Vitamin-D-dependent calcium-binding protein (CaBP-9K) in rat growth cartilage

Summary The presence of vitamin-D-dependent calcium-binding protein (CaBP-9K) in tibial growth-plate cartilage was immunohistochemically demonstrated using a specific antibody to rat duodenal CaBP-9K. The protein was found to be mainly localized in the cytoplasm of maturing chondrocytes. In hypertrophic chondrocytes, CaBP-9K concentrations decreased, and the protein was found in the cytoplasmic processes. No CaBP-specific immunoreactivity was seen in the hypertrophic chondrocytes of the lower calcified hypertrophic zone; in contrast, the protein was found in the extracellular lateral edges of longitudinal septa, i.e. where matrix vesicles are preferentially localized and where cartilage mineralization is initiated. These findings suggest that vitamin D has a direct function in this tissue. It also seems likely that CaBP-9K is an indicator of chondrocyte maturation, and that it is involved in the matrix vesicle-associated process of cartilage calcification.     

Evidence for the presence of Calbindin-D 28K (CaBP-28K) in the tibial growth cartilages of rats

Summary The distribution of the vitamin-D dependent calcium-binding protein (Calbindin-D 28K) (CaBP-28K) in the tibial growth plate cartilage of the rat has been studied immunohistochemically using an antibody raised against rat renal CaBP-28K. The protein was detected mainly in the nuclei of chondrocytes and occasionally in the juxta-nuclear cytoplasm. The distribution was not uniform throughout the growth plate, but concentrated in the proliferatively active chondrocytes of the resting and proliferative zones. These findings raise the possibility that CaBP-28K may be involved in the mitotic activity of the chondrocytes, acting as a regulator of the proliferative process, perhaps via intranuclear calcium.     

Immunohistochemical detection of cathepsin D, K, and L in the process of endochondral ossification in the human

Cathepsins D, K, and L were immunolocalized in tissue undergoing endochondral ossification in the human. Cathepsins D, K, and L were localized in osteoclasts and chondroclasts attached to bone matrix and cartilage matrix, respectively. Cathepsins D and L were immunostained in chondrocytes. Immunolocalization of cathepsin D was limited to hypertrophic chondrocytes adjacent to the osteochondral junction. In contrast, cathepsin L was immunolocalized in both proliferating and hypertrophic chondrocytes. In the bone marrow space, cathepsins D, K, and L were localized in multinucleated cells. Cathepsin D was diffusely detected in mononuclear bone marrow cells which were negative for cathepsins K and L. The present findings indicated that cathepsins K, D, and L were associated with the process of endochondral ossification in the human, and suggested that these cathepsins share roles in bone and cartilage turnover in the human.     

Persistence of cartilage proteoglycan and link protein during matrix-induced endochondral bone development: An immunofluorescent study

Monospecific antibodies to cartilage proteoglycan monomer and link protein were employed with immunofluorescence microscopy to determine the tissue distribution of these constituents during matrix-induced endochondral bone development. Subcutaneous implantation of demineralized diaphyseal bone matrix resulted in new endochondral bone formation. On Day 3, the implant consisted of mesenchymal tissue which did not contain any demonstrable cartilage-related proteoglycan or link protein. With the onset of early chondrogenesis on Day 5, cartilage proteoglycan monomer and link protein were first localized together in the cartilage matrix, particularly around chondrocytes in territorial sites. Progressively more staining around cells was observed at Days 7 and 9. On Day 9, when mineralization was first observed, there was no evidence of a net loss of these molecules prior to mineralization of the cartilage matrix. On Day 11 and thereafter, bone formation was observed by appositional growth on calcified cartilage spicules. Whereas the osteoblasts and bone matrix were devoid of any staining for cartilage proteoglycan and link components, the residual, partly mineralized cartilage spicules still reacted with antibodies to cartilage proteoglycan monomer and link protein in territorial sites, but in reduced amounts, indicating a loss of these molecules associated with a loss of hypertrophic chondrocytes. Since mineral prevented the access of Fab' antibody subunits, demineralization after fixation was routinely employed. The results reveal that cartilage proteoglycan monomer and link protein are present around chondrocytes in hyaline cartilage during the early stages of endochondral bone formation and that there is no net loss of these molecules prior to mineralization of this cartilage matrix as was previously thought.     

Association of an extracellular protein (chondrocalcin) with the calcification of cartilage in endochondral bone formation

We examined bovine fetal epiphyseal and growth plate cartilages by immunofluorescence microscopy and immunoelectron microscopy using monospecific antibodies to a newly discovered cartilage-matrix calcium-binding protein that we now call chondrocalcin. Chondrocalcin was evenly distributed at relatively low concentration in resting fetal epiphyseal cartilage. In growth plate cartilage, it was absent from the extracellular matrix in the zone of proliferating chondrocytes but was present in intracellular vacuoles in proliferating, maturing and upper hypertrophic chondrocytes. The protein then disappeared from the lower hypertrophic chondrocytes and appeared in the adjoining extracellular matrix, where it was selectively concentrated in the longitudinal septa in precisely the same location where amorphous mineral was deposited in large amounts as demonstrated by von Kossa staining and electron microscopy. Mineral then spread out from these "nucleation sites" to occupy much of the surrounding matrix. Matrix vesicles were identified in this calcifying matrix but they bore no observable morphological relationship to these major sites of calcification where chondrocalcin was concentrated. Since chondrocalcin is a calcium-binding protein and has a strong affinity for hydroxyapatite, these observations suggest that chondrocalcin may play a fundamental role in the creation of nucleation sites for the calcification of cartilage matrix in endochondral bone formation.     

Age-related changes in the expression of gelatinase and tissue inhibitor of metalloproteinase genes in mandibular condylar,growth plate,and articular cartilage in rats

Summary Mandibular condylar cartilage acts as both articular and growth plate cartilage during growth, and then becomes articular cartilage after growth is complete. Cartilaginous extracellular matrix is remodeled continuously via a combination of production, degradation by matrix metalloproteinases (MMPs), and inhibition of MMP activity by tissue inhibitors of metalloproteinases (TIMPs). This study attempted to clarify the age-related changes in the mRNA expression patterns of MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 in mandibular condylar cartilage in comparison to tibial growth plate and articular cartilage using an in situ hybridization method in growing and adult rats. MMP-2 and MMP-9 were expressed in a wide range of condylar cartilage cells during growth, and their expression domains became limited to mature chondrocytes in adults. The patterns of TIMP-1 and TIMP-2 expression were similar to those of MMP-2 and MMP-9 during growth, and were maintained until adulthood. TIMP-3 was localized to hypertrophic chondrocytes throughout the growth stage. Therefore, we concluded that TIMP-1 and TIMP-2 were general inhibitors of MMP-2 and MMP-9 in condylar cartilage, while TIMP-3 regulates the collagenolytic degradation of the hypertrophic cartilage matrix.     

Biochemical and immunohistochemical evidence that in cartilage an alkaline phosphatase is a Ca2+-binding glycoprotein

A glycoprotein that exhibits alkaline phosphatase activity and binds Ca2+ with high affinity has been extracted and purified from cartilage matrix vesicles by fast protein liquid chromatography. Antibodies against this glycoprotein were used to analyze its distribution in chondrocytes and in the matrix of calcifying cartilage. Under the light microscope, using immunoperoxidase or immunofluorescence techniques, the glycoprotein is localized in chondrocytes of the resting zone. At this level, the extracellular matrix does not show any reaction. In the cartilage plate, between the proliferating and the hypertrophic region, a weak immune reactivity is seen in the cytoplasm, whereas in the intercolumnar matrix the collagen fibers appear clearly stained. Stained granular structures, distributed with a pattern similar to that of matrix vesicles, are also visible. Calcified matrix is the most stained area. These results were confirmed under the electron microscope using both immunoperoxidase and protein A-gold techniques. In parallel studies, enzyme activity was also analyzed by histochemical methods. Whereas resting cartilage, the intercellular matrix of the resting zone, and calcified matrix do not exhibit any enzyme activity, the zones of maturing and hypertrophic chondrocytes are highly reactive. Some weak reactivity is also shown by chondrocytes of the resting zone. The observation that this glycoprotein (which binds Ca2+ and has alkaline phosphatase activity) is synthesized in chondrocytes and is exported to the extracellular matrix at the time when calcification begins, suggests that it plays a specific role in the process of calcification.     

Galectin-3 is a downstream regulator of matrix metalloproteinase-9 function during endochondral bone formation

Endochondral bone formation is characterized by the progressive replacement of a cartilage anlagen by bone at the growth plate with a tight balance between the rates of chondrocyte proliferation, differentiation, and cell death. Deficiency of matrix metalloproteinase-9 (MMP-9) leads to an accumulation of late hypertrophic chondrocytes. We found that galectin-3, an in vitro substrate of MMP-9, accumulates in the late hypertrophic chondrocytes and their surrounding extracellular matrix in the expanded hypertrophic cartilage zone. Treatment of wild-type embryonic metatarsals in culture with full-length galectin-3, but not galectin-3 cleaved by MMP-9, mimicked the embryonic phenotype of Mmp-9 null mice, with an increased hypertrophic zone and decreased osteoclast recruitment. These results indicate that extracellular galectin-3 could be an endogenous substrate of MMP-9 that acts downstream to regulate hypertrophic chondrocyte death and osteoclast recruitment during endochondral bone formation. Thus, the disruption of growth plate homeostasis in Mmp-9 null mice links galectin-3 and MMP-9 in the regulation of the clearance of late chondrocytes through regulation of their terminal differentiation.     

Immunohistochemistry of chondromodulin-I in the human intervertebral discs with special reference to the degenerative changes

The expression of the matrix protein chondromodulin-I has been studied in human intervertebral discs of 101 people using immunohistochemical analyses. The purpose of this report is to present data on the metabolic changes that were found to occur in the chondrocytes of intervertebral discs during development and aging. Chondromodulin-I was highly expressed during the gestational period and gradually decreased after maturation. It was detected in both the extracellular matrix and chondrocytes in the zone of hypertrophic cartilage, the zone of proliferative cartilage and the zone of resting cartilage in fetal discs. It was also present in the annulus fibrosus, nucleus pulposus and end-plate cartilage in mature discs. In degenerative discs, chondromodulin-I immunoreactivity tended to be elevated in the remaining chondrocytes. Our findings suggest that the expression of the protein is developmentally regulated and upregulated through a defense mechanism against the degenerative processes of the aged intervertebral disc.     

Retention of the matricellular protein SPARC in the endoplasmic reticulum of chondrocytes from patients with pseudoachondroplasia.

Pseudoachondroplasia (PSACH) is an autosomal dominant disease characterized by dwarfism, morphological irregularities of long bones and hips, and early-onset osteoarthritis. This disease has been attributed to mutations in a structural protein of the cartilage extracellular matrix (ECM), cartilage oligomeric matrix protein (COMP), which result in its selective retention in the chondrocyte rough endoplasmic reticulum (ER). Accumulation of excessive amounts of mutated COMP might reflect a defect in protein trafficking by PSACH chondrocytes. Here we identify the matricellular protein SPARC as a component of this trafficking deficit. SPARC was localized to the hypertrophic chondrocytes in the normal human tibial growth plate and in cultured control cartilage nodules. In contrast, concentrated intracellular depots of SPARC were identified in nodules cultured from three PSACH patients with mutations in COMP. The accumulated SPARC was coincident with COMP and with protein disulfide isomerase, a resident chaperone of the rough ER, whereas SPARC and COMP were not coincident in the ECM of control or PSACH nodules. SPARC-null mice develop severe osteopenia and degenerative intervertebral disc disease, and exhibit attenuation of collagenous ECM. The retention of SPARC in the ER of chondrocytes producing mutant COMP indicates a new intracellular function for SPARC in the trafficking/secretion of cartilage ECM.     

Contributions of matrix metalloproteinases toward Meckel’s cartilage resorption in mice: immunohistochemical studies,including comparisons with developing endochondral bones

The middle portion of Meckel’s cartilage (one of four portions that disappear with unique fate) degrades via hypertrophy and the cell death of chondrocytes and via the resorption of cartilage by chondroclasts. We have examined the immunolocalization of matrix metalloproteinase-2 (MMP-2), MMP-9, MMP-13, and MMP-14 (members of the MMP activation cascade) and galectin-3 (an endogenous substrate for MMP-9 and an anti-apoptotic factor) during resorption of Meckel’s cartilage in embryonic mice and have compared the results with those of developing endochondral bones in hind limbs. MMP immunoreactivity, except for MMP-2, is present in nearly all chondrocytes in the middle portion of Meckel’s cartilage. On embryonic day 15 (E15), faint MMP-2-immunoreactive and intense MMP-13-immunoreactive signals occur in the periosteal bone matrix deposited by periosteal osteoblasts on the lateral surface, whereas MMP-9 and MMP-14 are immunolocalized in the peripheral chondrocytes of Meckel’s cartilage. The activation cascade of MMPs by face-to-face cross-talk between cells may thus contribute to the initiation of Meckel’s cartilage degradation. On E16, immunopositive signaling for MMP-13 is detectable in the ruffled border of chondroclasts at the resorption front, whereas immunostaining for galectin-3 is present at all stages of chondrocyte differentiation, especially in hypertrophic chondrocytes adjacent to chondroclasts. Galectin-3-positive hypertrophic chondrocytes may therefore coordinate the resorption of calcified cartilage through cell-to-cell contact with chondroclasts. In metatarsal specimens from E16, MMPs are detected in osteoblasts, young osteocytes, and the bone matrix of the periosteal envelope, whereas galectin-3 immunoreactivity is intense in young periosteal osteocytes. In addition, intense MMP-9 and MMP-14 immunostaining has been preferentially found in pre-hypertrophic chondrocytes, although galectin-3 immunoreactivity markedly decreases in hypertrophic chondrocytes. These results indicate that the degradation of Meckel’s cartilage involves an activation cascade of MMPs that differs from that in endochondral bone formation.     

VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation.

Hypertrophic chondrocytes in the epiphyseal growth plate express the angiogenic protein vascular endothelial growth factor (VEGF). To determine the role of VEGF in endochondral bone formation, we inactivated this factor through the systemic administration of a soluble receptor chimeric protein (Flt-(1-3)-IgG) to 24-day-old mice. Blood vessel invasion was almost completely suppressed, concomitant with impaired trabecular bone formation and expansion of hypertrophic chondrocyte zone. Recruitment and/or differentiation of chondroclasts, which express gelatinase B/matrix metalloproteinase-9, and resorption of terminal chondrocytes decreased. Although proliferation, differentiation and maturation of chondrocytes were apparently normal, resorption was inhibited. Cessation of the anti-VEGF treatment was followed by capillary invasion, restoration of bone growth, resorption of the hypertrophic cartilage and normalization of the growth plate architecture. These findings indicate that VEGF-mediated capillary invasion is an essential signal that regulates growth plate morphogenesis and triggers cartilage remodeling. Thus, VEGF is an essential coordinator of chondrocyte death, chondroclast function, extracellular matrix remodeling, angiogenesis and bone formation in the growth plate.     

Histochemical localization of alkaline phosphatase activity in decalcified bone and cartilage.

We have developed methodology that enables alkaline phosphatase (ALP) to be histochemically stained reproducibly in decalcified paraffin-embedded bone and cartilage of rodents. Proximal tibiae and fourth lumbar vertebrae were fixed in periodate-lysine-paraformaldehyde (PLP) fixative, decalcified in an EDTA-G solution, and embedded in paraffin. In the articular cartilage of the proximal tibia, ALP activity was localized to the hypertrophic chondrocytes and cartilage matrix of the deep zone and the maturing chondrocytes of the intermediate zone. The cells and matrix in the superficial zone did not exhibit any enzyme activity. In tibial and vertebral growth plates, a progressive increase in ALP expression was seen in chondrocytes and cartilage matrix, with activity being weakest in the proliferative zone, higher in the maturing zone, and highest in the hypertrophic zone. In bone tissue, ALP activity was detected widely in pre-osteoblasts, osteoblasts, lining cells on the surface of trabeculae, some newly embedded osteocytes, endosteal cells, and subperiosteal cells. In areas of new bone formation, ALP activity was detected in osteoid. In the bone marrow, about 20% of bone marrow cells expressed ALP activity. In adult rats, the thickness of the growth plates was less and ALP activity was enhanced in maturing and hypertrophic chondrocytes, cartilage matrix in the hypertrophic zone, and primary spongiosa. This is the first time that ALP activity has been successfully visualized histochemically in decalcified, paraffin-embedded mineralized tissues. This technique should prove to be a very convenient adjunct for studying the behavior of osteoblasts during osteogenesis.     

Modification of plasma membrane of differentiating preosseous chondrocytes: Evidence for a degradative process in the mechanism of matrix vesicle formation

Chondrocytes of the growth plate are differentiating cells. Their evolution leads to matrix vesicle formation and to cartilage mineralization. This is an in vitro study of the plasma membrane of chondrocytes at two differentiation stages. Differences in protein and glycoprotein components, increased membrane fluidity, and responsiveness to PTH indicate that hypertrophic ("ossifying") chondrocytes possess a plasma membrane widely different from that of resting chondrocytes. Their plasma membrane is particularly enriched in alkaline phosphatase (Mr 70K). Purified matrix vesicles contain the 70K form of alkaline phosphatase, but a 50K species is also detectable, a signal of degradative process. In fact, proteins and glycoproteins of matrix vesicles are less numerous than those of cell plasma membranes. It is suggested that, in vivo, matrix vesicle formation may be mediated by Ca2(+)-activated neutral proteases.     

Epidermal Growth Factor Receptor (EGFR) Signaling Regulates Epiphyseal Cartilage Development through β-Catenin-dependent and -independent Pathways

The epidermal growth factor receptor (EGFR) is an essential player in the development of multiple organs during embryonic and postnatal stages. To understand its role in epiphyseal cartilage development, we generated transgenic mice with conditionally inactivated EGFR in chondrocytes. Postnatally, these mice exhibited a normal initiation of cartilage canals at the perichondrium, but the excavation of these canals into the cartilage was strongly suppressed, resulting in a delay in the formation of the secondary ossification center (SOC). This delay was accompanied by normal chondrocyte hypertrophy but decreased mineralization and apoptosis of hypertrophic chondrocytes and reduced osteoclast number at the border of marrow space. Immunohistochemical analyses demonstrated that inactivation of chondrocyte-specific EGFR signaling reduced the amounts of matrix metalloproteinases (MMP9, -13, and -14) and RANKL (receptor activator of NF-κB ligand) in the hypertrophic chondrocytes close to the marrow space and decreased the cartilage matrix degradation in the SOC. Analyses of EGFR downstream signaling pathways in primary epiphyseal chondrocytes revealed that up-regulation of MMP9 and RANKL by EGFR signaling was partially mediated by the canonical Wnt/β-catenin pathway, whereas EGFR-enhanced MMP13 expression was not. Further biochemical studies suggested that EGFR signaling stimulates the phosphorylation of LRP6, increases active β-catenin level, and induces its nuclear translocation. In line with these in vitro studies, deficiency in chondrocyte-specific EGFR activity reduced β-catenin amount in hypertrophic chondrocytes in vivo. In conclusion, our work demonstrates that chondrocyte-specific EGFR signaling is an important regulator of cartilage matrix degradation during SOC formation and epiphyseal cartilage development and that its actions are partially mediated by activating the β-catenin pathway.     

Role of proteoglycans in endochondral ossification: immunofluorescent localization of link protein and proteoglycan monomer in bovine fetal epiphyseal growth plate

The hypothesis is widely held that, in growth plate during endochondral ossification, proteoglycans in the extracellular matrix of the lower hypertrophic zone are degraded by proteases and removed before mineralization, and that this is the mechanism by which a noncalcifiable matrix is transformed into a calcifiable matrix. We have evaluated this hypothesis by examining the immunofluorescent localization and concentrations of proteoglycan monomer core protein and link protein, and the concentrations of glycosaminoglycans demonstrated by safranin 0 staining, in the different zones of the bovine fetal cartilage growth plate. Monospecific antibodies were prepared to proteoglycan monomer core protein and to link protein. The immunofluorescent localization of these species was examined in decalcified and undecalcified sections containing the zones of proliferating and hypertrophic chondrocytes and in sections containing the zones of proliferating and hypertrophic chondrocytes and the metaphysis, decalcified in 0.5 M EDTA, pH 7.5, in the presence of protease inhibitors. Proteoglycan monomer core protein and link protein are demonstrable without detectable loss throughout the extracellular matrix of the longitudinal septa of the hypertrophic zone and in the calcified cartilage of the metaphysis. In fact, increased staining is observed in the calcifying cartilage. Contrary to the prevailing hypothesis, our results indicate that there is no net loss of proteoglycans during mineralization and that the proteoglycans become entombed in the calcified cartilage which provides a scaffolding on which osteoid and bone are formed. Proteoglycans appear to persist unaltered in the calcified cartilage core of the trabeculae, until at last the entire trabeculae are eroded from their surfaces and removed by osteoclasts, when the primary spongiosa is replaced by the secondary spongiosa.     

Hypertrophic Chondrocytes in the Rabbit Growth Plate Can Proliferate and Differentiate into Osteogenic Cells when Capillary Invasion Is Interposed by a Membrane Filter

The fate of hypertrophic chondrocytes during endochondral ossification remains controversial. It has long been thought that the calcified cartilage is invaded by blood vessels and that new bone is deposited on the surface of the eroded cartilage by newly arrived cells. The present study was designed to determine whether hypertrophic chondrocytes were destined to die or could survive to participate in new bone formation. In a rabbit experiment, a membrane filter with a pore size of 1 µm was inserted in the middle of the hypertrophic zone of the distal growth plate of ulna. In 33 of 37 animals, vascular invasion was successfully interposed by the membrane filter. During 8 days, the cartilage growth plate was enlarged, making the thickness 3-fold greater than that of the nonoperated control side. Histological examination demonstrated that the hypertrophic zone was exclusively elongated. At the terminal end of the growth plate, hypertrophic chondrocytes extruded from their territorial matrix into the open cavity on the surface of the membrane filter. The progenies of hypertrophic chondrocytes (PHCs) were PCNA positive and caspase-3 negative. In situ hybridization studies demonstrated that PHCs did not express cartilage matrix proteins anymore but expressed bone matrix proteins. Immunohistochemical studies also demonstrated that the new matrix produced by PHCs contained type I collagen, osteonectin, and osteocalcin. Based on these results, we concluded that hypertrophic chondrocytes switched into bone-forming cells after vascular invasion was interposed in the normal growth plate.