Trans-differentiation of hypertrophic chondrocytes into cells capable of producing a mineralized bone matrix

Trans-differentiation of hypertrophic chondrocytes into bone-forming cells was observed when femurs from 14-day-old chick embryos were cut through the region of hypertrophic cartilage and the separated pieces were cultured for 2-18 days. Inside many chondrocytic lacunae a new matrix was present which had the staining characteristics of bone matrix including birefringence and the capacity to mineralize. The cells within the lacunae had the characteristics of osteoblasts, such as alkaline phosphatase activity and positive immunocytochemical staining for osteocalcin, osteonectin, osteopontin and type I collagen. Chondrocyte necrosis and empty lacunae were only observed immediately at the cut edge, and in that region no bone-forming cells were present inside the lacunae. Where bone-matrix was present, the lacunae had remained intact, the cells were viable and no evidence of cell migration was observed. This suggested that the bone-forming cells had originated from the hypertrophic chondrocytes. The temporal sequence of events was followed closely. Two days following the cut only a few chondrocytes showed a positive reaction for osteocalcin, osteonectin, osteopontin and the type I collagen. At that time no such reaction product was observed in the chondrocytes of uncut femurs. Many hypertrophic chondrocytes divided, as shown by tritiated thymidine incorporation. The rate of cell division increased between 2-6 days, when several smaller basophilic cells were present inside the lacuna instead of the single hypertrophic chondrocyte. These cells expressed alkaline phosphatase activity, were positive for fibronectin, the above non-collagenous bone proteins and type I collagen. The bone matrix that was observed after 6-18 days was initially confined to the inside of the chondrocytic lacunae, but later spread beyond the lacunar confines. The bone proteins were still associated with the bone-forming cells, but fibronectin was absent when matrix formation was evident. Mineralization of the intra-lacunar osteoid took place after 12-18 days. It is speculated that the trans-differentiation was initiated by disruptions of the normal cell-cell associations.