Showing papers in "Connective Tissue Research in 1986"

In vitro Formation of Hybrid Fibrils of Type v Collagen and Type I Collagen Limited Growth of Type I Collagen Into Thick Fibrils by Type V Collagen

Abstract: Type V collagen and type I collagen were obtained from human placentas by pepsin treatment, followed by salt fractionation. The precipitates formed at 37°c from a mixed solution of type V collagen and type I collagen, reacted with antibodies to either type V collagen or type I collagen. The precipitates seen by electron microscopy were fine flexible fibrils, with a D-periodic banding pattern. The average diameter of hybrid fibrils was smaller than 50nm, when the proportion of type V collagen exceeded that of type I collagen. Type V collagen directly interacts with type I collagen in forming hybrid fibrils, resulting in limitation of the growth of type I collagen fibrils into thicker fibrils. We propose that the fibrils with a predominant type V collagen content may occur in the pericellular environment of various tissues, as a basic structure in connecting basal laminae with interstitial collagen fibrils.

Proteoglycan-collagen relationships in developing chick and bovine tendons. Influence of the physiological environment.

Abstract: Developing and mature flexor digitorum tendons of chick and bovine origin were analyzed for nucleic acid, hydroxyproline, hyaluronate, chondroitin sulfate and sulfated glycosaminoglycan hexuronate Collagen fibril diameters were determined by electron microscopy Results are compared with similar data from rat tail tendon Tissue hydroxyproline (mg/g) increased rapidly in early embryogenesis, while collagen fibril diameters remained relatively constant and small Hyaluronate and chondroitin sulfate were present in considerable quantities, (phase I) A rapid increase in fibril diameters in midterm ‘pregnancy’ coincided with a decrease in hyaluronate and chondroitin sulfate contents (phase II) The transition from phase Ito phase II was associated with the first loading by active muscles of the tendon, independently of whether the animal was pre- or post partum The probable existence of a developmental program intrinsic to tendon itself, is discussed The data are compared with predictions from three diffe

Differences in the Fibril Structure of Corneal and Tendon Collagen. An Electron Microscopy and X-Ray Diffraction Investigation

Abstract: A detailed analysis of the D-period and axial electron density distribution of cornea and tendon collagen was carried out by means of X-ray diffraction and electron microscopy.Ultrastructural observations were made on replicas of freeze fractured and deep-ctched specimens. Synchrotron radiation was used to obtain high resolution small angle X-ray diffraction patterns.The data provide evidence that D-period and intraperiod distances in cornea are shorter than in tendon collagen fibrils. The observed different banding observed is interpreted on the basis of the different morphological arrangement of the microfibrils in the two tissues: “helicoidal” in cornea and “straight” in tendon microfibrils.

The Catabolic Fate of Hyaluronic Acid

Abstract: Part of the hyaluronic acid (HA) synthesized in peripheral tissues enters the blood circulation through the lymph. It is rapidly taken up by the endothelial cells in the liver (half-life in blood is 2.5–5.5 minutes) and degraded. Pure primary cultures of liver endothelial cells were obtained by a newly developed technique and used to follow the metabolism of the polysacharide. At 7°C the cells exhibit a saturable binding of HA indicating a specific receptor for the polysaccharide on the cell surface. At 37°C the HA is effectively endocytosed and degraded to acetate and lactate. A radioassay specific for HA and sensitive in the nanogram range has been developed to follow the concentration of HA in serum. The normal level in man is 10 to l00ug/l. Elevated serum levels of HA are seen in liver cirrhosis, rheumatoid arthritis and scleroderma indicating that both an impaired catabolism in the liver and an increased synthesis in the peripheral tissues can modify the HA level.

Relationships between the biomechanical properties, composition and molecular structure of connective tissues.

Abstract: Biomechanical, biochemical and morphological methods have been used in combination as analytical tools in the study of normal and pathological tissue functions. The biochemical studies described have been performed in vitro on whole tissue preparations and on individual components of connective tissues. Enzymatic degradation has been used to selectively remove components from the tissues. Collagen is the main load-bearing component in muscle tendon and skin. The mechanical properties of the aorta appear to depend on an interaction between collagen and elastin. Membranes of purified, reconstituted collagen type I itself exhibits the visco-elastic behavior which is characteristic for tissues like muscle tendon and skin. Applicability of the methods is shown in two examples: The strength of skin samples from patients with osteogenesis imperfecta was found to be reduced, and the skin from the patients with the lowest strength contained the highest proportions of collagen type III. Moreover, the stability of molecular collagen type I was decreased. In samples of the intracranial arteria cerebri media obtained at autopsy from patients with rupture of intracranial aneurysms the wall thickness, dry defatted weight and ratio collagen type I/type III were increased. The diameter of the arteries was reduced.

Distribution of type III collagen in bovine skin of various ages.

Abstract: The changes in the distribution of type III collagen in bovine skin samples of increasing age have been examined. Analysis of layers from bovine skin of various ages has shown that type III collagen was present in all samples and that the proportion of type III collagen present was greatest in the youngest tissues and in the papillary dermis. The proportion of type III collagen in the papillary dermis decreased until 18 months and then remained constant; the proportion in the reticular dermis continued to decline up to at least 112 months.

Identification of dentin phosphophoryn localization by histochemical stainings.

Abstract: Phosphophoryn, the most abundant of the dentin non-collagenous proteins, has been considered to be related in function to the mineralization process. In the present study, identification of dentin phosphophoryn localization was attempted using newly developed, precautionary histological methods by which phosphophoryn was retained in the sections during the specimen preparation and stained selectively in situ. Phosphophoryn was found to be present widely in all of the calcified dentin except the mantle dentin, the external, first-formed portion of dentin, but was not found in the predentin, the inner, uncalcified layer of dentin. These results indicate that phosphophoryn is apparently related to the mineral phase of calcified dentin and that the mineralization process of mantle dentin, which is formed before the odontoblasts are fully differentiated, may be different from that of circumpulpal dentin.

In-vitro culture of human chondrocytes from adult subjects.

Abstract: An agarose gel matrix was utilized to grow chondrocytes from human donors of various ages in cell culture. The chondrocytes produced the pericellular matrix characteristic for such cells and synthesized collagen type II as well as glyco-saminoglycans. The latter exhibit the typical distribution pattern of the respective articular cartilage matrix. The electron-microscopic appearance of the cultured chondrocytes closely resembles that of chondrocytes in sections of the original cartilage.

Collagen and elastin synthesis in human stroma and breast carcinoma cell lines: modulation by the extracellular matrix.

Abstract: The desmoplastic response to breast carcinoma is being studied. The stimulation of stromal cell proliferation by a preformed breast tumor matrix was shown. An additional mechanism for stimulating scleroprotein deposition is described here. On a per-cell basis, the synthesis of collagen and elastin was increased by 50% and 70%, respectively, in fibroblasts grown on the preformed breast tumor matrix compared to the same cells grown on plastic or on their own preformed matrix. Breast tumor cells themselves synthesized small amounts of collagen and elastin compared to fibroblasts. These levels were unchanged when breast tumor cells were grown on the preformed matrix of fibroblasts. Addition of steroid hormones to cultured cells grown on plastic or on preformed matrices in various combinations, did not change the levels of either collagen or elastin synthesis. The matrix of human breast tumor cells exerts a dual effect; it is mitogenic for fibroblasts, and also stimulates the level of collagen and elastin synt...

Laminin M is found in placental basement membranes, but not in basement membranes of neoplastic origin.

Abstract: Laminin is a high molecular weight glycoprotein found in all basement membranes studied to date. Two subunits of laminin, A and B, have been isolated and characterized from a variety of tumor matrices. Recently we have reported the finding, in human placenta, of a new laminin subunit which we termed M. In the present study we report on the presence of laminin M in placentae of other species such as bovine, rat and mouse. In addition, we have examined laminin extracted from mouse EHS-tumor, rat ED-PYS carcinoma and three human carcinoma cell lines. The laminin subunits were detected by the electroimmunoblot technique using antibodies against mouse, rat and human laminin. Laminin M could not be demonstrated either in the two murine tumors, or in the three different human neoplastic cell lines studied. If the absence of laminin M is the consequence of neoplastic transformation, then studies of the metabolism of this subunit may provide new information on neoplastic transformation and invasion, and a useful m...

Biosynthesis of collagen crosslinks: in vivo labelling of neonatal skin, tendon, and bone in rats.

Abstract: Collagen crosslinks in neonatal rats were labelled in vivo by a single intraperitoneal injection of 200 uCi of [14C]lysine. Rats were killed at times ranging from 30 minutes to 10 weeks after injection. Whole skin, tendon, and bone were analyzed, after reduction and hydrolysis, for collagen crosslink content by HPLC. Crosslinks and amino acids were visualized by their incorporation of radioactivity from [14C]lysine and also fluorometrically by post-column derivatization with o-phthalaldehyde. The incorporation of 14C from labelled lysine into the principal difunctional reducible crosslinks, N6.6'-dehydro-5,5'-dihydroxylysinonorleucine and N6.6'-dehydro-5-hydroxylysinonorleucine, increased most rapidly between 4 and 12 hours after injection, results similar to those observed by others studying crosslink biosynthesis in vitro. Incorporation of 14C into the tetrafunctional crosslink histidinohydroxymerodesmosine proceeded more slowly than it did for the difunctional crosslinks. Values for the amount of radio...

Comparison of Lysyl Oxidase From Bovine Lung And Aorta

Abstract: A prior report had concluded that bovine lung lysyl oxidase displayed an unusual resistance to inhibition by β-aminopropionitrile (BAPN) in contrast to the enzyme isolated from other connective tissues. Therefore, lysyl oxidase was purified from fetal bovine lung and from aorta of young calves by parallel procedures, and key chromatographic and catalytic properties of these enzymes were directly compared. The enzymes prepared from both tissues each demonstrated the same multiplicity of enzyme species which resolve on DEAE-cellulose and otherwise demonstrated the same chromatographic behavior on gel exclusion media and on collagen-Sepharose and Cibacron blue-Sepharose columns. The activities of the unresolved but partially purified enzyme species of lung and of aortic lysyl oxidase were each fully inhibitable by approximately the same low (μmolar) concentrations of BAPN. Thus, the enzymes of both tissues were found to be very similar to each other by several criteria.

The effect of tension on collagen remodelling by fibroblasts: a stereological ultrastructural study.

Abstract: Peak levels of collagen phagocytic activity by fibroblasts during scar maturation have been shown to coincide with a reorientation of major fibre bundles in a process analogous to Wolff's Law. This activity has been shown to increase when greater tension exists across the scar. In this study, the influences of reduced tension on collagen remodeling was studied.In two groups of five rats the right achilles tendon was either: a) transected at its muscle insertion or b) was exposed in a similar fashion but left intact. The tendon samples were retrieved five days following initial surgery with a third group of five rats serving as a source of control tendon. Relative volume of collagen phagocytosis was measured at an ultrastructural level using stereological methods.No significant influence of reduced tension on collagen phagocytosis was observed. The average relative volume of fibroblast cytoplasm was significantly increased in the transected tendon. This may represent an inductive influence of as yet undete...

Ultrastructural and Biochemical Aspects of the Sanfilippo Syndrome,—Type III Genetic Mucopolysaccharidosis

Abstract: The Sanfilippo Syndrome (SS) is a recessively inherited connective tissue disorder expressed in early life. It is classified as a genetic mucopolysaccharidosis (MPS) because the underlying defect involves the catabolism of heparan sulfate (HS), one of the glycosaminoglycans (GAG). Four variant forms, i.e., type A, B, C, and D, each associated with a different enzymatic defect, have been recognized in affected children.Biochemical studies show that characteristically HS accounts for most of the inereascd amounts of GAG excreted in the urine and those stored in viscera and brain. Gangliosides GM2, GM3 and GD2 are elevated considerably in the brain.Morphologically, the very water-soluble substanccs accumulating in thc viseera are metachromatic, and consist ultrastructurally of finely granulo-floccular (or filamentous) material which is bound in cytoplasmic vacuoles. These substances are considered to represent the GAG. In the central nervous system (CNS) the stored substances are not soluble in water or alco...

Connective tissue as an integral system: role of cell-cell and cell-matrix interactions.

Abstract: We have applied the systems approach, to the analysis of mechanisms whereby connective tissue (CT) is integrated into one functional system. The primary CT functions in health and in disease (biomechanical, trophic, protective, reparative, and morphogenetic) are carried out by means of cell-cell, cell-matrix and intertissue interactions based on feedback between these components. Both CT as a whole and its cellular and extracellular elements exhibit structural and functional heterogeneity which increases the capacity of CT for adaptation. The intercellular interactions take place via soluble and insoluble mediators, direct cellular contacts, and cell/matrix degradation products. Results of this study support the concept of internal “network” regulation of the CT composition, functions and growth through intercellular interactions at different structural levels. Feedback mechanisms between degradation and production of collagen and interactions between collagen, macrophages and fibroblasts play a special r...

Comparison of the use of isotopic proline vs leucine to measure protein synthesis in cultured fibroblasts.

Abstract: Compartmentation of the amino acid precursor pools for protein synthesis in cultured cells can substantially complicate measurements of synthesis rates. This is particularly true for nonessential amino acids such as proline, an amino acid often used in isotopic form to measure collagen synthesis. We have made a comparative study of this problem in cultured IMR-90 fibroblasts using isotopic proline and leucine to measure total protein and collagen synthesis. 3H-leucine in the extracellular (EC) medium equilibrates with tRNA-leucine at an EC concentration of 0.4 mM in both dividing and stationary cells. Thus, under these experimental conditions there is no complicating compartmentation of leucine for protein synthesis. Equilibration of EC and tRNA-bound 3H-proline, however, does not occur even when the EC concentration is in the mM range, based upon simultaneous measurements of synthesis rates using 3H-proline and 3H-leucine together. Furthermore, significant changes in EC proline concentration and specific activity occur over short time intervals (2 hr) if the initial EC proline concentration is below 0.2 mM. Thus, the use of isotopic proline to measure protein synthesis introduces substantial interpretive problems. Serum deprivation causes changes in both total collagen synthesis and the percent of protein synthesis devoted to collagen when measured with either 14C-leucine or 3H-proline. At the same time, isotopic proline remains the better choice for measuring percent collagen synthesis.

Variability in Proteoglycan Synthesis Among Six Strains of Normal Human Adult Skin Fibroblasts

Abstract: Several parameters of proteoglycan synthesis (incorporation of precursor isotope, distribution in culture compartments, sensitivity to chondroitinase ABC and Sepharose CL-2B elution profile) were investigated for six strains of normal human adult skin fibroblasts in culture in order to determine the range of variability in these parameters that could be expected in a normal population. When proteoglycan accumulation was measured in all cultures the uptake of Na235SO4 expressed per 106 cells had a coefficient of variation ranging from 17% to 50% in different experiments. Variability was always less when expressed per mg cell protein. The coefficient of variation was lowest when isotopic incorporation per mg cell protein was expressed as a percent of total incorporation of that isotope. Replicate tests indicated that the variability could not be explained by the assay methodologies alone. The protocol followed was designed to determine whether any consistent distinctions for individual cell strains could be...

“In Vitro” Fibril Formation of type I Collagen from Different Sources: Biochemical and Morphological Aspects

Abstract: Acid soluble type I collagen was prepared from foetal and adult bovine tendon and skin and from adult bovine cornea. The degree of hydroxylysine glycosylation and the hydroxylysine di-to monoglycoside ratio as well as the “in vivo” fibril diameters, were shown to be tissue and age-dependent. Fibrils of type I collagens were reconstituted “in vitro” monitoring at 313 nm. The fibrils obtained were examined by electron microscopy. It was shown that the “in vitro” lateral growth of collagen fibrils leads to the formation of fibrils with maximum diameters which may be correlated to those of the corresponding native fibrils. Moreover it is suggested that one of the factors controlling the lateral growth of collagen may be at the level of hydroxylysine glycosylation.

Collagen type distribution in healing of synthetic arterial prostheses.

Abstract: Layers of tissue encapsulating vascular prostheses recovered from humans were extracted and analyzed by SDS-polyacrylamide gel electrophoresis to determine the distribution of genetically distinct collagen types. Type V collagen was in maximal concentration in extracts of tissues nearest to the prosthesis lumen, type III in extracts of chronically inflamed tissue filling the interstices of the porous prosthesis, and type I in extracts of fibrous occlusive or outer capsule tissue. This pattern of distribution of collagen types across the prosthesis wall may have arisen due to the influence of modulating factors originating in the blood flowing through the prosthesis, and factors produced by inflammatory cells chronically present at the tissue-biomaterial interface. The increased proportion of type V collagen at or near the lumen may contribute to the recognized antithrombogenic properties of human pseudointima.

Undersulfated chondroitin sulfate in cartilage from a miniature poodle with spondyloepiphyseal dysplasia

Abstract: In order to determine if either the proteoglycans or collagen in the cartilagenous epiphyses of a Miniature Poodle with spondyloepiphyseal dysplasia were abnormal, the cartilage was dissociatively extracted in 4M guanidine HC1 in the presence of protease inhibitors and subjected to isopycnic cesium chloride dissociative density gradient ultracentrifugation. Dissociative extraction solubilized 97% of the uronic acid and 88% of the protein. Uronic acid distributed anomalously in the density gradient in that about 1/3 was recovered in each of the D1 (l.58g/ml), D2 (1.49g/ml) and D3 (1.44g/ml) fractions. Proteoglycans in the D1, D2 and D3 fractions also eluted from Sepharose CL-2B columns in a manner indicative of monomers of a smaller apparent hydrodynamic size than those from normal canine growth plate or articular cartilage. D1, D2 and D3 monomers subjected to the sodium borohydride reaction followed by chromatography on a Sepharose CL-6B column yielded glycosaminoglycan chain molecular weights of 10,200 (...

Degradation of cartilage by macrophages in culture: evidence for the involvement of an enzyme which is associated with the cell surface.

Abstract: A cell culture system is described in which purified mononuclear phagocytes may be cultured with a cartilage substrate which is radiolabelled in its proteoglycan. Resident mouse peritoneal macrophages degraded this substrate, and did so more avidly if cultured in direct contact with it. There was no evidence for complete intralysosomal degradation of the proteoglycan of the cartilage. Lysates were found to contain considerable activity at pH 7, which was inhibited by the presence of 10% serum, or by boiling the lysate. Proximity of macrophages to the substrate did not induce selective release of the lysosomal marker enzyme hexosaminidase, and concentrated enzymes secreted from the macrophages after treatment with the lysosomotropic agent ammonium chloride were ineffective in degrading cartilage at neutral pH. The active enzyme in macrophage lysates at neutral pH was found to be sedimentable by 100,000 X g centrifugation for 1 hour, in absence of lysosomal protective agents. There is evidence for a cell membrane-associated process in the degradation of cartilage by these cells, which may be a proteolytic, endoglycosidic or free radical-mediated event.

Isolation from cultured porcine gingival explants of a neutral proteinase with collagen telopeptidase activity.

Abstract: A neutral proteinase, distinct from collagenase, was isolated by gel-filtration from medium that had been conditioned by the culture of explanted porcine gingiva. This enzyme was shown to cleave the α1 (I) chain carboxyterminal telopeptide in native collagen proximal to (i.e. nearer to the helix than) the lysyl residue at position 17 which is known to be important in the formation of covalent intermolecular cross-links. We refer to this activity as ‘telopeptidase’. The enzyme had an apparent Mr of 70,000, as determined by gel-filtration. It was inhibited by ethylene diamine tetraacetic acid but not by N-ethyl-maleimide nor by phenyl-methylsulphonyl fluoride. It is therefore probably a metalloproteinase. The pH optimium for this activity was 7.0–7.5. Incubation of the enzyme with fibrillar (acid-insoluble) calf-skin collagen resulted in solublization of collagen in which shortening of the carboxy-terminal telopeptides could be demonstrated. It is suggested that the telopeptidase, acting within a region of ...

The structure and elastic properties of arterial junctions.

Abstract: Light microscopic studies showed marked differences in the elastin pattern at aorto-branch junctions on the proximal and distal lips of the junctions, both for small elastic branches such as the intercostals, and for the large muscular branches arising from the abdominal aorta.A light-pipe method showed that the internal apical curvature of translucent human cerebral arteries became flatter as the pressue increased. Longitudinal strips of aorto-branch junctions from sheep and dogs were stretched at known strain rates. There appear to be differences in the two species as the flow divider is least distensible in sheep and most distensible in dogs.Scanning electron microscopy of NaOH-digested arteries showed that the internal elastic membrane is a fenestrated sheet, the adventitia has fibrous elastin, and the medial layers are transitional. There are many interlamellar connections.

Skin lysyl oxidase activity is not rate limiting for collagen crosslinking in the glucocorticoid-treated rat.

Abstract: Lysyl oxidase activity in the skin of rats receiving triamcinolone diacetate (12 mg/kg) for three consecutive days was decreased by sixty-four percent as compared to control values. A decrease of lysyl oxidase activity was observed twelve hours after the initial glucocorticoid injection. The decreased lysyl oxidase activity was accompanied by a forty-nine percent decrease of acetic acid extractable collagen. There was also a forty-two percent decrease in the α/β ratio of the acetic acid soluble skin collagen of glucocorticoid-treated animals. These data indicate that although skin lysyl oxidase activity is decreased by glucocorticoid treatment. the crosslinking of acid extracted collagen as measured by the α/β ratio and collagen solubility is increased. Accordingly lysyl oxidase activity is not rate limiting for collagen crosslink formation in the skins of rats treated with glucocorticoids.

Contraction of scar tissue in the rabbit vitreous.

Abstract: Sheets of vitreous membrane (scar tissue) and associated retinal detachment were produced in the right eye of 86 adult New Zealand white rabbits by intravitreal injection of cultured autologous skin fibroblasts. The membranes were examined by light and electron microscopy and time-lapse cinephotomicrography. Immuno-histochemistry was used to demonstrate alterations in the distribution of cytoplasmic contractile proteins. While retinal detachment and membrane contraction were taking place, there was pronounced increase in the numbers of fibroblasts with an elongated spindle shape. These spindle-shaped cells had some similarities to myofibroblasts including the presence of ‘stress cables’. However, the myofibroblast-like cells stained much less avidly for cytoplasmic (actin) microfilaments than migratory fibroblasts seen at early stages of membrane development. The significance of migrating fibroblasts and myofibroblasts in scar contraction is discussed.

A comparison of proteoglycan from chick cartilage of different types and a study of the effect of vitamin D on proteoglycan structure.

Abstract: Normal chick growth cartilage contains mainly large proteoglycans that interact well with hyaluronic acid. In contrast normal articular and sternal cartilages contain mainly smaller molecules that interact poorly with hyaluronic acid. Similar smaller proteoglycans are present in all the cartilages in the rachitic state. The smaller proteoglycans all appear to be synthesized as such, and not to arise from degradation of larger molecules unless such degradation occurs extremely rapidly after synthesis. Supplementation of the vitamin D-deplete animals with calcium resulted in the production of large proteoglycans by the growth cartilage, but did not affect the size of the proteoglycan produced by the articular or sternal cartilages. It would appear that in these chicks the chondrocytes of the growth cartilage may be unique in their ability to produce large aggregating proteoglycans, and in their response to plasma calcium levels.

Physicochemical and ultrastructural properties of cholesterol esters bound to elastin

Abstract: Physicochemical and ultrastructural properties of cholesterol ester complexes of fibrous elastin and of K-elastin were studied using differential scanning calorimetry (DSC) and electron-microscopy. The number of molecules of the different fatty acids retained in these elastin complexes varied between large limits according to the nature of the fatty acid of the cholesterol ester, ranging from 0.1 μg/ 200mg of elastin for cholesterol arachidonate to 48μg/200mg elastin for cholesterol palmitate. The ultrastructural studies confirmed the association of cholesterol esters with fibrous elastin and soluble K-elastin. The DSC-data showed that the temperature of transition between the crystalline and the liquid crystalline state shifts towards higher temperatures (well above body temperature) when cholesterol esters are bound to elastin: no liquid crystalline mesophase was observed in cholesterol oleate or linoleate-elastin complexes at 37°C; melting of the crystalline structures took place at 51°C and at 42°C fo...

Collagen fibril formation in the presence of dexamethasone disodium phosphate

Abstract: Dexamethasone disodium phosphate was found to inhibit in vitro fibrillogenesis in a buffered collagen solution that otherwise formed in vivo like fibrils. A nonlinear relationship was observed between the steroid salt concentration and the kinetic parameter half transition time. Full fibril inhibition occurred at dexamethasone phosphate concentrations above 15 mM. At lower concentrations, sample buffers that also contained inorganic phosphate were not different from the control in activation energy of 224.3 +/- 29.3 kJ/mol (53.6 +/- 7.0 kcal/mol). The idea is advanced that the soluble steroid salt associates directly to the collagen and prevents the formation of lateral, hydrophobic interactions between adjacent collagen aggregates.