Active gelatinase B is identified by histozymography in the cartilage resorption sites of developing long bones

In order to determine which proteinases mediate the resorption of endochondral cartilage in the course of long bone development, a novel assay called "histozymography" has been developed. In this assay, frozen sections of tibial head from 21-day-old rats are placed for 4 hr at room temperature on light-exposed photographic emulsion (composed of silver grains embedded in gelatin). We report a localized but complete digestion of emulsion gelatin facing two tissue sites which are, therefore, presumed to contain an active proteinase. One of the sites is localized at the growth plate surface forming the epiphysis/metaphysis interface. The other consists of small patches located within the epiphysis at the edge of the marrow space. Both sites are engaged in the resorption of endochondral cartilage. In both sites, inhibitor tests have established that the involved proteinase is a gelatinase. Furthermore, the use of neutralizing antibodies against gelatinase A or B have demonstrated that only those that are specific for the latter block the reaction. That gelatinase B is present in the two sites has been confirmed by light microscopic immunohistochemistry. Finally, when immunoelectron microscopy is used for fine localization of the cartilage structures that form the epiphysis/metaphysis interface, the enzyme is detected within the 0.5-microm thick edge of the cartilage, and outside the cartilage, it is present in debris composed of type II collagen-rich fibrils in various states of digestion. It is concluded that gelatinase B attacks the edge of an endochondral cartilage and helps to solubilize the type II-collagen-rich fibrillar framework, which is then released as debris for further digestion. This final step opens the way to invasion by capillaries, thereby making possible the replacement of cartilage by bone. Dev Dyn 1999;215:190-205.