Cell- and animal-based models of bone formation reveal novel mechanisms involved in the cartilage-to-bone phenotype transition

Animal studies have shown that some cartilage cells can transition to a bone-like phenotype, challenging the belief that bone cells arise solely from stem cells in the bone marrow and growth plate. However, the molecular mechanisms driving this process remain unclear. Researchers have now developed in vitro and in vivo models of bone formation that enable tracking of cartilage-to-bone transition, providing new insights into the mechanisms and signaling pathways involved in cartilage-derived bone formation.

The skeletal system, composed primarily of bone and cartilage, forms the structural framework of vertebrates. Progenitor and stem cells located in the bone growth plate and bone marrow give rise to cells that form bone and cartilage tissues. Long bones develop through a process called ‘endochondral ossification’, traditionally described as a process in which stem cells first differentiate into chondrocytes that form a cartilage template, which is later replaced by bone-forming osteoblasts.

However, recent evidence suggests that not all chondrocytes undergo cell death during bone formation. Lineage-tracing studies in developing mice have shown that a subset of chondrocytes can transition into cells with a bone-like phenotype. As a result, the newly formed bone may be chimeric, arising from both bone-marrow-derived progenitor cells and cartilage cells that have undergone this phenotypic transition. However, most of these findings have been derived from mouse studies, and the molecular mechanisms underlying the cartilage-to-bone phenotype transition during bone formation remain poorly understood.

To bridge this gap, an international team of researchers developed in vitro and in vivo tools capable of modeling bone formation, enabling them to track the fate of cartilage cells.

“We generated a series of modelling tools and methods that, together with reporter mouse models, helped us define the molecular events triggering chondrocyte-derived osteoblasts formation to identify the key signaling pathways and transcription factors related to this process,” explains Dr. Ander Abarrategi, a research scientist at the Department of Cell Biology and Histology, University of the Basque Country, Spain, and the corresponding author of the study. Their findings were published in volume 14 of Bone Research on February 9, 2026.

The researchers examined long bones from mice using conventional histological techniques to characterize the cellular architecture of the bone growth plate and bone marrow during late developmental stages. Notably, their analysis revealed cartilage extracellular matrix markers within elongated structures that extended into the trabecular and cortical bone, suggesting the persistence of non-resorbed cartilage tissue.

Next, the researchers extracted cartilage progenitor cells from mouse pups and implanted fluorescent tagged cells subcutaneously in secondary mice. This allowed them to track the fate of the implanted cells over time. Using computed tomography, the researchers observed the formation of calcified tissue at the implant sites, suggesting that the implanted cartilage cells contributed to bone formation.

In vitro, the researchers mimicked the cartilage-derived bone formation process by sequentially inducing cartilage and bone differentiation of chondrogenic progenitor cells. Notably, this sequential differentiation led to the formation of calcified extracellular matrix in pellets of the progenitor cells, a feature that was not observed when only bone differentiation was induced. Gene expression analysis further supported this progression, with an initial increase in cartilage marker genes followed by a rise in bone marker expression.

Among the signaling pathways examined, MAPK, NOTCH, and BMP signaling pathways were the most significantly modulated during the early stages of the cartilage-to-bone transition. The researchers also identified Mesp1, Alx1, Grhl3, and Hmx3 as key transcription factors driving this transition. Silencing these genes disrupted the bone formation process, further confirming their critical role in regulating the cartilage-to-bone phenotype switch.

“To our knowledge, no direct or well-established interactions among these genes have been reported. Nevertheless, they participate in overlapping developmental pathways and stages. Our data suggest that their coordinated expression and function may be indispensable for successful cartilage-to-bone transition, leading to tissue formation and vascularization,” adds Dr. Abarrategi.

Overall, the study provides new evidence on the cartilage-to-bone transition process, offering insights that could help guide future research in bone development and regenerative medicine.