Chondrocytes in the endochondral growth cartilage are not hypoxic

Abstract

From an anatomic viewpoint, the blood supply to chondrocytes in the growth cartilage is limited. As a result, it has been suggested that these cells are hypoxic and that this condition regulates chondrocyte maturation and cartilage mineralization. We examined the state of chondrocyte oxygenation in the chick growth plate using a hypoxia-sensing drug, EF5. EF5 is a pentafluorinated derivative of the 2-nitroimidazole, etanidazole, that is metabolically reduced by oxygen-inhibitable nitroreductase(s). Reduced EF5 covalently forms adducts with cellular macromolecules that can be visualized with a highly specific fluorochrome-conjugated monoclonal antibody. When EF5 was injected into chicks and tissues were subsequently examined by immunohistochemical techniques, chondrocytes in articular, proliferating, and hypertrophic cartilage exhibited a low level of fluorescence-detectable binding, suggesting the absence of significant hypoxia. We confirmed that the results were not confounded by tissue-specific factors related to low-chondrocyte nitroreductase activity or problems from drug diffusion into cartilage. Using in vitro systems, we showed that, under conditions of imposed hypoxia, EF5 diffused into the tissue and was bound to chondrocytes. With the use of an in vivo model in which hypoxia was artificially induced by death, chick chondrocytes were found to bind the drug. Although the EF5-binding method is not optimally suited for determining the precise oxygen partial pressure in heterogeneous tissues, such as the growth plate, we concluded that chick chondrocytes are not oxygen deficient in vivo.