Immunolocation analysis of glycosaminoglycans in the human growth plate.

Monoclonal antibodies were used in this study to immunolocate glycosaminoglycans throughout the human growth plate. Chondroitin-4-sulfate, chondroitin-6-sulfate, and keratan sulfate were observed in the extracellular matrix of all zones of the growth plate and persisted into the cartilage trabeculae of newly formed metaphyseal bone. Also present in the extracellular matrix was an oversulfated chondroitin/dermatan sulfate glycosaminoglycan which appeared to be specific to the proliferative and hypertrophic zones of the growth plate. As with the other extracellular matrix molecules, this epitope persisted into the cartilage trabeculae of the metaphyseal bone. Zonal differences between the extracellular and pericellular or lacunae matrix were also observed. The hypertrophic chondrocytes appeared to synthesize chondroitin sulfate chains containing a non-reducing terminal 6-sulfated disaccharide, which were located in areas immediately adjacent to the cells. This epitope was not found to any significant extent in the other zones. The pericellular region around hypertrophic chondrocytes also contained a keratan sulfate epitope which was also observed in the resting zone but not in the proliferative zone. These cell-associated glycosaminoglycans were not found in the cartilage trabeculae of metaphyseal bone, indicating their removal as the terminal hypertrophic chondrocytes and their lacunae are removed by invading blood vessels. These changes in matrix glycosaminoglycan content, both in the different zones and within zones, indicate constant subtle alterations in chondrocyte metabolic products as they proceed through their life cycle of proliferation, maturation, and hypertrophy.     

Localization of CD44 (hyaluronan receptor) and hyaluronan in rat mandibular condyle.

CD44 is a multifunctional adhesion molecule that binds to hyaluronan (HA), type I collagen, and fibronectin. We investigated localization of CD44 and HA in mandibular condylar cartilage compared with the growth plate and the articular cartilage, to clarify the characteristics of chondrocytes. We also performed Western blotting using a lysate of mandibular condyle. In mandibular condyle, CD44-positive cells were seen in the surface region of the fibrous cell layer and in the proliferative cell layer. Western blotting revealed that the molecular weight of CD44 in condyle was 78 to 86 kD. Intense reactivity for HA was detected on the surface of the condyle and the lacunae of the hypertrophic cell layer. Moderate labeling was seen in cartilage matrix of the proliferative and maturative layer. Weak labeling was also seen in the fibrous cell layer. In growth plate and articular cartilage, HA was detected in all cell layers. However, chondrocytes of these cartilages did not exhibit reactivity for CD44. These results suggest that chondrocytes in the mandibular condylar cartilage differ in expression of CD44 from those in tibial growth plate and articular cartilage. Cell-matrix interaction between CD44 and HA may play an important role in the proliferation of chondrocytes in the mandibular condyle.     

Internalization of the Hyaluronan Receptor CD44 by Chondrocytes

Chondrocytes express CD44 as a primary receptor for the matrix macromolecule hyaluronan. Hyaluronan is responsible for the retention and organization of proteoglycan within cartilage, and hyaluronan-chondrocyte interactions are important for the assembly and maintenance of the cartilage matrix. Bovine articular chondrocytes were used to study the endocytosis and turnover of CD44 and the effects of receptor occupancy on this turnover. Matrix-intact chondrocytes exhibit approximately a 6% internalization of cell surface CD44 by 4 h. Treatment with Streptomyces hyaluronidase to remove endogenous pericellular matrix increased internalization to approximately 20% of cell surface CD44 at 4 h. This turnover could be partially inhibited by the addition of exogenous hyaluronan to these matrix-depleted chondrocytes. Cell surface biotin-labeled CD44 was internalized by chondrocytes and this internalization was decreased in the presence of hyaluronan. Colocalization of internalized CD44 and fluorescein-labeled hyaluronan in intracellular vesicles correlates with the previous results of receptor-mediated endocytosis pathway for the degradation of hyaluronan by acid hydrolases. Taken together, our results indicate that CD44 is internalized by chondrocytes and that CD44 turnover is modulated by occupancy with hyaluronan.     

Perlecan,the multidomain HS-proteoglycan of basement membranes,is a prominent pericellular component of ovine hypertrophic vertebral growth plate and cartilaginous endplate chondrocytes

The aim of this study was to immunolocalise perlecan in ovine vertebral growth plate (VGP) and cartilaginous endplate (CEP) cartilages using a monoclonal antibody (MAb A76) directed to a core protein epitope in perlecan domain-I, and to compare and contrast its localisation patterns with known cartilage matrix components. Perlecan was a prominent pericellular component of mature hypertrophic chondrocytes in the VGP and CEP in newborn 2- to 5-day-old sheep. Type I, II, VI and X collagen, chondroitin-4 and 6-sulphate, 7-D-4 chondroitin sulphate isomer proteoglycan epitope, keratan sulphate, aggrecan core protein, hyaluronan (HA) and hyaluronan binding proteins (HABPs) each had distinct localisation patterns in the VGP and CEP. Type X collagen was a prominent component of the VGP but was undetectable in the CEP. Aggrecan was strongly localised extracellularly throughout the VGP and CEP but increased cell-associated staining was also evident. In contrast to the aforementioned matrix components, HA, HABPs and perlecan were localised strongly to the pericellular matrices of the hypertrophic VGP and CEP chondrocytes apparently indicating an important role for these components in terminal chondrocyte differentiation.     

Electron microscopic study of condylar cartilage of rat mandible stained with ruthenium red: proteoglycans and hypertrophic chondrocytes

Ultrastructure of hypertrophic chondrocytes and extracellular matrix in condylar cartilage of rat mandible was studied in conjunction with ruthenium red staining. Special care was given to the preservation of proteoglycans in the extracellular matrix. Ruthenium red-positive granules were observed in the pericellular matrix of condylar chondrocytes, and their size and number increased around the hypertrophic cells. However, these granules disappeared in the lowest hypertrophic zone, in which uncalcified cartilage matrix was also disintegrated prior to initiation of ossification. Moreover, hypertrophic chondrocytes observed at the lowest zone appeared intact in their ultrastructural features, i.e., containing numbers of lysosomes and coated vesicles in the cytoplasm facing the blood capillaries. The results strongly suggest that the lowest hypertrophic chondrocytes in rat condylar cartilage may have an active role in the degradation and resorption of the pericellular matrix, especially proteoglycans, and uncalcified matrix, which changes seem an essential step for the initiation of endochondral ossification.     

Phagocytosis of dying chondrocytes by osteoclasts in the mouse growth plate as demonstrated by annexin-V labelling

Endochondral ossification in the epiphyseal growth plate of long bones is associated with programmed cell death (PCD) of a major portion of the chondrocytes. Here we tested the hypothesis that at the ossification front of the epiphyseal growth plate osteoclasts preferentially phagocytose chondrocytes that are undergoing PCD. We injected biotin-labelled annexin-V (anx-V-biotin, an early marker of PCD) intravenously in young adult mice. After 30 min of labelling, long bones were recovered and the tissue distribution examined of anx-V-biotin-labelled cells in the growth plate using ABC-peroxidase histochemistry. Positive staining for anx-V-biotin was detected in hypertrophic chondrocytes still present in closed lacunae at some distance from the ossification front. At the ossification front, chondrocyte lacunae were opened and close contacts were seen between tartrate-resistant acid phosphatase-positive osteoclasts and hypertrophic cartilage cells. Osteoclasts were significantly more frequently in contact with anx-V-biotin-labelled chondrocytes than with unlabelled chondrocytes. Osteoclasts also contained labelled and unlabelled phagocytic fragments within their cytoplasm. We conclude that in the growth plate osteoclasts preferentially phagocytose hypertrophic chondrocytes that are dying, suggesting these dying cells may signal osteoclasts for their removal.     

SIK3 is essential for chondrocyte hypertrophy during skeletal development in mice

Chondrocyte hypertrophy is crucial for endochondral ossification, but the mechanism underlying this process is not fully understood. We report that salt-inducible kinase 3 (SIK3) deficiency causes severe inhibition of chondrocyte hypertrophy in mice. SIK3-deficient mice showed dwarfism as they aged, whereas body size was unaffected during embryogenesis. Anatomical and histological analyses revealed marked expansion of the growth plate and articular cartilage regions in the limbs, accumulation of chondrocytes in the sternum, ribs and spine, and impaired skull bone formation in SIK3-deficient mice. The primary phenotype in the skeletal tissue of SIK3-deficient mice was in the humerus at E14.5, where chondrocyte hypertrophy was markedly delayed. Chondrocyte hypertrophy was severely blocked until E18.5, and the proliferative chondrocytes occupied the inside of the humerus. Consistent with impaired chondrocyte hypertrophy in SIK3-deficient mice, native SIK3 expression was detected in the cytoplasm of prehypertrophic and hypertrophic chondrocytes in developing bones in embryos and in the growth plates in postnatal mice. HDAC4, a crucial repressor of chondrocyte hypertrophy, remained in the nuclei in SIK3-deficient chondrocytes, but was localized in the cytoplasm in wild-type hypertrophic chondrocytes. Molecular and cellular analyses demonstrated that SIK3 was required for anchoring HDAC4 in the cytoplasm, thereby releasing MEF2C, a crucial facilitator of chondrocyte hypertrophy, from suppression by HDAC4 in nuclei. Chondrocyte-specific overexpression of SIK3 induced closure of growth plates in adulthood, and the SIK3-deficient cartilage phenotype was rescued by transgenic SIK3 expression in the humerus. These results demonstrate an essential role for SIK3 in facilitating chondrocyte hypertrophy during skeletogenesis and growth plate maintenance.     

The osmotic sensitivity of rat growth plate chondrocytes in situ; clarifying the mechanisms of hypertrophy

Bone elongation is predominantly driven by the volume expansion of growth plate chondrocytes. This mechanism was initially believed to be "hypertrophy", describing a proportional increase of cell water and organelles. However, morphometrical analysis subsequently assumed the increase to be "swelling", resulting in a disproportionate increase of cell water (osmotically active fraction). Histological approaches were performed on fixed tissue, and for the "swelling" assumption to be valid, the osmotic sensitivity of living cells before and during volume increase should differ. To test this, analysis of images acquired by 2-photon laser scanning microscopy (2PLSM) were used to determine the osmotic sensitivity, and osmotically active/inactive proportions of in situ chondrocytes from 15 living rat growth plates exposed to varying media osmolarities ( approximately 0-580 mOsm). The dimensions of cell volume swelling in hypotonic media were different to the preferential lengthening seen in vivo, confirming the complexity of directional cell volume increase. Boyle-van't Hoff analysis of cell volume over the range of media osmolarity indicated no significant difference (Student's t-test) in the osmotically inactive fraction, 39.5 +/- 2.9% and 47.0 +/- 4.3% (n = 13) for proliferative and hypertrophic zones, respectively, or the sensitivity of volume to changes in media osmolarity (proliferative 15.5 +/- 0.8 and hypertrophic zone 15.5 +/- 1.2%volume . Osm). The osmotic fractions did not change as chondrocytes progress from proliferative to hypertrophic regions of the growth plate. Our data suggest cell volume increase by hypertrophy may play a greater role in cell enlargement than swelling, and should be re-evaluated as a mechanism responsible for growth plate chondrocyte volume increase and hence bone elongation.     

Latrunculin and cytochalasin decrease chondrocyte matrix retention.

The proteoglycan-rich extracellular matrix (ECM) directly associated with the cells of articular cartilage is anchored to the chondrocyte plasma membrane via interaction with the hyaluronan receptor CD44. The cytoplasmic tail of CD44 interacts with the cortical cytoskeleton. The objective of this study was to determine the role of the actin cytoskeleton in CD44-mediated matrix assembly by chondrocytes and cartilage matrix retention and homeostasis. Adult bovine articular cartilage tissue slices and isolated chondrocytes were treated with latrunculin or cytochalasin. Tissues were processed for histology and chondrocytes were examined for CD44 expression and pericellular matrix assembly. Treatments that disrupt the actin cytoskeleton reduced chondrocyte pericellular matrix assembly and the retention of proteoglycan within cartilage explants. There was enhanced detection of a neoepitope resulting from proteolysis of aggrecan. Cytoskeletal disruption did not reduce CD44 expression, as monitored by flow cytometry, but detergent extraction of CD44 was enhanced and hyaluronan binding was decreased. Thus, disruption of the cytoskeleton reduces the anchorage of CD44 in the chondrocyte membrane and the capacity of CD44 to bind its ligand. The results suggest that cytoskeletal disruption within cartilage uncouples chondrocytes from the matrix, resulting in altered metabolism and deleterious changes in matrix structure.     

Intracellular Domain Fragment of CD44 Alters CD44 Function in Chondrocytes

The hyaluronan receptor CD44 undergoes sequential proteolytic cleavage at the cell surface. The initial cleavage of the CD44 extracellular domain is followed by a second intramembranous cleavage of the residual CD44 fragment, liberating the C-terminal cytoplasmic tail of CD44. In this study conditions that promote CD44 cleavage resulted in a diminished capacity to assemble and retain pericellular matrices even though sufficient non-degraded full-length CD44 remained. Using stable and transient overexpression of the cytoplasmic domain of CD44, we determined that the intracellular domain interfered with anchoring of the full-length CD44 to the cytoskeleton and disrupted the ability of the cells to bind hyaluronan and assemble a pericellular matrix. Co-immunoprecipitation assays were used to determine whether the mechanism of this interference was due to competition with actin adaptor proteins. CD44 of control chondrocytes was found to interact and co-immunoprecipitate with both the 65- and 130-kDa isoforms of ankyrin-3. Moreover, this interaction with ankyrin-3 proteins was diminished in cells overexpressing the CD44 intracellular domain. Mutating the putative ankyrin binding site of the transiently transfected CD44 intracellular domain diminished the inhibitory effects of this protein on matrix retention. Although CD44 in other cells types has been shown to interact with members of the ezrin/radixin/moesin (ERM) family of adaptor proteins, only modest interactions between CD44 and moesin could be demonstrated in chondrocytes. The data suggest that release of the CD44 intracellular domain into the cytoplasm of cells such as chondrocytes exerts a competitive or dominant-negative effect on the function of full-length CD44.     

Light- and electron-microscopic observations on the mandibular condylar cartilages in growing rats on a low-calcium diet.

In order to obtain more insight into the physiologic mechanism of endochondral ossification, histological changes occurring in the mandibular condylar cartilage of growing rats fed on a low-calcium diet were investigated by light and electron microscopy. Twenty-three-day-old rats were fed on a normal diet or a low-calcium diet for 8 weeks. For the histological observations the mandibular condyles were dissected from each animal at 1, 2, 4, 5 and 8 weeks after the initiation of the experiment. Histological changes occurring in the mandibular condylar cartilages of the rats fed on a low-calcium diet were as follows: (1) narrow proliferative and mature cell zones and a wide hypertrophic cell zone, (2) inhibition of development of cell organelles in the mature chondrocytes, (3) decrease in dead cells in the proliferative zone, (4) decrease in glycogen accumulation in the chondrocytes and (5) inhibition of calcification in the extracellular matrix of the hypertrophic cell zone. Additionally at the end of the experimental period, the following findings were observed: (1) appearance of small light cells in the mature cell zone and the hypertrophic cell zone and (2) decrease in proteoglycan granules and appearance of large collagen fibrils in the pericellular region of the hypertrophic cell zone.     

CD44-Anchored Hyaluronan-Rich Pericellular Matrices: An Ultrastructural and Biochemical Analysis

The chondrocyte pericellular matrix is an essential zone for cartilage matrix assembly and turnover. Electron micrographs of native endogenous and composition-defined exogenous pericellular matrices, both preserved via ruthenium hexaminetrichloride fixation procedures, depict strikingly similar networks of hyaluronan and proteoglycan extending out from the cell surface. Biochemical and morphological analyses of matrix regrowth show that monoclonal antibodies directed against the hyaluronan receptor CD44 blocked chondrocyte pericellular matrix assembly. Immunoperoxidase electron microscopy was used to display regular repeating spacing patterns of hyaluronan/proteoglycan assembly at the cell surface. These patterns compared well with the ultrastructural immunolocalization of CD44 at the cell surface. All of these data suggest that the hyaluronan receptor CD44 retains and participates in the assembly of the chondrocyte pericellular matrix.     

Hyaluronan fragments activate nitric oxide synthase and the production of nitric oxide by articular chondrocytes

Chondrocyte CD44 receptors anchor hyaluronan to the cell surface, enabling the assembly and retention of proteoglycan aggregates in the pericellular matrix. Hyaluronan-CD44 interactions also provide signaling important for maintaining cartilage homeostasis. Disruption of chondrocyte-hyaluronan contact alters CD44 occupancy, initiating alternative signaling cascades. Treatment with hyaluronan oligosaccharides is one approach to uncouple CD44 receptors from its native ligand, hyaluronan. In bovine articular chondrocytes, treatment with hyaluronan oligosaccharides or purified hyaluronan hexasaccharides induced the production of nitric oxide that mirrored nitric oxide production following interleukin-1 treatment. In contrast, 120 and 1,260 kDa hyaluronan did not induce production of nitric oxide. Human chondrocytes responded similarly to treatment with hyaluronan or hyaluronan oligosaccharides. Nitric oxide production from chondrocytes was mediated by activation of the inducible nitric oxide synthase, as confirmed by mRNA expression and inhibition of nitric oxide production by diphenyleneiodonium. Co-treatment of chondrocytes with hyaluronan oligosaccharides and interleukin-1 did not demonstrate additive effects. Blocking interleukin-1 receptors with an antagonist did not abolish the production of nitric oxide induced by treatment with hyaluronan oligosaccharides. Moreover, only COS-7 following transfection with a pCD44, not the CD44-null parental cells, responded to treatment with hyaluronan oligosaccharides by releasing nitric oxide. This study demonstrates a novel signaling potential by hyaluronan fragments, in lieu of endogenous hyaluronan-chondrocyte interactions, resulting in the activation of inducible nitric oxide synthase.     

Suppression of mammalian bone growth by membrane transport inhibitors

Bone lengthening during skeletal growth is driven primarily by the controlled enlargement of growth plate (GP) chondrocytes. The cellular mechanisms are unclear but membrane transporters are probably involved. We investigated the role of the Na⁺/H⁺ antiporter (NHE1) and anion exchanger (AE2) in bone lengthening and GP chondrocyte hypertrophy in Sprague–Dawley 7‐day‐old rat (P7) bone rudiments using the inhibitors EIPA (5‐(N‐ethyl‐N‐isopropyl)amiloride) and DIDS (4,4‐diidothiocyano‐2,2‐stilbenedisulphonate), respectively. We have also determined cell‐associated levels of these transporters along the GP using fluorescent immunohistochemistry (FIHC). Culture of bones with EIPA or DIDS inhibited rudiment growth (50% at approx. 250 and 25 µM, respectively). Both decreased the size of the hypertrophic zone (P < 0.05) but had no effect on overall length or cell density of the GP. In situ chondrocyte volume in proliferative and hypertrophic zones was decreased (P < 0.01) with EIPA but not DIDS. FIHC labeling of NHE1 was relatively high and constant along the GP but declined steeply in the late hypertrophic zone. In contrast, AE2 labeling was relatively low in proliferative zone cells but increased (P < 0.05) reaching a maximum in the early hypertrophic zone, before falling rapidly in the late hypertrophic zone suggesting AE2 might regulate the transition phase of chondrocytes between proliferative and hypertrophic zones. The inhibition of bone growth by EIPA may be due to a reduction to chondrocyte volume set‐point. However the effect of DIDS was unclear but could result from inhibition of AE2 and blocking of the transition phase. These results demonstrate that NHE1 and AE2 are important regulators of bone growth. J. Cell. Biochem. 114: 658–668, 2013. © 2012 Wiley Periodicals, Inc.     

Electron microscopic observations on the fate of hypertrophic chondrocytes in condylar cartilage of rat mandible

The present study focused on the hypertrophic cell zone and the adjacent region of primary spongiosa in the mandibular condylar cartilage in growing rats (3 to 7 weeks old). In this cartilage, chondrocytes were not arranged in columns, and there was no clear distinction between longitudinal and transverse septum. The hypertrophic chondrocytes were not surrounded entirely by calcified matrix, and capillaries were in close contact with cartilage cells. The staining intensity of the pericellular matrix decreased in the lower hypertrophic cell zone in comparison with that in the upper part of the hypertrophic cell zone. Electron microscopic examinations indicated that the lowest hypertrophic cells contained lysosomes and pinocytotic vesicles. Some hypertrophic chondrocytes appeared to have been released from their lacunae and were observed in the region of the primary spongiosa. Hence it is suggested that the lowest hypertrophic chondrocytes in the rat mandibular condyle do not die but are released from their lacunae into the bone marrow. Further study is needed to determine whether or not these cells do indeed become osteoblasts and/or chondroclasts.     

Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation

To elucidate the role of PTHrP in skeletal development, we examined the proximal tibial epiphysis and metaphysis of wild-type (PTHrP-normal) 18- 19-d-old fetal mice and of chondrodystrophic litter mates homozygous for a disrupted PTHrP allele generated via homologous recombination in embryonic stem cells (PTHrP-depleted). In the PTHrP-normal epiphysis, immunocytochemistry showed PTHrP to be localized in chondrocytes within the resting zone and at the junction between proliferative and hypertrophic zones. In PTHrP-depleted epiphyses, a diminished [3H]thymidine-labeling index was observed in the resting and proliferative zones accounting for reduced numbers of epiphyseal chondrocytes and for a thinner epiphyseal plate. In the mutant hypertrophic zone, enlarged chondrocytes were interspersed with clusters of cells that did not hypertrophy, but resembled resting or proliferative chondrocytes. Although the overall content of type II collagen in the epiphyseal plate was diminished, the lacunae of these non-hypertrophic chondrocytes did react for type II collagen. Moreover, cell membrane-associated chondroitin sulfate immunoreactivity was evident on these cells. Despite the presence of alkaline phosphatase activity on these nonhypertrophic chondrocytes, the adjacent cartilage matrix did not calcify and their persistence accounted for distorted chondrocyte columns and sporadic distribution of calcified cartilage. Consequently, in the metaphysis, bone deposited on the irregular and sparse scaffold of calcified cartilage and resulted in mixed spicules that did not parallel the longitudinal axis of the tibia and were, therefore, inappropriate for bone elongation. Thus, PTHrP appears to modulate both the proliferation and differentiation of chondrocytes and its absence alters the temporal and spatial sequence of epiphyseal cartilage development and of subsequent endochondral bone formation necessary for normal elongation of long bones.     

Hyaluronan receptor-directed assembly of chondrocyte pericellular matrix

Initial assembly of extracellular matrix occurs within a zone immediately adjacent to the chondrocyte cell surface termed the cell- associated or pericellular matrix. Assembly within the pericellular matrix compartment requires specific cell-matrix interactions to occur, that are mediated via membrane receptors. The focus of this study is to elucidate the mechanisms of assembly and retention of the cartilage pericellular matrix proteoglycan aggregates important for matrix organization. Assembly of newly synthesized chondrocyte pericellular matrices was inhibited by the addition to hyaluronan hexasaccharides, competitive inhibitors of the binding of hyaluronan to its cell surface receptor. Fully assembled chondrocyte pericellular matrices were displaced using hyaluronan hexasaccharides as well. When exogenous hyaluronan was added to matrix-free chondrocytes in combination with aggrecan, a pericellular matrix equivalent in size to an endogenous matrix formed within 30 min of incubation. Addition of hyaluronan and aggrecan to glutaraldehyde-fixed chondrocytes resulted in matrix assembly comparable to live chondrocytes. These matrices could be inhibited from assembling by the addition of excess hyaluronan hexasaccharides or displaced once assembled by subsequent incubation with hyaluronan hexasaccharides. The results indicate that the aggrecanrich chondrocyte pericellular matrix is not only on a scaffolding of hyaluronan, but actually anchored to the cell surface via the interaction between hyaluronan and hyaluronan receptors.