Unidirectional porous beta-tricalcium phosphate supports three-dimensional osteogenic differentiation of human iPS cells without surface coating.

Abstract

Beta-tricalcium phosphate (βTCP) is widely used as a bone graft substitute, but it lacks intrinsic osteogenic cells. Human induced pluripotent stem cells (iPSCs) are a promising cell source for bone regeneration; however, the suitability of clinically available, uncoated βTCP as a scaffold for iPSC osteogenic differentiation remains unclear. We compared interconnected porous and unidirectional porous βTCP combined with either negative-pressure or atmospheric-pressure seeding. Metabolic activity was assessed using a CCK-8 assay, and the selected condition was further evaluated by RT-qPCR, scanning electron microscopy, and immunofluorescence staining during osteogenic induction. In vivo osteogenic progression was examined after subcutaneous transplantation into immunodeficient rodents. Negative-pressure seeding was associated with lower metabolic activity, whereas atmospheric-pressure seeding onto unidirectional porous βTCP yielded the highest values among the tested conditions. Under this condition, undifferentiated markers decreased after osteogenic induction, while osteoblast- and osteocyte-related genes showed time-dependent changes consistent with osteogenic lineage progression. Scanning electron microscopy demonstrated cell attachment and three-dimensional extension within the oriented pores, and immunofluorescence staining showed increasing type I collagen deposition during culture. After transplantation, human cell-derived RNA remained detectable, and several osteogenic markers increased over time. These findings indicate that clinically available, uncoated unidirectional porous βTCP can support the survival and osteogenic differentiation of human iPSCs in the absence of additional scaffold coating. This system may provide a simple platform for further studies of iPSC-based bone regenerative strategies.