Hypertrophic chondrocytes contribute directly to the osteoblast and osteocyte lineage in endochondral bones in vivo

Abstract

It is widely accepted that in vertebrates, bone formation occurs via one of two processes - membranous bone formation in which osteoblasts directly differentiate from mesenchymal cells or via endochondral ossification (EO), a multistep process wherein chondrocytes differentiate from mesenchymal condensations, to form a cartilaginous template, proliferate, exit the cell cycle to undergo hypertrophy and terminal differentiation. Vascular invasion occurs, and hypertrophic chondrocytes (HCs) are thought to undergo apoptosis, while bone is laid down by osteoblasts from the periosteum and bone collar surrounding the hypertrophic zone, replacing cartilage. However whether in vivo, all HCs undergo apoptosis in EO or can become osteoblasts has been a subject of considerable controversy. To address this controversy we have followed the fate of HCs using the Cre-loxP system in mice. We used homologous recombination to generate two cre lines, Col10a1-cre and Col10a1-creERTM, which express Cre recombinase under the control of HC-specific Col10a1 gene. We genetically tagged HCs by crossing Col10a1-cre mice to Cre-reporter mice (Rosa-26R-LacZ or Rosa-26R-YFP) and performed cell-lineage analyses to track the fate of HCs in fetal and postnatal stages. These experiments in combination with pulse-chase experiments using tamoxifen induction of Cre activity in Col10a1-creERTM; Rosa-26R-LacZ mice, show that in vivo, HCs contribute directly to the osteoblast and osteocyte lineage of ALL endochondral bones. These osteoblasts of HC origin (HCObs) contribute to about a third of bone cells in long bones. In a bone-injury model we further show that HCObs contribute to new bone formation in the healing process. These discoveries impact on our current understanding of the origin of bone cells and have translational implications for regenerative medicine.