Mutable Collagenous Tissue Research Articles

A starfish-inspired 4D self-healing morphing structure

Inspired by the starfish's unique ability to achieve flexibility and posture-holding with minimal energy expenditure, we present a novel bioinspired morphing structure. Our two-component design, consisting of a thermoplastic mesh and elastomeric jacket, effectively mimics the functions of the starfish's ossicles, mutable collagenous tissues, and derma. This structure exhibits a remarkable combination of self-healing, time-dependent shape memory, and self-posture-holding properties. Systematic variations in mesh geometry demonstrate precise control over structural stiffness and thermal response, enabling customization for specific applications. The structure's scalability and ease of fabrication further enhance its adaptability. We experimentally demonstrate the potential of our biomimetic morphing structure using several prototypes. This work lays the foundation for developing a new type of versatile morphing structures with applications in diverse fields, including robotics, biomedical devices, and adaptive structures.

Functional morphology of the stem in the Lower Paleozoic crinoid Macrostylocrinus Hall from Scotland

Two species of the closely related monobathrid crinoid from the Lower Paleozoic of Scotland, namely Macrostylocrinus cirrifer Ramsbottom (Upper Ordovician, Katian) and Macrostylocrinus silurocirrifer Brower (Lower Silurian, Telychian), are similar in having elongate, unbranched radices proximally. These were not cirri, as suggested by their names, but were radices that were more or less inflexible, lacking contractile tissues. The function of these radices was uncertain. In the absence of contractile tissues, they could not have been for grasping other upright structures and crinoids do not need help to balance, their posture being maintained by mutable collagenous tissues. It is possible, but unlikely, that they may have acted to direct feeding currents towards the crown. Most probably, in an analogy to the post-Paleozoic isocrinids, the stem acted like a ‘conveyor belt’, the proximal, radicular and upright part being carried away from the cup as further columnals are inserted, eventually forming a distal, recumbent attachment structure. The elongate radices would have stabilized the dististele, but, unlike isocrinids, the arms of Macrostylocrinus spp. were not adapted for crawling and thus escaping predators. Both M. silurocirrifer (type species) and M. cirrifer are included in Macrostylocrinus ( Scotimacrostylocrinus ) subgen. nov.

Mutable Collagenous Tissue: A Concept Generator for Biomimetic Materials and Devices.

Echinoderms (starfish, sea-urchins and their close relations) possess a unique type of collagenous tissue that is innervated by the motor nervous system and whose mechanical properties, such as tensile strength and elastic stiffness, can be altered in a time frame of seconds. Intensive research on echinoderm 'mutable collagenous tissue' (MCT) began over 50 years ago, and over 20 years ago, MCT first inspired a biomimetic design. MCT, and sea-cucumber dermis in particular, is now a major source of ideas for the development of new mechanically adaptable materials and devices with applications in diverse areas including biomedical science, chemical engineering and robotics. In this review, after an up-to-date account of present knowledge of the structural, physiological and molecular adaptations of MCT and the mechanisms responsible for its variable tensile properties, we focus on MCT as a concept generator surveying biomimetic systems inspired by MCT biology, showing that these include both bio-derived developments (same function, analogous operating principles) and technology-derived developments (same function, different operating principles), and suggest a strategy for the further exploitation of this promising biological resource.

Mutable Collagenous Tissue Isolated from Echinoderms Leads to the Production of a Dermal Template That Is Biocompatible and Effective for Wound Healing in Rats.

The mutable collagenous tissue (MCT) of echinoderms possesses biological peculiarities that facilitate native collagen extraction and employment for biomedical applications such as regenerative purposes for the treatment of skin wounds. Strategies for skin regeneration have been developed and dermal substitutes have been used to cover the lesion to facilitate cell proliferation, although very little is known about the application of novel matrix obtained from marine collagen. From food waste we isolated eco-friendly collagen, naturally enriched with glycosaminoglycans, to produce an innovative marine-derived biomaterial assembled as a novel bi-layered skin substitute (Marine Collagen Dermal Template or MCDT). The present work carried out a preliminary experimental in vivo comparative analysis between the MCDT and Integra, one of the most widely used dermal templates for wound management, in a rat model of full-thickness skin wounds. Clinical, histological, and molecular evaluations showed that the MCDT might be a valuable tool in promoting and supporting skin wound healing: it is biocompatible, as no adverse reactions were observed, along with stimulating angiogenesis and the deposition of mature collagen. Therefore, the two dermal templates used in this study displayed similar biocompatibility and outcome with focus on full-thickness skin wounds, although a peculiar cellular behavior involving the angiogenesis process was observed for the MCDT.

Physicochemical and Biochemical Characterization of Collagen from Stichopus cf. horrens Tissues for Use as Stimuli-Responsive Thin Films.

The mutable collagenous tissue (MCT) of sea cucumber, with its ability to rapidly change its stiffness and extensibility in response to different environmental stress conditions, serves as inspiration for the design of new smart functional biomaterials. Collagen, extracted from the body wall of Stichopus cf. horrens, a species commonly found in the Philippines, was characterized for its suitability as stimuli-responsive films. Protein BLAST search showed the presence of sequences commonly found in type VII and IX collagen, suggesting that Stichopus horrens collagen is heterotypic. The maximum transition temperature recorded was 56.0 ± 2 °C, which is higher than those of other known sources of marine collagen. This suggests that S. horrens collagen has better thermal stability and durability. Collagen-based thin films were then prepared, and atomic force microscopy (AFM) imaging showed the visible collagen network comprising the films. The thin films were subjected to thermomechanical analysis with degradation starting at >175 °C. At 100-150 °C, the collagen-based films apparently lose their translucency due to the removal of moisture. Upon exposure to ambient temperature, instead of degrading, the films were able to revert to the original state due to the readsorption of moisture. This study is a demonstration of a smart biomaterial developed from S. cf. horrens collagen with potential applications in food, pharmaceutical, biomedical, and other collagen-based research.

Morphological and Physiological Aspects of Mutable Collagenous Tissue at the Autotomy Plane of the Starfish Asterias rubens L. (Echinodermata, Asteroidea): An Echinoderm Paradigm.

The mutable collagenous tissue (MCT) of echinoderms has the capacity to undergo changes in its tensile properties within a timescale of seconds under the control of the nervous system. All echinoderm autotomy (defensive self-detachment) mechanisms depend on the extreme destabilisation of mutable collagenous structures at the plane of separation. This review illustrates the role of MCT in autotomy by bringing together previously published and new information on the basal arm autotomy plane of the starfish Asterias rubens L. It focuses on the MCT components of breakage zones in the dorsolateral and ambulacral regions of the body wall, and details data on their structural organisation and physiology. Information is also provided on the extrinsic stomach retractor apparatus whose involvement in autotomy has not been previously recognised. We show that the arm autotomy plane of A. rubens is a tractable model system for addressing outstanding problems in MCT biology. It is amenable to in vitro pharmacological investigations using isolated preparations and provides an opportunity for the application of comparative proteomic analysis and other "-omics" methods which are aimed at the molecular profiling of different mechanical states and characterising effector cell functions.

Morphological, Physiological and Mechanical Features of the Mutable Collagenous Tissues Associated with Autotomy and Evisceration in Dendrochirotid Holothuroids.

Evisceration in dendrochirotid sea cucumbers leads to expulsion of the digestive tract, pharyrngeal complex and coelomic fluid through rupture of the anterior body wall. This process involves failure of three mutable collagenous tissue (MCT) structures, the introvert, the pharyngeal retractor muscle tendon, and the intestine-cloacal junction. These are complex structures composed of several tissue strata. The MCT in the three autotomy structures contains collagen fibrils, unstriated microfibrils, and interfibrillar molecules. Neurosecretory-like processes (juxtaligamental-type) with large dense vesicles (LDVs) are prominent in the autotomy structures. Biomechanical tests show that these structures are not inherently weak. Failure of the autotomy structures can be elicited by manipulating the ionic environment and the changes are blocked by anaesthetics. Autotomy and evisceration are under neural control, but local neural elements and neurosecretory-like processes do not appear to be a source of factors that cause MCT destabilisation. The LDVs remain intact while the tissue destabilises. The coelomic fluid contains an evisceration inducing factor indicating a neurosecretory-like mediation of autotomy. This factor elicits muscle contraction and MCT destabilisation. As the autotomy structures are completely or partially surrounded by coelomic fluid, the agent(s) of change may be located in the coelom (systemic origin) as well as originate from cells within the MCT. The biochemistry and mechanism(s) of action of the evisceration factor are not known. This factor is a promising candidate for biodiscovery investigation.

Quality assessment of variable collagen tissues of sea cucumber (Stichopus japonicus) body wall under different heat treatment durations by label-Free proteomics analysis

The microstructure of the body wall, body wall composition, and collagen fibers of sea cucumber (Stichopus japonicus) under different heating times (1 h, 4 h, 12 h, and 24 h) was investigated based on heat treatment at 80 °C. A Label-Free proteomics technique was applied to study the proteomic changes in the body wall of sea cucumbers under 4 and 12 h of heat treatment. Compared with the fresh group, 981 proteins were found to be differentially expressed proteins (DEPs) after heat treatment at 80 °C (4 h), and 1110 DEPs were observed after heat treatment at the same temperature for 12 h. There were 69 DEPs associated with mutable collagenous tissues (MCTs) structures. The results of correlation analysis showed that 55 DEPs were correlated with sensory properties, among which A0A2G8KRV2 was significantly correlated with hardness and SEM image texture features (SEM_Energy, SEM_Correlation, SEM_Homogeneity, and SEM_Contrast). These findings could be conducive to further comprehension of the structural changes and mechanisms of quality loss in the body wall of sea cucumbers at different heat treatment times.

EchinoDB: an update to the web-based application for genomic and transcriptomic data on echinoderms

BackgroundHere we release a new version of EchinoDB, EchinoDB v2.0 (https://echinodb.uncc.edu). EchinoDB is a database of genomic and transcriptomic data on echinoderms. The initial database consisted of groups of 749,397 orthologous and paralogous transcripts arranged in orthoclusters by sequence similarity.ResultsThe updated version of EchinoDB includes two new major datasets: the RNA-Seq data of the brittle star Ophioderma brevispinum and the high-quality genomic assembly data of the green sea urchin Lytechinus variegatus. In addition, we enabled keyword searches for annotated data and installed an updated version of Sequenceserver to allow Basic Local Alignment Search Tool (BLAST) searches. The data are downloadable in FASTA format. The first version of EchinoDB appeared in 2016 and was implemented in GO on a local server. The new version has been updated using R Shiny to include new features and improvements in the application. Furthermore, EchinoDB now runs entirely in the cloud for increased reliability and scaling.ConclusionEchinoDB serves a user base drawn from the fields of phylogenetics, developmental biology, genomics, physiology, neurobiology, and regeneration. As use cases, we illustrate the function of EchinoDB in retrieving components of signaling pathways involved in the tissue regeneration process of different echinoderms, including the emerging model species Ophioderma brevispinum. Moreover, we use EchinoDB to shed light on the conservation of the molecular components involved in two echinoderm-specific phenomena: spicule matrix proteins involved in the formation of stereom endoskeleton and the tensilin protein that contributes to the capacity of the connective tissues to quickly change its mechanical properties. The genes involved in the former had been previously studied in echinoids, while gene sequences involved in the latter had been previously described in holothuroids. Specifically, we ask (a) if the biomineralization-related proteins previously reported only in sea urchins are also present in other, non-echinoid, echinoderms and (b) if tensilin, the protein responsible for the control of stiffness of the mutable collagenous tissue, previously described in sea cucumbers, is conserved across the phylum.

Molecular mechanisms mediating stiffening in the mechanically adaptable connective tissues of sea cucumbers

Mutable collagenous tissues (MCTs) from echinoderms (e.g., sea stars, sea urchins) possess the remarkable ability to change their mechanical properties rapidly and reversibly thanks to the release of effector molecules regulating the number of cross-links between collagen fibrils. Among these effector molecules, tensilin has been identified as a stiffening factor in sea cucumber MCTs. Since its discovery and description twenty years ago, tensilin orthologs have been identified in a few sea cucumber species but no novel information about its molecular mode of action has been reported. In this study, using a combination of in silico analyses, we identified the tensilin present in the dermis of Holothuria forskali, Hf-(D)Tensilin. Anti-peptide antibodies showed that this protein is localised in the secretory granules of type 2 juxtaligamental-like cells, a MCT specific cell type. We then used the bacterium E. coli to produce recombinantly Hf-(D)Tensilin and confirmed its stiffening effect on pieces of the dermis and its aggregation effect on collagen fibrils extracted from the sea cucumber dermis. To investigate how tensilin can cross-bridge collagen fibrils, truncated recombinant tensilins were also produced and used in combination with various compounds. Results suggest that two types of interactions contribute to the aggregation effect of tensilin on the fibrils: (1) the N-terminal NTR TIMP like domain of the protein interacts strongly with sulfated GAGs attached to the surface of the collagen fibrils, and (2) the C-terminal part of the protein is involved in its dimerisation/oligomerisation through ionic but possibly also cation-π and hydrophobic interactions.

Sea star wasting disease pathology in Pisaster ochraceus shows a basal-to-surface process affecting color phenotypes differently.

Sea star wasting disease (SSWD) refers to a suite of poorly described non-specific clinical signs including abnormal posture, epidermal ulceration, and limb autotomy (sloughing) causing mortalities of over 20 species of sea stars and subsequent ecological shifts throughout the northeastern Pacific. While SSWD is widely assumed to be infectious, with environmental conditions facilitating disease progression, few data exist on cellular changes associated with the disease. This is unfortunate, because such observations could inform mechanisms of disease pathogenesis and host susceptibility. Here, we replicated SSWD by exposing captive Pisaster ochraceus to a suite of non-infectious organic substances and show that development of gross lesions is a basal-to-surface process involving inflammation (e.g. infiltration of coelomocytes) of ossicles and mutable collagenous tissue, leading to epidermal ulceration. Affected sea stars also manifest increases in a heretofore undocumented coelomocyte type, spindle cells, that might be a useful marker of inflammation in this species. Finally, compared to purple morphs, orange P. ochraceus developed more severe lesions but survived longer. Longer-lived, and presumably more visible, severely-lesioned orange sea stars could have important demographic implications in terms of detectability of lesioned animals in the wild and measures of apparent prevalence of disease.

A programmable powerful and ultra-fast water-driven soft actuator inspired by the mutable collagenous tissue of the sea cucumber

Composition/dimensional adjustment of a bulk PNIPAAm-based water-driven soft actuator tuned actuation force/speed in wide ranges at maximum of 2 N and 3 s−1, respectively, demonstrating immediate potentials in soft robotics and biomedical appliance.

A farewell to arms: using X-ray synchrotron imaging to investigate autotomy in brittle stars

Autotomy, the self-induced loss of a body structure, occurs in every living class of echinoderms and is related to the remarkable regeneration capabilities of the group. It is particularly prevalent in brittle stars (Class Ophiuroidea). Autotomy is facilitated by mutable collagenous tissue, which undergoes nervous system-mediated changes in tensile stiffness, tensile strength, and viscosity. The previous investigations of autotomy have been based on observations of the external surface, surgical manipulation of internal structures, or data on the morphology of structures post-autotomy. We used fast phase-contrast X-ray synchrotron imaging to visualize full autotomy events in vivo in the arms of specimens of the brittle star Ophioderma brevispina. This method requires no chemical or surgical manipulation and enabled us to identify several key stages in the autotomy process. We used this methodology to observe critical changes within the internal structure of the arm as it transitions from a functional mechanical apparatus to a dysfunctional disarticulated state. This method is the first in which the full intersegmental plane of the arm can be observed during autotomy. It can be applied to visualize autotomy and motion in vivo in other brittle star taxa, as well as in other groups such as asteroids and arthropods.

Unlocking the secrets of mutable collagenous tissue

The mutable collagenous tissue (MCT) of echinoderms (e.g. sea cucumbers, starfish and sea urchins) is unique because of its ability to ‘switch’ mechanical states rapidly and reversibly – from stiff to soft and vice versa. This kind of tissue in humans, for example, in skin, tendons and ligaments, does not have this property. So what are the molecular-level secrets by which MCT achieves this transformative ability? New real-time ultrastructural investigations are beginning to shed light on this question. Synchrotron X-ray measurements of dynamic molecular conformational changes point to the key factor being the gel-like matrix between the collagen fibrils. These findings could have applications for developing treatments for collagen-based disorders.

Bioinspiring Chondrosia reniformis (Nardo, 1847) Collagen-Based Hydrogel: A New Extraction Method to Obtain a Sticky and Self-Healing Collagenous Material.

Collagen is a natural and abundant polymer that serves multiple functions in both invertebrates and vertebrates. As collagen is the natural scaffolding for cells, collagen-based hydrogels are regarded as ideal materials for tissue engineering applications since they can mimic the natural cellular microenvironment. Chondrosia reniformis is a marine demosponge particularly rich in collagen, characterized by the presence of labile interfibrillar crosslinks similarly to those described in the mutable collagenous tissues (MCTs) of echinoderms. As a result single fibrils can be isolated using calcium-chelating and disulphide-reducing chemicals. In the present work we firstly describe a new extraction method that directly produces a highly hydrated hydrogel with interesting self-healing properties. The materials obtained were then biochemically and rheologically characterized. Our investigation has shown that the developed extraction procedure is able to extract collagen as well as other proteins and Glycosaminoglycans (GAG)-like molecules that give the collagenous hydrogel interesting and new rheological properties when compared to other described collagenous materials. The present work motivates further in-depth investigations towards the development of a new class of injectable collagenous hydrogels with tailored specifications.

Collagenous Extracellular Matrix Biomaterials for Tissue Engineering: Lessons from the Common Sea Urchin Tissue.

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.

Mechanical adaptability of sea cucumber Cuvierian tubules involves a mutable collagenous tissue.

Despite their soft body and slow motion, sea cucumbers present a low predation rate, reflecting the presence of efficient defence systems. For instance, members of the family Holothuriidae rely on Cuvierian tubules for their defence. These tubules are normally stored in the posterior coelomic cavity of the animal, but when the sea cucumber is threatened by a potential predator, they are expelled through the cloacal aperture, elongate, become sticky and entangle and immobilise the predator in a matter of seconds. The mechanical properties (extensibility, tensile strength, stiffness and toughness) of quiescent (i.e. in the body cavity) and elongated (i.e. after expulsion) Cuvierian tubules were investigated in the species Holothuria forskali using traction tests. Important mechanical differences were measured between the two types of tubules, reflecting adaptability to their operating mode: to ease elongation, quiescent tubules present a low resistance to extension, while elongated tubules present a high toughness to resist tractions generated by the predator. We demonstrate that a mutable collagenous tissue (MCT) is involved in the functioning of these organs: (1) some mechanical properties of Cuvierian tubules are modified by incubation in a cell-disrupting solution; (2) the connective tissue layer encloses juxtaligamental-like cells, a cell type present in all MCTs; and (3) tensilin, a MCT stiffening protein, was localised inside these cells. Cuvierian tubules thus appear to enclose a new type of MCT which shows irreversible stiffening.

Functional Morphology of the Arm Spine Joint and Adjacent Structures of the Brittlestar Ophiocomina nigra (Echinodermata: Ophiuroidea).

The skeletal morphology of the arm spine joint of the brittlestar Ophiocomina nigra was examined by scanning electron microscopy and the associated epidermis, connective tissue structures, juxtaligamental system and muscle by optical and transmission electron microscopy. The behaviour of spines in living animals was observed and two experiments were conducted to establish if the spine ligament is mutable collagenous tissue: these determined (1) if animals could detach spines to which plastic tags had been attached and (2) if the extension under constant load of isolated joint preparations was affected by high potassium stimulation. The articulation normally operates as a flexible joint in which the articular surfaces are separated by compliant connective tissue. The articular surfaces comprise a reniform apposition and peg-in-socket mechanical stop, and function primarily to stabilise spines in the erect position. Erect spines can be completely immobilised, which depends on the ligament having mutable tensile properties, as was inferred from the ability of animals to detach tagged spines and the responsiveness of isolated joint preparations to high potassium. The epidermis surrounding the joint has circumferential constrictions that facilitate compression folding and unfolding when the spine is inclined. The interarticular connective tissue is an acellular meshwork of collagen fibril bundles and may serve to reduce frictional forces between the articular surfaces. The ligament consists of parallel bundles of collagen fibrils and 7–14 nm microfibrils. Its passive elastic recoil contributes to the re-erection of inclined spines. The ligament is permeated by cell processes containing large dense-core vesicles, which belong to two types of juxtaligamental cells, one of which is probably peptidergic. The spine muscle consists of obliquely striated myocytes that are linked to the skeleton by extensions of their basement membranes. Muscle contraction may serve mainly to complete the process of spine erection by ensuring close contact between the articular surfaces.

Interfibrillar stiffening of echinoderm mutable collagenous tissue demonstrated at the nanoscale

The mutable collagenous tissue (MCT) of echinoderms (e.g., sea cucumbers and starfish) is a remarkable example of a biological material that has the unique attribute, among collagenous tissues, of being able to rapidly change its stiffness and extensibility under neural control. However, the mechanisms of MCT have not been characterized at the nanoscale. Using synchrotron small-angle X-ray diffraction to probe time-dependent changes in fibrillar structure during in situ tensile testing of sea cucumber dermis, we investigate the ultrastructural mechanics of MCT by measuring fibril strain at different chemically induced mechanical states. By measuring a variable interfibrillar stiffness (EIF), the mechanism of mutability at the nanoscale can be demonstrated directly. A model of stiffness modulation via enhanced fibrillar recruitment is developed to explain the biophysical mechanisms of MCT. Understanding the mechanisms of MCT quantitatively may have applications in development of new types of mechanically tunable biomaterials.

Ultrastructural Changes Associated with Reversible Stiffening in Catch Connective Tissue of Sea Cucumbers.

The dermis of sea cucumbers is a catch connective tissue or a mutable collagenous tissue that shows rapid, large and reversible stiffness changes in response to stimulation. The main component of the dermis is the extracellular material composed of collagen fibrils embedded in a hydrogel of proteoglycans. The stiffness of the extracellular material determines that of the dermis. The dermis has three mechanical states: soft (Sa), standard (Sb) and stiff (Sc). We studied the ultrastructural changes associated with the stiffness changes. Transverse sections of collagen fibrils in the dermis showed irregular perimeters with electron-dense protrusions or arms that cross-bridged between fibrils. The number of cross-bridges increased in stiffer dermis. The distance between the fibrils was shorter in Sc than that in other states, which was in accord with the previous report that water exuded from the tissue in the transition Sb→Sc. The ultrastructure of collagen fibrils that had been isolated from the dermis was also studied. Fibrils aggregated by tensilin, which causes the transition Sa→Sb possibly through an increase in cohesive forces between fibrils, had larger diameter than those dispersed by softenin, which antagonizes the effect of tensilin. No cross-bridges were found in isolated collagen fibrils. From the present ultrastructural study we propose that three different mechanisms work together to increase the dermal stiffness. 1.Tensilin makes collagen fibrils stronger and stiffer in Sa→Sb through an increase in cohesive forces between subfibrils that constituted fibrils; 2. Cross-bridging by arms caused the fibrils to be a continuous network of bundles both in Sa→Sb and in Sb→Sc; 3. The matrix embedding the fibril network became stiffer in Sb→Sc, which was produced by bonding associated with water exudation.