Osteoclast Incorporation in an In Vitro 3D Model of Endochondral Ossification

In vitro models aim to recapitulate human physiological processes, improving upon and replacing the need for animal-based models. Modeling bone formation via endochondral ossification in vitro 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 in vitro 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 in vitro-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 in vivo. To identify suitable in vitro 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 in situ in regions of both mineralized and unmineralized cartilages. Notably, in vitro-generated osteoclasts showed the ability to form tunnels in the chondrogenic matrix and infiltrate the mineralized matrix. Addition of osteoclasts in human in vitro models of endochondral ossification increases the physiological relevance of these models. This will allow for the development of robust 3D human in vitro systems for the study of bone formation, disease modeling, and drug discovery, further reducing the need for animal models in the future. Impact Statement In vitro 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 in vitro 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.