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The echinoderm collagen fibril: A hero in the connective tissue research of the 1990s
Abstract
Collagen fibrils are some of the most-abundant and important extracellular structures in our bodies, yet we are unsure of their shape and size. This is largely due to an inherent difficulty in isolating them from their surrounding tissues. Echinoderms have collagenous tissues that are similar to ours in many ways, yet they can be manipulated to easily relinquish their collagen fibrils, providing an excellent opportunity to study native fibrillar structure. In the early 1990s, they were found to defy the commonly accepted fibrillar model of the time in that they were much shorter, they were shaped like double-ended spindles, and their centers exhibited a reversal in molecular polarity. Realization of these features helped to reform the questions that were being asked about vertebrate fibrils, shifting the focus toward shape and size. Since then, researchers working with both groups (echinoderms and vertebrates) have worked together to find the structure of native fibrils. This information will be fundamental in understanding what holds collagenous tissues together at the fibrillar level, and could have important implications for people with Ehlers-Danlos syndrome.
... The tissues can extensively change their mechanical properties, such as elasticity and viscosity, within a few minutes under the regulation of their nervous systems [1][2][3]. The tissues contain a large amount of extracellular matrix consisting mainly of collagen fibrils, proteoglycans and microfibrils [4][5][6][7][8]. The unique properties of these collagenous tissues might be due to lack of permanent associations between the collagen fibrils and the surrounding extracellular matrix because it is easy to isolate collagen fibrils from catch connective tissues, though not from the collagenous tissues of adult vertebrates [4], [5], [8][9][10]. ...
... The tissues contain a large amount of extracellular matrix consisting mainly of collagen fibrils, proteoglycans and microfibrils [4][5][6][7][8]. The unique properties of these collagenous tissues might be due to lack of permanent associations between the collagen fibrils and the surrounding extracellular matrix because it is easy to isolate collagen fibrils from catch connective tissues, though not from the collagenous tissues of adult vertebrates [4], [5], [8][9][10]. It seems that crosslinks between the collagen fibrils and other components of the extracellular matrix are formed or broken during changes in mechanical properties. ...
The dermis in the holothurian body wall is a typical catch connective tissue or mutable collagenous tissue that shows rapid changes in stiffness. Some chemical factors that change the stiffness of the tissue were found in previous studies, but the molecular mechanisms of the changes are not yet fully understood. Detection of factors that change the stiffness by working directly on the extracellular matrix was vital to clarify the mechanisms of the change. We isolated from the body wall of the sea cucumber Stichopus chloronotus a novel protein, softenin, that softened the body-wall dermis. The apparent molecular mass was 20 kDa. The N-terminal sequence of 17 amino acids had low homology to that of known proteins. We performed sequential chemical and physical dissections of the dermis and tested the effects of softenin on each dissection stage by dynamic mechanical tests. Softenin softened Triton-treated dermis whose cells had been disrupted by detergent. The Triton-treated dermis was subjected to repetitive freeze-and-thawing to make Triton-Freeze-Thaw (TFT) dermis that was softer than the Triton-treated dermis, implying that some force-bearing structure had been disrupted by this treatment. TFT dermis was stiffened by tensilin, a stiffening protein of sea cucumbers. Softenin softened the tensilin-stiffened TFT dermis while it had no effect on the TFT dermis without tensilin treatment. We isolated collagen from the dermis. When tensilin was applied to the suspending solution of collagen fibrils, they made a large compact aggregate that was dissolved by the application of softenin or by repetitive freeze-and-thawing. These results strongly suggested that softenin decreased dermal stiffness through inhibiting cross-bridge formation between collagen fibrils; the formation was augmented by tensilin and the bridges were broken by the freeze-thaw treatment. Softenin is thus the first softener of catch connective tissue shown to work on the cross-bridges between extracellular materials.
... Catch connective tissues or mutable collagenous tissues of echinoderms can extensively change their mechanical properties such as elasticity and viscosity within a few minutes under the regulation of their nervous system (Motokawa, 1984;Motokawa, 1988;Wilkie, 2002). The tissues contain a large amount of the extracellular matrix, mainly consisting of collagen fibrils, proteoglycans and microfibrils (Trotter and Koob, 1989;Trotter et al., 1994;Thurmond and Trotter, 1996;Thurmond et al., 1997;Szulgit, 2007). The unique properties of these collagenous tissues might be due to lack of permanent associations between the collagen fibrils and the surrounding extracellular matrix because it is easy to isolate collagen fibrils from catch connective tissues -unlike collagenous tissues of adult vertebrates (Matsumura, 1974;Trotter and Koob, 1989;Trotter et al., 1994;Tamori et al., 2006;Szulgit, 2007). ...
... The tissues contain a large amount of the extracellular matrix, mainly consisting of collagen fibrils, proteoglycans and microfibrils (Trotter and Koob, 1989;Trotter et al., 1994;Thurmond and Trotter, 1996;Thurmond et al., 1997;Szulgit, 2007). The unique properties of these collagenous tissues might be due to lack of permanent associations between the collagen fibrils and the surrounding extracellular matrix because it is easy to isolate collagen fibrils from catch connective tissues -unlike collagenous tissues of adult vertebrates (Matsumura, 1974;Trotter and Koob, 1989;Trotter et al., 1994;Tamori et al., 2006;Szulgit, 2007). It seems that crosslinking of the collagen fibrils with adjacent ones and other components of the extracellular matrix is formed or broken during changes in the mechanical properties of catch connective tissues. ...
The dermis of sea cucumbers is a catch connective tissue or mutable collagenous tissue that shows large changes in stiffness. Extensive studies on the dermis revealed that it can adopt three different states having different mechanical properties that can be reversibly converted. These are the stiff, standard and soft states. The standard state is readily produced when a dermal piece is immersed in the sea water containing Ca²+, whereas the soft state can be produced by removal of Ca²+. A stiffening protein, tensilin, has been isolated from some sea cucumbers (Cucumaria frondosa and Holothuria leucospilota). Although tensilin converts the state of the dermis from soft to standard, it cannot convert from standard to stiff. In this study, we isolated and partially purified a novel stiffening factor from the dermis of Holothuria leucospilota. The factor stiffened the dermis in normal artificial sea water (ASW) but did not stiffen the soft dermis in Ca²+-free ASW. It also stiffened the dermis that had been converted to the standard state in Ca²+-free ASW by the action of tensilin. These results suggest that the factor produces the stiff dermis from the standard state but cannot work as a stiffener on the soft dermis. Its addition to longitudinal muscles of the sea cucumber produced no effects, suggesting that its effect is specific to the catch connective tissue. Its stiffening activity was susceptible to trypsin, meaning that it is a polypeptide, and its molecular mass estimated from gel filtration chromatography was 2.4 kDa.
... The ability to separate body parts is carried out by changing of the mechanical properties of the connective tissue referred to as mutable collagenous tissue (MCT) [13] or catch connective tissue [14]. An extracellular matrix (ECM) with MCT properties can form various anatomical structures, in particular, ligaments and connective body wall tissue [8,15]. Another widely known peculiarity of echinoderms is their regenerative abilities [16][17][18][19]. ...
Echinoderms are one of the most ancient groups of invertebrates. The study of their genomes has made it possible to conclude that these animals have a wide variety of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The phylogenetic analysis shows that the MMPs and TIMPs underwent repeated duplication and active divergence after the separation of Ambulacraria (Echinodermata+Hemichordata) from the Chordata. In this regard the homology of the proteinases and their inhibitors between these groups of animals cannot be established. However, the MMPs of echinoderms and vertebrates have a similar domain structure. Echinoderm proteinases can be structurally divided into three groups—archetypal MMPs, matrilysins, and furin-activatable MMPs. Gelatinases homologous to those of vertebrates were not found in genomes of studied species and are probably absent in echinoderms. The MMPs of echinoderms possess lytic activity toward collagen type I and gelatin and play an important role in the mechanisms of development, asexual reproduction and regeneration. Echinoderms have a large number of genes encoding TIMPs and TIMP-like proteins. TIMPs of these animals, with a few exceptions, have a structure typical for this class of proteins. They contain an NTR domain and 10–12 conservatively located cysteine residues. Repeated duplication and divergence of TIMP genes of echinoderms was probably associated with an increase in the functional importance of the proteins encoded by them in the physiology of the animals.
... Matrix metalloproteinase-16 (MMP-16), also called MT3-MMP, can degrade various components of the ECM, such as collagen type III, gelatin, casein, and fibronectin. The AJBW dermis is a typical catch connective tissue (or called mutable collagenous tissue) that contains a large amount of ECM consisting mainly of collagen fibrils, proteoglycans and microfibrils [33]. Approximately 70% of the total body wall protein was insoluble collagen fibers and the collagen protein belonged to collagen type I formed by (α1) 2 α2 [34]. ...
Sea cucumber (Apostichopus japonicus) is an economically significant species in China having great commercial value. It is challenging to maintain the textural properties during thermal processing due to the distinctive physiochemical structure of the A. japonicus body wall (AJBW). In this study, the gene expression profiles associated with tenderization in AJBW were determined at 0 h (CON), 1 h (T_1h), and 3 h (T_3h) after treatment at 37 °C using Illumina HiSeq™ 4000 platform. Seven-hundred-and-twenty-one and 806 differentially expressed genes (DEGs) were identified in comparisons of T_1h vs. CON and T_3h vs. CON, respectively. Among these DEGs, we found that two endogenous proteases—72 kDa type IV collagenase and matrix metalloproteinase 16 precursor—were significantly upregulated that could directly affect the tenderness of AJBW. In addition, 92 genes controlled four types of physiological and biochemical processes such as oxidative stress response (3), immune system process (55), apoptosis (4), and reorganization of the cytoskeleton and extracellular matrix (30). Further, the RT-qPCR results confirmed the accuracy of RNA-sequencing analysis. Our results showed the dynamic changes in global gene expression during tenderization and provided a series of candidate genes that contributed to tenderization in AJBW. This can help further studies on the genetics/molecular mechanisms associated with tenderization.
... Szulgit has suggested that the fibrils in the ligaments may be required to possess a certain q in order to be able to transmit the appropriate level of force [135]. How high should the aspect ratio be in order to enable collagen fibrils in MCTs to provide reinforcement to the MCT? Goh and co-workers have investigated the stress uptake in a fibril at varying q by finite element analysis and analytical modelling [81]. ...
Scaffolds for tissue engineering application may be made from a collagenous extracellular matrix (ECM) of connective tissues because the ECM can mimic the functions of the target tissue. The primary sources of collagenous ECM material are calf skin and bone. However, these sources are associated with the risk of having bovine spongiform encephalopathy or transmissible spongiform encephalopathy. Alternative sources for collagenous ECM materials may be derived from livestock, e.g., pigs, and from marine animals, e.g., sea urchins. Collagenous ECM of the sea urchin possesses structural features and mechanical properties that are similar to those of mammalian ones. However, even more intriguing is that some tissues such as the ligamentous catch apparatus can exhibit mutability, namely rapid reversible changes in the tissue mechanical properties. These tissues are known as mutable collagenous tissues (MCTs). The mutability of these tissues has been the subject of on-going investigations, covering the biochemistry, structural biology and mechanical properties of the collagenous components. Recent studies point to a nerve-control system for regulating the ECM macromolecules that are involved in the sliding action of collagen fibrils in the MCT. This review discusses the key attributes of the structure and function of the ECM of the sea urchin ligaments that are related to the fibril-fibril sliding action—the focus is on the respective components within the hierarchical architecture of the tissue. In this context, structure refers to size, shape and separation distance of the ECM components while function is associated with mechanical properties e.g., strength and stiffness. For simplicity, the components that address the different length scale from the largest to the smallest are as follows: collagen fibres, collagen fibrils, interfibrillar matrix and collagen molecules. Application of recent theories of stress transfer and fracture mechanisms in fibre reinforced composites to a wide variety of collagen reinforcing (non-mutable) connective tissue, has allowed us to draw general conclusions concerning the mechanical response of the MCT at specific mechanical states, namely the stiff and complaint states. The intent of this review is to provide the latest insights, as well as identify technical challenges and opportunities, that may be useful for developing methods for effective mechanical support when adapting decellularised connective tissues from the sea urchin for tissue engineering or for the design of a synthetic analogue.
... On the other hand, certain species of echinoderms also possess collagen fibrils to fulfill their functional purpose. Echinoderms have collagenous tissues that are similar to vertebrate collagens; however, they can be manipulated to easily relinquish their collagen fibrils, which provides an excellent opportunity for the exploration of the native fibrillar structure (Szulgit 2007). The tissues of these animals have the unique capacity to rapidly and reversibly alter their mechanical properties, resembling the collagenous tissues of other phyla consisting of collagen fibrils in a nonfibrillar matrix (Trotter et al. 1994). ...
... MCTs are present in all five living echinoderm classes in several anatomical forms (dermal connective tissue, interossicular ligaments and tendons). Most MCT structures resemble mammalian connective tissues in that they consist largely of collagen fibril arrays, proteoglycans (PGs), fibrillin-containing microfibrils and water [2,91011121314151617. MCTs are also characterized by the invariable presence of specialized neurosecretory-like cells known as juxtaligamental cells (JLCs) [2,18]. ...
Mutable collagenous tissues (MCTs) of echinoderms show reversible changes in tensile properties (mutability) that are initiated and modulated by the nervous system via the activities of cells known as juxtaligamental cells. The molecular mechanism underpinning this mechanical adaptability has still to be elucidated. Adaptable connective tissues are also present in mammals, most notably in the uterine cervix, in which changes in stiffness result partly from changes in the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). There have been no attempts to assess the potential involvement of MMPs in the echinoderm mutability phenomenon, apart from studies dealing with a process whose relationship to the latter is uncertain. In this investigation we used the compass depressor ligaments (CDLs) of the sea-urchin Paracentrotus lividus. The effect of a synthetic MMP inhibitor - galardin - on the biomechanical properties of CDLs in different mechanical states ("standard", "compliant" and "stiff") was evaluated by dynamic mechanical analysis, and the presence of MMPs in normal and galardin-treated CDLs was determined semi-quantitatively by gelatin zymography. Galardin reversibly increased the stiffness and storage modulus of CDLs in all three states, although its effect was significantly lower in stiff than in standard or compliant CDLs. Gelatin zymography revealed a progressive increase in total gelatinolytic activity between the compliant, standard and stiff states, which was possibly due primarily to higher molecular weight components resulting from the inhibition and degradation of MMPs. Galardin caused no change in the gelatinolytic activity of stiff CDLs, a pronounced and statistically significant reduction in that of standard CDLs, and a pronounced, but not statistically significant, reduction in that of compliant CDLs. Our results provide evidence that MMPs may contribute to the variable tensility of the CDLs, in the light of which we provide an updated hypothesis for the regulatory mechanism controlling MCT mutability.
... energy storage, transmission and dissipation), MCT provides mechanisms for the detachment of appendages or body parts in response to disease, trauma or predator attack [2] and for the energy-sparing maintenance of posture [3]. Most mutable collagenous structures consist largely of parallel aggregations of collagen fibrils to which proteoglycans are covalently and non-covalently attached, as in mammalian connective tissue234567891011. An elastomeric network of microfibrils surrounds and separates collagen fibers (bundles of fibrils), maintaining their organization and providing a long-range restoring force [2,12,13]. ...
The mutable collagenous tissue (MCT) of echinoderms has the ability to undergo rapid and reversible changes in passive mechanical properties that are initiated and modulated by the nervous system. Since the mechanism of MCT mutability is poorly understood, the aim of this work was to provide a detailed morphological analysis of a typical mutable collagenous structure in its different mechanical states. The model studied was the compass depressor ligament (CDL) of a sea urchin (Paracentrotus lividus), which was characterized in different functional states mimicking MCT mutability. Transmission electron microscopy, histochemistry, cryo-scanning electron microscopy, focused ion beam/scanning electron microscopy, and field emission gun-environmental scanning electron microscopy were used to visualize CDLs at the micro- and nano-scales. This investigation has revealed previously unreported differences in both extracellular and cellular constituents, expanding the current knowledge of the relationship between the organization of the CDL and its mechanical state. Scanning electron microscopies in particular provided a three-dimensional overview of CDL architecture at the micro- and nano-scales, and clarified the micro-organization of the ECM components that are involved in mutability. Further evidence that the juxtaligamental cells are the effectors of these changes in mechanical properties was provided by a correlation between their cytology and the tensile state of the CDLs.
... Type I collagen fibrils were isolated from the dermis of sea cucumber, Cucumaria frondosa (28,29). This structure is several dozen microns long, has cross-sectional dimensions ;10-500 nm, and is easily obtainable as an isolated fibril. ...
Collagen, a molecule consisting of three braided protein helices, is the primary building block of many biological tissues including bone, tendon, cartilage, and skin. Staggered arrays of collagen molecules form fibrils, which arrange into higher-ordered structures such as fibers and fascicles. Because collagen plays a crucial role in determining the mechanical properties of these tissues, significant theoretical research is directed toward developing models of the stiffness, strength, and toughness of collagen molecules and fibrils. Experimental data to guide the development of these models, however, are sparse and limited to small strain response. Using a microelectromechanical systems platform to test partially hydrated collagen fibrils under uniaxial tension, we obtained quantitative, reproducible mechanical measurements of the stress-strain curve of type I collagen fibrils, with diameters ranging from 150-470 nm. The fibrils showed a small strain (epsilon < 0.09) modulus of 0.86 +/- 0.45 GPa. Fibrils tested to strains as high as 100% demonstrated strain softening (sigma(yield) = 0.22 +/- 0.14 GPa; epsilon(yield) = 0.21 +/- 0.13) and strain hardening, time-dependent recoverable residual strain, dehydration-induced embrittlement, and susceptibility to cyclic fatigue. The results suggest that the stress-strain behavior of collagen fibrils is dictated by global characteristic dimensions as well as internal structure.
Responsive polymer materials possessing variable mechanical properties have shown promising practical applications, whereas water has clear advantages among the triggers owing to its wide abundance, green characteristics, as well as mild conditions involved. However, ubiquitous water‐induced softening would prevent polymer materials from applications with high humidity or aqueous environment. Herein, an unprecedented polymer gel material is reported that exhibits a dramatic and reversible water‐induced stiffening base on phase separation, differing from traditional ones that are usually weakened upon hydration due to the plasticizing effect. The material shows a large stiffness change in Young's modulus (as much as 104 times), which is much larger than that induced by glass transition and comparable to that caused by crystallization‐melting process. The polymer materials are fabricated in a facile way by introducing an ionic liquid and a lithium salt into a poly(benzyl methacrylate) network. Moreover, the volume remains almost unchanged during the reversible soft–stiff transition. A universal approach of water‐induced stiffening is proposed and verified on various systems. As for demonstration, this material is used for humidity‐induced shape memory. This work offers an effective method for developing water‐induced stiffened material and will pave the way toward potential applications for water‐responsive polymer materials. An unprecedented polymer material with a dramatic and reversible water‐induced stiffening (stiffness increase as much as 104 times) is introduced based on phase separation, differing from traditional ones that are usually weakened upon hydration. A universal approach for water‐induced stiffening is proposed and verified on various systems. This work would pave the way for the design and development of water‐responsive polymer materials.
Dynamic soft materials that have the ability to expand and contract, change stiffness, self-heal or dissolve in response to environmental changes, are of great interest in applications ranging from biosensing and drug delivery to soft robotics and tissue engineering. This book covers the state-of-the-art and current trends in the very active and exciting field of bioinspired soft matter, its fundamentals and comprehension from the structural-property point of view, as well as materials and cutting-edge technologies that enable their design, fabrication, advanced characterization and underpin their biomedical applications.
The book contents are supported by illustrated examples, schemes, and figures, offering a comprehensive and thorough overview of key aspects of soft matter. The book will provide a trusted resource for undergraduate and graduate students and will extensively benefit researchers and professionals working across the fields of chemistry, biochemistry, polymer chemistry, materials science and engineering, nanosciences, nanotechnologies, nanomedicine, biomedical engineering and medical sciences.
Collagens represent the suprafamily of more than 28 main types which have been isolated and described in diverse marine organisms including invertebrates. Especially collagens from glass sponges (Hexactinellida) and horny sponges (Demospongiae) attract attention because of their very ancient origin and unique structural features. Domains typical for collagen have been detected as main structural segments in other structural marine proteins including spongin, gorgonin, byssus, conchiolin. Collagen is to be found as crucial player in diverse mineral-based biocomposites as well as within non-mineralized, cartilage-like tissues of invertebrates. Due to their biocompatibility, marine collagens remain to be biological materials in trend for applications in biomedicine, regenerative medicine, wound healing, cartilage and hard tissue engineering. Soft corals collagens have been recently recognized as a new potential group pf marine collagens for biomedicine.
Echinoderms are capable of asexual reproduction by fission. An individual divides into parts due to changes in the strength of connective tissue of the body wall. The structure of connective tissue and the mechanisms of variations in its strength in echinoderms remain poorly studied. An analysis of transcriptomes of individuals during the process of fission provides a new opportunity to understand the mechanisms of connective tissue mutability. In the holothurian Cladolabes schmeltzii, we have found a rather complex organization of connective tissue. Transcripts of genes encoding a wide range of structural proteins of extracellular matrix, as well as various proteases and their inhibitors, have been discovered. All these molecules may constitute a part of the mechanism of connective tissue mutability. According to our data, the extracellular matrix of echinoderms is substantially distinguished from that of vertebrates by the lack of elastin, fibronectins, and tenascins. In case of fission, a large number of genes of transcription factors and components of different signaling pathways are expressed. Products of these genes are probably involved in regulation of asexual reproduction, connective tissue mutability, and preparation of tissues for subsequent regeneration. It has been shown that holothurian tensilins are a special group of tissue inhibitors of metalloproteinases, which has formed within the class Holothuroidea and is absent from other echinoderms. Our data can serve a basis for the further study of the mechanisms of extracellular matrix mutability, as well as the mechanisms responsible for asexual reproduction in echinoderms.
Collagen is widely studied in the pharmaceutical field as a biomaterial for drug delivery systems. Generally, for this purpose, collagen is used in the cross-linked form. However, substances such as glutaraldehyde and formaldehyde, which can present cytotoxicity, have been used to achieve cross-linking in these molecules. The objective of the present paper was the extraction and purification of the collagen and its modification by the Maillard reaction. This reaction uses reducing sugars as cross-linking agents with the purpose of eliminating the use of toxic aldehydes. The unmodified collagen and glycated collagen were characterized by electrophoresis in polyacrylamide gel (SDS-PAGE), FTIR spectroscopy, thermal analysis, and by the determination of the NH2-linking degree. The glycated collagen and unmodified collagen were also evaluated in the gel form for their rheological behavior and drug delivery profiles. The SDS-PAGE results showed that the extraction and purification were efficient. Additionally, the thermogravimetric analysis and NH2-linking degree indicated that the reaction had occurred. The rheological assays indicated a higher viscosity for the glycated collagen gel with an increase in the yield at 25 °C. Related to the drug delivery, the results suggested that the increase in collagen concentration in the gel promotes a higher retention time of acetaminophen in the system. The unmodified collagen gel showed a better drug delivery profile than the glycated collagen gel.
Advancements in the areas of natural biomaterials are not a new branch of science, and it has existed from many decades. The application of natural materials as biomaterials is recently undergoing a revival in the biomedical engineering. The major limitations of natural biomaterials are due to the uncontrolled degradation that can occur following in vivo implantation and a lot of variabilities in molecular structure associated with animal sourcing. The main applications of natural biomaterials as materials in medicine are, namely, tissue engineering, wound management products, and drug delivery systems. In recent times, a significant number of biomaterials with biomedical significance have been discovered from the sponges. This book chapter is going to enlighten few important outcomes and their applications in the field of biomedical sciences.
Collagenous tissues (e.g. bone and tendon) have well organized hierarchical structures. To improve understanding of the mechanical behavior of collagenous tissues and to guide the development of multiscale models, mechanical testing at different length scales is required. Whole tissues, fascicles, and fibril bundles have been studied extensively, but little is known at the fibrillar level. Using microelectromechanical systems (MEMS) technology, tensile mechanical testing was performed on type I collagen fibril specimens isolated from the dermis of sea cucumbers. In air uniaxial tensile tests showed that the fibrils had a small strain elastic modulus of 860 ± 450 MPa, a yield stress of 220 ± 140 MPa, and a yield strain of 21% ± 13%. In vitro fracture tests showed that the fibrils had an elastic modulus of 470 ± 410 MPa, a fracture strength of 230 ± 160 MPa, and a fracture strain of 80% ± 44%. The fibrils displayed significantly lower elastic modulus in vitro than in air. Both the fracture strength/strain obtained in vitro and in air were significantly larger than those obtained in vacuo, indicating that the difference arises from the lack of intrafibrillar water molecules produced by vacuum drying. Fracture strength/strain of fibril specimens were different from those reported for collagenous structures of higher hierarchical levels, indicating the importance of obtaining these properties at the fibrillar level for multiscale modeling. In vitro coupled creep and stress relaxation tests demonstrated the intrinsic viscoelastic behavior of collagen fibrils. The stress-strain-time data were fitted using a Kelvin model consisting of a spring and a dashpot in parallel. The fibrils showed an elastic modulus of 180 ± 100 MPa, a viscosity of 4.7 ± 3.2 GPa*sec, and a relaxation time of 29 ± 16 sec. The fibrillar relaxation time was smaller than the tissue-level relaxation time, suggesting tissue relaxation is dominated by non-collagenous components (e.g. proteoglycans). To our knowledge, in vitro fracture and viscoelastic properties of isolated collagen fibrils were measured for the first time. The mechanical properties obtained in this work can be used as input parameters for multiscale modeling and help guide the development of synthetic biomaterials.
As a family of proteins with unique structural features, marine invertebrate collagens have been a focus of structure–function
correlation studies as well as studies interrelating successive levels of structural organization, from the amino acid sequence
to the anatomically defined fibril. Structural and biochemical peculiarities of marine invertebrates collagens isolated from
sponges, jellyfishes, molluscs, and echinoderms as well as perspectives of their applications are described and analyzed here.
Genetically engineered spider silk‐like block copolymers were studied to determine the influence of polyalanine domain size on secondary structure. The role of polyalanine block distribution on β ‐sheet formation was explored using FT‐IR and WAXS. The number of polyalanine blocks had a direct effect on the formation of crystalline β ‐sheets, reflected in the change in crystallinity index as the blocks of polyalanines increased. WAXS analysis confirmed the crystalline nature of the sample with the largest number of polyalanine blocks. This approach provides a platform for further exploration of the role of specific amino acid chemistries in regulating the assembly of β ‐sheet secondary structures, leading to options to regulate material properties through manipulation of this key component in spider silks.
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Collagen type I is the most abundant extracellular matrix protein in the human body, providing the basis for tissue structure and directing cellular functions. Collagen has complex structural hierarchy, organized at different length scales, including the characteristic triple helical feature. In the present study, the relationship between collagen structure (native vs. denatured) and sensitivity to UV radiation was assessed, with a focus on changes in primary structure, changes in conformation, microstructure and material properties. A brief review of free radical reactions involved in collagen degradation is also provided as a mechanistic basis for the changes observed in the study. Structural and functional changes in the collagens were related to the initial conformation (native vs. denatured) and the energy of irradiation. These changes were tracked using SDS-PAGE to assess molecular weight, Fourier transform infrared (FTIR) spectroscopy to study changes in the secondary structure, and atomic force microscopy (AFM) to characterize changes in mechanical properties. The results correlate differences in sensitivity to irradiation with initial collagen structural state: collagen in native conformation vs. heat-treated (denatured) collagen. Changes in collagen were found at all levels of the hierarchical structural organization. In general, the native collagen triple helix is most sensitive to UV-254 nm radiation. The triple helix delays single chain degradation. The loss of the triple helix in collagen is accompanied by hydrogen abstraction through free radical mechanisms. The results received suggest that the effects of electromagnetic radiation on biologically relevant extracellular matrices (collagen in the present study) are important to assess in the context of the state of collagen structure. The results have implications in tissue remodeling, wound repair and disease progression.
Invertebrate animals have been used as medicinals for 4,000 years and have served as models for research and teaching since the late 1800s. Interest in invertebrate models has increased over the past several decades as the research community has responded to public concerns about the use of vertebrate animals in research. As a result, invertebrates are being evaluated and recognized as models for many diseases and conditions. Their use has led to discoveries in almost every area of biology and medicine--from embryonic development to aging processes. Species range from terrestrial invertebrates such as nematodes and insects to freshwater and marine life including planarians, crustaceans, molluscs, and many others. The most often used models are the fruit fly Drosophila melanogaster and the minuscule nematode Caenorhabditis elegans. Topics in this article are categorized by biologic system, process, or disease with discussion of associated invertebrate models. Sections on bioactive products discovered from invertebrates follow the models section, and the article concludes with uses of invertebrates in teaching. The models reviewed can serve as references for scientists, researchers, veterinarians, institutional animal care and use committees (IACUCs), and others interested in alternatives to vertebrate animals.
Collagen fibrils generated in vitro at 37 degrees C by enzymic removal of C-terminal propeptides from type I pC-collagen (an intermediate in the normal processing of type I procollagen to collagen containing the C-terminal propeptides but not the N-terminal propeptides) display shape polarity, with one tip fine tapered and the other coarse tapered. Mass measurements by scanning transmission electron microscopy show that the mass per unit length along both kinds of tip increases roughly linearly over distances of approximately 100 D periods from the fibril end [D (axial periodicity) = 67 nm]. The fine tips of fibrils of widely differing lengths exhibit near-identical mass distributions, the mass in all cases increasing at the rate of approximately 17 molecules per D period, irrespective of fibril length. Coarse tips display less regular behavior. These results show that (i) the shape of a fine tip is not conical but resembles more closely a paraboloid of revolution, and (ii) for this shape to be maintained throughout growth, accretion (rate of mass uptake per unit area) cannot everywhere be the same on the surface of the tip but must decrease as the diameter increases. To a first approximation, accretion alpha (diameter)-1.
Growth of collagen fibrils was examined in a system in which collagen monomers are generated by specific enzymic cleavage of type IpCcollagen with procollagen C-proteinase. Fibrils formed at 37 degrees C had highly tapered and symmetrical pointed tips. The pattern of cross-striations in the pointed tips indicated that all the molecules were oriented so that the N-termini were directed towards the tip. At 29 degrees C and 32 degrees C, the fibrils formed were thicker. One end of fibrils formed at 29 degrees C was blunt, and the other was pointed. Growth of the fibrils was exclusively from pointed tips. Occasionally a spear-like projection appeared at a blunted end. The spear-like projection then became a new pointed tip for growth in the opposite direction. The results suggested a model for fibril growth with at least three distinct binding sites for monomers. In the model, the pointed tip is the site with the highest affinity for the binding of monomers and most probably defines the critical concentration for fibril assembly. The main shaft of the fibril is a site with very low affinity for binding. The blunted end defines a low-affinity binding site where monomers can bind in opposite orientation to produce growth from a new pointed end.
The assembly of discontinuous fibril segments and bundles was studied in 14-day chicken embryo tendons by using serial sections, transmission electron microscopy, and computer-assisted image reconstruction. Fibril segments were first found in extracytoplasmic channels, the sites of their polymerization; they also were found within fibril bundles. Single fibril segments were followed over their entire length in consecutive sections, and their lengths ranged from 7 to 15 microns. Structural differences in the ends of the fibril segments were identified, suggesting that the amino/carboxyl polarity of the fibril segment is reflected in its architecture. Our data indicate that fibril segments are precursors in collagen fibril formation, and we suggest that postdepositional fusion of fibril segments may be an important process in tendon development and growth.
The formation of collagen fibrils, fibril bundles, and tissue-specific collagen macroaggregates by chick embryo tendon fibroblasts was studied using conventional and high voltage electron microscopy. During chick tendon morphogenesis, there are at least three extracellular compartments responsible for three levels of matrix organization: collagen fibrils, bundles, and collagen macroaggregates. Our observations indicate that the initial extracellular events in collagen fibrillogenesis occur within narrow cytoplasmic recesses, presumably under close cellular regulation. Collagen fibrils are formed within these deep, narrow recesses, which are continuous with the extracellular space. Where these narrow recesses fuse with the cell surface, it becomes highly convoluted with folds and processes that envelope forming fibril bundles. The bundles laterally associate and coalesce, forming aggregates within a third cell-defined extracellular compartment. Our interpretation is that this third compartment forms as cell processes retract and cytoplasm is withdrawn between bundles. These studies define a hierarchical organization within the tendon, extending from fibril assembly to fascicle formation. Correlation of different levels of extracellular compartmentalization with tissue architecture provides insight into the cellular controls involved in collagen fibril and higher order assembly and a better understanding of how collagen fibrils are collected into structural groups, positioned, and woven into functional tissue-specific collagen macroaggregates.
The form and integrity of the animal body is largely dependent upon the composition and spatial arrangement of the structural composite known as connective tissue. Each member of this complex family of tissues contains the same building blocks — collagen, glycosaminoglycans, glycoproteins, elastic fibres, cellular constituents, water and minerals — but they are assembled in a multitude of different ways. Ultimately, the interactions of these components with one another bestow the appropriate mechanical attributes upon the tissue.
1. The mechanical properties of the mesogloea of the sea anemone Metridium senile were investigated. An amorphous polymer network in the matrix was found to play a major role in determining the mechanical properties of the tissue.
2. The matrix network provides an elastic mechanism based on ‘rubber elasticity’ of the folded matrix molecules. The properties of the matrix network alone account for the extensibility and elasticity of mesogloea.
3. The collagen acts as a reinforcing filler providing short-term rigidity to the flimsy polymer network.
4. The collagen fibres are not directly cross-linked to one another but are tied together through the amorphous matrix.
5. The extensibility and elasticity of the tissue appear to be dependent on a very low degree of cross-linking in the mesogloeal system. Inorganic ions mask ionized groups on the collagen and matrix polymer chains and block electrostatic interactions which could cross-link the system.
The Nature of Viscoelastic Behavior. Illustrations of Viscoelastic Behavior of Polymeric Systems. Exact Interrelations among the Viscoelastic Functions. Approximate Interrelations among the Linear Viscoelastic Functions. Experimental Methods for Viscoelastic Liquids. Experimental Methods for Soft Viscoelastic Solids and Liquids of High Viscosity. Experimental Methods for Hard Viscoelastic Solids. Experimental Methods for Bulk Measurements. Dilute Solutions: Molecular Theory and Comparisons with Experiments. Molecular Theory for Undiluted Amorphous Polymers and Concentrated Solutions Networks and Entanglements. Dependence of Viscoelastic Behavior on Temperature and Pressure. The Transition Zone from Rubberlike to Glasslike Behavior. The Plateau and Terminal Zones in Uncross-Linked Polymers. Cross-Linked Polymers and Composite Systems. The Glassy State. Crystalline Polymers. Concentrated Solutions, Plasticized Polymers, and Gels. Viscoelastic Behavior in Bulk (Volume) Deformation. Applications to Practical Problems. Appendices. Author & Subject Indexes.
The mutable collagenous tissue (MCT) of echinoderms can
undergo extreme changes in passive mechanical properties within a timescale
of less than 1 s to a few minutes, involving a mechanism that is under direct
neural control and coordinated with the activities of muscles. MCT occurs at
a variety of anatomical locations in all echinoderm classes, is involved in
every investigated echinoderm autotomy mechanism, and provides a mechanism
for the energy-sparing maintenance of posture. It is therefore crucially
important for the biology of extant echinoderms. This chapter summarises
current knowledge of the physiology and organisation of MCT, with particular
attention being given to its molecular organisation and the molecular
mechanism of mutability. The biotechnological potential of MCT is discussed.
It is argued that MCT could be a source of, or inspiration for, (1) new pharmacological
agents and strategies designed to manipulate therapeutically connective
tissue mechanical properties and (2) new composite materials with
biomedical applications.
The catch apparatus (CA) of the sea-urchin spine has been known to have a muscle-like holding property, though it is composed mainly of extra-cellular collagen fibres. An electron microscopic study has been made on the CA of the sea urchin, Anthocidaris crassispina, with special reference to its content of muscle cells and to structural changes of the collagen components on elongation of the CA. The stretch resistance of the CA in a highly extensible state and in a very inextensible state was also measured. Although very thin smooth muscle cells were found scattered among the collagen fibres in the CA, the difference in the passive tension was greater than the estimated stress which could be generated by the muscle cells in the CA by three orders of magnitude. The collagen fibrils remained undeformed but slid along one another during the length change of the CA. The present results suggest that the cohesive force between the collagen fibrils rather than the contractile activity of the muscle cells plays a significant role in determining the mechanical properties of the CA.
The catch apparatus (CA), a collagenous connective tissue which connects the sea-urchin spine to the test, is known to undergo a remarkable change in its mechanical properties. Effects of various physico-chemical factors on the mechanical properties of the CA have been investigated in order to charac-terize the linkages which are responsible for determining the mechanical properties of the CA. The stress-strain relations obtained by stretching the specimen at a slow constant speed, 7 jxm s" 1 , were taken as a measure of the viscous resistance of the CA. The viscous resistance of the CA depended largely on the pH and the ionic strength of the medium. It increased with increasing pH and with decreasing ionic strength. The viscous resistance was markedly reduced in a Ca-free medium containing 5mM-EGTA. It is suggested that linkages sensitive to low pH and those mediated by Ca 2+ play a significant role in determining the mechanical properties of the CA.
Certain echinoderm collagenous structures can sustain rapid changes in their mechanical properties. In some cases the structure can switch reversibly between stiff and compliant states; in other cases, usually associated with autotomy mechanisms, the structure disintegrates irreversibly.The ultrastructure and microarchitecture of the extracellular elements of such mutable collagenous tissues (MCTs) are unremarkable. The only unusual aspect of their biochemistry so far discovered is a high glycoprotein content. All MCTs are permeated by granule‐containing processes belonging to neurosecretory‐like peri‐karya which may be in synaptic contact with motor neurons.In their maximally stiffened condition, most parallel‐fibred MCTs behave as if their collagen fibrils are strongly crosslinked by ground substance macromolecules. Mechanical and ultrastructural data suggest that their compliant state is achieved through the destabilization of interfibrillar linkages which permits slippage between adjacent collagen fibrils. Interfibrillar cohesion appears to be highly dependent on electrostatic interactions. Physicochemical and morphological data support the hypothesis that variable tensility involves the active control of extracellular pH or Ca‐ion availability.Evidence for the nervous control of variable tensility is fragmentary but accumulatively convincing. The coelomic fluid of a wide range of echinoderms contains factors that can influence the mechanical behaviour of MCTs, but whose physiological significance awaits elucidation.
1.1. Collagen fibrils were isolated and purified from the body wall tissue of the starfish, Asterias amurensis.2.2. The fibrils were elongated spindle-shaped and heterogeneous in size. They were similar in shape, while different in size and chemical composition, to sea cucumber fibrils (obtained from Stichopus japonicus).3.3. Variety in the properties of body wall tissue among echinodermal animals presumably depends on variety in the properties of the constituent collagen fibrils.
Zusammenfassung Nachdem an der Hand einer Kurve für die Sperrschwelle die Eigenschaften der reinen Sperrmuskeln erläutert wurden, werden sie am Sperrmuskel der Holoturien aufgesucht, unter Anwendung neuer Methoden, wie dem Umkrempeln des ganzen Tieres und der Zerstörung der Epidermis durch Verschleimung.
(1) Catch connective tissue is defined as the collagenous connective tissue whose mechanical properties can be changed rapidly (in seconds or minutes) under nervous control.
(2) Catch connective tissues are found in all five classes of Echinodermata. They function in tone control of the tissues and in autotomy.
(3) The change in mechanical properties occurs in viscosity.
(4) Muscle cells are not responsible for the viscosity change.
(5) The viscosity change is controlled by nervous activities. Neurosecretory-like cells with large electron-dense granules are found in all the catch connective tissues so far studied.
(6) The viscosity change is quite likely caused by the change in the ionic environment in the connective tissues, which alters the weak (non-covalent) interactions between extracellular macromolecules in the tissue.
In the regular echinoid Diadema setosum Leske the central ligament that connects the spine to its tubercle is mainly composed of closely-packed collagen fibres which are arranged in the longitudinal axis of the ligament. The mechanical properties of the ligament are quite different from ligament to ligament: the viscosity determined by creep tests ranges from 0·02 to 6 GPa·s. The viscosity of a ligament changes reversibly in response to stimulation. Adrenaline and noradrenaline (10-6-10-3 M) decrease the viscosity. Acetylcholine (10-8-10-3 M), artificial sea water containing high (100 MM) potassium concentration, and electrical stimulation increase the viscosity. As the ligament contains no muscle cells in it, the viscosity change cannot be attributable to muscle activities. The functional significance of having a central ligament with variable and controllable viscosity is that it binds the spine base onto the articulation surface of the tubercle, so as to reduce the possibility of spine dislocation, while being flexible enough to allow the spine considerable freedom of movement when necessary.
The collagenous tissues of echinoderms, which have the unique capacity to rapidly and reversibly alter their mechanical properties, resemble the collagenous tissues of other phyla in consisting of collagen fibrils in a nonfibrillar matrix. Knowledge of the composition and structure of their collagen fibrils and interfibrillar matrix is thus important for an understanding of the physiology of these tissues. In this report it is shown that the collagen molecules from the fibrils of the spine ligament of a seaurchin and the deep dermis of a sea-cucumber are the same length as those from vertebrate fibrils and that they assemble into fibrils with the same repeat period and gap/overlap ratio as do those of vertebrate fibrils. The distributions of charged residues in echinoderm and vertebrate molecules are somewhat different, giving rise to segment-long-spacing crystallites and fibrils with different banding patterns. Compared to the vertebrate pattern, the banding pattern of echinoderm fibrils is characterized by greatly increased stain intensity in the c3 band and greatly reduced stain intensity in the a3 and b2 bands. The fibrils are spindle-shaped, possessing no constant-diameter region throughout their length. The shape of the fibrils is mechanically advantageous for their reinforcing role in a discontinuous fiber-composite material.
1.1.The interaction of collagen fibrils and the interfibrillar matrix of tendon has been studied.2.2.Treatment of tendon with chelating agents at pH 7.5 results in the subsequent dispersion of collagen fibrils in dilute acetic acid.3.3.The dispersed collagen fibrils have an amino acid composition which is almost identical with the best preparations of tendon gelatin, indicating the chemical purity of the collagen.4.4.The action of chelating agents can be reversed by equilibration of the treated tendon with calcium acetate prior to acetic acid extraction.5.5.The interfibrillar matrix can be isolated in a purified form virtually free of collagen.6.6.It is suggested that metal ions play a part in the interaction of collagen fibrils and the interfibrillar matrix in tendon.
S.-A. aus Jena'sche Zeitschrift, für Naturwissenschaft. Bd. 34. Zürich. Phil. Diss. II. S. 1900/1901. Die Tafeln fehlen in den Tauschexemplaren.
The collagens of all major invertebrate phyla have been studied, but characterization has been thorough in only a few classes and in no case in the detail (such as sequence analysis) known for vertebrate collagen. Biochemical data on insect collagen are particularly sparse. Invertebrate and vertebrate collagens are strikingly similar, with some notably unique features in annelids and nematodes. Present data do not support the suggestion that invertebrate collagens resemble vertebrate basement membrane collagen. In invertebrates, as in vertebrates, collagens of specific tissues show differenes that probably reflect individual tissue requirements.
The growth of native type fibrils from acetic-acid-soluble collagen has been investigated by quantitative dark-field electron microscopy. Axial mass distributions of early D-periodic fibrils were measured and expressed as plots of na against axial position (na is the number of molecules in a fibril cross section). Measurements were made on entire fibrils in the length range 15 D to 250 D. At the end regions of fibrils these plots were linear, with na increasing by 4 – 5 molecules per D-period at the N-terminal end and 8 – 10 at the C-terminal. These results were not dependent on fibril length and were observed over a range of precipitating conditions.
Electron microscopic autoradiographic observations on collagen fibrils grown in vitro allow growth rates in the N- and C-terminal directions to be measured on individual fibrils. Such observations, made on normal and iodinated collagen, show that normal fibrils grow at both ends (although rather more rapidly at the N-terminal end), whereas fully-iodinated collagen fibrils grow only at the N-terminal end.
Measurements of growth rates at different temperatures provide estimates of the activation enthalpy (ΔH≠) and entropy (ΔS≠) of precipitation for the two types of collagen. Solubility measurements have also yielded values for the thermodynamic enthalpy (ΔH) and entropy (ΔS) of precipitation. Results show that the activated (rate-limiting) state is characterized by a large positive ΔH≠ and ΔS≠ similar in magnitude to the ΔH and ΔS of transition from solution to fibril. It is also concluded that the different rates of precipitation of normal and iodinated collagen cannot be explained in terms of fibril formation requiring ionization of the tyrosine residues.
A model was developed to account for the recent observations indicating that type I collagen fibrils assembled in vivo grow from symmetrical pointed tips. The essential features of the model are (i) a distinctive structural nucleus forms at each end of a growing fibril and growth of the fibril then proceeds by propagation of the two structural nuclei, (ii) the two structural nuclei have similar spiral or helical conformations, and (iii) assembly of each structural nucleus requires two kinds of specific binding steps defined as 3.4 D-period and 0.4 D-period overlaps, but propagation of the nucleus requires only the 3.4 D-period binding step.
A theoretical expression has been derived for the mean collagen fibril length in tendon based on the assumption that collagen fibrils originate in cell surface invaginations and terminate either at some remote cell surface or another collagen fibril bundle. The expression thus determined requires knowledge of the effective lengths of the fibrocytes (or fibrocyte assemblies) and the cellular content of the tendon. Both of these parameters have been measured experimentally as a function of age for rat-tail tendon using a combined light microscope and electron microscope approach. The results obtained for immature tendon suggest that the mean collagen fibril length is at least equal to the critical length required to maintain the appropriate tensile properties. In the most mature tissue studied, however, the mean-collagen fibril length is in excess of 100 times the critical length.
The "problematic ligament" of sea urchins is a connective tissue which crosses the ball-and-socket joint between spine and body wall. The problem of this ligament is that it is composed of parallel collagen fibrils, yet normally undergoes rapid and dramatic alterations in mechanical properties and in length. Previous work has suggested that the collagen fibrils of the ligament are able to slide past one another during length changes but are inhibited from sliding when the ligament is in "catch". In this model of the ligament both the collagen fibrils and the interfibrillar matrix are mechanically important. We have found that the collagen fibrils of the spine ligament of the pencil urchin Eucidaris tribuloides are discontinuous and end by tapering within the body of the ligament. Intact fibrils that have been isolated from the ligament vary by more than an order of magnitude in length and in radius but have a constant length/radius (aspect) ratio of about 5,300. This is the first determination of the aspect ratio of collagen fibrils from any source. The constant aspect ratio of the fibrils is consistent with their functioning as the discontinuous fiber phase in a fiber-reinforced composite material, while the high value of the aspect ratio indicates that the nonfibrillar matrix, which must act to transfer stress between fibrils, can produce a stiff and strong ligament even if it is several orders of magnitude weaker and more compliant than the fibrils. Moreover, the tensile properties of the ligament may be determined by the properties of the matrix. A prominent component of the interfibrillar matrix is a proteoglycan which associates with specific bands at the surface of the collagen fibrils through noncovalent binding of its core protein. The glycosaminoglycan moiety of this proteoglycan is partly comprised of chondroitin sulfate/dermatan sulfate polymers. These results are consistent with the "sliding fibril" hypothesis and suggest that the proteoglycan may be an important component of the stress-transfer matrix.
Transmission of contractile tension from skeletal muscle fibers to connective tissue elements is thought to occur at the muscle-tendon junctions, specialized regions at the extreme ends of the fibers. Previous stereological studies on adult mouse and chicken fibers have shown that, with reference to equal cross-sectional areas of myofibrils, the muscle-tendon junctions of faster fibers have significantly more surface membrane devoted to force transmission than do those of slower fibers (Trotter et al.: Anat. Rec. 213:16-25, 1985a; Trotter et al.: Anat. Rec. 213:26-32,1985b; Trotter and Baca: Anat. Rec. 218:256-266, 1987). In the present study we have analyzed the muscle-tendon junctions of 30-month-old mice, employing techniques for scanning and transmission electron microscopy and for ultrastructural stereology which are identical to those previously used to study the same muscles in 4-month-old mice. Whereas the principal structural features of the muscle-tendon junctions of fibers from adult and aged mice are indistinguishable, stereological analyses of the fiber-tendon interfaces indicate that, in aging animals, the interfacial ratio (the ratio of the surface area of force-transmitting membrane to the cross-sectional area of force-generating myofibrils) is significantly reduced. While the interfacial ratio of fast-twitch fibers of the adult plantaris is about 14.5, the corresponding ratio in aged animals is about ten. In the predominantly slow-twitch fibers of the adult soleus, the interfacial ratio is about ten at the insertion and about 12.7 at the origin, whereas the corresponding ratios in the aged animals are both about ten.(ABSTRACT TRUNCATED AT 250 WORDS)
Tension-induced molecular rearrangements in wet native fibres of rat-tail tendons and human finger flexor tendons are registered with the help of time-resolved diffraction spectra using synchrotron radiation. The tension-induced increase of the 67 nm D period is combined with changes in the intensities of some orders of the meridional small angle reflection. Both effects are reversible when unloading the fibre, but are preserved when the load is held constant until the fibre tears. The increase in the D period is partly due to a sliding of the triple helices relative to each other and partly due to a stretching of the triple helices themselves. The sliding of the triple helices results in an alteration of the D stagger, leading to a change in the length of the gap and overlap regions, and to a stretching of the cross-linked telopeptides. This interpretation is supported by comparison with the relative intensities derived from a model with varying length of gap and overlap regions, as well as by comparison with model calculations that include the telopeptides.
Collagen fibrils were isolated from the body wall of the sea cucumber, Stichopus japonicrts, and purified by centrifugation. Phase contrast and electronmicroscopy showed elongated, spindle shaped fibrils. Their average diameter was calculated to be 1.44 D, where 1 D = the length of unit striation of the fibril b̃ 640 Å The number-average contour length was 154 μm. Heterogeneity of size was demonstrated by differential centrifugation and diameter measurements. © 1974 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
In order to study the nature of highly organized components of connective tissue, preparation of fibrous components of collagen in their intact form was attempted by chemical treatment. By use of a solution consisting of 0.5M NaCl, 0.1M Tris-HCl buffer, pH 8.0, 0.05M EDTA-Na, and 0.2M β-mercaptoethanol the body wall of sea cucumber, Stichopus japonicus , and calf skin chips were found to be disaggregated into collagen fibril and fiber bundle, respectively. The collagen fiber-bundle of calf skin was further disaggregated into collagen fiber by stirring in acetic acid solution in the cold. The nature of the assembly of fibrous components of connective tissues was investigated with reference to animal and tissue sources.
Crude bacterial α-amylase and ethylenediaminetetraacetic acid act on the interfibrillar matrix of the fiber to weaken the fibril—fibril interaction and allow the constituent fibrils to disperse in dilute acetic acid. The morphology, range of diameter, amino acid composition, carbohydrate content, and stability of the polymeric collagen fibrils thus obtained is identical to that of native fibrils.
Stress-strain and relaxation tests were conducted on human tendon, aorta and bovine ligamentum nuchae which have different arrangements and proportions of elastin, collagen and ground substance. The removal of the ground substance with an enzyme or a chelating agent induced a decrease in stress level, stiffness, relaxation, hysteresis and other time-dependent effects in all three tissues. These changes could be largely explained by a reduction in the effective viscosity of the interfibre matrix although some other factor seemed to be present in the case of tendon, possibly a change in the relationship between the collagen fibres and the glycoproteins in the matrix.Treating aorta and nuchal ligament with formic acid to remove collagen and other material produced a marked decrease in stress level and time-dependence and also a decrease in the stress and strain at rupture which may be due to the removal of collagen that normally prevents premature rupture at weak points in the elastic network.
1. 1.The interaction of collagen fibrils and the interfibrillar matrix of tendon has been studied. 2. 2.Treatment of tendon with chelating agents at pH 7.5 results in the subsequent dispersion of collagen fibrils in dilute acetic acid. 3. 3.The dispersed collagen fibrils have an amino acid composition which is almost identical with the best preparations of tendon gelatin, indicating the chemical purity of the collagen. 4. 4.The action of chelating agents can be reversed by equilibration of the treated tendon with calcium acetate prior to acetic acid extraction. 5. 5.The interfibrillar matrix can be isolated in a purified form virtually free of collagen. 6. 6.It is suggested that metal ions play a part in the interaction of collagen fibrils and the interfibrillar matrix in tendon.