Tissue-engineered hypertrophic chondrocyte grafts enhanced long bone repair

Bone has innate ability to regenerate following injury. However, large and complex fractures exceed bone's natural repair capacity and result in non-unions, requiring external intervention to facilitate regeneration. One potential treatment solution, tissue-engineered bone grafts, has been dominated by recapitulating intramembranous ossification (bone formation by osteoblasts), although most serious bone injuries heal by endochondral ossification (bone formation by remodeling of hypertrophic cartilaginous anlage). The field has demonstrated that using endochondral ossification-based strategies can lead to bone deposition. However, stem cell differentiated hypertrophic chondrocytes, the key cell type in endochondral ossification, have not been studied for long bone defect repair. With translation in mind, we created tissue-engineered grafts using human adipose stem cells (ASC), a clinically relevant stem cell source, differentiated into hypertrophic chondrocytes in decellularized bone scaffolds, and implanted these grafts into critical-size femoral defects in athymic rats. Over 12 weeks of implantation, these grafts were compared to acellular scaffolds and grafts engineered using ASC-derived osteoblasts. Grafts engineered using hypertrophic chnodrocytes recapitulated endochondral ossification, as evidenced by the expression of genes and proteins associated with bone formation. Markedly enhanced bone deposition was associated with extensive bone remodeling and the formation of bone marrow, and with the presence of pro-regenerative M2 macrophages within the hypertrophic grafts. As a result, hypertrophic chondrocyte grafts bridged 7/8 defects, as compared to only 1/8 for osteoblast grafts and 3/8 acellular scaffolds. These data suggest that ASC-derived hypertrophic chondrocytes in osteogenic scaffolds can improve long bone repair.