Helical superstructures between amyloid and collagen in cardiac fibrils from a patient with AL amyloidosis.

Systemic light chain (LC) amyloidosis (AL) is a disease where organs are damaged by an overload of a misfolded patient-specific antibody-derived LC, secreted by an abnormal B cell clone. The high LC concentration in the blood leads to amyloid deposition at organ sites. Indeed, cryogenic electron microscopy (cryo-EM) has revealed unique amyloid folds for heart-derived fibrils taken from different patients. Here, we present the cryo-EM structure of heart-derived AL amyloid (AL59) from another patient with severe cardiac involvement. The double-layered structure displays a u-shaped core that is closed by a ß-arc lid and extended by a straight tail. Noteworthy, the fibril harbours an extended constant domain fragment, thus ruling out the variable domain as sole amyloid building block. Surprisingly, the fibrils were abundantly concatenated with a proteinaceous polymer, here identified as collagen VI (COLVI) by immuno-electron microscopy (IEM) and mass-spectrometry. Cryogenic electron tomography (cryo-ET) showed how COLVI wraps around the amyloid forming a helical superstructure, likely stabilizing and protecting the fibrils from clearance. Thus, here we report structural evidence of interactions between amyloid and collagen, potentially signifying a distinct pathophysiological mechanism of amyloid deposits.

Central versus peripheral thickness in the human cornea explained.

PURPOSE: The human cornea is thicker in the periphery than the center and it has been suggested that this must be due to greater numbers of lamellae in the peripheral corneal stroma. The purpose of this study was to use high-resolution ultrastructural imaging to determine if the greater thickness of the peripheral cornea is due to the presence of more lamellae or if there is some other anatomical explanation. METHODS: In this study, full thickness corneas from three human donors were processed for light microscopy (LM) and transmission electron microscopy (TEM). Images were taken in three distinct stromal regions (anterior, middle, and posterior) from the central and peripheral cornea. Stromal thickness was evaluated by LM while TEM was used to evaluate numbers and thicknesses of lamellae, mean collagen fibril diameter, and mean collagen fibril density. RESULTS: Mean stromal thickness was significantly thinner in the central (415 ± 34 µm) compared to the peripheral (536 ± 29 µm) cornea (P = 0.009). Numbers of lamellae were not significantly different between central (246 ± 14) and peripheral (251 ± 14) cornea. Average lamellar thickness was not different across all regions of the cornea, except for the peripheral posterior where the lamellae were approximately 50 % thicker (P < 0.05). Collagen fibril diameters were larger in the peripheral cornea by approximately 30 % when compared to the central cornea, in all regions (P < 0.01). CONCLUSIONS: This study shows that it is an increase peripheral posterior lamellar thickness, rather than an increase in the number of lamellae, that accounts for the increase in corneal stromal thickness in the periphery of the human cornea. While collagen fibril diameters are greater throughout the peripheral stroma, the lamellae in the mid and anterior peripheral stroma are not thicker than centrally.

Transmission electron microscopic study of the surface layer of surgical resected disc specimens in human temporomandibular joint.

In this study, we investigated specific and characteristic findings of the surface layer of surgical resected disc specimens in human temporomandibular joint osteoarthritis cases by transmission electron microscopy (TEM).Specimens were surgically removed from the TMJ of 5 cases (4 female patients: 5 cases) clinically osteoarthritis. Following findings were observed by TEM. Images were photographed on a JEM1400-Flash Electron microscope (JEOL, Japan) equipped with an EM-14661FLASH high-sensitivity digital complementary metal-oxide-semiconductor camera.Following findings were observed by TEM. 1) The surface is covered with plump fibroblastic and histiocytoid cells. 2) Collagen fiber bundles and collagenous matrix are exposed onto the eroded disc surface. 3) Fibrinous dense material is observed on the eroded disc surface. 4) Bundles of collagen fibers are densely observed. 5) Collagen bundles are rich around capillary vessels. 6) Synovial surface cells reveal features of activated macrophages with vacuole formation. Especially, plump fibroblastic and histiocytoid cells, and activated macrophages with vacuole, which were significant findings of the surface layer. These findings might have a significant effect on the regulation of synovial fluid.

Three-dimensional fine structures in deep fascia revealed by combined use of cryo-fixed histochemistry and low-vacuum scanning microscopy.

Recent physiological studies have shown that the deep fascia has received much attention concerning clinical medicine; however, histological examination of the deep fascia has not been well established. In this study, we aimed to clarify and visualize the structure of the deep fascia by taking advantage of cryofixation techniques and low-vacuum scanning electron microscopy. As a result, the ultrastructural observations revealed three-dimensional stratification of the deep fascia composed of three layers: the first superficial layer consisting of collagen fibers extending in various directions with blood vessels and peripheral nerves; the second intermediate layer formed by single straight and thick collagen fibers with flexibility; and the third deepest layer, consisting of relatively straight and thin collagen fibers. We explored the use of two hooks to hold a piece of deep fascia in place through the course of cryo-fixation. A comparative observation with or without the hook-holding procedure would indicate the morphological adaptation to physiological stretch and contraction of the deep fascia. The present morphological approach paves the way to visualize three-dimensional ultrastructures for future biomedical studies including clinical pathophysiology.

Ultrastructure abnormalities of collagen and elastin in Arab patients with arterial tortuosity syndrome.

Arterial tortuosity syndrome (ATS) is a rare autosomal recessive disease characterized by elongation and tortuosity of the large- and medium-sized arteries. ATS patients display features that are also found in Ehlers-Danlos syndrome (EDS) patients. ATS is caused by pathogenic mutations in the SLC2A10 gene, which encodes for the glucose transporter, GLUT10. This study aimed at examining the ultrastructure of skin for abnormalities that can explain the loose skin and arterial phenotypes of Arab patients with the p.S81R mutation in SLC2A10. Forty-eight patients with SLC2A10 mutation were recruited for this study. Skin biopsy specimens from three children with ATS and a healthy child were examined by electron microscopy to determine the ultrastructure of collagen and elastin. Histopathologic staining of sections from tissue biopsy specimens was also performed. Large spaces were observed among the collagen fibrils in the skin biopsy specimens obtained from ATS patients, suggesting disorganization of the collagen structures. Furthermore, elastin fiber contents and their thickness are reduced in the skin. In small muscular arteries in the skin from ATS patients, discontinuous internal elastic lamina, lack of myofilaments, and disorganized medial smooth muscle cells with vacuolated cytoplasm are present. The disorganization of collagen fibrils and reduced elastin contents in the skin may explain the loose skin phenotype of ATS patients similar to the EDS patients. The lack of elastin in small muscular arteries may have contributed to the development of arterial tortuosity in these patients.

Light-Sheet Scattering Microscopy to Visualize Long-Term Interactions Between Cells and Extracellular Matrix.

Visualizing interactions between cells and the extracellular matrix (ECM) mesh is important to understand cell behavior and regulatory mechanisms by the extracellular environment. However, long term visualization of three-dimensional (3D) matrix structures remains challenging mainly due to photobleaching or blind spots perpendicular to the imaging plane. Here, we combine label-free light-sheet scattering microcopy (LSSM) and fluorescence microscopy to solve these problems. We verified that LSSM can reliably visualize structures of collagen matrices from different origin including bovine, human and rat tail. The quality and intensity of collagen structure images acquired by LSSM did not decline with time. LSSM offers abundant wavelength choice to visualize matrix structures, maximizing combination possibilities with fluorescently-labelled cells, allowing visualizing of long-term ECM-cell interactions in 3D. Interestingly, we observed ultrathin thread-like structures between cells and matrix using LSSM, which were not observed by normal fluorescence microscopy. Transient local alignment of matrix by cell-applied forces can be observed. In summary, LSSM provides a powerful and robust approach to investigate the complex interplay between cells and ECM.

An ultrastructural 3D reconstruction method for observing the arrangement of collagen fibrils and proteoglycans in the human aortic wall under mechanical load.

An insight into changes of soft biological tissue ultrastructures under loading conditions is essential to understand their response to mechanical stimuli. Therefore, this study offers an approach to investigate the arrangement of collagen fibrils and proteoglycans (PGs), which are located within the mechanically loaded aortic wall. The human aortic samples were either fixed directly with glutaraldehyde in the load-free state or subjected to a planar biaxial extension test prior to fixation. The aortic ultrastructure was recorded using electron tomography. Collagen fibrils and PGs were segmented using convolutional neural networks, particularly the ESPNet model. The 3D ultrastructural reconstructions revealed a complex organization of collagen fibrils and PGs. In particular, we observed that not all PGs are attached to the collagen fibrils, but some fill the spaces between the fibrils with a clear distance to the collagen. The complex organization cannot be fully captured or can be severely misinterpreted in 2D. The approach developed opens up practical possibilities, including the quantification of the spatial relationship between collagen fibrils and PGs as a function of the mechanical load. Such quantification can also be used to compare tissues under different conditions, e.g., healthy and diseased, to improve or develop new material models. STATEMENT OF SIGNIFICANCE: The developed approach enables the 3D reconstruction of collagen fibrils and proteoglycans as they are embedded in the loaded human aortic wall. This methodological pipeline comprises the knowledge of arterial mechanics, imaging with transmission electron microscopy and electron tomography, segmentation of 3D image data sets with convolutional neural networks and finally offers a unique insight into the ultrastructural changes in the aortic tissue caused by mechanical stimuli.

Diaphragm muscle fibrosis involves changes in collagen organization with mechanical implications in Duchenne muscular dystrophy.

In Duchenne muscular dystrophy (DMD), diaphragm muscle dysfunction results in respiratory insufficiency, a leading cause of death in patients. Increased muscle stiffness occurs with buildup of fibrotic tissue, characterized by excessive accumulation of extracellular matrix (ECM) components such as collagen, and prevents the diaphragm from achieving the excursion lengths required for respiration. However, changes in mechanical properties are not explained by collagen amount alone and we must consider the complex structure and mechanics of fibrotic tissue. The goals of our study were to 1) determine if and how collagen organization changes with the progression of DMD in diaphragm muscle tissue and 2) predict how collagen organization influences the mechanical properties of the ECM. We first visualized collagen structure with scanning electron microscopy (SEM) images and then developed an analysis framework to quantify collagen organization and generate image-based finite-element models. Image analysis revealed increased collagen fiber straightness and alignment in mdx over wild type (WT) at 3 mo (straightness: mdx = 0.976 ± 0.0108, WT = 0.887 ± 0.0309, alignment: mdx = 0.876 ± 0.0333, WT = 0.759 ± 0.0416) and 6 mo (straightness: mdx = 0.942 ± 0.0182, WT = 0.881 ± 0.0163, alignment: mdx = 0.840 ± 0.0315, WT = 0.759 ± 0.0368). Collagen fibers retained a transverse orientation relative to muscle fibers (70°-90°) in all groups. Mechanical models predicted an increase in the transverse relative to longitudinal (muscle fiber direction) stiffness, with stiffness ratio (transverse/longitudinal) increased in mdx over WT at 3 mo (mdx = 5.45 ± 2.04, WT = 1.97 ± 0.670) and 6 mo (mdx = 4.05 ± 0.985, WT = 1.96 ± 0.506). This study revealed changes in diaphragm ECM structure and mechanics during disease progression in the mdx muscular dystrophy mouse phenotype, highlighting the need to consider the role of collagen organization on diaphragm muscle function.NEW & NOTEWORTHY Scanning electron microscopy images of decellularized diaphragm muscle from WT and mdx, Duchenne muscular dystrophy model, mice revealed that collagen fibers in the epimysium are oriented transverse to muscle fibers, with age- and disease-dependent changes in collagen arrangement. Finite-element models generated from these images predicted that changes in collagen arrangement during disease progression influence the mechanical properties of the extracellular matrix. Thus, changes in collagen fiber-level structure are implicated on tissue-level properties during fibrosis.

Identification of remodeled collagen fibers in tumor stroma by FTIR Micro-spectroscopy: A new approach to recognize the colon carcinoma.

The tumor stroma plays a pivotal role in colon cancer genesis and progression. It was observed that collagen fibers in the extracellular matrix (ECM) of cancer stroma, undergo a strong remodeling. These fibrous proteins result more aligned and compact than in physiological conditions, creating a microenvironment that favors cancer development. In this work, micro-FTIR spectroscopy was applied to investigate the chemical modifications in the tumor stroma. Using Fuzzy C-means clustering, mean spectra from diseased and normal stroma were compared and collagen was found to be responsible for the main differences between them. Specifically, the modified absorptions at 1203, 1238, 1284 cm-1 and 1338 cm-1 wavenumbers, were related to the amide III band and CH2 bending of side chains. These signals are sensitive to the interactions between the α-chains in the triple helices of collagen structure. This provided robust chemical evidence that in cancer ECM, collagen fibers are more parallelized, stiff and ordered than in normal tissue. Principal Component Analysis (PCA) applied to the spectra from malignant and normal stroma confirmed these findings. Using LDA (Linear Discriminant Analysis) classification, the absorptions 1203, 1238, 1284 and 1338 cm-1 were examined as spectral biomarkers, obtaining quite promising results. The use of a PCA-LDA prediction model on samples with moderate tumor degree further showed that the stroma chemical modifications are more indicative of malignancy compared to the epithelium. These preliminary findings have shown that micro-FTIR spectroscopy, focused on collagen signals, could become a promising tool for colon cancer diagnosis.

The ultrastructure of cartilage tissue and its swelling response in relation to matrix health.

This multi-length scale anatomical study explores the influence of mild cartilage structural degeneration on the tissue swelling response. While the swelling response of cartilage has been studied extensively, this is the first study to reveal and correlate tissue microstructure and ultrastructure, with the swelling induced cartilage tissue strains. Cartilage sample strips (n = 30) were obtained from the distal-lateral quadrant of thirty mildly degenerate bovine patellae and, following excision from the bone, the cartilage strips were allowed to swell freely for 2 h in solutions of physiological saline and distilled water successively. The swelling response of this group of samples were compared with that of healthy cartilage, with (n = 20) and without the surface layer (n = 20). The subsequent curling response of cartilage showed that in healthy tissue it was highly variable, and with the surface removed some samples curved in the opposite direction, while in the mildly degenerate tissue group, virtually all tissue strips curved in a consistent upward manner. A significant difference in strain was observed between healthy samples with surface layer removed and mildly degenerate samples, illustrating how excision of the surface zone from pristine cartilage is insufficient to model the swelling response of tissue which has undergone natural degenerative changes. On average, total tissue thickness increased from 940 µm (healthy) to 1079 µm (mildly degenerate), however, looking at the zonal strata, surface and transition zone thicknesses both decreased while deep zone thickness increased from healthy to mildly degenerate tissue. Morphologically, changes to the surface zone integrity were correlated with a diminished surface layer which, at the ultrastructural scale, correlated with a decreased fibrillar density. Similarly, fibrosity of the general matrix visible at the microscale was associated with a loss of later interconnectivity resulting in large, aggregated fibril bundles. The microstructural and ultrastructural investigation revealed that the key differences influencing the tissue swelling strain response was (1) the thickness and extent of disruption to the surface layer and (2) the amount of fibrillar network destructuring, highlighting the importance of the collagen and tissue matrix structure in restraining cartilage swelling.

Ablation of mpeg+ Macrophages Exacerbates mfrp-Related Hyperopia.

Purpose: Proper refractive development of the eye, termed emmetropization, is critical for focused vision and is impacted by both genetic determinants and several visual environment factors. Improper emmetropization caused by genetic variants can lead to congenital hyperopia, which is characterized by small eyes and relatively short ocular axial length. To date, variants in only four genes have been firmly associated with human hyperopia, one of which is MFRP. Zebrafish mfrp mutants also have hyperopia and, similar to reports in mice, exhibit increased macrophage recruitment to the retina. The goal of this research was to examine the effects of macrophage ablation on emmetropization and mfrp-related hyperopia. Methods: We utilized a chemically inducible, cell-specific ablation system to deplete macrophages in both wild-type and mfrp mutant zebrafish. Spectral-domain optical coherence tomography was then used to measure components of the eye and determine relative refractive state. Histology, immunohistochemistry, and transmission electron microscopy were used to further study the eyes. Results: Although macrophage ablation does not cause significant changes to the relative refractive state of wild-type zebrafish, macrophage ablation in mfrp mutants significantly exacerbates their hyperopic phenotype, resulting in a relative refractive error 1.3 times higher than that of non-ablated mfrp siblings. Conclusions: Genetic inactivation of mfrp leads to hyperopia, as well as abnormal accumulation of macrophages in the retina. Ablation of the mpeg1-positive macrophage population exacerbates the hyperopia, suggesting that macrophages may be recruited in an effort help preserve emmetropization and ameliorate hyperopia.

Study of collagen remodeling in structural disorders of the temporomandibular joint using second-harmonic generation microscopy.

Structural disorder of the temporomandibular joint (TMJ) is a progressive disease with poor prognosis due to its physiological three-dimensional anatomical position and the complicated relationship among muscles, ligaments, and cartilage. The lack of detection methods for changes in the collagen structure of the TMJ disc makes the diagnosis untimely and unclear. This work aimed to explore the feasibility of using a promising detection technique, second-harmonic generation (SHG), to characterize collagen fibers in a TMJ disc with structural disorders. The TMJ discs with structural disorder were observed using SHG microscopy, and assessment of collagen orientation was conducted by analyzing digitized images. The SHG images were also compared with the scanning electron microscopy images and microscopic images acquired after hematoxylin and eosin and Masson's trichrome staining. The SHG imaging showed that the collagen fibers in diseased TMJ were distributed in a disorderly manner, and pixel intensities in diseased TMJ discs were significantly different from those acquired in healthy TMJs. Moreover, the three dimensions of collagen fibers and dynamic images acquired by SHG nonlinear optical microscopy showed the structural disorder of the collagen fibers in a diseased TMJ. In summary, SHG imaging could provide three-dimensional and quantitative data, with dynamic and critical pathological information for clinical diagnosis, showing its potential value in the diagnosis and evaluation of structural disorders of the TMJ disc.

Effect of luminal surface structure of decellularized aorta on thrombus formation and cell behavior.

Due to an increasing number of cardiovascular diseases, artificial heart valves and blood vessels have been developed. Although cardiovascular applications using decellularized tissue have been studied, the mechanisms of their functionality remain unknown. To determine the important factors for preparing decellularized cardiovascular prostheses that show good in vivo performance, the effects of the luminal surface structure of the decellularized aorta on thrombus formation and cell behavior were investigated. Various luminal surface structures of a decellularized aorta were prepared by heating, drying, and peeling. The luminal surface structure and collagen denaturation were evaluated by immunohistological staining, collagen hybridizing peptide (CHP) staining, and scanning electron microscopy (SEM) analysis. To evaluate the effects of luminal surface structure of decellularized aorta on thrombus formation and cell behavior, blood clotting tests and recellularization of endothelial cells and smooth muscle cells were performed. The results of the blood clotting test showed that the closer the luminal surface structure is to the native aorta, the higher the anti-coagulant property. The results of the cell seeding test suggest that vascular cells recognize the luminal surface structure and regulate adhesion, proliferation, and functional expression accordingly. These results provide important factors for preparing decellularized cardiovascular prostheses and will lead to future developments in decellularized cardiovascular applications.

A method to analyze the influence of mechanical strain on dermal collagen morphologies.

Collagen fibers and their orientation play a major role in the mechanical behavior of soft biological tissue such as skin. Here, we present a proof-of-principle study correlating mechanical properties with collagen fiber network morphologies. A dedicated multiphoton stretching device allows for mechanical deformations in combination with a simultaneous analysis of its collagen fiber network by second harmonic generation imaging (SHG). The recently introduced Fiber Image Network Evaluation (FINE) algorithm is used to obtain detailed information about the morphology with regard to fiber families in collagen network images. To demonstrate the potential of our method, we investigate an isotropic and an anisotropic ex-vivo dorsal pig skin sample under quasi-static cyclic stretching and relaxation sequences. Families of collagen fibers are found to form a partially aligned collagen network under strain. We find that the relative force uptake is accomplished in two steps. Firstly, fibers align within their fiber families and, secondly, fiber families orient in the direction of force. The maximum alignment of the collagen fiber network is found to be determined by the largest strain. Isotropic and anisotropic samples reveal a different micro structural behavior under repeated deformation leading to a similar force uptake after two stretching cycles. Our method correlates mechanical properties with morphologies in collagen fiber networks.

Comparative Study of The Yield and Physicochemical Properties of Collagen from Sea Cucumber (Holothuria scabra), Obtained through Dialysis and the Ultrafiltration Membrane.

Collagen was extracted from the body wall of sea cucumber (Holothuria scabra) using the pepsin-solubilized collagen method followed by isolation using dialysis and the ultrafiltration membrane. The yield and physicochemical properties of the collagen obtained from both isolation methods, denoted as D-PSC and UF-PSC, were compared. The ultrafiltration method affords a higher yield of collagen (11.39%) than that of the dialysis (5.15%). The isolated collagens have almost the same amino acid composition, while their functional groups, referred to as amide A, B, I, II, and III bands, were in accordance with commercial collagen, as verified by Fourier Transform Infrared (FT-IR) spectroscopy. The UV-Vis absorption peaks at 240 nm and 220 nm, respectively, indicated that the collagens produced are type-I collagen. The D-PSC showed interconnecting sheet-like fibrils, while the UF-PSC exhibited a flaky structure with flat-sheets arranged very close to each other. The higher yield and comparable physicochemical properties of the collagen obtained by ultrafiltration as compared with dialysis indicate that the membrane process has high potential to be used in large-scale collagen production for food and pharmaceutical applications.

Fabrication of hybrid scaffolds obtained from combinations of PCL with gelatin or collagen via electrospinning for skeletal muscle tissue engineering.

The creation of skeletal muscle tissue in vitro is a major topic of interest today in the field of biomedical research, due to the lack of treatments for muscle loss due to traumatic accidents or disease. For this reason, the intrinsic properties of nanofibrillar structures to promote cell adhesion, proliferation, and cell alignment presents an attractive tool for regenerative medicine to recreate organized tissues such as muscle. Electrospinning is one of the processing techniques often used for the fabrication of these nanofibrous structures and the combination of synthetic and natural polymers is often required to achieve optimal mechanical and physiochemical properties. Here, polycaprolactone (PCL) is selected as a synthetic polymer used for the fabrication of scaffolds, and the effect of protein addition on the final scaffolds' properties is studied. Collagen and gelatin were the proteins selected and two different concentrations were analyzed (2 and 4 wt/vol%). Different PCL/protein systems were prepared, and a structural, mechanical and functional characterization was performed. The influence of fiber alignment on the properties of the final scaffolds was assessed through morphological, mechanical and biological evaluations. A bioreactor was used to promote cell proliferation and differentiation within the scaffolds. The results revealed that protein addition produced a decrease in the fiber size of the membranes, an increase in their hydrophilicity, and a softening of their mechanical properties. The biological study showed the ability of the selected systems to harbor cells, allow their growth and, potentially, develop musculoskeletal tissues.

Animal model with structural similarity to human corneal collagen fibrillar arrangement.

Rabbit and porcine corneas have been used in scientific research due to their structural similarity to the human cornea. Currently, there are no studies that have compared corneal collagen fibrillar diameter, interfibrillar distance and interlamellar distance between human and animal models. Ten pairs of porcine, rabbit, and human corneas were used. These were analysed using light and Transmission Electron microscopy. The collagen fibrillar diameter, interfibrillar distance and interlamellar distance were statistically compared between porcine, rabbit and human corneas. The human, porcine and rabbit; mean collagen fibrillar diameters were: 24.52 ± 2.09 nm; 32.87 ± 0.87 nm; and 33.67 ± 1.97 nm. The mean interfibrillar distances were: 46.10 ± 2.44 nm; 53.33 ± 2.24 nm; and 52.87 ± 2.73 nm, respectively. The collagen fibrillar diameter and interfibrillar distance of porcine and rabbit corneas were significantly different (p < 0.001) to the human corneal values but not form each other. The interlamellar distance of human, porcine and rabbit corneas was: 2190 ± 820 nm; 6460 ± 1180 nm; and 4410 ± 1330 nm, respectively. All the comparisons were statistically different, in porcine versus rabbit at the p < 0.01 level and both porcine and rabbit versus human at the p < 0.001 level. Histologically, all five layers (epithelium, Bowman's layer, stroma, Descemet membrane and endothelium) of the cornea were visible in all the three species. While neither animal model was structurally identical to the human cornea, they are both relatively close to being used as models to study the biomechanical effects of external insults/treatments to be extrapolated to the human cornea.

Liquid-Exfoliated Mesostructured Collagen from the Bovine Achilles Tendon as Building Blocks of Collagen Membranes.

Mesoscaled assemblies are organized in native collagen tissues to achieve remarkable and diverse performance and functions. In this work, a facile, low-cost, and controllable liquid exfoliation method was applied to directly extract these collagen mesostructures from bovine Achilles tendons using a sodium hydroxide (NaOH)/urea aqueous system with freeze-thaw cycles and sonication. A series of collagen fibrils with diameters of 26-230 nm were harvested using this process, and in situ observations under polarizing microscopy (POM) and using molecular dynamics simulations revealed the influence of the NaOH/urea system on the tendon collagen. FTIR and XRD results confirmed that these collagen fibrils preserved typical structural characteristics of type I collagen. These isolated collagen fibrils were then utilized as building blocks to fabricate free-standing collagen membranes, which exhibited good stability in solvents and outstanding mechanical properties and transparency, with potential for utility in optical and electronic sensors. Moreover, in vitro and vivo evaluations demonstrated that these new resulting collagen membranes had good cytocompatibility, biocompatibility, and degradability for potential applications in biomedicine. This work provides a new approach for collagen processing by liquid exfoliation with utility for the formation of robust collagen materials that consist of native collagen mesostructures as building blocks.

Effects of ultrasound and thermal treatment on the ultrastructure of collagen fibers from bovine tendon using atomic force microscopy.

As the most important tenderness related protein in mammal, there are few studies on how the nanoscale morphology of collagen I in tissues is related to traditional meat processing. The ultrastructure and mechanical characteristics of collagen fibers in tendon with different treatments have been explored in this study. Collagen fibers in homogenate group and acetic acid group were treated with ultrasound and thermal treatment. The nanoscale morphology of collagen fiber in homogenate group became granular at 60 °C and gelatin was formed at 70 °C. The collagen fibers extracted from acetic acid are unstable and easier to break under the same processing parameters, when compared with homogenated collagen fibers in both ultrasound and thermal treatment. The results suggested that acetic acid can disassemble the salt bond and Schiff-base in collagen, and the collagen fibers became loose but the triple helix structure remained integrity.

Development of New Collagen/Clay Composite Biomaterials.

The fabrication of collagen-based biomaterials for skin regeneration offers various challenges for tissue engineers. The purpose of this study was to obtain a novel series of composite biomaterials based on collagen and several types of clays. In order to investigate the influence of clay type on drug release behavior, the obtained collagen-based composite materials were further loaded with gentamicin. Physiochemical and biological analyses were performed to analyze the obtained nanocomposite materials after nanoclay embedding. Infrared spectra confirmed the inclusion of clay in the collagen polymeric matrix without any denaturation of triple helical conformation. All the composite samples revealed a slight change in the 2-theta values pointing toward a homogenous distribution of clay layers inside the collagen matrix with the obtaining of mainly intercalated collagen-clay structures, according X-ray diffraction analyses. The porosity of collagen/clay composite biomaterials varied depending on clay nanoparticles sort. Thermo-mechanical analyses indicated enhanced thermal and mechanical features for collagen composites as compared with neat type II collagen matrix. Biodegradation findings were supported by swelling studies, which indicated a more crosslinked structure due additional H bonding brought on by nanoclays. The biology tests demonstrated the influence of clay type on cellular viability but also on the antimicrobial behavior of composite scaffolds. All nanocomposite samples presented a delayed gentamicin release when compared with the collagen-gentamicin sample. The obtained results highlighted the importance of clay type selection as this affects the performances of the collagen-based composites as promising biomaterials for future applications in the biomedical field.