Cartilage Macromolecules Research Articles

Repair of intra-membranous bone fractures and defects in rats: Immunolocalization of bone and cartilage proteins and proteoglycans

Osseous healing of experimental fractures and defects in membranous bone was studied in an animal model and the appearance and localization of selected bone and cartilage proteins and proteoglycans determined by polyclonal antibodies. The bone lesions were made in the parietal bone of young rats and subsequently studied on days 3, 5, 8, 15, 30, and 100 after surgery. The bone matrix proteins investigated (62 kDa, bone sialoprotein I and II, and osteopontin) appeared early, adjacent to the periosteal surfaces (pericranium and dura mater) and the marginal bone. The staining reactions were maximal at days 8 or 15 after trauma. Similar patterns were discerned for some cartilage macromolecules studied (58 kDa, 59 kDa, and chondrocalcin), although others showed no labelling whatsoever (148 kDa, and 400 kDa proteins). The proteoglycans PG-S1, PG-S2, and PG-LA were not identified. No callus or cartilage formation were associated with the bone healing process, and the differences between the regenerative pattern of the fractures and defects were limited. The findings emphasize the importance of rigid fixation in craniomaxillofacial surgery.

Cell density dependent effect of a tumor promoter on proliferation and chondrogenesis of limb bud mesenchymal cells

When limb bud mesenchymal cells are cultured at high density, chondrogenesis takes place in vitro. Treatment of such cultures with the tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) resulted in complete inhibition of chondrogenesis as indicated from staining the cultures for proteoglycans and from RNA hybridization to cDNA probes specific for four cartilage macromolecules. The effect of TPA varied depending on the initial plating density. At high density, TPA inhibited cell proliferation. At low density, cell proliferation was stimulated by TPA and above a certain cell density, chondrogenesis took place even in the presence of TPA. These results are interpreted to mean that the effect of TPA on chondrogenesis is indirect, possibly through its influence on cell proliferation.

Structure and biology of cartilage and bone matrix noncollagenous macromolecules.

Over recent years a number of cartilage and bone matrix molecules have been identified and characterized. These include major constituents such as collagens and proteoglycans as well as a number of less-abundant matrix proteins. In several cases these proteins have been characterized by cloning and sequence analysis of the corresponding cDNA. Some properties of the macromolecules have been studied and an understanding of their functions in the structure, assembly, and breakdown of connective tissue matrix is emerging. It appears that some of these molecules have structural roles whereas others participate in the assembly of the tissue. In this paper we attempt to give a current picture of the organization and role of the noncollagenous matrix macromolecules in cartilage and bone.

Cartilage macromolecules and the calcification of cartilage matrix.

The calcification of cartilage matrix in endochondral bone formation occurs in an extracellular matrix composed of fibrils of type II collagen with which type X collagen is closely associated. Also present within this matrix are the large proteoglycans containing chondroitin sulfate which aggregate with hyaluronic acid. In addition, the matrix contains matrix vesicles containing alkaline phosphatase. There is probably a concentration of calcium as a result of its binding to the many chondroitin sulfate chains. At the time of calcification, these proteoglycans become focally concentrated in sites where mineral is deposited. This would result in an even greater focal concentration of calcium. Release of inorganic phosphate, as a result of the activity of alkaline phosphatase, can lead to the displacement of proteoglycan bound calcium and its precipitation. The C-propeptide of type II collagen becomes concentrated in the mineralizing sites, prior to which it is mainly associated with type II collagen fibrils and is present in dilated cisternae of the enlarged hypertrophic chondrocytes. The synthesis of type II collagen and the C-propeptide, together with alkaline phosphatase, are regulated by the vitamin D metabolites 24,25(OH)2 cholecalciferol and 1,25 (OH)2 cholecalciferol. At the time of calcification, type X collagen remains associated with type II collagen fibrils. It may play a role in preventing the initial calcification of these fibrils focusing mineral formation in focal interfibrillar sites. This process of calcification is clearly very complex, and involves different interacting matrix molecules and is carefully regulated at the cellular level.

Immune mechanisms in osteoarthritis

0 STEOARTHRITIS (OA) is generally thought of as a disease of articular cartilage failure induced by a variety of genetic, metabolic, and biomechanical factors. Coexisting with the cartilage abnormalities, most affected joints exhibit synovial inflammatory responses that may range from minimal lining cell layer hyperplasia to severe acute and chronic synovitis. The synovial response may be responsible for the pain symptomatology associated with early OA. Although it is unlikely that immune processes play an important primary role in initiating synovial inflammation, cellular and humoral immune responses to cartilage macromolecules may constitute important factors in the maintenance and severity of synovitis in OA. This review focuses on the specific immunologic mechanisms that may be operative in OA. The physiopathogenesis of inflammation induced by immunologic mechanisms is not discussed in detail because it is likely that once the antigenspecific interactions occur, the development of the inflammatory process results from the generation of cellular and humoral proinflammatory factors that constitute a final common pathway to most inflammatory processes, regardless of the disease entity under consideration.

Freeze-substitution staining of rat growth plate cartilage with alcian blue for electron microscopic study of proteoglycans.

To selectively stain polyanionic macromolecules of growth plate cartilage and to prevent artifacts induced by aqueous fixation, proximal tibial growth plates were excised from rats, slam-frozen, and freeze-substituted in 100% methanol containing the cationic dye Alcian blue. Electron microscopic examination showed the tissue stained with Alcian blue to be comparable in ultrastructural preservation to tissues slam-frozen and freeze-substituted in the absence of Alcian blue. The extracellular matrix exhibited a characteristic staining pattern when stained by this method. The pericellular rim was identified as a band of varying width encircling the chondrocyte and its cell processes. Peripheral to the pericellular rim the heterogeneity of staining within the extracellular matrix increased, taking the form of polymorphic densities. X-ray microanalysis showed that the visual interpretation of electron density was related to the concentration of copper present, and that the concentration of sulfur was variable in the pericellular rim and in the interterritorial matrix. The difficulties associated with aqueous fixation and staining procedures are discussed in contrast to the improved preservation achieved by cryogenic methods.

Evidence that a humoral immune response to autologous cartilage proteoglycan can participate in the induction of cartilage pathology.

We examined antiproteoglycan antibodies as an autoimmune response for induction of synovitis. This hypothesis was studied by monitoring humoral antiproteoglycan antibody following IgG induction of experimental immune synovitis, localization in the articular cartilage of an autologous immune response, and loss of proteoglycan from cartilage following intravenous administration of antiproteoglycan monoclonal antibodies. The data support the hypothesis that autoimmunity to cartilage macromolecules may play a role in the etiopathogenesis of arthritis.

Minireview Interleukin-1 and osteoarthritis

Il-1, a multifunctional monokine, can stimulate both synoviocytes and articular chondrocytes to release neutral proteases and prostaglandin E 2. It is also capable of promoting bone resorption. Therefore, this molecule (or family of molecules) is likely to play an important role in the mechanism of articular cartilage destruction that occurs in degenerative arthropathies. The synovial tissue itself can produce Il-1 (Catabolin) in some conditions, such as a slight traumatism, so that the presence of local inflammation is not necessary for “Il-1-cartilage” interaction to occur. Fundamental macromolecules of cartilage (collagens, proteoglycans) exert a stimulatory effect on Il-1 production, either as such or in the form of immune complexes. Some activated complement fractions (C3a and C5a) may also be actively involved. Studies on the mechanisms which regulate Il-1 synthesis and release, as well as investigations on the response of target cells to Il-1, are presently fascinating goals that could lead to new strategies in therapeutic research.

The in vitro behavior of fetal condylar cartilage in serum-free hormone-supplemented medium

Mandibular condyles of fetal mice 19–20 days in utero were kept in a serum-free organ culture system for up to 14 days and were investigated for their capacity to develop osteoid and to mineralize in vitro. After 3 days in culture, the cartilage of the mandibular condyle appeared to have maintained all its inherent structural characteristics, including its various cell layers: chondroprogenitor, chondroblastic, and hypertrophic. After 7–9 days in culture, no chondroblasts could be seen; instead, most of the cartilage consisted of hypertrophic chondrocytes. In addition, various areas throughout the explant revealed the appearance of osteoidlike material. The process of matrix mineralization progressed with time, and by the 14th day new bonelike material was found to occupy a larger portion of the explant. The newly formed matrix reacted positively with antibodies against type I and type III collagens, as well as against bone alkaline phosphatase. Electron microscopic examination showed that the mineralization appeared to be associated with collagen fibers as well as the matrix vesicles. In composition, the in vitro-formed mineral deposits resembled hydroxyapatite crystals. Biochemical assays indicated that the newly formed tissue reacted strongly for alkaline phosphatase and incorporated 45Ca. The findings of the present study imply that fetal condylar cartilage possesses the potential to develop in vitro osseouslike tissue even in a system that is serum-free. Due to the fact that the newly formed extracellular matrix mineralized and reacted positively to bone markers as well as to cartilage macromolecules, it would seem justifiable to define the new tissue as chondroid bone.

Stimulation of uridine, leucine, and sulfate incorporation into rat cartilage macromolecules by adenosine 3',5'-monophosphate.

Conflicting findings on the ability of cAMP analogs or phophodiesterase inhibitors to stimulate precursor incorporation into macromolecules of rat cartilage have been reported. Therefore, the effects of these compounds on the incorporation of uridine into RNA, leucine into proteins, and sulfate into proteoglycans have been reexamined in cartilage from normal and hypophysectomized rats. When cartilage was incubated for 24 h in a medium with the test agents and then pulsed for 2 h in the basal medium containing labeled precursors, both monobutyryl cAMP (BucAMP) and methylisobutylxanthine (MIX) enhanced the ability of the tissue to incorporate precursors into macromolecules. The effect of BucAMP was significant in most cases at a concentration of 30 microM, optimal at concentrations of 100-300 microM, and diminished at a concentration of 1000 microM. Similar stimulation was produced by dibutyrul cAMP [(Bu)2cAMP] or 8-dimethylamino cAMP, but monobutyryl cGMP was ineffective. MIX in a concentration of 20 microM increased precursor incorporation in most cases, and a concentration of 100 microM was optimal; at a concentration of 500 microM, MIX had no significant effect on leucine or sulfate incorporation. When cartilage from hypophysectomized rats was incubated in a medium with the test agents for 4-6 h and the labeled precursors were added for the last 2 h, BucAMP did not increase incorporation of any of the precursors. MIX was also ineffective, even though tissue cAMP levels were increased. However, precursor incorporation was increased by exposure to partially purified rat somatomedin for the same periods. The degree of stimulation of sulfate incorporation induced by either BucAMP or MIX was proportional to the time of exposure to these agents. Preincubation of cartilage in basal medium alone for 22 h or longer increased basal sulfate incorporation, but caused only a slight enhancement of the action of BucAMP. The addition of synthetic bovine PTH-(1-34) (1 microM) to the incubation medium increased sulfate incorporation into hypophysectomized rat cartilage by 24 h, and this effect was potentiated by MIX (10 microM). No stimulation was detectable by 6 h, even with MIX in the medium. PTH-(1-34) increased the cartilage cAMP level, and this effect was also potentiated by MIX. In the presence of MIX, PTH-(1-34) increased the level of cAMP within 30 min, while the rat somatomedin preparation had no effect on the cAMP level during 60 min of incubation. The level of cartilage cGMP was not raised by either PTH or somatomedin.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro induction of cartilage-specific macromolecules by a bone extract.

An in vitro system has been developed to study the onset of chondrogenesis. Embryonic rat muscle mesenchymal cells, when treated in suspension culture with an extract of bovine bone matrix, synthesized cartilage-specific proteoglycan and type II collagen. The synthesis of these two macromolecules was assayed by the enzyme-linked immunosorbent assay inhibition technique. Further evidence of chondrogenesis was demonstrated by morphological changes of treated cells when cultured in firm agarose and stained for metachromatic matrix. Even with crude bone matrix extracts, the assay was sensitive at the microgram level and significant differences in cartilage macromolecules compared with controls were observed in 2-3 d. In vivo the same extract induced first cartilage and then bone.

Induction of prenatal toxicity in the rat by diethylstilbestrol, zeranol, 3,4,3',4',-tetrachlorobiphenyl, cadmium, and lead.

A teratological study was conducted in pregnant Sprague-Dawley rats dosed orally with diethylstilbestrol (DES), zeranol (ZN), 3,3',4,4'-tetrachlorobiphenyl (4CB), cadmium, or lead on days 6-18 of gestation. Fetuses were examined on day 19 for growth retardation, resorption, gross malformations, and organ-level anomalies. Synthesis of protein, DNA, and proteoglycan in fetal limb cartilage was also studied by measuring the incorporation of labeled precursors in vitro. DES, ZN, and 4CB produced a dose-dependent increase in embryolethality. Treatment with DES caused an increase in cryptorchidism and edema, and reduced average fetal weight. Zeranol also decreased fetal weight, but was not teratogenic nor did it alter rates of synthesis of macromolecules in cartilage. 4CB caused severe intestinal lesions that were associated with accumulation of blood in the digestive tract and amniotic fluid. 4CB also decreased overall fetal size and total and ossified tibia lengths. Cadmium produced no malformations although incorporation of [3H]amino acids by limb cartilage was slightly increased. Lead was not teratogenic.

Radioimmunoassay of the 148-kilodalton cartilage protein. Distribution of the protein among bovine tissues.

A sensitive and specific radioimmunoassay for the 148 kDa cartilage protein (previously referred to as 'cartilage matrix protein') was developed. The protein is insoluble in conventional buffers and was therefore dissolved in sodium dodecyl sulphate. Excess sodium dodecyl sulphate was bound in mixed micelles with Triton X-100 before assay. Antibodies raised against the 148 kDa cartilage protein did not react with other cartilage macromolecules. Neither did they react with guanidinium chloride extracts of a number of non-cartilaginous tissues. The protein, then, is unique for cartilage. It was demonstrated in tracheal, nasal-septum, xiphisternal, auricular and epiphysial cartilage. Surprisingly, the protein was detected neither in extracts of articular cartilage nor in extracts of the anulus fibrosus or the nucleus pulposus of the intervertebral disc.

Abnormalities in the biosynthesis of cartilage and bone proteoglycans in experimental diabetes.

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.

Neutral proteinases from articular chondrocytes in culture that degrade synthetic substrates and cartilage macromolecules

Neutral proteinases from articular chondrocytes in culture that degrade synthetic substrates and cartilage macromolecules

Biochemical and metabolic abnormalities in articular cartilage from osteoarthritic human hips. III. Distribution and metabolism of amino sugar-containing macromolecules.

Since 1960, numerous studies have supported the thesis that the synthetic activity of articular chondrocytes is increased in osteoarthritis, but several recent reports have challenged this concept. To clarify this problem fully and also to define further the products of this increased synthesis, three experiments were performed in which the distribution and rates of synthesis of amino sugar-containing macromolecules in normal and osteoarthritic cartilage from the human femoral head were assessed by biochemical analysis and studies of the incorporation of 3H-glucosamine and 35SO4. The biochemical data obtained clearly demonstrated the previously noted significant decrease in hexosamine content in osteoarthritic tissue. This decrease was principally due to a diminution in glucosamine concentration and correlated inversely with the severity of the disease process (as measured by a previously described histological-histochemical grading system). Metabolic studies showed a marked increment in the rates of incorporation of 3H-glucosamine into both the glucosamine and the galactosamine fractions of the cartilage. The increased synthesis correlated directly in a non-linear fashion with the severity of the disease. The ratio of the rate of incorporation of 3H-glucosamine into the glucosamine fraction to the rate of its incorporation into the galactosamine fraction was the same in normal and osteoarthritic samples, suggesting that the decline in glucosamine concentration was not related to a qualitative alteration of synthetic activity.

Adenosine 3′,5′-Monophosphate Effects on Cartilage Ribonucleic Acid Synthesis*

We have previously shown that N6-monobutyryl-cAMP stimulates the incorporation of radiolabeled precursors into the macromolecules of embryonic chicken cartilage incubated in organ culture. The present study assessed the specific effects of N6-monobutyryl-cAMP on RNA synthesis in the cartilage organ culture system. The data show that treatment of cartilage with N6-monobutyryl-cAMP results in a coordinated stimulation of mRNA, rTNA, and tRNA synthesis. Incubation of cartilage with N6-monobutyryl-cAMP causes an increase of [5-3H]uridine incorporation into poly A-containing cartilage RNA and 28S, 18S, and 4S RNA as well. Increased incorporation of the radiolabeled nucleotide occurs without a measurable change in uridine triphosphate specific activity or in the rate of RNA degradation. Further, the isolated poly A-containing RNA directs protein synthesis in an in vitro cell-free system, and stimulation of cartilage mRNA by N6-monobutyryl-cAMP does not alter the size distribution of the mRNA species synthesized from that present in control tissue. These data indicate that cAMP enhances cartilage transcriptional activity. In addition, the increased synthesis of mRNA occurs antecedent to demonstrable effects on protein synthesis. Thus, the effects of cAMP on protein synthesis may be secondary to action at the nucleus.

Articular Damage in Arthritis and Its Control

Erosion of joint surfaces in arthritic conditions is always associated with the degradation of the two principal matrix macromolecules of cartilage, proteoglycan and collagen. The major enzymes thought to be involved in articular catabolism have not been isolated, purified, and their properties studied, but precise spatial and temporal activities are still imperfectly understood. We are beginning to understand some aspects of cellular function in the control of catabolic enzyme function. Recent studies on molecular, cellular, and tissue mechanisms in this process are discussed here. Of particular interest is a possible natural control mechanism involving a recently discovered inhibitor of collagenase. A newly developed pharmacologic method of inflammation control utilizes the properties of liposomes in the closed environment of the joint cavity. Very low doses of modified steroids encapsulated within liposomes are capable of substantially reducing inflammation in experimentally arthritic animals and may be applicable to man.

Stimulation of cartilage macromolecule synthesis by adenosine 3′,5′-monophosphate

The role of cyclic AMP in the regulation of cartilage macromolecule synthesis in vitro was studied in pelvic cartilage from 10–12 day chick embryos. Incubation of cartilages in medium containing 0.5 mM cyclic AMP resulted in a 30% inhibition of 35SO 4 −2, [ 3H]leucine and [ 3H]uridine incorporation into proteoglycan, total protein and RNA, respectively. Higher concentrations of cyclic AMP had no greater effects. In contrast, butyrylated cyclic AMP derivatives (0.5–5.0 mM) added to the incubation medium stimulated (50–100%) the incorporation of these radiolabeled precursors into cartilage macromolecules. Theophylline, in concentrations (0.1–0.5 mM) which raise intracellular cyclic AMP, also increases the incorporation of radiolabeled precursors into macromolecules. The data indicate that exogenous cyclic AMP and butyrylated cyclic AMP derivatives have paradoxical effects on cartilage macromolecule synthesis. Butyrylated cyclic AMP derivatives, not exogenous cyclic AMP, mimic the effects of intracellular cyclic AMP. Incubation of embryonic chicken cartilage with exogenous cyclic AMP results in the extracellular degradation of the cyclic AMP to adenosine. Adenosine (0.125 mM) inhibits precursor incorporation into cartilage macromolecules. The metabolism of exogenous cyclic AMP generates sufficient adenosine to account for the observed inhibitory effects of exogenous cyclic AMP on cartilage macromolecule synthesis. Butyrylated cyclic AMP derivatives are not degraded during incubation with cartilage. The data indicate that cartilage is a tissue in which the effect of cyclic AMP is to stimulate anabolic processes.

Characterisation of the Major CNBr-Derived Peptides of Porcine Type II Collagen

As part of a general study on the matrix macromolecules of pig cartilage, the CNBr-derived peptides of porcine type II collagen have been isolated from laryngeal cartilage and characterized. Type II was the only molecular type of collagen detected in laryngeal cartilage from 6-9 month old pigs. The major CNBr-peptides of this collagen were prepared by ion exchange and molecular sieve chromatography and characterized by SDS-polyacrylamide disc electrophoresis and amino acid analysis. Six peptides were recovered in high yield and were shown to be closely homologous to similar peptides previously recovered from bovine and human type II collagens. The largest peptide alpha1(II)CB10 appeared by SDS-disc electrophoresis to be slightly larger than previously reported. The amino acid composition of alpha1(II)CB10 supported this finding of a higher molecular weight.