Showing papers on "Cartilage published in 1976"
TL;DR: The concentration of GAG gradually increases from the articular surface to the deep zone, and it is suggested that this particular profile is adapted to the physiological function and mechanical properties of cartilage.
Abstract: ARTICULAR cartilage contains a high concentration of acid glycosaminoglycans (GAG), reaching 6% by wet weight and associated with fixed charge densities up to 0.2 mEq g−1. This leads to considerable swelling pressure within cartilage, due to, first, the strongly non-ideal osmotic pressure, characteristic of polymer solutions, which increases sharply with concentration and, second, the ionic contribution, in accordance with the Gibbs-Donnan equilibrium. Ogston and Wells1–3 have estimated the values of these two components and I have calculated them from my experimental data on cartilage4,5. The two components of swelling pressure are approximately of the same order of magnitude and can reach values as high as 1.7kgcm−2 (refs 4 and 5). Since normal cartilage does not swell in solution, even when it is removed from the joint and cut into thin (250 µm) slices (lowest curve, Fig. 1), this implies that its high swelling pressure must be counteracted by considerable elastic forces within the collagen fibre network. It has been known for some time that the concentration of GAG gradually increases from the articular surface to the deep zone (for example, refs 6 and 7). A typical variation of total GAG content with depth, measured as fixed charge density, is shown in Table 1. I now suggest that this particular profile is adapted to the physiological function and mechanical properties of cartilage.
705 citations
TL;DR: The repair of articular cartilage removed from the femora of adult rabbits at the knee was studied, and by twelve months there was complete resurfacing of the joint, although the material lost its hyaline appearance after eight months and at one year it resembled dense collagenous tissue.
Abstract: The repair of articular cartilage removed from the femora of adult rabbits at the knee was studied. When multiple perforations were made through the subchondral bone a cartilaginous material, staining heavily with safranin O, first filled the holes. This material resembled hyaline cartilage, and by twelve months there was complete resurfacing of the joint, although the material lost its hyaline appearance after eight months and at one year it resembled dense collagenous tissue.
527 citations
TL;DR: The results suggest that in response to altered mechanical stresses the chondrocytes synthesise proteoglycans that contain more chondroitin sulphate relative to keratin sulphate than normally, as in immature articular cartilage.
Abstract: Biochemical changes in the articular cartilage of the knees of mature dogs, one with natural and four with surgically induced osteoarthritis, have been investigated. The four dogs were killed three, six, nine and forty-eight weeks after division of the right anterior cruciate ligament, the left knees serving as controls. The cartilage of the joints operated on was thicker and more hydrated than the control cartilage; the proteoglycans were more easily extracted and had higher galactosamine/glucosamine molar ratios. The proportion of proteoglycans firmly associated with collagen, and hence not extractable, diminished before fibrillation was demonstrable by indian ink staining of the surface. These biochemical changes were present throughout the entire cartilage of the joints operated on of the dogs killed more than three weeks later, and of the dog with natural osteoarthritis. The results suggest that in response to altered mechanical stresses the chondrocytes synthesise proteoglycans that contain more chondroitin sulphate relative to keratin sulphate than normally, as in immature articular cartilage.
406 citations
TL;DR: Specific antibodies against skin and bone collagen and cartilage collagen are prepared for the study of differential collagen synthesis during development of the chick embryo by immunofluorescence.
364 citations
TL;DR: Findings imply a role for extracellular collagenous matrix in cell differentiation in rat diaphyseal bone formation and the onset and maintenance of erythropoiesis in the induced bone marrow were monitored by 59Fe incorporation into protein-bound heme.
Abstract: Transplantation of collagenous matrix from the rat diaphyseal bone to subcutaneous sites resulted in new bone formation by an endochondral sequence. Functional bone marrow develops within the newly formed ossicle. On day 1, the implanted matrix was a discrete conglomerate with fibrin clot and polymorphonuclear leukocytes. By day 3, the leukocytes disappeared, and this event was followed by migration and close apposition of fibroblast cell surface to the collagenous matrix. This initial matrix-membrane interaction culminated in differentiation of fibroblasts to chondroblasts and osteoblasts. The calcification of the hypertrophied chondrocytes and new bone formation were correlated with increased alkaline phosphatase activity and 45Ca incorporation. The ingrowth of capillaries on day 9 resulted in chondrolysis and osteogenesis. Further remodelling of bony trabeculae by osteoclasts resulted in an ossicle of cancellous bone. This was followed by emergence of extravascular islands of hemocytoblasts and their differentiation into functional bone marrow with erythropoietic and granulopoietic elements and megakaryocytes in the ossicle. The onset and maintenance of erythropoiesis in the induced bone marrow were monitored by 59Fe incorporation into protein-bound heme. These findings imply a role for extracellular collagenous matrix in cell differentiation.
340 citations
TL;DR: A new classification is proposed and osteoarthritis of the hip is divided into three pathogenetic groups: 1) failure of essentially normal cartilage subjected to abnormal or incongruous loading for long periods; 2) damaged or defective cartilage failing under normal conditions of loading; 3) break-up of articular cartilage due to defective subchondral bone.
Abstract: The division of osteoarthritis into primary and secondary varieties implies that these are aetiologically distinct entities, the former being due to some intrinsic defect of cartilage and the latter resulting from previous articular damage. This traditional concept is questioned and the hypothesis is advanced that osteoarthritis is always secondary to some underlying abnormality of the joint. A detailed clinical, radiographic and morbid anatomical study of 327 cases of osteoarthritis of the hip is presented. In all but twenty-seven some predisposing abnormality of the joint was diagnosed: 107 (33%) were associated with major pathology such as Perthes' disease or epiphysiolysis; minor acetabular dysplasia was present in sixty-seven (20%), with a male: female ratio of 1:10; minimal femoral head tilt was demonstrated in fifty-nine (18%), the male: female ratio being 14:1; and in forty-three (13%) there were features suggesting an underlying inflammatory arthritis. On the basis of this study a new classification is proposed and osteoarthritis of the hip is divided into three pathogenetic groups: 1) failure of essentially normal cartilage subjected to abnormal or incongruous loading for long periods; 2) damaged or defective cartilage failing under normal conditions of loading; 3) break-up of articular cartilage due to defective subchondral bone.
312 citations
TL;DR: A cartilage fraction isolated by guanidine extraction and purified by affinity chromatography inhibits tumor-induced vascular proliferation and consequently restricts tumor growth.
Abstract: A cartilage fraction isolated by guanidine extraction and purified by affinity chromatography inhibits tumor-induced vascular proliferation and consequently restricts tumor growth. This fraction contains several different proteins; the major one has a molecular weight of about 16,000. The fraction strongly inhibits protease activity.
307 citations
TL;DR: From previous work it was postulated that joint lubrication is accomplished first by fluid exudation into the joint space, and second by imbibition of the expelled fluid back into the cartilage toward the trailing edge of the contact area caused by the "elastic" recovery of the tissue.
Abstract: The permeability of bovine articular cartilage was measured in an apparatus designed to permit this measurement while the fluid pressure gradient across the cartilage and the axial compressive strain applied to the cartilage were varied independently. For all of the pressure gradients tested the permeability of the cartilage decreased as the compressive strain increased. From previous work, it was postulated that joint lubrication is accomplished first by fluid exudation into the joint space. both at the leading edge of the moving contact area and between portions of the opposing cartilaginous surfaces, and second by imbibition of the expelled fluid back into the cartilage toward the trailing edge of the contact area caused by the "elastic" recovery of the tissue. The present work extends this model to include the condition that the permeability of cartilage is dependent on the extent to which it is deformed.
242 citations
TL;DR: The preliminary results obtained by incubating cartilage for 24 h with clostridial collagenase showed that both the tensile stiffness and the fracture stress were considerably lower than the corresponding values for the untreated tissue.
160 citations
TL;DR: Using the immunohistological method it was possible to identify specific collagen types in areas undergoing rapid proliferation and collagen transition, such as diaphyseal and epiphyseal perichondrium, or in enchondral osteogenesis, in chick embryo.
157 citations
TL;DR: Sedimentation, gel chromatography, sodium dodecyl sulfate-gel electrophoresis, and immuno-diffusion studies of digests of isolated proteoglycan fraction produced by the partially purified cartilage extract at neutral and acid pH confirmed that the cartilage enzymes act only on the protein component of proteogly can subunit, producing fragments with 5 to 12 chondroitin sulfate chains.
Abstract: Extracts of human articular cartilage contain proteases capable of degrading the proteoglycan component of cartilage matrix at neutral and acid pH. These enzymes have been partially purified by ion exchange chromotography and characterized by disc electrophoresis, inhibition patterns, and action of proteoglycan. Three distinct metalloproteases are described. A neutral protease that digests proteoglycan subunit optimally at pH 7.25 has been purified up to 900-fold. It is strongly inhibited by o-phenanthroline, alpha-2-macroglobulin, and egg white, and to a lesser extent by D-penicillamine and EDTA. Inhibition by chelating agents is reversed by cobalt, zinc, and ferrous ions. Two acid metalloproteases, distinct from cathespins B1, D, and F, digest proteoglycan subunit at pH 4.5 and 5.5. Both are inhibited by o-phenanthroline and activity is restored by cobalt, zinc, or ferrous ions. With electron microscopy, it was found that cartilage slices were depleted of ruthenium red-staining matrix proteoglycan after incubation in vitro with a partially purified cartilage extract at neutral pH. Sedimentation, gel chromatography, sodium dodecyl sulfate-gel electrophoresis, and immuno-diffusion studies of digests of isolated proteoglycan fraction produced by the partially purified cartilage extract at neutral and acid pH confirmed that the cartilage enzymes act only on the protein component of proteoglycan subunit, producing fragments with 5 to 12 chondroitin sulfate chains. The link proteins were not digested.
TL;DR: Extracts of human peripheral blood polymorphonuclear leukocyte granules, and two purified proteases derived from such extracts, an elastase and a chymotrypsin-like enzyme, degrade isolated bovine nasal cartilage proteoglycan at neutral pH.
Abstract: Extracts of human peripheral blood polymorphonuclear leukocyte granules, and two purified proteases derived from such extracts, an elastase and a chymotrypsin-like enzyme, degrade isolated bovine nasal cartilage proteoglycan at neutral pH. Viscosity studies indicate that the leukocyte granule extracts lack hyaluronidase activity and that their degradative effect on proteoglycan at physiological pH is due entirely to proteolytic action. Sepharose 4B gel chromatography and SDS-polyacrylamide gel electrophoresis of proteoglycan fractions treated with leukocyte granule enzymes at pH 7.0 indicate that they degrade one of the proteoglycan link proteins, release a fragment from the hyaluronic acid-binding portion of the proteoglycan subunit core protein, and break down the remainder of the proteoglycan subunit molecule into peptide fragments with varying numbers of chondroitin sulfate chains. Immunodiffusion studies indicate that the antigenic determinants of the proteoglycan subunit core protein and the link proteins survive treatment with granule proteases. Similar degradation of human articular cartilage proteoglycan by granule neutral proteases can be presumed to occur, in view of the similarity of structure of human articular and bovine nasal cartilage proteoglycans. The release of granule enzymes in the course of neutrophil-mediated inflammation can thus result in the degradation of cartilage matrix proteoglycan, leading to cartilage destruction and joint injury.
TL;DR: It is concluded that that fall in somatomedin activity and cartilage growth activity associated with STZ-induced diabetes appears to be due to insulin deficiency and growth failure in diabetes, as in malnutrition, may beDue to decreased somatOMin activity.
Abstract: Since diabetes mellitus is a condition in which poor growth occurs despite elevation of plasma GH, we have attempted to determine if poor growth in diabetes, as in malnutrition, could be associated with a decrease in somatomedin activity. Young male rats were rendered diabetic with intravenous streptozotocin (STZ). The growth activity of their cartilage was estimated by 35 SO 4 incorporation in vitro, and somatomedin (SM) activity in their serum was determined by the stimulation of SO 4 incorporation by cartilage from hypophysectomized rats or normal young pigs. Cartilage growth activity was significantly decreased 24 hours after STZ and fell to hypopituitary levels after 48 hours. The decreased growth activity could not be attributed to decreased cartilage responsiveness to SM, since incubation of diabetic cartilage with normal rat serum (normal SM) resulted in significant stimulation of cartilage SO 4 incorporation. SM in diabetic serum decreased to hypopituitary levels 24 hours after STZ, and decreased further after 48 hours. The decrease in SM and cartilage growth activity was not prevented by the administration of high doses of bovine GH. The fall in bioassayable SM appeared to be due in part to the presence of an SM inhibitor in the diabetic serum, since addition of diabetic serum to normal serum decreased to measurable SM in the normal serum. Administration of insulin to diabetic rats 48 hours after STZ led to significant increases in SM and cartilage growth activity, and insulin therapy 24 hours after STZ prevented the decreases in SM and cartilage growth activity which occurred without insulin. Thus, acute STZ-induced diabetes in rats was associated with a significant decrease in both serum SM and cartilage growth activity; these changes were not ameliorated by administration of GH, and insulin therapy could both prevent and reverse the fall in SM and cartilage growth activity. From these observations, we conclude that (1) that fall in somatomedin activity and cartilage growth activity associated with STZ-induced diabetes appears to be due to insulin deficiency and (2) growth failure in diabetes, as in malnutrition, may be due to decreased somatomedin activity.
TL;DR: Findings provide the first evidence for a requirement for silicon in articular cartilage and connective tissue formation and that the site of action of silicon is in the glycosaminoglycan-protein complexes of the ground substance.
Abstract: Studies were undertaken to determine further effects of silicon deficiency in the chick. The diet and experimental conditions were the same as those used in previous studies to demonstrate the essentiality of silicon for growth and development. Skeletal and other abnormalities involving glycosaminoglycans in formation of articular cartilage and comb connective tissue were found to be associated with silicon deficiency. The bones of 1 day-old deutectomized cockerels fed a silicon supplemented diet and killed at 4 weeks of age had significantly greater amounts of articular cartilage and water as compared with the silicon deficient group and also a greater proportion of hexosamine in the cartilage. The greater water content in bones of the silicon supplemented chicks coincided with a larger content of glycosaminoglycans in the articular cartilage. A similar relationship was obtained in cockerel comb. In addition to larger amounts of connective tissue and of total hexosamine in combs of the supplemented group, a higher percentage of hexosamine and a higher silicon content was found. These findings provide the first evidence for a requirement for silicon in articular cartilage and connective tissue formation and that the site of action of silicon is in the glycosaminoglycan-protein complexes of the ground substance.
TL;DR: The studies reported here indicate that mutant, like normal cartilage, synthesizes type II collagen and contains normal quantities of glycosaminoglycans as judged by uronic acid content, however, the glycosamination from the mutant differ from the normal in their chromatographic and electrophoretic properties.
01 Jan 1976
TL;DR: In this article, two purified proteases derived from human peripheral blood polymorphonuclear leukocyte granules, an elastase and a chymotrypsin-like enzyme, degrade isolated bovine nasal cartilage proteoglycan at neutral pH.
Abstract: A B S T R A C T Extracts of human peripheral blood polymorphonuclear leukocyte granules, and two purified proteases derived from such extracts, an elastase and a chymotrypsin-like enzyme, degrade isolated bovine nasal cartilage proteoglycan at neutral pH. Viscosity studies indicate that the leukocyte granule extracts lack hyaluronidase activity and that their degradative effect on proteoglycan at physiological pH is due entirely to proteolytic action. Sepharose 4B gel chromatography and SDS-polyacrylamide gel electrophoresis of proteoglycan fractions treated with leukocyte granule enzymes at pH 7.0 indicate that they degrade one of the proteoglycan link proteins, release a fragment from the hyaluronic acid-binding portion of the proteoglycan subunit core protein, and break down the remainder of the proteoglycan subunit molecule into peptide fragments with varying numbers of chondroitin sulfate chains. Immunodiffusion studies indicate that the antigenic determinants of the proteoglycan subunit core protein and the link proteins survive treatment with granule proteases. Similar degradation of human articular cartilage proteoglycan by granule neutral proteases can be presumed to occur, in view of the similarity of structure of human articular and bovine nasal cartilage proteoglycans. The release of granule enzymes in the course of neutrophil-mediated inflammation can thus result in the
TL;DR: It is concluded that the presence of matrix compounds and the cellmatrix interaction as well are necessary to maintain synthesis of type II collagen in chondrocytes.
Abstract: Synthesis of collagen by chondrocytes was studied by immunofluorescence using antibodies specific for type I, II and III collagen. The following tissues and culture conditions were chosen for this immunohistological study: normal articular cartilage, epiphyseal growth cartilage, cartilage undergoing osteoarthrotic degeneration, suspension culture and monolayer culture. While type II collagen is the unique collagen all over hyaline cartilage, type I collagen is produced by hypertrophic chondrocytes in the growth plate. In addition, chondrocytes in osteoarthrotic areas of articular cartilage synthesize type I collagen. Under in vitro culture conditions, chondrocytes initially produce type II collagen and synthesize later on type I collagen. The change of synthesis from type II to type I collagen is more rapid in monolayer than in suspension culture. It is concluded that the presence of matrix compounds and the cellmatrix interaction as well are necessary to maintain synthesis of type II collagen in chondrocytes. Alterations in the cell-matrix interactions are shown to occur in the hypertrophic zone of the epiphyseal growth plate, in cartilage undergoing osteoarthrotic degeneration as well as in chondrocytes grown in culture. Thus, change in the control of gene activity may subsequently lead to change in collagen synthesis. It is possible that the synthesis of type I collagen, which cannot fulfil the physiological function of a structural element in cartilageneous tissue, is a crucial factor in the process of osteoarthrosis.
TL;DR: The toluidine blue O fixation method, unlike conventional fixation and staining, retained proteoglycan in the pericellular areas of actively synthesizing cells and made it visible by light and electron microscopy.
Abstract: The simultaneous localization of proteoglycan by light and electron microscopy was demonstrated by fixing epiphyseal cartilage in a glutaraldehyde toluidine blue O solution. Sections cut for light microscopy viewing and those cut for electron microscopy required no further staining, although, in the latter case, staining with uranyl acetate and lead improved the overall contrast. By this technique, electron-dense structures were seen concentrated about the cells which were actively synthesizing matrix, and these structures appeared to bind collagen fibrils. Similar structures were not seen in conventionally fixed tissue. They could also not be identified when the specimens were previously incubated with the proteoglycan-digesting enzyme, papain, prior to toluidine blue O fixation. The toluidine blue O fixation method, unlike conventional fixation and staining, retained proteoglycan in the pericellular areas of actively synthesizing cells and made it visible by light and electron microscopy. It appears tha...
TL;DR: Safranin O was added to the fixation process in an attempt to retain the soluble proteoglycans and to increase the electron density of these polyanions following fixation in the presence of this cationic dye, proteoglycan localization was visualized by light microscopy.
TL;DR: Histologic study of the bone disclosed lack of organized cartilage columnar formation, large lacuni containing hypertrophied cells, and lack of trabecular formation with uninterrupted areas of calcified cartilage.
TL;DR: Findings suggest a model of cartilage matrix degradation by PMN neutral proteases in which local protease-antiprotease imbalance, coupled with different rates of penetration of protease and antiprotease into target tissue, plays a key role in accounting for matrix damage.
Abstract: The present work was undertaken to explore the effect of two purified neutral proteases derived from human peripheral blood polymorphonuclear leukocytes (PMN) on articular cartilage as a model of joint injury. Human leukocyte elastase and chymotrypsin-like enzyme, purified by affinity chromatography, released 32SO4 from labeled rabbit articular cartilage slices in vitro. Release of isotope was initially delayed, suggesting that either a lag in enzyme penetration occurs or that size of degradation fragments is a limiting factor in diffusion of label out of the tissue. The release of 35SO4 was inhibited by preincubation of elastase and chymotrypsin-like enzyme with human alpha 1-anti-trypsin, or with their specific chloromethyl ketone inactivators, and the action of elastase was also inhibited by a monospecific antiserum to PMN elastase, freed of major serum proteinase inhibitors. Immunohistochemical staining procedures revealed the presence of PMN elastase inside the matrix of cartilage slices after a 20-min exposure of tissue to either the pure enzyme or crude PMN granule extract. Serum alpha 1-antitrypsin failed to penetrate into the cartilage slices under identical in vitro conditions. In association with the results reported in the accompanying paper, these findings suggest a model of cartilage matrix degradation by PMN neutral proteases in which local protease-antiprotease imbalance, coupled with different rates of penetration of protease and antiprotease into target tissue, plays a key role in accounting for matrix damage.
TL;DR: Remodelling processes were seen in all components of the joints and the significance of the remodelling seen in the fossa and on the mandibular neck is discussed with relation to condylar and periosteal growth of the mandible.
Abstract: Temporomandibular joints from 61 humans, aged 2 days to 27 years, were examined histologically. Four layers of the condyle were studied in detail. The outermost layer was richly vascularised in new-borns but by 3 years of age it had become avascular and contained few cells. In neonates the cartilage layer constituted a large part of the condyle but soon decreased in thickness and by 5-6 years of age it constituted only a thin zone of the top of the condyle. In the proliferative zone, mitoses occurred up to 13-15 years of age. This zone then decreased in thickness; the number of cells decreased, while the amount of intercellular substance increased. At birth, the temporal component was flat and was lined by vascularised connective tissue which became richer in collagen with increasing age. The cartilage layer was lacking in the fossa but was present on the tuberculum. A proliferative zone in this cartilage could be seen up to the age of 17-18 years and cartilage having only few cells was found in adults. Remodelling processes were seen in all components of the joints. The significance of the remodelling seen in the fossa and on the mandibular neck is discussed with relation to condylar and periosteal growth of the mandible.
TL;DR: It was concluded that the fatigue properties of human articular cartilage decrease with age to such an extent that fatigue failure of the material could occur in life.
TL;DR: Initial studies indicated that bone and cartilage, when treated with a hypertonic glutaraldehyde fixative for a short period, retained significant enzyme activity for histochemistry and also maintained excellent fine structure.
TL;DR: Sodium salicylate and indomethacin at higher doses produced significant inhibition of sulphated glycosaminoglycan synthesis in all femoral heads studied, reflecting the variable response of human articular cartilage to anti-inflammatory drugs.
Abstract: The anti-inflammatory drugs, sodium salicylate, indomethacin, hydrocortisone, ibuprofen, and flurbiprofen, were examined for their effects on sulphated glycosaminoglycan synthesis in aged human cartilage in vitro. Cartilage was obtained from femoral heads removed during surgery and drug effects were found to vary significantly from one head to another. Statistical analysis of the results showed that sodium salicylate exhibits concentration-dependent inhibition of glycosaminoglycan synthesis over the concentration range used. Indomethacin, hydrocortisone, and ibuprofen, at concentrations comparable to those attained in man, caused a statistically significant depression of sulphated glycosaminoglycan synthesis in cartilage from some femoral heads but not others, reflecting the variable response of human articular cartilage to anti-inflammatory drugs. Sodium salicylate and indomethacin at higher doses produced significant (Pless than 0-005) inhibition of sulphated glycosaminoglycan synthesis in all femoral heads studied. The results for flurbiprofen were less conclusive; this compound appears not to inhibit glycosaminoglycan synthesis over the concentration range used.
TL;DR: This chapter describes the hormonal regulation of cartilage growth and metabolism and demonstrates that serum sulfation factor increases cartilage cAMP content as well as α-aminoisobutyrate transport and that the correlation between the two effects is striking.
Abstract: Publisher Summary This chapter describes the hormonal regulation of cartilage growth and metabolism. The central role of cartilage in linear growth has been appreciated as the process of endochondral ossification was recognized. Cartilage is a specialized connective tissue whose primary function is to make a matrix that is resilient and resists deformation and compression. Several different types of cartilage exist in animals, and each appears to be adapted to its own specific function by the chemical composition of its matrix. The matrix compositions of the three major types of cartilage, elastic, fibrous, and hyaline are studied. Most of the studies of the effect of serum sulfation factor on cartilage metabolism in vitro have been conducted using serum containing sulfation factor activity or a partially purified fraction of serum containing such activity. It is shown that serum sulfation factor probably stimulates cartilage amino acid transport through a CAMP-dependent mechanism. It is demonstrated that serum sulfation factor increases cartilage cAMP content as well as α-aminoisobutyrate transport and that the correlation between the two effects is striking.
TL;DR: Wherever the perichondrium was grafted in the presence of clotted blood new cartilage was produced, irrespective of age and weight of the rabbit, whereas when blood was withheld, little or no cartilage formed.
TL;DR: It has been found that chick embryo cartilageproteoglycan is reactive with types I, II, and III soluble collagen and that both the core-protein and chondroitin sulfate chains of the proteoglycan are necessary for this interaction.
TL;DR: Biochemical and ultrastructural analysis of the sternal cartilage of chick embryos homozygous for the autosomal recessive gene nanomelia suggest that the mutant cells are functional chondrocytes in all respects except in proteochondroitin sulfate synthesis.