LYSOZYME IN EPIPHYSEAL CARTILAGE : IV. Embryonic Chick Cartilage Lysozyme-Its Localization and Partial Characterization.

THE LOCALIZATION OF CHICK EMBRYONIC LYSOZYME WAS DETERMINED BY TWO TECHNIQUES: by studying the rate of release from the tissue during sequential enzymatic digestion and by immunocytochemistry. Both techniques indicate that, in this tissue, lysozyme is primarily extra-cellular. Cartilage lysozyme was isolated and partially characterized and found to be identical with egg white lysozyme in its immunologic and enzymatic behavior. In addition, a method for the isolation of large numbers of viable chondrocytes is described.

ENZYME ACTIVITIES IN CHICK EMBRYONIC CARTILAGE : Their Subcellular Distribution in Isolated Chondrocytes.

Some characteristic enzymatic activities were determined in chick embryonic cartilage and compared with the analogous activities in bone and liver. Chondrocytes were isolated, broken by sonication, and subjected to subcellular fractionation to yield a nuclear pellet, the mitochondrial, lysosomal, and microsomal fractions, and the high speed supernatant solution. It was established that these fractions are characterized by enzymatic activities usually associated with similar fractions in other tissues, but with some quantitative differences. Lysozyme, a particulate-associated enzyme in other tissues, was not detected in any subcellular fraction even by the sensitive technique of microzone electrophoresis and is therefore considered to be primarily extracellular in cartilage.

Synthesis of cartilage matrix by mammalian chondrocytes in vitro. III. Effects of ascorbate.

Chondrocytes isolated from bovine articular cartilage were plated at high density and grown in the presence or absence of ascorbate. Collagen and proteoglycans, the major matrix macromolecules synthesized by these cells, were isolated at times during the course of the culture period and characterized. In both control and ascorbate-treated cultures, type II collagen and cartilage proteoglycans accumulated in the cell-associated matrix. Control cells secreted proteoglycans and type II collagen into the medium, whereas with time in culture, ascorbate-treated cells secreted an increasing proportion of types I and III collagens into the medium. The ascorbate-treated cells did not incorporate type I collagen into the cell-associated matrix, but continued to accumulate type II collagen in this compartment. Upon removal of ascorbate, the cells ceased to synthesize type I collagen. Morphological examination of ascorbate-treated and control chondrocyte culture revealed that both collagen and proteoglycans were deposited into the extracellular matrix. The ascorbate-treated cells accumulated a more extensive matrix that was rich in collagen fibrils and ruthenium red-positive proteoglycans. This study demonstrated that although ascorbate facilitates the formation of an extracellular matrix in chondrocyte cultures, it can also cause a reversible alteration in the phenotypic expression of those cells in vitro.

Synthesis of cartilage matrix by mammalian chondrocytes in vitro. I. Isolation, culture characteristics, and morphology.

We describe the isolation and the ultrastructural characteristics of adult bovine articular chondrocytes in vitro. Slices of bovine articular cartilage undergo sequential digestions with pronase and collagenase in order to release cells. Chondrocytes are plated at high density (1 x 10(5) cells/cm2) in culture dishes or roller bottles with Ham's F-12 medium, supplemented with 10% fetal bovine serum. Before culture, chondrocytes are freed of surrounding territorial matrix. Within the first few days of culture they re-establish a territorial matrix. As time progresses, chondrocytes synthesize both territorial and extraterritorial matrices. The matrices are rich in collagen fibrils and ruthenium red-positive proteoglycans. These features are most apparent in mass roller cultures in which aggregates of cells and matrix appear as long streaks and nodules. This morphology reveals an organization of chondrocytes and their matrices that is similar to that of the parent articular cartilage in vivo.

Synthesis of cartilage matrix by mammalian chondrocytes in vitro. II. Maintenance of collagen and proteoglycan phenotype.

The in vitro phenotype of bovine articular chondrocytes is described. Chondrocytes plated at high density in roller-bottle and dish cultures were maintained in vitro. The major matrix macromolecules, collagen and proteoglycan, synthesized by these cells were characterized during the course of the culture period. The chondrocytes synthesized mainly Type II collagen, which was found predominantly in the cell-associated matrix. The media contained a mixture of Type II and Type III collagens. Type I collagen was detectable in neither the medium nor the cell-associated matrix. The proteoglycan monomers found in media and cell-associated matrix had the same hydrodynamic sizes as monomers synthesized by cartilage slices or those extracted from adult articular cartilage. The majority of proteoglycans synthesized by the cells were found in high molecular weight aggregates which were readily recovered from the media and were extractable from cell-associated matrix with low ionic strength buffers. The results demonstrate the long-term in vitro phenotypic stability of the bovine articular chondrocytes. The advantages of the in vitro system as a model for studying the effects of external agents, such as drugs and vitamins, are discussed.

Selective emergence of differentiated chondrocytes during serum-free culture of cells derived from fetal rat calvaria.

Cells dispersed from the chondrocranial portions of fetal rat calvaria proliferated and performed specialized functions during primary culture in a chemically defined medium. Mature cultures were typified by multilayered clusters of redifferentiating cartilage cells. Flattened cells that lacked distinguishing features occupied areas between the clusters. Alkaline phosphate-enriched, ultrastructurally typical chondrocytes within the clusters were encased in a dense extracellular matrix that stained prominently for chondroitin sulfate proteoglycans. This matrix contained fibrils measuring 19 nm in diameter, which were associated with proteoglycan granules that preferentially bound ruthenium red. A progressive increase in the number of cells indicated the proliferation of certain elements in the primary culture. The cells in primary culture were biochemically as well as morphologically heterogeneous since they were found to synthesize type I and type II collagens. Homogeneous populations of redifferentiated chondrocytes were recovered as floating cells and were shown to express the chondrocyte phenotype in secondary culture. Subcultured cells synthesized type II collagen and its precursors almost exclusively and incorporated 35SO4 into proteoglycan monomer and aggregates to a greater degree than the cells in primary culture. The pattern of proteoglycan monomer and aggregate labeling resembled that of intact cartilage segments and bovine articular chondrocytes. Skin fibroblasts harvested from the same rat fetuses failed to proliferate when maintained under identical conditions. Hence, exogenous hormones, growth factors, and protein are not required for chondrocyte growth and maturation.

Lysozyme in epiphyseal cartilage. II. The effect of egg white lysozyme on mouse embryonic femurs in organ cultures.

Embryonic mouse femoral cartilage, like the epiphyseal cartilage of the calf scapula, contains large amounts of lysozyme. The addition of egg white lysozyme to organ cultures of embryonic mouse femurs induces unique alterations in the gross and microscopic morphology of the femurs. The sites of these alterations are precisely related to the natural distribution of lysozyme in calf scapula. If the exogenous lysozyme is withdrawn from the culture, the morphological changes disappear, accompanied by a resumption or derepression of growth. The effect on growth is evident only in 17-day embryos. These observations support the idea that lysozyme has a physiological role in cartilage, perhaps related to a regulatory mechanism in bone formation.

Inhibition of bone resorption in vitro by a cartilage-derived anticollagenase factor.

A cartilage-derived factor containing a specific collagenous inhibitor was found to block reversibly parathyroid hormone-stimulated 45Ca release from fetal rat bone in vitro. Morphologic and quantitative histometric examination revealed that this factor modulates osteoclastic activities.

Tumor cell collagenase and its inhibition by a cartilage-derived protease inhibitor.

Human osteosarcoma and mammary carcinoma cells were cultured separately in a medium supplemented with fetal calf serum, until they were confluent. The medium was then replaced by serum-free medium supplemented with heparin. Both cell cultures secreted collagenase, and this activity was inhibited by a cartilage-derived protein of low molecular weight. Since cartilage is rarely invaded by neoplasms, the presence of this inhibitor may play an important role in the regulation of tumor invasion.

Regulation of tumor invasion by cartilage-derived anti-invasion factor in vitro.

The resistance of cartilage to tumor invasion was studied with the use of a novel in vitro culture system. Articular cartilage obtained from fresh metacarpophalangeal joints of preadolescent bovines was used as a growth surface for human TE-85 osteosarcoma cells and foreskin fibroblasts. Cartilage disks formed the bottoms of stainless-steel cylinders, providing closed growth chambers for these cells. Both invasive osteosarcoma cells and normal fibroblasts were unable to penetrate viable, unextracted cartilage during a 2-week culture period. When cartilage was devitalized by freezing and thawing, the tissue remained resistant to invasion. Cartilage, extracted with either 1 or 3 M guanidine hydrochloride, was invaded by osteosarcoma cells, but not by control fibroblasts. Invasion by osteosarcoma cells into salt-extracted cartilage was abolished when low concentrations of a cartilage-derived, anti-invasion factor were added to the culture medium. These data provided evidence that the resistance of cartilage to tumor invasion is regulated in part by tissue-derived proteinase inhibitors.

In vitro determination of tumor invasiveness using extracted hyaline cartilage.

Our previous observation that salt-extracted, devitalized cartilage could be penetrated by malignant tumor cells but was nonpermissive to fibroblastic ingrowth led us to postulate that this matrix might be used as a test connective tissue to discriminate in vitro between noninvasive and invasive tumor cell lines. In a novel in vitro system, salt-extracted, bovine articular cartilage was therefore used as a growth surface for defined noninvasive, invasive, and metastatic carcinoma cell lines, derived from chemical carcinogen-induced tumors of the rat urinary bladder. As monitored by thin-section electron microscopy, salt-extracted cartilage was readily penetrated by the invasive and metastatic rat bladder carcinoma cell lines. The metastatic cell line could be differentiated from the invasive, nonmetastatic cell line by its greater depth of invasion. In contrast, noninvasive carcinoma cells as well as normal bladder epithelial cells lacked the capacity to erode and penetrate the extracted matrix of the articular cartilage. Using these defined cell lines, salt-extracted cartilage can be used to reproducibly discriminate between carcinomas having different invasive potentials. This assay system may have diagnostic application for the in vitro staging of tumors.

Development of an in vitro and in vivo epithelial tumor model for the study of invasion.

Three continuous cell lines were isolated from N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT)-induced carcinoma of the Fischer rat urinary bladder by standard explant techniques. RBTCC-2 carcinoma cells were derived from a noninvasive FANFT tumor of Stage 0, RBTCC-5 carcinoma cells were from an invasive FANFT tumor of Stage B2, and RBTCC-8 carcinoma cells were from a s.c. metastasis of a FANFT tumor of Stage D2. Invasive and metastatic carcinoma cells were differentiated from their noninvasive counterparts by cellular and nuclear pleomorphism, cell size, nuclear:cytoplasmic ratio, number of nucleoli, and abnormalities of occludens junctions. Using low (less than 10) and high (greater than 80) passages of these cell strains, tumorigenicity experiments in syngeneic rats showed that the normal in vivo progression of FANFT tumors was interrupted by the isolation of carcinoma cells to cell culture. Histological appearance and biological behavior of tumor isografts closely resembled those of the original FANFT tumors. This was best demonstrated when tumor cells were inoculated adjacent to rat femurs. The destruction of bone, monitored radiographically and histologically, served as a measure of the invasive potential of the tumor cells. Destruction and deep invasion were observed only with isografts of invasive and metastatic carcinoma cells, presumably due to collagenolytic activity. Despite rapid degradation of bone by these isografts, the natural resistance of cartilage to tumor invasion could not be overcome. These carcinoma cell lines, together with their normal epithelial counterparts and the major supporting cells of connective tissue characterized previously by our laboratory, provide a unique system to study tumor invasion.

The properties of the neutral proteinase released by primary chondrocyte cultures and its action on proteoglycan aggregate.

The enzymatic mechanism of proteoglycan breakdown is of major interest, since it has been proposed that osteoarthritis involves increased proteolytic breakdown of proteoglycans. This paper describes the properties of the proteoglycan-degrading enzymes released into the extracellular milieu by chondrocyte cultures that produce cartilage-specific type II collagen but no detectable type I collagen. Attention has been focused on enzymes active at neutral pH, since the pH of the extracellular matrix is around neutrality. Biogel P-60 chromatography of concentrated culture medium showed a major peak of enzyme activity on proteoglycan monomer entrapped in polyacrylamide beads as well as on native proteoglycan aggregates. The enzyme yields a specific limit digestion peptide from the aggregate of approximately 55,000 daltons (in the presence of SDS). This limit peptide is probably derived from the hyaluronic acid-binding region of proteoglycan. The proteolytic enzyme is latent but can be activated by aminophenylmercuric acetate or trypsin. The molecular weight of both the active and latent forms, determined by gel filtration, is approximately 33,000. The activity is not inhibited by phenylmethylsulfonyl fluoride or pepstatin but is completely inhibited by o-phenanthroline; the activity is restored by Zn or Co ions in the presence of calcium chloride. Removal of calcium by dialysis results in a reversible loss of activity. The release of such a metalloproteinase by chondrocytes into the extracellular milieu, its activity at physiological pH and its ability to degrade native proteoglycans are consistent with a role of the enzyme in proteoglycan metabolism.

Characterization of the metalloproteinase inhibitor produced by bovine articular chondrocyte cultures.

Primary cultures of bovine articular chondrocytes release a latent metalloproteinase which is activated by incubation with organomercurials to degrade proteoglycans. All the enzyme present in the culture medium is latent and binds to columns of heparin-Sepharose. The yield of activity from the heparin-Sepharose columns (measured after organomercurial treatment) is approximately 300-1000% depending on the chondrocyte culture batch. Recombination of column fractions shows that the increase in activity is due to the separation of an inhibitor of the metalloproteinase by the chromatographic step. The metalloproteinase inhibitor has a molecular weight of approximately 35000 (determined by Bio-Gel P-60 chromatography) and binds reversibly to columns of concavalin A-Sepharose. It is relatively heat stable (30 min at 60 degrees C) and resistant to inactivation by trypsin (2 h, 37 degrees C, 10 microgram/ml trypsin). The inhibitor is active against rat uterine collagenase and gelatinase but does not affect bacterial metalloproteinases such as thermolysin and Clostridium histolyticum collagenase.

The potential role of fibroblasts in periprosthetic osteolysis: fibroblast response to titanium particles.

Periprosthetic osteolysis with or without aseptic loosening is a major clinical problem in total hip arthroplasty. While the macrophage response to prosthetic wear debris and its role in periprosthetic osteolysis has been extensively studied, information regarding other cell types (fibroblasts, osteoblasts) is limited. This study explored the response of fibroblasts to particulate wear debris. Fibroblasts isolated from interfacial membranes of patients with failed total hip replacements and normal synovial tissue, when challenged with small-sized ( < 3 microns) titanium (Ti) particles, responded with significantly enhanced expressions of collagenase, stromelysin and, to a much lesser extent, their tissue inhibitor of metalloproteinases (TIMP). These "regulated" expressions at both mRNA and protein levels were correlated with the size and composition of particles. De novo protein synthesis was required for the regulation of these mRNAs. A similar effect could be induced by the treatment of the cells with particle-free conditioned medium from Ti particle-stimulated fibroblasts. Furthermore, this conditioned medium significantly suppressed the mRNA levels of procollagen alpha 1 (I) and alpha 1 (III) in osteoblast-like MG-63 cells. It is concluded that fibroblasts stimulated with certain particle debris may play an important role in periprosthetic osteolysis by releasing bone-resorbing metalloproteinases and mediator(s) which resulted in suppressed collagen synthesis in osteoblasts.

The ground substance of the arterial wall. Part 1. Extractability of glycosaminoglycans and the isolation of a proteoglycan from bovine aorta.

Water and glycosaminoglycan contents were measured in upper and lower thoracic aortas of claves and steers. The ability of various molarities of guanidine hydrochloride to extract glycosaminoglycans from these tissues was assessed. Some glycosaminoglycans seem to be more resistant to extraction than others. A procedure is described for the isolation of a proteoglycan. The molecule appears to contain both dermatan sulfate and chondroitin sulfate. It also seems to be less dense than cartilage proteoglycans extracted by similar methods as assessed by its behavior in centrifugal fields. The properties, locus and biological activities of this molecule are currently being studied.

ADAM-10 message is expressed in human articular cartilage.

The depletion of the pericellular and territorial matrices in articular cartilage is considered to be one of the earliest events in pathobiology of osteoarthritis (OA). A newly discovered family of proteins with a disintegrin-like and metalloproteinase-like domain (ADAM) may be involved in matrix degradation as well as in cell-cell and cell-matrix interactions. The purpose of this study was to determine by in situ hybridization whether human articular chondrocytes from newborn, normal adult, and OA cartilages express messenger RNA for ADAM-10, one of the members of this family, and by semiquantitative RT-PCR to compare the levels of this expression. The results confirmed the expression of ADAM-10 by human articular chondrocytes and revealed the highest levels of expression in the continuously remodeling cartilage of newborns and the most fibrillated areas of OA cartilage, especially the regions of cell clusters. Importantly, ADAM-10 mRNA expression was evident in tissues with the greatest loss of Safranin O staining from the territorial and interterritorial matrix of the chondrocytes. Messenger RNA was upregulated in OA tissue compared to the age-matched normal cartilage, as detected by RT-PCR. Upregulated levels of ADAM-10 mRNA expression appear to be related to the degree of cartilage damage and/or degradation, which suggests a potential role for at least one member of this new family in the cartilage matrix destruction accompanying OA.

Human articular chondrocytes express osteogenic protein-1.

This study demonstrates for the first time that human articular chondrocytes express osteogenic protein-1 (OP-1). OP-1 was originally purified from bone matrix and was shown to induce cartilage and bone formation. Both OP-1 protein and message were present in human normal and osteoarthritic (OA) cartilages. OP-1 mRNA was upregulated in OA cartilage compared with normal adult tissues. However, the level of mature OP-1 protein in the same OA tissues was downregulated, whereas the pro-OP-1 remained high. Moreover, these two forms of OP-1 were localized in an inverted manner. Mature OP-1 was primarily detected in the superficial layer, whereas the pro-form was mostly in the deep layer of cartilage. The presence of pro- and mature OP-1 in extracts of normal and OA cartilages was confirmed by Western blotting. These findings imply that articular chondrocytes continue to express and synthesize OP-1 throughout adulthood. The observed patterns of the distribution of pro- and mature OP-1 also suggest differences in the processing of this molecule by normal and OA chondrocytes and by the cells in the superficial and deep layers. Distinct distribution of OP-1 and its potential activation in deep zones and regions of cloning in OA cartilages may provide clues to the potential involvement of endogenous OP-1 in repair mechanisms. (J Histochem Cytochem 48:239-250, 2000)

Osteoarthritis in ankle and knee joints.

Ankle and knee joints differ in their susceptibility to osteoarthritis (OA). This article reviews literature on differences between these joints. A Medline search and search of bibliographies of review articles was conducted. Knee cartilage degeneration leads to the development of OA with clinical symptoms, whereas the ankle cartilage develops fissures that do not appear to progress to later stages of OA. Epidemiological studies support these findings. Factors that might explain this phenomena include differences in joint motion, cartilage thickness, congruency, mechanical forces, and even evolutionary changes. Data suggest that chondrocytes from the two joints may respond differently to stimuli. Comparisons of cartilage from the knee and ankle joint of the same donor may provide a better understanding of the biochemical and molecular processes that induce the pathogenesis of OA and may provide new approaches to early detection and treatment.

Molecular characteristics of the inhibition of human neutrophil elastase by nonsteroidal antiinflammatory drugs.

Nonsteroidal antiinflammatory drugs(NSAIDs) are known as clinically effective agents for treatment of inflammatory diseases. Inhibition of cyclooxygenase has been thought to be a major facet of the pharmacological mechanism of NSAIDs. However, it is difficult to ascribe the antiinflammatory effects of NSAIDs solely to the inhibition of prostaglandin synthesis. Human neutrophil elastase (HNElastase; HNE, EC 3.4.21.37) has been known as a causative factor in inflammatory diseases. To investigate the specific relationship between HNElastase inhibition and specificity of molecular structure of several NSAIDs, HNElastase was purified by Ultrogel AcA54 gel filtration, CM-Sephadex ion exchange, and HPLC (with TSK 250 column) chromatography. HNElastase was inhibited by aspirin and salicylate in a competitive manner and by naproxen, ketoprofen, phenylbutazone, and oxyphenbutazone in a partial competative manner, but not by ibuprofen and tolmetin. HNElastase-phenylbutazone-complex showed strong Raman shifts at 200, 440, 1124, 1194, 1384, 1506, and 1768 cm(-1). The Raman bands 1194, 1384, and 1768 cm(-1) may represent evidences of the conformational change at -N=N-phi radical, pyrazol ring, and -C=O radical of the elastase-drug complex, respectively. Phenylbutazone might be bound to HNElastase by ionic and hydrophobic interaction, and masked the active site. Inhibition of HNElastase could be another mechanism of action of NSAIDs besides cyclooxygenase inhibition in the treatment of inflammatory diseases. Different inhibition characteristics of HNE-lastase by NSAIDs such as aspirin, phenylbutazone-like drugs and ineffective drugs could be important points for drawing the criteria for appropriate drugs in clinical application.