Bundles Of Collagen Fibrils Research Articles

Ultrastructural features of skin pigmentation in the lizardHeloderma suspectumwith emphasis on xanto-melanophores

The morphological origin of the dark and pink-orange areas in the skin of the venomous lizard Heloderma suspectum is not known. Histology and electron microscopy show that dark-grey areas of the skin derived from dermal chromatophores localized in specific areas present underneath the epidermis. A dynamic chromatophoric unit in the dermis is absent. In the darkest areas of the skin, the accumulation of melanosomes in cells of the beta-layer contributes to increase the black intensity. In the orange-pink areas, the superficial dermis contains xantophores storing numerous carotenoid vesicles, rare or absent lamellated pterinosomes and a variable number of melanosomes. These xanto-melanophores predominate over the remaining chromatophores and form a continuous stratum underneath the epidermis. Beneath this lipoid-rich stratum, iridophores are infrequent and do not form a continuous layer in the dermis. In the paler areas of the skin, melanophores are sparse in both superficial and deeper part of the dermis where irregularly oriented bundles of collagen fibrils are present. The prevalent xanto-melanophores localized in the pink-orange areas of the skin contribute to an effective sunlight protection in desert conditions in addition to the darker regions occupied by melanophores.

Immunocytochemical localization of cysteine‐rich beta‐proteins in the extensible epidermis of the dewlap in the lizard Anolis carolinensis

AbstractThe dewlap in the lizard Anolis carolinensis is made of scales separated by large interscale regions capable of broad stretching during fan extension. This indicates that the skin contains proteins that allow extension of interscale regions. The immunocytochemical analysis of the epidermis indicates that HgG5, a glycine‐rich hydrophobic beta‐protein poor in cysteine is localized only in the stiff beta‐layer of the outer scale surface, but is completely absent in mesos and alpha‐layers and in hinge regions. HgGC10, a cysteine‐medium‐rich beta‐protein is present in beta‐layers but especially in alpha‐layers of interscale epidermis that presents folds and lacks a beta‐layer. HgGC3 is weakly localized in the alpha‐layer, but is mainly found in hinge regions. HgGC8 and HgG13 are low to absent in the alpha‐ and beta‐layer. The immunolocalization of cysteine‐rich beta‐proteins such as HgGC10/3 in alpha‐layers and interscale epidermis suggests that these small proteins are involved in the formation of a corneous material compatible with dewlap extension. The basement membrane underneath scales is joined to bundles of collagen fibrils in the dermis through anchoring fibrils that likely determine flattening of the epidermis during the extension of the throat fan.

Cartilage, bone, and intermandibular connective tissue in the australian lungfish,Neoceratodus forsteri(Osteichthyes: Dipnoi)

The connective tissue that links the bones of the mandible in the Australian lungfish, Neoceratodus forsteri, has been described as an intermandibular cartilage, and as such has been considered important for phylogenetic analyses among lower vertebrates. However, light and electron microscopy of developing lungfish jaws demonstrates that the intermandibular tissue, like the connective tissue that links the bones of the upper jaw, contains fibroblasts and numerous bundles of collagen fibrils, extending from the trabeculae of the bones supporting the tooth plates. It differs significantly in structure and in staining reactions from the cartilage and the bone found in this species. In common with the cladistian Polypterus and with actinopterygians and some amphibians, lungfish have no intermandibular cartilage. The connective tissue linking the mandibular bones has no phylogenetic significance for systematic grouping of lungfish, as it is present in a range of different groups among lower vertebrates.

Accelerating repaired basement membrane after bevacizumab treatment on alkali-burned mouse cornea

To understand the corneal regeneration induced by bevacizumab, we investigated the structure changes of stroma and basement membrane regeneration. A Stick soaked in 0.5 N NaOH onto the mouse cornea and 2.5 mg/ml of bevacizumab was delivered into an alkali-burned cornea (2 μl) by subconjunctival injections at 1 hour and 4 days after injury. At 7 days after injury, basement membrane regeneration was observed by transmission electron microscope. Uneven and thin epithelial basement membrane, light density of hemidesmosomes, and edematous collagen fibril bundles are shown in the alkali-burned cornea. Injured epithelial basement membrane and hemidesmosomes and edematous collagen fibril bundles resulting from alkali-burned mouse cornea was repaired by bevacizumab treatment. This study demonstrates that bevacizumab can play an important role in wound healing in the cornea by accelerating the reestablishment of basement membrane integrity that leads to barriers for scar formation. [BMB Reports 2013; 46(4): 195-200]

Advanced Glycation End Products and Ultrastructural Changes in Corneas of Long-term Streptozotocin-Induced Diabetic Monkeys

The purpose of this study was to investigate the ultrastructural corneal changes of chronic diabetic monkeys and explore the relationship between advanced glycation end products and ultrastructural changes in diabetic corneas. A total of 8 cynomolgus monkeys were used in this experiment. Four monkeys were induced into insulin-dependent diabetes mellitus for 4 years. Four age-matched healthy monkeys were used as the controls. Ultrathin sections obtained from the corneas were examined by transmission electron microscopy. Advanced glycation end product immunoreactivity was observed in the epithelial cells, epithelial basement membrane, and stromal keratocytes of diabetic corneas, whereas advanced glycation end product immunoreactivity was not found in the corresponding area in normal corneas. Abnormal collagen fibril bundles of variable thickness were identified in corneal stroma in all diabetic monkeys. Epithelial and endothelial cell degeneration was also observed in 1 diabetic monkey. Abnormal aggregates of collagen fibrils in stromal matrix were common among long-term diabetic monkeys, and the formation of the abnormal collagen fibril aggregates might result from excessive nonenzymatic glycosylation.

Fibrosis-related gene expression in the prostate is modulated by doxazosin treatment

AimsTo gain new insights into the molecular mechanisms of action of doxazosin, we investigated the prostatic stroma ultrastructure and the expression of genes involved with fibrosis, such as collagen type I and III (COL1A1 and COL3A1, respectively) and TGF-beta 1, in the rat ventral prostate. Main methodsAdult Wistar rats were treated with doxazosin (25mg/kg/day), and the ventral prostates were excised at 7 and 30days after treatment. Untreated rats were controls. Ventral prostates were subjected to ultrastructural, immunohistochemical, biochemical and molecular analyses. Key findingsDoxazosin-treated prostates showed thickened bundles of collagen fibrils, activated fibroblasts, enlarged neurotransmitter vesicles and increased tissue immunostaining for collagen type I and type III when compared to untreated prostates. After 7 and 30days of doxazosin treatment mRNA expression of COL1A1 and COL3A1 was significantly increased and reduced, respectively, compared to the control group. TGF-beta 1 mRNA and protein levels were increased after 7days of doxazosin treatment, whereas only mRNA levels remained increased after 30days of treatment. SignificanceOur data suggest that relaxation of smooth muscle cells by alpha-blockers interferes with the mechanical dynamics of the prostatic stroma extracellular matrix components, generating a pro-fibrotic effect probably via the TGF-beta 1 signaling pathway. Long term treatment with doxazosin may also lead to a reduced turnover of extracellular matrix components. Our results add to a better understanding of the molecular mechanisms behind the effects of alpha-blockade on prostatic histoarchitecture and the response to treatment for benign prostatic hyperplasia.

Recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) could accelerate burn wound healing in hamster skin

Burns are one of the most devastating forms of trauma and wound healing is a complex and multicellular process, which is executed and regulated by signaling networks involving numerous growth factors, cytokines, and chemokines. Recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) was specifically produced from rice cell culture through use of a recombinant technique in our laboratory. The effect of rhGM-CSF on promotion of deep second-degree burn wound healing on the back skin of a hamster model was evaluated through a randomized and double-blind trial. As macroscopic results, hamster skins of the experimental groups showed earlier recovery by new epidermis than the control groups. Immunohistochemical reactions of proliferating cell nuclear antigen and transforming growth factor-β1, which are indicators of cell proliferation, were more active in the experimental group, compared with the control group. On electron microscopy, basal cells in the epidermis of the experimental group showed oval nuclei, prominent nucleoli, numerous mitochondria and abundant free ribosomes. In addition, fibroblasts contained well-developed rough endoplasmic reticulum with dilated cisternae. Bundles of collagen fibrils filled the extracellular spaces. Particularly, ultrastructural features indicating active metabolism for regeneration of injured skin at 15 days after burn injury, including abundant euchromatin, plentiful free ribosomes, and numerous mitochondria, were observed. These findings suggest that use of rhGM-CSF could result in accelerated deep second-degree burn wound healing in animal models.

Effect of desmin knockout on skeletal muscle extracellular matrix organization

The genetic deletion of desmin, an intermediate filament in skeletal muscle, leads to lower muscle stress production, muscle stiffness, and fibrosis. The extracellular matrix (ECM) of desmin knockout muscle was studied to determine how its structure and organization change in fibrotic muscle. The abundance and organization of perimysial collagen was quantified by stereological analysis of transmission electron micrographs, and perimysial collagen structure was observed by scanning electron microscopy and confocal imaging of fiber bundles stained for type I collagen. The organization of perimysial collagen termination on muscle fibers was determined by serial block face imaging (3View, Gatan, Inc.). An endomysial mesh was identified, as well as perimysium arranged in large bundles of collagen fibrils. These perimysial “cables” were ~1μm in diameter and tortuous at resting length, but straightened in response to muscle elongation. The apparent load bearing differences of endomysium and perimysium seem to be based on structure, and this structural specialization has not previously been appreciated. Increased stiffness observed in desmin‐null muscle may be partially due to altered ECM organization or material properties in addition to a generalized increase in ECM quantity. Supported by Allergan, Inc. and NIH grants R24 HD05083 and R01 AR057393.

Formation and structure of scales in the Australian lungfish, neoceratodus forsteri (Osteichthyes: Dipnoi)

The large elasmoid scales of the Australian lungfish, Neoceratodus forsteri, are formed within the dermis by unpigmented scleroblasts, growing within a collagenous dermal pocket below a thick glandular epidermis. The first row of scales, on the trunk of the juvenile lungfish, appears below the lateral line of the trunk, single in this species, at around stage 53. The scales, initially circular in outline, develop anteriorly and posteriorly from the point of initiation in the mid-trunk region, and rows are added alternately below the line, and above the line, until they reach the dorsal or ventral midline, or the margins of the fins. Scales develop later on the ventral surface of the head, from a separate centre of initiation. Scales consist of three layers, all produced by scleroblasts of dermal origin. The outermost layer of interlocking plates, or squamulae, consists of a mineralised matrix of fine collagen fibrils, covered by unmineralised collagen and a single layer of cells. Squamulae of the anterior and lateral surfaces are ornamented with short spines, and the mineralised tissue of the posterior surface is linked to the pouch by collagen fibrils. The innermost layer, known as elasmodin, consists of bundles of thick collagen fibrils and cells arranged in layers. An intermediate layer, made up of collagen fibrils, links the outer and inner layers. The elasmoid scales of N. forsteri can be compared with scale types among other osteichthyan groups, although the cellsand canaliculi in the mineralised squamulae bear little resemblance to typical bone.

Structural arrangement of collagen fibrils in the periarterial connective tissue of the kidney: their functional relevance as a structural stabilizer against arterial pressure

Periarterial connective tissue with a moderate amount of collagen fibrils is known to be a specialized domain in the renal interstitium. This study aimed to clarify the microscopic architecture of the periarterial connective tissue as a mechanical supportive structure of the intrarenal arteries. Transmission and scanning electron microscopy revealed two populations of collagen fibrils in the periarterial connective tissue. The major one was composed of many bundles of collagen fibrils running in longitudinal directions, whereas the minor one was represented by a few circumferential bundles adjacent to the smooth muscles. The amount of collagen fibrils was obviously variable and correlated with the arterial caliber. The correlation between abundance of collagen fibrils and the arterial caliber was confirmed by morphometric analysis of the collagen fibril area per arterial perimeter on electron micrographs. The size of individual collagen fibrils was measured in periarterial connective tissue of arteries with various calibers. A positive correlation between the diameter of collagen fibrils and arterial caliber was confirmed, indicating the supportive function of collagen fibrils in the periarterial connective tissue. The accumulated morphological findings supported the hypothesis that the collagen fibrils in the periarterial connective tissue develop longitudinal tension with their tensile strength, whereas the smooth muscle cells in the media develop circumferential tension with active regulation of contracting force.

The Layered Structure of Coronary Adventitia under Mechanical Load

The mechanical loading-deformation relation of elastin and collagen fibril bundles is fundamental to understanding the microstructural properties of tissue. Here, we use multiphoton microscopy to obtain quantitative data of elastin and collagen fiber bundles under in situ loading of coronary adventitia. Simultaneous loading-imaging experiments on unstained fresh coronary adventitia allowed morphometric measurements of collagen and elastin fibril bundles and their individual deformation. Fiber data were analyzed at five different distension loading points (circumferential stretch ratio λθ = 1.0, 1.2, 1.4, 1.6, and 1.8) at a physiological axial stretch ratio of λaxial = 1.3. Four fiber geometrical parameters were used to quantify the fibers: orientation angle, waviness, width, and area fraction. The results show that elastin and collagen fibers in inner adventitia form concentric densely packed fiber sheets, and the fiber orientation angle, width, and area fraction vary transmurally. The extent of fiber deformation depends on the initial orientation angle at no-distension state (λθ = 1.0 and λaxial = 1.3). At higher distension loading, the orientation angle and waviness of fibers decrease linearly, but the width of collagen fiber is relatively constant at λθ = 1.0–1.4 and then decrease linearly for λθ ≥ 1.4. A decrease of the relative dispersion (SD/mean) of collagen fiber waviness suggests a heterogeneous mechanical response to loads. This study provides fundamental microstructural data for coronary artery biomechanics and we consider it seminal for structural models.

Implications of obesity for tendon structure, ultrastructure and biochemistry: A study on Zucker rats

The extracellular matrix consists of collagen, proteoglycans and non-collagen proteins. The incidence of obesity and associated diseases is currently increasing in developed countries. Obesity is considered to be a disease of modern times, and genes predisposing to the disease have been identified in humans and animals. The objective of the present study was to compare the morphological and biochemical aspects of the deep digital flexor tendon of lean (Fa/Fa or Fa/fa) and genetically obese (fa/fa) Zucker rats. Ultrastructural analysis showed the presence of lipid droplets in both groups, whereas disorganized collagen fibril bundles were observed in obese animals. Lean animals presented a larger amount of non-collagen proteins and glycosaminoglycans than obese rats. We propose that the overweight and lesser physical activity in obese animals may have provoked the alterations in the composition and organization of extracellular matrix components but a genetic mechanism cannot be excluded. These alterations might be related to organizational and structural modifications in the collagen bundles that influence the mechanical properties of tendons and the progression to a pathological state.

Sub-lamellar microcracking and roles of canaliculi in human cortical bone

Bone is a tough biological material. It is generally accepted that bone’s toughness arises from its unique hierarchical structure, which in turn facilitates distributed microcracking prior to fracture. Yet, there has been limited progress on the detailed roles of the structural elements in the microcracking process. The present study examines the structure–microcracking relations at the lamellar and sub-lamellar levels of human cortical bone subjected to compressive loading. Laser scanning confocal microscopy revealed a clear influence of the local structure and porosity of the Haversian systems’ lamellae on microcrack development. In particular, crack initiation and growth under transverse compression were associated with stress concentration at canaliculi. Later stages of microcracking showed extensive sub-lamellar cracks forming cross-hatched patterns and regularly spaced 0.5–1.7μm apart. The density, size and regularity of the crack patterns suggest enhanced inelastic deformation capacity through cracking control at the level of mineralized collagen fibril bundles. The present study thus improves the current understanding of the nature of inelastic deformation and microcracking in bone and further suggests that bone’s resistance to fracture is achieved through microcrack control at multiple length scales.

Ultrastructural organisation of stingray and shark corneal stroma

AbstractPurpose Collagen fibrils and proteoglycans are the main components of the corneal stroma. We report here ultrastructural organisation of collagen fibrils and proteoglycans of corneal stroma in stingray and shark.Methods Four corneas of stingray and shark were fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and processed for electron microscopy. Tissues were embedded in TAAB 031 resin. Measurements were carried out by using the ‘AnalySIS LS Professional program’.Results The corneal stroma of shark is thicker (336µm) compared to stingray stroma (143µm). The corneal stroma in both fish consists of parallel running lamellae containing collagen fibrils and proteoglycans. The lamellae in sharks are thicker in the anterior stroma (12.14µm), middle stroma (18.3µm) and posterior stroma (13.0µm) compared to stingray lamellae in anterior stroma (7.6µm), middle stroma (8.9µm) and posterior stroma (5.2µm). In both fish lamellae of middle stroma was thicker compared to anterior and posterior stroma. In both fish, lamellae were obliquely crossed by bundles of collagen fibrils, which are known as sutures. Proteoglycans are larger in stingray compared to shark.Conclusion The presence of sutures is the unique feature of these fish. It has been reported that these sutures inhibit the swelling of the cornea in sharks.

An Anisotropic Microsphere-Based Approach for Fiber Orientation Adaptation in Soft Tissue

Evolutionary processes in biological tissue, such as adaptation or remodeling, represent an enterprising area of research. In this paper, we present a multiscale model for the remodeling of fibered structures, such as bundles of collagen fibrils. With this aim, we introduce a von Mises statistical distribution function to account for the directional dispersion of the fibrils, and we remodel the underlying fibrils by changing their orientation. To numerically compute this process, we make use of the microsphere approach, which provides a useful multiscale tool for homogenizing the microstructure behavior, related to the fibrils of the bundle, in the macroscale of the problem. The results show how the fibrils respond to the stimulus by reorientation of their structure. This process leads to a stiffer material eventually reaching a stationary state. These results are in agreement with those reported in the literature, and they characterize the adaptation of biological tissue to external stimuli.

Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy

In this protocol, we describe a 3D imaging technique known as 'volume electron microscopy' or 'focused ion beam scanning electron microscopy (FIB/SEM)' applied to biological tissues. A scanning electron microscope equipped with a focused gallium ion beam, used to sequentially mill away the sample surface, and a backscattered electron (BSE) detector, used to image the milled surfaces, generates a large series of images that can be combined into a 3D rendered image of stained and embedded biological tissue. Structural information over volumes of tens of thousands of cubic micrometers is possible, revealing complex microanatomy with subcellular resolution. Methods are presented for tissue processing, for the enhancement of contrast with osmium tetroxide/potassium ferricyanide, for BSE imaging, for the preparation and platinum deposition over a selected site in the embedded tissue block, and for sequential data collection with ion beam milling; all this takes approximately 90 h. The imaging conditions, procedures for alternate milling and data acquisition and techniques for processing and partitioning the 3D data set are also described; these processes take approxiamtely 30 h. The protocol is illustrated by application to developing chick cornea, in which cells organize collagen fibril bundles into complex, multilamellar structures essential for transparency in the mature connective tissue matrix. The techniques described could have wide application in a range of fields, including pathology, developmental biology, microstructural anatomy and regenerative medicine.

Nanostructural investigation of frontalis sling biomaterial surfaces

This study examined the nanostructural surface of three frontalis sling biomaterials: autogenous fascia lata, preserved fascia lata and silicone rod. The morphological characteristics of the sling biomaterial surfaces were examined qualitatively and quantitatively by scanning electron microscopy and atomic force microscopy, respectively. The autogenous fascia lata showed well-arranged nanostructures of parallel fascia collagen fibrils with clear 67 nm axial periodicity, whereas the preserved fascia lata showed tangled nanostructures of damaged collagen fibril bundles. The silicone rod showed a substantial amount of debris with some scratches and the smoothest roughness compared with the other sling biomaterials, followed by preserved fascia lata. Autogenous fascia lata showed the highest surface roughness. The association between the roughness and cell adhesion suggests that the nanostructure of autogenous fascia lata biomaterials is the best for frontalis sling and that of the silicone rod biomaterials is the worst.

Dense Fibrillar Collagen Matrices Sustain Osteoblast PhenotypeIn Vitroand Promote Bone Formation in Rat Calvaria Defect

Two pure collagen materials were prepared from acidic collagen solutions at 5 and 40 mg/mL. Benefits of collagen concentration on bone repair were evaluated in vitro with human calvaria cells and in vivo in a rat cranial defect. Both materials exhibited specific structures, 5 mg/mL was soft with an open porous network of fibrils; 40 mg/mL was stiffer with a plugged surface and bundles of collagen fibrils. Osteoblasts seeded on 5 mg/mL formed an epithelioid layer with ultrastructural characteristics of mature osteoblasts and induced mineralization. Numerous osteoblasts migrated inside 5 mg/mL, triggering reorganization of their actin cytoskeleton, whereas on 40 mg/mL osteoblasts remained in a resting state. In rat calvaria defects, both materials induced active bone formation. Dual-energy X-ray absorption bone area measures after 4 weeks averaged 84.0% with 5 mg/mL, 88.4% with 40 mg/mL, and 36.7% in the controls (p < 0.05). Tartrate-resistant acid phosphatase-positive giant cells releasing amounts of metalloproteinase-2 progressively degraded the implants at 76.5% with 5 mg/mL and 38.2% with 40 mg/mL (p < 0.05), whereas alkaline phosphatase-positive osteoprogenitors invaded collagen remnant. Hence, the dense structure of collagen materials allowed cell invasion and raise their mechanical behavior without addition of chemical cross-linkers. Collagen concentration can be tuned to form 3D matrices for in vitro investigations or to fit degradation rate to different bone repair purposes.

Characteristics of newly‐formed cementum following emdogain application

Periodontal regenerative techniques have been proposed; however, the outcomes remain debatable. The present investigation assessed the regenerated cementum following enamel matrix derivative application in dehiscence-type defects. Buccal osseous dehiscences were surgically created on the maxillary cuspid, and the second and fourth premolars in five female beagle dogs. The treatment group (n=15 sites) received the enamel matrix derived application, whereas the control groups (n=15) did not. The dogs were sacrificed 4 months following treatment and the specimens were histologically and histometrically examined. The newly formed cementum was uneven in thickness and mineralization, overlapped the old cementum and exhibited functional orientation, cementocyte lacunae and collagen fibril bundles. Most of the histological specimens showed the presence of a gap between the newly formed cementum and the underlying dentin. Control sites did not exhibit any cementum formation. The present study concluded that newly formed cementum is of cellular type and exhibits multiple characteristics.

Keratocyte‐mediated establishment of lamellar structure in corneal development

Abstract Purpose Mechanisms by which presumptive keratocytes in the developing cornea engineer the 3‐dimensional lamellar matrix of the transparent stroma remain unknown. We used Volume Scanning Electron Microscopy to observe lamella formation in embryonic avian cornea in order to understand better corneal stromal biosynthesis, transparency and repair. Methods Corneas were obtained from chick embryos at E10 and E14, when a loose structure of collagen fibril bundles condenses into the lamellar organisation of the mature stroma. Aldehyde‐fixed corneas were contrast‐enhanced in osmium ferricyanide and tannic acid and embedded in Durcupan resin. Toluidine blue‐stained sections were used to locate suitable sites, subsequently examined in unstained ultrathin sections in a Jeol 1010 transmission electron microscope. The polished faces of the resin blocks were then imaged using a BSE detector in a Quanta 3D FEG FIB/SEM via an alternate slice‐and‐view protocol. 50nm slicing was performed with a focused gallium ion beam and image series were collected over 18h. Image stacks were processed using ImageJ software. Results Transmission and volume scanning electron microscopy reveal complex compartmentalisation of developing chick corneal stroma. Keratocyte filopodia extend many microns from cells into the extracellular space. These invest collagen fibrils, with varying orientations, and persist as the stroma condenses, appearing to orchestrate the spatial and directional organisation of the component lamellae. Conclusion Volume SEM reveals in 3D the complex cellular morphology of prospective keratocytes in developing chick cornea with extensive filopodia, which presumably channel and direct collagen fibril bundles into an emergent lamellar architecture.