Although bone has an innate capacity for repair, clinical situations such as comminuted fracture, open fracture, or the surgical resection of bone tumors produce critical-sized bone defects that exceed the capacity and require external intervention. Initiating endochondral ossification (EO) by the implantation of a cartilaginous template into the bone defect is a relatively new approach to cure critical-sized bone defects. The combination of chondrogenically primed mesenchymal stromal/stem cells and artificial scaffolds has been the most extensively studied approach for inducing endochondral bone formation in bone defects. In this study, we prepared cartilage (human-induced pluripotent stem [hiPS]-Cart) from hiPS cells (hiPSCs) in a scaffoldless manner and implanted hiPS-Cart into 3.5 mm large defects created in the femurs of immunodeficient mice to examine the repair capacity. For the control, nothing was implanted into the defects. The implantation of hiPS-Cart significantly induced more new bone in the defect compared with the control. Culture periods for the chondrogenic differentiation of hiPSCs significantly affected the speed of bone induction, with less time resulting in faster bone formation. Histological analysis revealed that hiPS-Cart induced new bone formation in a manner resembling EO of the secondary ossification center, with the cartilage canal, which extended from the periphery to the center of hiPS-Cart, initially forming in unmineralized cartilage, followed by chondrocyte hypertrophy at the center. In the newly formed bone, the majority of osteocytes, osteoblasts, and adipocytes expressed human nuclear antigen (HNA), suggesting that these types of cells mainly derived from the perichondrium of hiPS-Cart. Osteoclasts and blood vessel cells did not express HNA and thus were mouse. Finally, integration between the newly formed bone and mouse femur was attained substantially. Although hiPS-Cart induced new bone that filled bone defects, the newly formed bone, which is a hybrid of human and mouse, had not remodeled to mature bone within the observation period of this study (28 weeks). Impact statement Although bone has an innate capacity for repair, critical-sized bone defects that exceed the capacity require external intervention. We prepared cartilage (human-induced pluripotent stem [hiPS]-Cart) from hiPS cells (hiPSCs) in a scaffoldless manner and examined whether implantation of hiPS-Cart heals critical-sized defects created in the femurs of immunodeficient mice. The implantation of hiPS-Cart induced new bone in the defect in a manner resembling endochondral bone formation of the secondary ossification center. Although hiPS-Cart induced new bone that filled bone defects, the newly formed bone, which is a hybrid of human and mouse, had not remodeled to mature bone within the observation period of this study (28 weeks).
Keywords: bone defect; cartilage; endochondral bone formation; fracture; induced pluripotent stem cells; regeneration.