Figure 1. Stages in the development of the secondary ossification center in the proximal tibial epiphysis of 4-21 day old rats (modified from 2).Before the center development begins, the epiphysis is solidly cartilaginous A.. Each one of the four panels (B-E) represents one of the successive stages in center development. The sites undergoing "ossification-dependent" cartilage resorption are indicated by the simple broken line under which are shown small ascending white arrows. (The broken line signifies true interruptions between adjacent remodeling sites). Alternatively, the sites undergoing "independent" cartilage resorption are continuous as indicated by the black line above which are shown small black descending arrows. (In fact each black line corresponds to a "pre-resorptive layer" which is composed of a modified cartilage that differs from normal cartilage in three ways: chondrocytes are degenerating, proteoglycan particles are rare or absent and collagen fibrils are damaged (2)).

Figure 2. Structure of the metaphyseal border. The metaphyseal border is a narrow region located between the cartilaginous epiphysis and the diaphysis (as depicted in figure 1A), where an "ossification-dependent" cartilage resorption process erodes the epiphyseal cartilage boundary (indicated by small, white arrows). The metaphyseal border is usually described as the zone of interaction between invading capillaries and the cartilage zone rich in hypertrophic chondrocytes. The dark gray signifies calcified cartilage (cc), the light gray, represents the matrix of the cartilaginous epiphysis that is not calcified. A- refers to the framed part - this is a diagrammatic representation of the metaphyseal border. The upper half of the figure is occupied by the hypertrophic chondrocyte (HC) - including-cartilage, with degeneration shown in the lowermost one, named terminal chondrocyte (TC). The lower half of the figure is the region of invading capillaries. In the center, a capillary where EC denotes an endothelial cell, the lumen includes red blood cells (rbc), while on each side, a calcified, longitudinal cartilage septum (cc) appears as a dark-looking band running vertically. At left, an osteoclast (OC) cuts through a longitudinal septum. On the other side of the capillary, two osteoblasts (OB) may be distinguished. At right, between a transverse septum (identified by small arrowheads) and capillary, a cell described under the name "septoclast" has a ruffled apex that erodes nearby cartilage. An enlargement of the cell at right B. shows the elongated shape of the septoclast as well as the abundance of lysosomes (indicated by brown shading). The "metaphyseal border" represents a cartilage remodeling site where the resorption progresses as follows (25; 26). The calcified longitudinal septae, extend from the epiphysis into the metaphysis and are resorbed by osteoclasts although about 50% of their cartilage persists to serve as a substrate for bone deposition (figure 2A) (26). The non-calcified transverse septae that lie directly at the interface are also resorbed (arrowheads, figure 2A), but the cell involved in this resorption was not known. The collective events were those indicative of the "ossification-dependent" cartilage resorption mode.

Figure 3. Septoclasts at the metaphyseal border of stage I epiphysis stained either with anti-cathepsin B antiserum (B and C) or with toluidine blue (D and E). The first row includes three panels, the first one A. has been exposed to non-immune IgG for control. The next panel B. displays the binding of the antiserum whose reaction is revealed by peroxidase as a brown reaction. The brown reaction is indicative of cathepsin B (as pro or active enzyme). The final panel at high power C. identifies three septoclasts (S) containing dot-like brown reactions which have been localized to lysosomes by electron microscopy (3). The white arrows identify a transverse septum that is unstained with the antibodies and is barely visible in the section. The second row's panels (D and E) also display septoclasts (S). This tissue has been exposed first to mixed aldehydes then treated with OsO4 before embedding in Epoxy resin. In D., at center and to the left, endothelial cells (EC) define the boundaries of two independent but adjacent vessels. Identified between the vessels are two septoclasts (S). The identified cells (arrows) have a single nucleus and a cytoplasm rich in lysosomes. The lysosomes, visualized here as blue dots, are varied in size and shape. In panel E. the distinctiveness of the septoclast is further emphasized by comparing its morphology to that of a neighboring osteoclast (OC). cc in E. denotes calcified cartilage. Bar in A = 50 µm (applies to B). Bar in D = 20 µm (applies to C and E).

Figure 4. Three aggrecan molecules anchored on a filament of hyaluronate (vertical line at left; modified from 2). A.. Intact aggrecan molecule composed of an aggrecan core protein attached to the hyaluronate filament. Along the core protein, three globular domains are respectively labeled G1 (bound to the hyaluronate), G2 (separated from G1 by the first "interglobular region" - arrow) and G3 (ending the core protein and separated from G2 by the second "interglobular region, along which are attached many glycosaminoglycan chains, represented by short lines). B. Aggrecan cleaved by a so far unidentified member of the matrix metalloproteinase (MMP) subfamily. The cleavage takes place in the first interglobular region at the N³⁴¹-F³⁴² bond. . C. Aggrecan cleaved by a member of the aggrecanase subfamily The hyaluronate-attached fragment ends in the amino acid sequence NITEGE. Antibodies against a peptide identical to the neoepitope are used to detect the cut and, at the same time, the enzyme subfamily that has made the cut.

Figure 5. Light microscopic immunostaining of the stage I canal blind end region after exposure to various antibodies, whose immunostaining is indicated by a brown reaction. The canal blind end is chiefly composed of the pre-resorptive cartilage layer. In every panel, the canal blind end forms a half circle that is oriented toward the picture top, as emphasized by strong staining in several panels. The canal content mostly consists of cells and capillaries. The first row includes three panels, the first one of which A. has been exposed to non-immune IgGs for control. The next panel B. displays intense immunostaining by anti-FVDIPEN antibodies. The final panel C. displays the binding of anti-NITEGE antibodies, which are directed to the G1-373 fragment. The second row's first panel (D) records immunostaining by anti-MMP-13-proenzyme antibodies. The second panel in the second row (E) shows immunostaining by anti-activated MMP-13 antibodies. Panel F. shows immunostaining by anti-LAGQR antibodies, directed to collagen 2-¼-long fragments. The third row's first panel (G) shows immunostaining by anti-QRGIV antibodies that are directed to the collagen 2-¼-short fragments. Note the immunostaining in panel E. is distributed in exactly the same way as the two types of fragments (F, G) produced when this enzyme cleaves collagen 2. The second panel of this row (H) records immunostaining by antibodies directed to the MMP-9 proenzyme. The canal blind end is not stained. The third row last panel (I) depicts immunostaining by anti-activated MMP-9 antibodies, that is, a fairly strong reaction of the canal blind end indicative of the presence of activated MMP-9 in the pre-resorptive layer. Bar in A = 50 µm (applies to B - I).

Figure 6. Structure of the pre-resorptive layer shown at low A. and high magnification B. A.. Normal cartilage is depicted in solid gray, the pre-resorptive layer is depicted in a dark tone, whereas the canal lumen with its light background occupies the upper half of panel A.. Only shown are two of the structures present in the lumen: a capillary in which an endothelial cell is pointed out and a canalyser cell showing the cytoplasmic components. B. Diagrammatic representation of the fine structure of the regions. (The relative positions of the enlarged regions are shown as inserts in A). B. Normal cartilage consists of gray rods representing collagen fibrils, a fine black filament represents the hyaluronate filaments and finally, bottle brush-like structures is indicative of the aggrecan molecule. The middle rectangle of panel B. depicts the pre-resorptive layer. The fine black filaments representing hyaluronate are easily recognizable, but the aggrecan molecules are no longer present except for a very small piece of them referred to as G1-341 fragment. Finally, the top rectangle depicts the layer's debris. In conclusion, the diagram summarizes the fact that MMPs cleavage of aggrecan core protein has been revealed as a key step in cartilage resorption (2).

Figure 7. A three-step description of the gelatin histozymography method. Before the procedure, "blackened emulsion slides" are prepared by dipping slides into photographic emulsion, such as the Kodak NTB-2 emulsion, exposing them to light for five minutes and finally developing, fixing and drying them. A.. The first step in histozymography. The first step is the cutting of a frozen section and its deposition on transparent Scotch tape 800. In the second step, the tape mounted section with section surface facing down, is deposited on a blackened emulsion slide, as shown at lower left. A blank glass slide such as seen as upper left is placed on top. And finally, the entire slide section sandwich complex is transferred to a humid chamber at 21 ⁰C. Section and emulsion are thus kept in contact for some time. (Much of our work was done using a four hour exposure).B. . The third step is the separation of section and emulsion after the exposure has been terminated. Thus the tape with the attached section is detached from the emulsion and stained using a routine histology stain such as toluidine blue. The section is then mounted on a glass slide in the same orientation as the emulsion image - referred to as histozymogram. Comparison of section and emulsion makes it possible to compare each light spot in the emulsion (indicative of gelatin digestion) with the section site that has produced it and, therefore, contains an enzyme capable of digesting the emulsion gelatin. An example is presented in figure 8.

Figure 8. Histozymograms of rat tibal epiphysis at the four stages in secondary ossification center formation. We have demonstrated that reactive histozymograms are produced by the presence in the adjacent tissue of the enzyme known as gelatinase B or MMP-9 (5, 6). A.. Before center formation begins, a clearly distinct reactive histozymogram corresponds to the junction between epiphysis and diaphysis referred to as "metaphyseal border" (described in figures 1 and 2). The metaphyseal border of corresponding histozymograms can be recognized in every other panel of this figure. B.. Stage I, when canals arise, there are positive, faint pale spots evident on the histozymogram corresponding to the blind ends of the small canals. C. . Stage II, when canals fuse to form the marrow space, there is a gelatin loss from the emulsion at sites corresponding to the distal marrow space wall at right and a large canal at left. D.. Stage III, when the initial signs of ossification appear, there is a reaction on the histozymogram produced along almost all the marrow space wall. E.. Stage IV, when the enlarged marrow space is associated with distinct primary and secondary growth plates, reactions are produced on the histozymogram by the proximal and lateral walls of the marrow space.

Figure 9. Diagram outlining enzyme's life-history. At left, an enzyme producer cell is elaborating the "proenzyme" - a functionally inactive form of the enzyme - which is then secreted by the cell to outside tissue. Eventually the secreted "proenzyme" loses its propeptide domain and thus becomes capable of functional activity as an "activated enzyme". Finally the "activated enzyme" binds to its substrate to carry out its function such as, shown here, peptide bond cleavage. The goal of the "protease neoepitope approach" was to reveal this life-history directly in the tissue.