Title: Engineering Hypertrophic Chondrocyte-based Grafts for Enhanced Bone Regeneration
Description: Bone formation occurs through two ossification processes, intramembranous and endochondral. Intramembranous ossification is characterized by the direct differentiation of stem cells into osteoblasts, which then create bone. Endochondral ossification involves an intermediate step, as stem cells first differentiate into chondrocytes and produce a cartilage anlage. The chondrocytes mature into hypertrophic chondrocytes, which transform the cartilage anlage into bone. Bone tissue engineering has predominantly mimicked intramembranous ossification, creating osteoblast-based grafts through the direct differentiation of stem cells. Though successful in specific applications, greater adoption of osteoblast-based grafts has failed due to incomplete integration, limited regeneration, and poor mechanical maintenance. To overcome these obstacles, inspiration was drawn from native bone fracture repair, creating tissue engineered bone grafts replicating endochondral ossification. Hypertrophic chondrocytes, the key cell in endochondral ossification, were differentiated from mesenchymal stem cell sources by first generating chondrocytes and then instigating maturation to hypertrophic chondrocytes.

Conditions influencing this differentiation were investigated, indicating the necessity of prolonged chondrogenic cultivation and elevated oxygen concentrations to ensure widespread hypertrophic maturation. Comparing the bone production performance of differentiated hypertrophic chondrocytes to differentiated osteoblasts revealed that hypertrophic chondrocytes deposit significantly greater volume of bone mineral at a higher density than osteoblasts, albeit in a more juvenile form. When implanted subcutaneously, the hypertrophic chondrocytes stimulated turnover of this juvenile template into compact-like bone, whereas osteoblasts proceeded with processes similar to bone remodeling, generating spongy-like bone. Implanting these tissue engineered constructs into an orthotopic, critical-sized femoral defect saw hypertrophic chondrocyte-based constructs integrate quickly with the femur and facilitate the creation of significantly more bone, resulting in a successful bridging of the defect. The success of hypertrophic chondrocyte-based grafts in overcoming the failures of tissue engineered bone grafts demonstrates the potential of endochondral ossification inspired bone strategies and prompts its further investigation towards clinical utilization.