Showing papers on "Cartilage published in 1981"

Parathyroid hormone stimulates bone formation and resorption in organ culture: evidence for a coupling mechanism.

Abstract: We have developed an in vitro system, using embryonic chicken tibiae grown in a serum-free medium, which exhibits simultaneous bone formation and resorption. Tibiae from 8-day embryos increased in mean (+/- SD) length (4.0 +/- 0.4 to 11.0 +/- 0.3 mm) and dry weight (0.30 +/- 0.04 to 0.84 +/- 0.04 mg) during 12 days in vitro. There was increased incorporation of [3H]proline into hydroxyproline (120 +/- 20 to 340 +/- 20 cpm/mg of bone per 24 hr) as a measure of collagen synthesis, as well as a 62 +/- 5% increase in total calcium and 45Ca taken up as an indication of active mineralization. A physiologic concentration (1 pM) of parathyroid hormone was found to stimulate bone resorption over control levels in this system. Parathyroid hormone stimulated the release of [3H]hydroxyproline from the bone shafts but not from the cartilage ends, indicating the specificity of the response. With 1 pM parathyroid hormone we observed an acute inhibition of bone formation, followed (after 12-16 hr) by a chronic stimulation of bone formation during the 12-day incubation. Both mineral uptake and matrix formation were enhanced at approximately the same rate during the 12-day incubation. The chronic enhancement of formation required parathyroid hormone only for the initial 8-10 hr of incubation. These results could be explained by the production or release of a factor from bone to stimulate formation in response to the acute increase in resorption--a "coupling factor." Indeed, dialyzed culture medium conditioned by actively resorbing bones stimulated bone formation over controls when added to organ cultures at a 1:20 dilution. The factor is larger than 12,000 daltons as determined by dialysis. The factor is specific for the bone shaft and did not affect the cartilage ends.

Appearance of fibronectin during the differentiation of cartilage, bone, and bone marrow.

Abstract: Fibronectin has been localized by indirect immunofluorescence during the various phases of endochondral bone formation in response to subcutaneously implanted demineralized bone matrix. Its histologic appearance has been correlated with results of biosynthetic experiments. (a) The implanted collagenous bone matrix was coated with fibronectin before and during mesenchymal cell proliferation. (b) During proliferation of mesenchymal precursor cells, the newly synthesized extracellular matrix exhibited a fibrillar network of fibronectin. (c) During cartilage differentiation, the fibronectin in the extracellular matrix was apparently masked by proteoglycans, as judged by hyaluronidase treatment. (d) Differentiating chondrocytes exhibited a uniform distribution of fibronectin. (e) Fibronectin was present in a cottony array around osteoblasts during osteogenesis. (f) The developing hematopoietic colonies revealed fibronectin associated with them. Therefore, it appears that fibronectin is ubiquitous throughout the development of endochondral bone and bone marrow.

Three-dimensional architecture of lumbar intervertebral discs.

Abstract: Three-dimensional observations of the collagen framework of lumbar intervertebral discs by scanning electron microscopy confirmed that the fibrillar framework of the annulus and the cartilage end-plate was made of nucleus as a closed pack system. The cartilage end-plate was made of dense collagen framework aligned horizontally. There was no interconnection between the cartilage end-plate and the lamellar subchondral collagen. In places, the vascular channels ran directly at the end-plate. In the inner one third of the annulus, obliquely oriented fibrillar lamellae interconnected with the cartilage end-plate. In the outer two thirds, the fibrillar lamellae were formed from fibrillar bundles and were firmly anchored into the vertebral bodies. Architectural relationships between the disc and the vertebral bodies are discussed.

Running Inhibits the Reversal of Atrophic Changes in Canine Knee Cartilage After Removal of a Leg Cast

Abstract: The effect of vigorous exercise on the reversibility of canine knee cartilage atrophy produced by immobilization of the leg was studied. In comparison to cartilage from the contralateral control knees, cartilage from knees which had been immobilized in a cast for 6 weeks showed an increase in water content and decreases in thickness, Safranin O staining of the matrix, uronic acid content, and net proteoglycan synthesis. In addition, the ability of both newly synthesized (35S) and total tissue proteoglycans to interact with hyaluronic acid to form aggregates was diminished; this was apparently due to an abnormality in the hyaluronate-binding region of the core proteins. If the casts were removed and the animals were then allowed to ambulate ad libitum for 3 weeks, all of these changes were reversed. However, knee cartilage from 3 dogs which had been run daily on a treadmill (6 miles/day) for 3 weeks after removal of the casts exhibited continuing decreases in thickness, Safranin O staining, and uronic acid content (mean 31%), even though net proteoglycan synthesis was increased (mean 16%) in comparison to that in control cartilage from the contralateral (nonimmobilized) knee. Furthermore, the abnormality in both 35S- and total tissue proteoglycans which precluded their interaction with high molecular weight hyaluronic acid persisted. In this respect, the proteoglycans were indistinguishable from those obtained from knee cartilage immediately following 6 weeks in a cast.

Structural changes during development in bovine fetal epiphyseal cartilage.

Abstract: Sedimentation coefficients of approximately 150 S show that proteoglycan aggregates from bovine fetal epiphyseal cartilage are exceptionally large. To determine the structural basis for the unusually large size of these proteoglycan aggregates, identify changes in proteoglycan structure with changing developmental age, and provide a basis for demonstrating the structural modifications which may occur in growth plate proteoglycan aggregates during endochondral ossification, we examined the molecular architecture and dimensions of fetal epiphyseal proteoglycans by electron microscopy. The eight bovine epiphyseal cartilages studied ranged in fetal age from 168 to 241 days. Proteoglycans were extracted in 4 M guanidinium hydrochloride containing protease inhibitors and isolated by equilibrium density gradient centrifugation under associative and dissociative conditions. Electron micrographs were made from monolayer preparations of proteoglycan-cytochrome c mixtures on nitrocellulose support films. The overall molecular architecture of the proteoglycan aggregates from fetal epiphyseal cartilages was similar to that of aggregates from other cartilages and showed a single, unbranched central hyaluronic acid filament to which many proteoglycan monomers were attached. However, the dimensions of the fetal proteoglycans differed strikingly from those of proteoglycans from mature cow nasal or immature calf nasal cartilage. Specifically, proteoglycan aggregates from bovine fetal epiphyseal cartilage showed: (a) longer hyaluronic acid central filaments; (b) greater numbers of proteoglycan monomers per aggregate; (c) closer spacing of proteoglycan monomers along the hyaluronic acid central filament; and (d) longer proteoglycan monomer core proteins. Proteoglycan monomers bound to hyaluronate consisted of two segments: (1) a peripheral thick segment, composed of the chondroitin sulfate chains condensed along the peripheral portion of the protein core, which corresponds to the chondroitin sulfate-rich region; and, (2) a central thin segment, devoid of visible glycosaminoglycan chains, which attaches directly to the hyaluronic acid central filament and contains the hyaluronic acid binding region and a portion of the keratan sulfate-rich region. The contribution of the thin segment to total monomer length decreased as total monomer length increased. Thus, in longer monomers the thick segment contributed more to total monomer length and the thin segment contributed less. Both the thin and thick segments of monomers from fetal epiphyseal cartilage were longer than the corresponding segments of calf nasal cartilage and mature bovine nasal cartilage monomers.(ABSTRACT TRUNCATED AT 400 WORDS)

Autoantibodies to cartilage and type II collagen in relapsing polychondritis and other rheumatic diseases.

Abstract: Cartilage antibodies were demonstrated by indirect immunofluorescence (IFL) on human fetal cartilage in 6 out of 9 patients with relapsing polychondritis (RPC), in 4 out of 260 patients with rheumatoid arthritis (RA), and in only 1 out of 1016 patients with other disorders The antibodies were specific for cartilage and evenly stained the whole cartilage matrix They were predominantly of IgG class and varied in titres from 1:1 to 1:320 Follow-up studies in the RPC patients indicated that higher titres were present during the early acute phase of the disease Five of the 6 positive cases had developed the disease within the past 12 months, and the 3 negative cases had had the disease for 3 to 7 years when tested The RA cases showing positive cartilage IFL had no clinical evidence of RPC Sequential measurements in 2 of the 4 cases showed that these antibodies became detectable some years after the onset of arthritis Absorption studies with human type II collagen and purified porcine proteoglycan failed to remove the cartilage IFL Antibodies to human native type II collagen were measured by an enzyme-linked immunosorbent assay The highest levels were found in the RA sera which also displayed cartilage IFL, but the 2 tests gave discordant results RPC sera showed the same antibody levels by this method, as did cartilage-IFL-negative RA sera, though both groups had higher mean levels than health controls The findings that cartilage antibodies are detected in the majority of cases of RPC and only rarely in other diseases suggests these antibodies may play an important role in the pathogenesis of cartilage destruction in RPC

Metalloproteinases from rabbit bone culture medium degrade types IV and V collagens, laminin and fibronectin

Abstract: Gel-filtration chromatography of culture medium from rabbit bone explants separates three latent metalloproteinases with activities against collagen, proteoglycan and gelatin respectively. The fractions degrading proteoglycan also degrade laminin, fibronectin and the polymeric products of pepsin-solubilized type IV collagen and can also solubilize insoluble type IV collagen. The fractions degrading gelatin are capable of degrading solubilized type V and 1 alpha,2 alpha,3 alpha (cartilage) collagens, as well as the lower-molecular-weight products of pepsin-solubilized type IV collagen. All activities can be inhibited by 1,10-phenanthroline and occur in either partially or totally latent forms that can be activated by 4-aminophenylmercuric acetate.

Cartilage proteoglycans inhibit fibronectin-mediated adhesion.

Abstract: Normal tissues and organs show, on histological examination, a pattern of cellular and acellular zones that is characteristic and unique for each organ or tissue. This pattern is maintained in health but is sometimes destroyed by disease. For example, in mobile joints, the articular surfaces consist of relatively acellular hyaline cartilage, and the joint space is enclosed by a capsule of loose connective tissue with a lining of fibroblasts and macrophages. In the normal joint these cells are confined to the synovial lining and the articular surface remains acellular. In in vitro culture, macrophages and their precursor monocytes are very adhesive, and fibroblasts can migrate and overgrow surfaces such as collagen or plastic used for tissue culture. The fibroblasts adhere to collagen by means of fibronectin, which they synthesize and secrete1. Because the collagen of cartilage is capable of binding serum fibronectin2 and fibronectin is present in cartilage during its development3, these cells should, in theory, slowly migrate from the synovial lining to the articular surface. It is their absence from the articular cartilage in normal circumstances, and then presence in such pathological states as rheumatoid arthritis, that is striking. We therefore set out to determine whether a component of cartilage could prevent fibroblast adherence in a defined adhesion assay. As normal cartilage is composed of 50% proteoglycans and 50% collagen by dry weight4, we tested the possibility that the proteoglycans in cartilage inhibit fibroblast adhesion to collagen. We present here evidence that fibroblast spreading and adhesion to collagenous substrates is inhibited by cartilage proteoglycans.

Human mononuclear cell factors mediate cartilage matrix degradation through chondrocyte activation.

Abstract: Human blood mononuclear cells (BMC) in short-term culture secrete one or more factors that induce degradation of matrix proteoglycan and collagen in cartilage explants in organ culture. Induction of matrix degradation took place both in nasal septum and articular cartilage explants in the presence of the mononuclear cell supernates. Cartilage degradation in this system was absolutely dependent on the presence of live chondrocytes. Matrix depletion did not occur in dead cartilage explants cultured with active supernates. Supernates obtained from unstimulated BMC showed variable cartilage matrix degrading activity (MDA). BMC stimulated with phytohemagglutinin (PHA) showed increased MDA, which in one dilution experiment was found to be five times higher than that in the unstimulated control supernate. Concanavalin A and pokeweed mitogen were also shown to stimulate release of MDA. Time experiments showed that most of the degrading activity was released by the mononuclear cells during the first day of culture. The cellular origin of MDA was investigated with the aid of partially purified BMC subpopulations. Removal of adherent cells resulted in a decrease of MDA release. Purified T lymphocytes failed to show enhanced MDA release in spite of their ability to mount a virtually intact proliferative response to PHA. Purified adherent cells also failed to show enhanced PHA-dependent MDA release. Nevertheless, restoration of PHA-dependent MDA release took place in reconstituted cell populations containing both T lymphocytes and monocytes. These experiments suggest that MDA may be released by adherent mononuclear cells, presumably monocytes, and that the PHA-dependent increase in MDA release may be mediated by T lymphocytes. Partial characterization of MDA by gel chromatography showed one active fraction corresponding to an apparent molecular weight ranging from 12,000 to 20,000. The fraction was also shown to degrade cartilage matrix only in the presence of live chondrocytes. These results demonstrate that factors released by human BMC mediate degradation of matrix proteoglycan and collagen in intact cartilage explants through chondrocyte activation. This pathogenic mechanism may play a role in in vivo cartilage destruction in chronic inflammatory joint diseases.

A novel low-molecular weight chondroitin sulphate proteoglycan isolated from cartilage.

Abstract: Proteoglycans were isolated from cartilage by extraction with 4M-guanidinium chloride followed by direct centrifugation in 4M-guanidinium chloride/CsCl at a low starting density, 1.34 g/ml. N-Ethylmaleimide was included in the extraction solvent as a precaution against contamination of proteoglycans with unrelated proteins mediated by disulphide exchange. A novel, discrete, low-buoyant-density proteoglycan (1.40--1.35 g/ml) was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Its proteoglycan nature was revealed by the shift in the molecular size observed on gel electrophoresis after treatment with chondroitinase ABC. The core protein was monodisperse. The proteoglycan was further purified by gel chromatography with and without addition of hyaluronate. The proteoglycan constitutes less than 2% (by weight) of the total extracted proteoglycans and is not capable of interacting with hyaluronate. The same proteoglycan was purified in larger quantities by sequential associative and dissociative CsCl-density-gradient centrifugation, zonal rate sedimentation in a sucrose gradient and gel chromatography on Sepharose CL-4B. The pure proteoglycan had a molecular weight of 76 300 determined by sedimentation-equilibrium centrifugation and an apparent partial specific volume of 0.59 ml/g. It contained about 25% protein (of dry weight) and had remarkably high contents of leucine and cysteine as compared with other proteoglycans. The proteoglycan contained two to three large chondroitin sulphate chains and some oligosaccharides.

Polymorphonuclear granulocytes in rheumatic tissue destruction. III. an electron microscopic study of PMNs at the pannus-cartilage junction in rheumatoid arthritis.

Abstract: Metatarsophalangeal and metacarpophalangeal joints from 3 patients with rheumatoid arthritis were investigated electron microscopically with regard to the occurrence of polymorphonuclear granulocytes (PMNs) at the pannus-cartilage junction. In all 3 cases PMNs could be detected at the junction and within the cartilaginous matrix. PMN cytoplasmic processes surrounded collagenous islands in the cartilage. From the morphological findings it is deduced that PMNs are cells capable of destroying cartilage in inflammatory joint diseases, in particular in rheumatoid arthritis.

Variations in the composition of bovine hip articular cartilage with distance from the articular surface.

Abstract: Punch biopsies of bovine hip articular cartilage was sectioned according to depth and the proteoglycans were isolated. The mid-sections of the cartilage contained more proteoglycans than did either the superficial or the deepest portions of the cartilage proteoglycans than did either the superficial or the deepest portions of the cartilage. The most superficial 40 micrometer of the cartilage contained relatively more glucosaminoglycans compared with the remainder of the cartilage. The proteoglycans recovered from the surface 200 micrometer layer contained less chondroitin sulphate, were smaller and almost all of these molecules were able to interact with hyaluronic acid to form aggregates. From about 200 micrometer and down to 1040 micrometer from the surface, the proteoglycans became gradually somewhat smaller, probably owing to decreasing size of the chondroitin sulphate-rich region. The proportion of molecules that were able to interact with the hyaluronic acid was about 90% and remained constant with depth. The proteoglycans from the deepest layer near the cartilage-bone junction contained a large proportion of non-aggregating molecules, and the average size of the proteoglycans was somewhat larger. The alterations of proteoglycan structure observed with increasing depth of the articular cartilage beneath the surface layer (to 200 micrometer) are of the same nature as those observed with increasing age in full-thickness articular cartilage. The articular-cartilage proteoglycans were smaller and had much higher keratan sulphate and protein contents that did molecules isolated from bovine nasal or tracheal cartilage.

Characterization of catabolin, the major product of pig synovial tissue that induces resorption of cartilage proteoglycan in vitro.

Abstract: 1. Pig synovium in organ culture produces material which induces living cartilage to resorb its proteoglycan in vitro. 2. The bioassay for this material was to measure glycosaminoglycan released from explants of bovine nasal-septal cartilage cultured for 8 days. The performance of the assay was greatly improved by adding cortisol succinate (0.1μg/ml). This decreased the release of glycosaminoglycan from unstimulated cartilage without inhibiting its response to catabolic factors from the synovium. 3. By using this improved assay it was shown that 90% of the active materials in synovial culture medium were retained by dialysis membrane. 4. An active protein was partially purified from synovial culture medium by (NH4)2SO4 precipitation, ion-exchange chromatography, gel filtration and preparative isoelectric focusing. 5. This protein, called catabolin, had mol.wt. 17000 and pI4.6. 6. Synovial culture medium concentrated in dialysis tubing was subjected to gel chromatography and found to contain one major active component, which was eluted at the same position as the partially purified catabolin. 7. The synovial culture medium was not inactivated by heating (70°C for 10min), nor were diluted preparations of partially purified catabolin, but concentrated crude preparations were thermolabile. 8. These results suggest that catabolin is the major substance produced by the synovial tissue in culture which induces resorption of proteoglycan of living cartilage in vitro. 9. Other cultured soft connective tissues produced catabolin-like activity. The example of sclera is shown, and production was inhibited by cortisol succinate (0.1μg/ml). 10. It is suggested that catabolin may be a general product of soft connective tissues in culture, and its physiological function may be to induce resorption of connective-tissue matrix after injury.

Comparison of phosphohydrolase activities from articular cartilage in Calcium Pyrophosphate deposition disease and primary osteoarthritis

Abstract: One abnormality in calcium pyrophosphate deposition disease (CPDD) which fosters consistently high synovial fluid pyrophosphate ion (PPi) and large accumulations of calcium pyrophosphate dihydrate crystals (Ca pyrophosphate) might be an aberration in chondrocytes involving elaboration of PPi and failure of its hydrolysis within cartilage matrix. Exploration of this hypothesis required further information on the phosphohydrolases in relevant human articular cartilages. Triton X-100 extracts of whole homogenized cartilage from 18 patients with primary osteoarthritis (OA), 10 patients with CPDD and secondary OA, as well as 6 "normal" subjects were partially purified by DE-52 chromatography and eluates studied for phosphohydrolase activity in a variety of substrates, inhibitors, and environmental conditions. Almost all the protein as well as crude alkaline phosphatase and pyrophosphatase activities were clustered in peaks designated I and II. Findings in CPDD cartilage not observed in OA controls were: 1) consistent alkaline phosphatase activity in the void volume of DE-52 columns, 2) high levels of 5'nucleotidase activity, 3) abundant generation of PPi by CPDD cartilage during in vitro incubation of cartilage extract fractions with ATP. This enzymatic behavior is likely to bear a regulatory relationship to PPi production by chondrocytes in CPDD.

Antigen-induced and zymosan-induced arthritis in mice: studies on in vivo cartilage proteoglycan synthesis and chondrocyte death.

Abstract: The influence of joint inflammation on patellar hyaline articular cartilage was studied in mice Antigen-induced and zymosan-induced arthritis were used as models for immunologically and non-immunologically induced joint inflammation The contribution of newly formed proteoglycan to the cartilage proteoglycan content, as measured by labelling of the cartilage after iv administration of 35S-sulphate, was decreased in parallel with the severity of inflammation during both zymosan-induced and antigen-induced arthritis The decreased 35S content of the cartilage was due to inhibition of synthesis rather than breakdown of newly synthesized proteoglycan, since no accelerated release of 35S from arthritic cartilage could be demonstrated in vitro Antigen-induced arthritis was associated with progressive chondrocyte damage Loss of chondrocytes was consistently found in the central part of the patella, without nearby presence of pannus It would appear that, in addition to enzymatic breakdown of cartilage, other phenomena are important in cartilage destruction: inhibition of proteoglycan synthesis and chondrocyte death, apparently unrelated to pannus formation

Neocartilage derived from transplanted perichondrium: what is it?

Abstract: The articular surfaces of rabbit patellae were completely eburnated and resurfaced with ear perichondrium or fascia lata or left unresurfaced. Both mature and immature animals were used and were sacrificed at 3, 6, and 12 months postoperatively. Only those joints resurfaced with perichondrium formed neocartilage. The neocartilage was composed of variable amounts of chondroitin sulfate; histologic appearances ranged from hyaline cartilage to fibrocartilage to fibrous tissue. Scanning electron microscopy revealed that the neocartilage surface was irregular, fibrillated, and disorganized. Biochemical analyses documented the variability of the neocartilage, one specimen being quite similar to normal articular cartilage. Cell-culture experiments with isolated rabbit periochondrocytes, chondrocytes, and fibroblasts were conducted to determine whether those cell types could produce cartilage matrix in vitro. The ability of sparse cultures of perichondrocytes to synthesize chondroitin sulfate under serum-free conditions is evidence that these cells are unlike fibroblasts and more like chondrocytes in their in vitro behavior. These in vitro and in vivo studies show that perichondrocytes are relatively differentiated cells with the potential to make cartilage. Elucidation of the factors contributing to the variable results of perichondral transplantation is essential before clinical applications will be predictably successful.

Abnormalities in the Biosynthesis of Cartilage and Bone Proteoglycans in Experimental Diabetes

Abstract: Proteoglycans synthesized in developing cartilage and bone were investigated in control and streptozotocin-induced (65 mg/kg, i.v.) diabetic rats. Ten days after streptozotocin injection, animals were implanted subcutaneously with demineralized bone matrix particles. This system induces formation of cartilage and bone on days 7 and 14, respectively. Two hours before they were killed, animals were injected with 35SO4 and the labeled proteoglycans were extracted from the explants and metaphyses by either a direct associative extraction (0.5 M GuCl2) or a direct dissociative extraction (4.0 M GuCl2). These procedures extract 80-90% of the total counts incorporated. To characterize the proteoglycans, extracts were subjected to cesium chloride density gradient centrifugation and molecular sieve chromatography. These data showed that (1) there is less proteoglycan made in diabetic bone; (2) the proteoglycan aggregate is of a smaller molecular weight in bone than in cartilage; (3) 10% of the proteoglycan synthesized in diabetic bone was in the form of aggregates compared with 48% of the control bone; (4) aggregates did form in the diabetic cartilage, and their molecular weight was smaller than in normal cartilage. This investigation shows that proteoglycans, structurally important macromolecules of cartilage and bone, are altered in experimental diabetes. This metabolic abnormality may be an important factor contributing to decreased bone formation observed in diabetes.

Early, asymptomatic stage of degenerative joint disease in canine hip joints.

Abstract: The early stages of degenerative joint disease were investigated in coxofemoral joints from dogs with a hereditary predisposition to hip dysplasia. Alterations observed included mild nonsuppurative synovitis, increased volume of both synovial fluid and the ligamentum teres, and focal degenerative articular cartilage lesions. On radiologic examination, subluxation of the femoral head was seen, but only in the most severely affected joints. Synovial inflammation with increased synovial fluid and ligament volumes were indicators of early degenerative joint disease in dogs. These changes seemed to coincide with, or perhaps to precede, microscopic evidence for articular cartilage degeneration and occurred before radiologic abnormalities were detected.

Evidence for polymorphonuclear-leucocyte-derived proteinases in arthritic cartilage.

Abstract: 1. An enzyme that degrades proteoglycan at neutral pH was extracted with 4 M-guanidine hydrochloride from the articular cartilage of rabbits with antigen-induced arthritis. 2. The enzyme had an apparent molecular weight on Ultrogel AcA 54 of about 8000 and was optimally active at pH 7.5 in Tris/HCl buffer containing 0.2 M-NaCl. The partially purified preparation was totally inhibited by 0.01 mM-N-acetyldialanylprolylvalylchloromethane, severely inhibited by 2 mM-phenylmethanesulphonyl fluoride and soya-bean trypsin inhibitor (200 microgram/ml) and slightly inhibited by 10 mM-EDTA. Marked inhibition was also obtained with a cytosolic fraction prepared from rabbit polymorphonuclear leucocytes. 3. All properties of the enzyme were virtually identical with those of an 'elastase-like' proteinase that was isolated from rabbit polymorphonuclear-leucocyte granules. 4. The results are consistent with the idea that cartilage proteoglycan degradation in acute joint inflammation is due at least partly to the diffusion into the cartilage of proteinases derived from synovial-fluid polymorphonuclear leucocytes.

Ectopic bone formation and aging.

Abstract: Six-week-old, 6-month-old, and 2-year-old female rats of the Wistar strain were implanted with decalcified bone powder prepared from rats of the same strain. An encapsulated deposit of cartilage and bone formed. The deposits were removed at intervals and studied histologically and after administration of 45Ca. Intramembranous bone formed in 14 days in the 6-week-old animals, but was not seen until the 23rd day in the 2-year-old rats. This implant system may provide a model for the study of the mechanisms for the decline of osteogenesis, or the low bone formation rate which occurs with aging.

Release of pyrophosphate by normal mammalian articular hyaline and fibrocartilage in organ culture.

Abstract: Calcium pyrophosphate dihydrate crystals are found most frequently in fibrocartilaginous tissue and to a lesser extent in hyaline articular cartilage. Previous investigators found that pyrophosphate (PPi) was released into the medium by immature rabbit hyaline cartilage and osteoarthritic human cartilage in organ culture but not by normal human or mature rabbit cartilage. By employing a sensitive fluorometric assay for PPi and correcting for hydrolysis of PPi during the incubations, we detected PPi release by all normal mammalian cartilage studied. PPi release per mg wet weight of lapine and canine cartilage was paralleled by uronic acid production. Meniscal fibrocartilage, the most common site of calcium pyrophosphate deposits, also elaborate PPi.

Simulation of the initial stage of endochondral ossification: in vitro sequential culture of growth cartilage cells and bone marrow cells.

Abstract: Growth cartilage cells were isolated from the ribs of young rats and cultured at high cell density in Ham's F-12 medium supplemented with 10% fetal calf serum. During 7 days, glycosaminoglycans and proteoglycans were actively synthesized and secreted, forming a metachromatic matrix. When cultured together with growth cartilage cells precultured and biosynthetically prelabeled with 35SO4(2-) in their glycosaminoglycans, bone marrow cells caused release of 35S-labeled material into the culture medium. Glycosaminoglycan was also released by addition of conditioned medium obtained from cultures of bone marrow cells or peritoneal macrophages to the growth cartilage cell cultures. Electron microscopic studies of the extracellular matrix of growth cartilage cells cocultured with bone marrow cells showed that needles of apatite mineral were deposited within and in close apposition to the surfaces of matrix vesicles. These findings suggest that enzymes released from bone marrow cells or macrophages removed glycosaminoglycan or proteoglycans, which may be inhibitors of mineral growth, and consequently mineralization was initiated. From these findings, sequential culture of growth cartilage cells and bone marrow cells is promising as an experimental system for investigating the mechanism of the initial stage of endochondral ossification.