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 endochond ral cartilage in the course of long bone development, a novel assay called "histozymography" has been developed. In this assay, frozen sections of tib ial head from 21-day-old rats are placed for 4 hr at room. temperature on l ight-exposed photographic emulsion (composed of silver grains embedded in g elatin), We report a localized but complete digestion of emulsion gelatin f acing two tissue sites which are, therefore, presumed to contain an active proteinase. One of the sites is localized at the growth plate surface formi ng the epiphysis/metaphysis interface. The other consists of small patches located within the epiphysis at the edge of the marrow space. Both sites ar e engaged in the resorption of endochondral cartilage. In both sites, inhib itor tests have established that the involved proteinase is a gelatinase, F urthermore, the use of neutralizing antibodies against gelatinase A or B ha ve 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 mic roscopy is used for fine localization of the cartilage structures that form the epiphysis/metaphysis interface, the enzyme is detected within the 0.5- mu m 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 di gestion. It is concluded that gelatinase B attacks the edge of an endochond ral cartilage and helps to solubilize the type II-collagen-rich fibrillar f ramework, which is then released as debris for further digestion, This fina l step opens the way to invasion by capillaries, thereby making possible th e replacement of cartilage by bone, (C) 1999 Wiley-Liss, Inc.