Osteoclast Incorporation in an <i>In Vitro</i> 3D Model of Endochondral Ossification

<i>In vitro</i> models aim to recapitulate human physiological processes, improving upon and replacing the need for animal-based models. Modeling bone formation via endochondral ossification <i>in vitro</i> is a very complex process due to the large number of cell types involved. Most current models are limited to mimicking the initial stages of the process (i.e., cartilage template formation and mineralization of the matrix), using a single cell type. Chondroclasts/osteoclasts are key players in cartilage resorption during endochondral ossification, but their introduction into <i>in vitro</i> models has thus far proven challenging. In this study, we aimed toward a new level of model complexity by introducing human monocyte-derived osteoclasts into 3D <i>in vitro-</i>cultured cartilage templates undergoing mineralization. Chondrogenic and mineralized chondrogenic pellets were formed from human pediatric bone marrow stromal cells and cultured in the presence of transforming growth factor-β3 (TGF-β) and TGF-β/β-glycerophosphate, respectively. These pellets have the capacity to form bone if implanted <i>in vivo.</i> To identify suitable <i>in vitro</i> co-culture conditions and investigate cell interactions, pellets were co-cultured with CD14+ monocytes in an indirect (transwell) or direct setting for up to 14 days, and osteoclastogenesis was assessed by means of histological stainings, osteoclast counting, and gene expression analysis. Upon direct co-culture, we achieved effective osteoclast formation <i>in situ</i> in regions of both mineralized and unmineralized cartilages. Notably, <i>in vitro</i>-generated osteoclasts showed the ability to form tunnels in the chondrogenic matrix and infiltrate the mineralized matrix. Addition of osteoclasts in human <i>in vitro</i> models of endochondral ossification increases the physiological relevance of these models. This will allow for the development of robust 3D human <i>in vitro</i> systems for the study of bone formation, disease modeling, and drug discovery, further reducing the need for animal models in the future. Impact Statement <i>In vitro</i> bone formation models of endochondral ossification are currently limited to the recapitulation of the initial stages of the process. In this article, we present a novel <i>in vitro</i> endochondral ossification model where osteoclasts were incorporated into mineralized hypertrophic cartilage templates, adding a new level of complexity toward the modeling of cartilage resorption during endochondral ossification.