(A) Diagram of the growth plate showing different cartilage zones.
(B) Histological sections of 2-week-old metatarsals stained with hematoxylin/fast green/safranin O. (ep) epiphysis; (c) reserve, proliferation, and maturation zones; (hc) hypertrophic cartilage; (tb) trabecular bone; (bm) bone marrow. Scale bar = 200 μm.
(C) Histological sections of 3-week-old metatarsals. Magnified view on the right lower panel shows cortical bone surrounding the hypertrophic cartilage in gelatinase B–null mice (outlined by white arrows). Black arrows point to capillaries invading from the cortical bone. (hc) hypertrophic cartilage; (tb) trabecular bone.
(D) Histological sections of 4-week-old metatarsals showing aberrant ossification process in the gelatinase B–null mice (arrows in upper right panel). The lower left panel shows a bone whose hypertrophic cartilage has been almost completely ossified giving the appearance of osteopetrosis (arrows). Magnified view on the lower right panel shows a vessel invading from the side (arrow).
(E) Von Kossa staining of 2-week-old metatarsal growth plates. Calcium deposits stain black.
(F) Histological sections of 8-week-old metatarsals.

(A and B) Propidium iodide (PI) and ApopTag (Apop) staining of 1- and 2-week-old metatarsal growth plates from wild-type and gelatinase B–null mice. Arrows point to terminal hypertrophic chondrocytes that are apoptotic. Apoptotic cells are also present in the bone marrow, perichondrium, and periosteum. (hc) hypertrophic cartilage.
(C and D) PI and Apop staining of 3-week-old (C) and 4-week-old (D) metatarsal growth plates. Arrows in the gelatinase B–null growth plates show apoptotic chondrocytes in the middle of the hypertrophic cartilage at three weeks (C) or in the vascularized and ossified hypertrophic cartilage at 4 weeks (D). Scale bars = 200 μm.

(A) Histological sections of metatarsals from untransplanted and bone marrow (BM) transplanted mice at 3 and 4 weeks after transplantation. Arrow points to area of ectopic ossification in the untransplanted 4-week-old growth plate. Scale bar = 200 μm.
(B) TRAP staining and in situ hybridization with an antisense mRNA gelatinase B probe showing that gelatinase B–expressing cells, some of which correspond to TRAP-positive cells, appear at the cartilage–bone junction and on the trabecular bone surfaces after transplantation (arrows).

(A) Genomic organization of the wild-type and targeted alleles after homologous recombination. The location of the fragment used as a probe in Southern blotting is shown, as well as the sizes of the two XbaI fragments detected for wild-type and targeted alleles. (B) Southern blot analysis of genomic DNA. Fragments corresponding to wild-type (8 kb) and targeted (5 kb) alleles are shown. (C) Gelatin zymographic analysis of speen cell lysates. Gelatinolytic activity corresponding to gelatinase B is shown.

Left column panels show metatarsals from 2-week-old wild type and right column panels show those from gelatinase B–null mice. Cells that have incorporated BrdU stain brown. Arrow points to the secondary site of osssification. Zones of expression of Indian hedgehog (ihh), bone morphogenetic protein-6 (bmp-6), collagen type II (col2A1), and collagen type X (colXA1) are shown by dark-field photography of in situ hybridization with antisense mRNA probes. (hc) hypertrophic cartilage. Scale bar = 200 μm.

(A–D) Bright-field (A and B) and dark-field (C and D) photographs of a section of 3-week-old wild-type metartarsal growth plate hybridized with a gelatinase B antisense mRNA probe. Arrows point to gelatinase B expressing cells at the cartilage–bone junction. (B) and (D) are magnified views of (A) and (C). (ep) epiphysis. Scale bar = 100 μm.
(E and F) TRAP staining of an adjacent section (E) and magnified view (F) showing TRAP positive (red) cells at the cartilage–bone junction (white arrows) and on the trabecular bone surfaces (black arrows). Note the distribution of TRAP-positive cells is similar to that of gelatinase B–expressing cells.
(G and H) Immunohistochemistry of 3-week-old wild type metatarsal growth plate with an antibody to gelatinase B, showing staining at the cartilage bone juction and trabecular surfaces. Magnified view (H) shows staining of ECM and stellate cells (arrow).
(I and J) Immunohistochemistry of 3-week-old wild-type and gelatinase B–null metatarsal growth plates with an antibody to PECAM-1 showing staining of blood vessels at the cartilage–bone junction (white arrows) and in between trabecular bones (black arrows).

(A–C) Hypertrophic cartilage from 2-week-old wild-type (A) and gelatinase B–Null (B) mice after one week in a collagen gel coculture with endothelial cells, showing an angiogenic response from the endothelial cells only to wild-type cartilage. Magnified view (C) shows tubular structures formed by endothelial cells surrounding the wild-type hypertrophic cartilage.
(D) Gelatinase B–Null hypertrophic cartilage after 2.5 weeks in coculture with endothelial cells, showing an angiogenic response. (E) Wild-type and gelatinase B–Null cartilage after one week side by side in coculture with endothelial cell, showing angiogenic response only to wild-type cartilage that is not abrogated by the presence of the gelatinase B–Null cartilage.