-precursor proximity and local confinement in precursor fusion and multinucleation. Here, we employed a DNA-directed cell patterning system to immobilize THP-1-derived macrophages within defined seeding environments and evaluated how spatial organization regulates osteoclastogenesis. THP-1-derived macrophages were immobilized in defined polyacrylamide (PA)-confined circular seeding areas of varying diameter and inter-precursor spacing. Reducing macrophage spacing increased osteoclast formation, with close and medium configurations yielding 8.41 ± 0.82% and 8.03 ± 1.39% total osteoclast conversion, compared with 4.84 ± 0.27% in the far design. Decreasing the seeding area diameter further increased osteoclastogenesis: a microenvironment of 900 μm diameter yielded 12.55 ± 0.58% total osteoclasts, representing 1.49-, 2.25-, and 2.27-fold increases over 1200, 1400, and 1600 μm, respectively. Density-matched unpatterned controls yielded a lower conversion rate across all conditions, indicating that environmental confinement and precursor proximity affect osteoclast differentiation through mechanisms beyond initial seeding density alone. These findings reveal precursor spatial organization as an underappreciated regulator of osteoclast fusion and a practical strategy for improving osteoclast conversion in in vitro models. This platform is extensible to osteoblasts, stromal, immune, endothelial, and tumor cells, offering a foundation for in vitro recapitulation of human bone marrow disease.