Evaluation of the Properties and Osteogenic Potential of a Novel Scaffold-Free Material, Spheroid Blocks Comprising Fused Spheroids of Human Periodontal Ligament Mesenchymal Stem Cells.

Periodontal ligament stem cell spheroids are reportedly valuable for tissue regeneration; however, their application in vivo requires the use of a scaffold, which may raise safety concerns. We moulded human periodontal ligament mesenchymal stem cell (hPDLMSC) spheroids into blocks as a scaffold-free method for handling spheroids. We then examined the characteristics and osteogenic capabilities of hPDLMSC spheroid blocks in vitro and in vivo. First, the hPDLMSCs were seeded into microwell chips to form homogeneous spheroids, which were then seeded into net mould devices and cultured with rotary shaking to form hPDLMSC spheroid blocks. Next, real-time polymerase chain reaction (PCR) analysis, a live/dead assay and histological analysis were performed to investigate the properties of the hPDLMSC spheroid blocks. Finally, bone defects were created in mouse calvaria, and the defects were transplanted with hPDLMSC spheroid blocks; the osteogenic potential of the spheroid blocks was evaluated using three-dimensional (3D) micro-X-ray computed tomography (µCT) analysis and histological techniques. We identified that the expression levels of stemness markers and osteogenesis-related genes were higher in hPDLMSC spheroid blocks than in monolayer and spheroid-cultured hPDLMSCs. The live/dead assay and histological analysis revealed that there was almost no central necrosis in the hPDLMSC spheroid blocks, and hPDLMSC spheroid blocks formed nodules under osteogenic differentiation induction. Furthermore, the hPDLMSC spheroid block treatment group (without the use of scaffolds) exhibited both the nearly complete closure of the mouse calvarial bone defect and significantly increased bone microstructural parameters compared with the group in which hPDLMSC spheroids were transplanted in combination with scaffolds. Together, these findings indicate that hPDLMSC spheroid blocks possess excellent stemness and osteogenic potential, and may contribute to the establishment of novel scaffold-free therapies.