Three-dimensional clumps of mesenchymal stem cells (MSCs)/extracellular matrix (ECM) complexes (C-MSCs) can be transplanted into tissue defect site with no artificial scaffold. Importantly, most bone formation in the developing process or fracture healing proceeds via endochondral ossification. Accordingly, this present study investigated whether C-MSCs generated with chondro-inductive medium (CIM) can induce successful bone regeneration and assessed its healing process. Human bone marrow-derived MSCs were cultured with xeno-free/serum-free (XF) growth medium. To obtain C-MSCs, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and then torn off. The sheet was rolled to make a round clump of cells. The cell clumps, i.e., C-MSCs, were maintained in XF-CIM. C-MSCs generated with XF-CIM showed enlarged round cells, cartilage matrix, and hypertrophic chondrocytes genes elevation in vitro. Transplantation of C-MSCs generated with XF-CIM induced successful bone regeneration in the SCID mouse calvaria defect model. Immunofluorescence staining for human-specific vimentin demonstrated that donor human and host mouse cells cooperatively contributed the bone formation. Besides, the replacement of the cartilage matrix into bone was observed in the early period. These findings suggested that cartilaginous C-MSCs generated with XF-CIM can induce bone regeneration via endochondral ossification.

Background: Cells typically function and behave within a three-dimensional (3D) environment. Mesenchymal stem cells (MSCs), known for their self-renewal, multi-lineage differentiation capabilities, and paracrine effects, have garnered significant medical interest. MSC spheroid culture is widely adopted to study the biological properties of MSCs in a 3D context. In contrast, we previously developed 3D clumps of MSC/ECM complexes termed C-MSCs. C-MSCs consisted of cells and self-produced ECM proteins, allowing grafting into tissue defects without any artificial scaffolds. This present study aimed to elucidate the fundamental biological distinctions between 3D MSC spheroids and C-MSCs. Methods: MSC spheroids and C-MSCs are generated from human bone-marrow-derived MSCs. The physical properties, histological structures, and gene expression patterns were compared in vitro. Results: Macroscopic and histological examinations revealed that, whereas MSC spheroids are dense cell clusters primarily formed through Cadherin-mediated cell–cell interactions, C-MSCs are cell aggregates anchored by the ECM component COL1, enabling them to form larger structures. Furthermore, transcriptome analysis showed that C-MSCs possess enhanced capacities to produce immunomodulatory and cytoprotective factors, a prominent biological characteristic of MSCs. Conclusion: Recognizing the distinct attributes of each cell aggregate offers insights into the potential evolution of 3D cell culture techniques and possible therapeutic implications.