Modulation of human adipose derived stromal cell chondrogenesis for controlled endochondral ossification and efficient bone formation

Endochondral ossification (ECO), is the primary ossification process during embryogenesis and fracture repair. It involves the formation of a cartilaginous tissue (CT) intermediate which progressively remodels into a mature bone organ. Adipose-derived stromal cells (ASC), non-skeletal multipotent progenitors derived from the stromal vascular fraction (SVF) of human adipose tissue, hold promise for bone regeneration via ECO. Few studies have shown the potential of ASC to recapitulate ECO and form a bone organ in vivo when pre-differentiated into cartilage tissue in vitro. However, the reproducibility of this process remains a challenge and the major triggers are not fully understood. Furthermore, ex vivo processing and expansion of ASC negatively impact their functionality. Therefore, a deeper understanding of ASC response to chondrogenic cues in vitro and how they modulate their microenvironment in vivo is crucial. Thus, this thesis aims to get better insights into the different stages of ASC chondrogenesis in vitro to ultimately control the associated endochondral ossification process and therefore, enhance their bone-forming capacity. We found that both SVF-cells and expanded-ASC can reproducibly generate cartilage tissue (CT) with varying degrees of maturation in a time-dependent manner. However, SVF cells exhibited accelerated maturation compared to expanded-ASC. Proteomic analysis revealed a quiescent state of SVF-ASC characterized by enrichment in lipid metabolism pathways, which transitions to a proliferative state upon monolayer expansion. We also showed that the metabolic signature can be restored by inducing quiescence at the end of the expansion phase. Furthermore, we could preserve and enhance the chondrogenic potential during monolayer expansion of ASC by modulating their metabolic signature (by quiescence induction) or by modulating their TGFb3 receptors expression profile (by TGFβ3 supplementation). Our findings indicate that the host response, specifically osteoclast recruitment, is dependent on the maturation level of the implanted CT, leading to various remodelling outcomes. Consequently, we identified a specific in vitro maturation window that leads to endochondral ossification and ultimately the formation of a mature bone organ in vivo. Moreover, we demonstrated that adipose-derived hypertrophic cartilage grafts (Adiscaf) outperformed clinical standard biomaterials in maxillofacial surgery, exhibiting enhanced bone formation and osteointegration. In summary, this work provides a detailed characterization of the stages of ASC endochondral ossification, highlighting the critical roles of monolayer expansion and chondrogenic maturation in modulating ECO. These findings offer a simple, yet reproducible and effective strategy for enhanced bone formation that warrants further investigation in clinical settings.