Clumps of Mesenchymal Stem Cells/Extracellular Matrix Complexes Generated with Xeno-Free Chondro-Inductive Medium Induce Bone Regeneration via Endochondral Ossification

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.