Development of a Complex Human In Vitro Model of Endochondral Ossification.

During development and regeneration, bone is formed by endochondral ossification (EO) through the remodeling of a cartilage template. This complex process involves multiple cell types and interactions that cannot currently be modeled in vitro. This study aimed to develop a novel tissue-engineered human in vitro model of certain aspects of the early stages of EO by integrating cartilage which undergoes mineralization, self-assembled vascular networks, and osteoclasts into a single system. We first studied the dynamics of osteoclastogenesis and vascularization in an in vivo model of stromal cell-mediated EO, to inform our in vitro system. Next, we aimed to develop a fully human cell-based three-dimensional model of EO by combining pediatric bone marrow stromal cells differentiating into chondrocytes, osteoclasts derived from human CD14+ monocytes, and human umbilical vein endothelial cells and adipose-derived stromal cells as vessel-forming cells. We investigated how mineralizing cartilage affects osteoclast and vessel formation in vitro through separate cartilage-osteoclasts and cartilage-vessels cocultures. Finally, we combined these elements and established a complex in vitro model that supports the functionality of all these cell types and recapitulates chondrogenesis, cartilage mineralization, vessel formation and osteoclastogenesis. This integrated approach reaches unprecedented complexity and will enable new tissue engineering strategies to model skeletal diseases or cancer metastasis to the bone.