Lack of transformation of the collagen substratum in collagen lattice cultures

Human dermal fibroblasts were seeded into collagen lattices (tridimensional meshwork) of two preparations: (1) acid-extracted, (2) pepsin-digested-calf skin collagens. Lattices prepared with pepsin-digested collagen retracted faster during the first 2 days, then the two preparations gave the same contraction pattern. Lattices of both preparations were contracted for up to 23 days and their collagens submitted to CNBr treatment. The patterns of CB-peptides were found identical for all the incubation periods tested. There is no formation of cross-links during the contraction process.     

Monitoring of collagen and collagen fragments in chromatography of protein mixtures

A new method for the detection of collagen and collagen peptides in the presence of other proteins is described. The procedure is based on alkaline hydrolysis of the proteins and monitoring of the free hydroxyproline after chromophore formation. The method is quick and sensitive and the color yield of hydroxyproline is not affected by chromatography solvents. As all steps of the assay take place in a single test tube the method is suitable for batch processing of column fractions. Due to the high sensitivity of the assay it can be used for the analysis of collagen and collagen peptides in extracts of small tissue samples or biopsies.     

Dysfunctional tendon collagen fibrillogenesis in collagen VI null mice

Tendons are composed of fibroblasts and collagen fibrils. The fibrils are organized uniaxially and grouped together into fibers. Collagen VI is a non-fibrillar collagen expressed in developing and adult tendons. Human collagen VI mutations result in muscular dystrophy, joint hyperlaxity and contractures. The purpose of this study is to determine the functional roles of collagen VI in tendon matrix assembly. During tendon development, collagen VI was expressed throughout the extracellular matrix, but enriched around fibroblasts and their processes. To analyze the functional roles of collagen VI a mouse model with a targeted inactivation of Col6a1 gene was utilized. Ultrastructural analysis of Col6a1−/− versus wild type tendons demonstrated disorganized extracellular micro-domains and associated collagen fibers in the Col6a1−/− tendon. In Col6a1−/− tendons, fibril structure and diameter distribution were abnormal compared to wild type controls. The diameter distributions were shifted significantly toward the smaller diameters in Col6a1−/− tendons compared to controls. An analysis of fibril density (number/μm²) demonstrated a ~ 2.5 fold increase in the Col6a1−/− versus wild type tendons. In addition, the fibril arrangement and structure were aberrant in the peri-cellular regions of Col6a1−/− tendons with frequent very large fibrils and twisted fibrils observed restricted to this region. The biomechanical properties were analyzed in mature tendons. A significant decrease in cross-sectional area was observed. The percent relaxation, maximum load, maximum stress, stiffness and modulus were analyzed and Col6a1−/− tendons demonstrated a significant reduction in maximum load and stiffness compared to wild type tendons. An increase in matrix metalloproteinase activity was suggested in the absence of collagen VI. This suggests alterations in tenocyte expression due to disruption of cell-matrix interactions. The changes in expression may result in alterations in the peri-cellular environment. In addition, the absence of collagen VI may alter the sequestering of regulatory molecules such as leucine rich proteoglycans. These changes would result in dysfunctional regulation of tendon fibrillogenesis indirectly mediated by collagen VI.     

Liquid crystallinity in collagen solutions and magnetic orientation of collagen fibrils

Studies of the optical birefringence of solutions of acid-soluble collagen from rat-tail tendon at 22°C in the pH range 1.0–6.0 show that collagen exhibits an isotropic to mesophase transition only between pH 2.4 and 3.0 at 10% weight concentration. Such liquid crystalline order is probably essential for the orientation of collagen in a magnetic field. When solutions of neutral salt-soluble collagen were precipitated at pH 7.0 by warming to 37°C (“heat gelling”) in a magnetic field of ca. 20 kG, the resulting fibrils wee oriented perpendicular to the direction of the field. Heat gelling is shown to be a useful technique for maintaining the orientation induced in precursor solutions even after the sample is removed from the magnetic field.     

Regulation of collagen synthesis in fibroblasts within a three-dimensional collagen gel

Fibroblasts cultivated within a three-dimensional collagen gel display an elongated, spindle-like morphology, reduce their proliferation rate, contact the gel to a very dense tissue, and modify their metabolic activity as compared to monolayer cultures. Collagen synthesis measured as protein-bound hydroxyproline is reduced to 5% of the values found in monolayer culture. The reduction involving type I and type III collagen is due to decreased de novo synthesis and not to enhanced degradation. Dot blot hybridization, Northern blot analysis, and in situ hybridization using collagen I- and III-specific cDNA probes demonstrate that reduced biosynthesis rates are reflected by a marked reduction of pro alpha 1 (I), pro alpha 2 (I), and pro alpha 1 (III) collagen mRNA indicating pretranslational regulation. A similar reduction was observed for actin mRNA whereas levels of tubulin mRNA were similar for fibroblasts in monolayer culture or cultivated within the three-dimensional collagen gels. The data suggest a specific reprogramming of various cellular activities in response to contact with the reconstituted extracellular matrix.     

Characterization of notochord collagen as a cartilage-type collagen

Sturgeon notochord and cartilage collagens have been characterized with respect to chromatographic properties, amino acid composition, carbohydrate content, and cyanogen bromide cleavage products of the component α chains. The data show that the collagen of both tissues is comprised of a single type of α chain and that the notochord and cartilage chains are identical. Further, the sturgeon chains bear a striking resemblance to previously characterized α1(II) chains from avian and mammalian hyaline cartilages. These observations strongly suggest that the data may be extrapolated to higher organisms and indicate that during development, a cartilage-type collagen is synthesized by notochord cells prior to the appearance of tissues classically identified as cartilage on the basis of morphology.     

Abnormal collagen deposition in synovia after collagen type V immunization in rabbits

Sinovitis in Scleroderma (SSc) is rare, usually aggressive and fully resembles rheumatoid arthritis. Experimental models of SSc have been used in an attempt to understand its pathogenesis. Previous studies done in our laboratory had already revealed the presence of a synovial remodeling process in rabbits immunized with collagen V. To validate the importance of collagen type V and to explore the quantitative relationship between this factor and synovia remodeling as well as the relationship between collagen type V and other collagens, we studied the synovial tissue in immunized rabbits. Rabbits (N=10) were immunized with collagen V plus Freund's adjuvant and compared with animals inoculated with adjuvant only (N=10). Synovial tissues were submitted to histological analysis, immunolocalization to collagen I, III and V and biochemical analysis by eletrophoresis, immunoblot and densitometric method. The synovial tissue presented an intense remodeling process with deposits of collagen types I, III and V after 75 and 120 days of immunization, mainly distributed around the vessels and interstitium of synovial extracellular matrix. Densitometric analysis confirmed the increased synthesis of collagen I, III and V chains (407.69+/-80.31; 24.46+/-2.58; 70.51+/-7.66, respectively) in immunized rabbits when compared with animals from control group (164.91+/-15.67; 12.89+/-1.05; 32+/-3.57) (p<0.0001). We conclude that synovial remodeling observed in the experimental model can reflect the articular compromise present in patients with scleroderma. Certainly, this experimental model induced by collagen V immunization will bring new insights in to pathogenic mechanisms and allow the testing of new therapeutic strategies to ameliorate the prognosis for scleroderma patients.     

Procoagulant activity of collagen. Effect of difference in type and structure of collagen

Procoagulant activities of different types and structures of collagen were examined. Collagens used were types I (including its methylated and succinylated forms), II, III, IV and V. Each collagen was coated on an inner surface of a glass tube. The change of fluidity during coagulation of blood in the tube was measured by means of a new rheological technique. For monomeric collagen, the procoagulant activity of the succinylated form (negatively charged) was higher than that of the methylated form (positively charged). The procoagulant activity of type IV (dry) was lower than that of other types of collagen. For fibrillar collagens, the initiation of coagulation for type V (non-banded) was fairly delayed compared to those for types I, II and III (banded). An increase in water content in both monomeric and fibrillar forms promoted procoagulant activity. For most of the collagen forms, the addition of factor XII inhibitor (Polybrene) to blood brought about a remarkable delay of the initiation of coagulation, suggesting that the activation of factor XII on the collagen surface is one of main factors governing procoagulant activity. In addition, our data suggest that large numbers of adherent platelets to the collagen surface promote activation of the intrinsic coagulation system.     

Biosynthesis of collagen

During the biosynthesis and assembly of collagen structures, disulfide links can serve several functions. During biosynthesis they successively stabilize intra-peptide folding and associations of three chains into one molecule. Studies on the refolding and reassociation of reduced and denatured carboxyl propeptides of procollagen I showed that successive interactions of folding and assembly are successively weaker. Disulfide bridges were reestablished within correctly refolded carboxyl propeptides. Rearrangements of disulfide bridges may occur during the processing of type V procollagen molecules as these collagens become incorporated into extracellular matrix. The basement membrane procollagen IV molecules become disulfide linked at each end into networks, and there are indications that further rearrangements of disulfide links may allow additional modulation.     

胶原蛋白与Ⅵ型胶原蛋白的结构概述

描述了胶原蛋白的结构和Ⅵ型胶原蛋白的结构以及胶原蛋白的应用 ,并对人类胶原蛋白的生产进行了展望。     

Stereochemistry of collagen

This review article, based on a lecture delivered in Madras in 1985, is an account of the author's experience in the working out of the molecular structure and conformation of the collagen triple-helix over the years 1952-78. It starts with the first proposal of the correct triple-helix in 1954, but with three residues per turn, which was later refined in 1955 into a coiled-coil structure with approximately 3.3 residues per turn. The structure readily fitted proline and hydroxyproline residues and required glycine as every third residue in each of the three chains. The controversy regarding the number of hydrogen bonds per tripeptide could not be resolved by X-ray diffraction or energy minimization, but physicochemical data, obtained in other laboratories during 1961-65, strongly pointed to two hydrogen bonds, as suggested by the author. However, it was felt that the structure with one straight NH...O bond was better. A reconciliation of the two was obtained in Chicago in 1968, by showing that the second hydrogen bond is via a water molecule, which makes it weaker, as found in the physicochemical studies mentioned above. This water molecule was also shown, in 1973, to take part in further cross-linking hydrogen bonds with the OH group of hydroxyproline, which occurred always in the location previous to glycine, and is at the right distance from the water. Thus, almost all features of the primary structure, X-ray pattern, optical and hydrodynamic data, and the role of hydroxyproline in stabilising the triple helical structure, have been satisfactorily accounted for. These also lead to a confirmation of Pauling's theory that vitamin C improves immunity to diseases, as explained in the last section.     

Schachtelhalm-structure of the octafibrils in collagen

X-ray diffraction patterns suggest that the microfibrils in the collagen of native rat tail tendon are eight-stranded ropes, quarter staggered and packed together in an orthorhombic lattice with paracrystalline (liquid-like) distortions between adjacent tropocollagen molecules. These so called octafibrils react under stress like synthetic elastomers with a 135 Å periodicity of hard segments.