RESUMO
Endochondral ossification is a developmental process in the skeletal system and bone marrow of vertebrates. During endochondral ossification, primitive cartilaginous anlages derived from mesenchymal stem cells (MSCs) undergo vascular invasion and ossification. In vitro regeneration of endochondral ossification is beneficial for research on the skeletal system and bone marrow development as well as their clinical aspects. However, to achieve the regeneration of endochondral ossification, a stem cell-based artificial cartilage (cartilage organoid, Cart-Org) that possesses an endochondral ossification phenotype is required. Here, we modified a conventional 3D culture method to create stem cell-based Cart-Org by mixing it with a basement membrane extract (BME) and further characterized its chondrogenic and ossification properties. BME enlarged and matured the bone marrow MSC-based Cart-Orgs without any shape abnormalities. Histological analysis using Alcian blue staining showed that the production of cartilaginous extracellular matrices was enhanced in Cart-Org treated with BME. Transcriptome analysis using RNA sequencing revealed that BME altered the gene expression pattern of Cart-Org to a dominant chondrogenic state. BME triggered the activation of the SMAD pathway and inhibition of the NK-κB pathway, which resulted in the upregulation of SOX9, COL2A1, and ACAN in Cart-Org. BME also facilitated the upregulation of genes associated with hypertrophic chondrocytes (IHH, PTH1R, and COL10A1) and ossification (SP7, ALPL, and MMP13). Our findings indicate that BME promotes cartilaginous maturation and further ossification of bone marrow MSC-based Cart-Org, suggesting that Cart-Org treated with BME possesses the phenotype of endochondral ossification.