Quantitative analysis of fibrillar collagen organization in the immediate proximity of embedded fibroblasts in 3D collagen hydrogels.

Fibroblasts embedded in a 3D matrix microenvironment can remodel the matrix to regulate cell adhesion and function. Collagen hydrogels are a useful in vitro system to study cell-matrix interactions in a 3D microenvironment. While major matrix reorganizations are easily recognizable, subtle changes in response to environmental or biochemical cues are challenging to discern in 3D hydrogels. Three-dimensional collagen gels at 1.0 mg/ml vs 1.5 mg/ml were labelled with DQ-collagen and imaged by confocal reflectance microscopy to evaluate these small changes. An image analysis pipeline was developed, hydrogel area and number of crosssections analysed were optimized, and fibrillar collagen properties (number of branches, number of junctions, and average branch length) were quantified. While no significant changes were seen in fibrillar collagen organization between 1.0 mg/ml and 1.5 mg/ml collagen hydrogels, embedded mouse fibroblasts caused a significant increase in collagen branching and organization. Using the phalloidin-labelled cells, this change was quantitated in immediate proximity of the cell. A distinct increase in branch and junction numbers was observed, significantly altered by small changes in collagen concentration (1.0 mg/ml vs 1.5 mg/ml). Together, this analysis gives a quantitative evaluation of how cells respond to and modify their immediate microenvironment in a 3D collagen hydrogel.

Decorin regulates collagen fibrillogenesis during corneal wound healing in mouse in vivo.

A characteristic rigid spatial arrangement of collagen fibrils in the stroma is critical for corneal transparency. This unique organization of collagen fibrils in corneal stroma can be impacted by the presence and interactions of proteoglycans and extracellular matrix (ECM) proteins in a corneal microenvironment. Earlier studies revealed that decorin, a leucine-rich proteoglycan in stroma, regulates keratocyte-collagen matrix assembly and wound healing in the cornea. This study investigated the role of decorin in the regulation of stromal fibrillogenesis and corneal transparency in vivo employing a loss-of-function genetic approach using decorin null (dcn-/-) and wild type (dcn+/+) mice and a standard alkali-injury model. A time-dependent ocular examinations with Slit lamp microscope in live animals assessed corneal clarity, haze, and neovascularization levels in normal and injured eyes. Morphometric changes in normal and injured dcn+/+ and dcn-/- corneas, post-euthanasia, were analyzed with Masson's Trichrome and Periodic Acid-Schiff (PAS) histology evaluations. The ultrastructure changes in all corneas were investigated with transmission electron microscopy (TEM). Injury to eye produced clinically relevant corneal haze and neovascularization in dcn-/- and dcn+/+ mice while corneas of uninjured eyes remained clear and avascular. A clinically significant haze and neovascularization appeared in injured dcn-/- corneas compared to the dcn+/+ corneas at day 21 post-injury and not at early tested times. Histological examinations revealed noticeably abnormal morphology and compromised collagen levels in injured dcn-/- corneas compared to the injured/normal dcn+/+ and uninjured dcn-/- corneas. TEM analysis exhibited remarkably uneven collagen fibrils size and distribution in the stroma with asymmetrical organization and loose packing in injured dcn-/- corneas than injured/normal dcn+/+ and uninjured dcn-/- corneas. The minimum and maximum inter-fibril distances were markedly irregular in injured dcn-/- corneas compared to all other corneas. Together, results of clinical, histological, and ultrastructural investigations in a genetic knockout model suggested that decorin influenced stromal fibrillogenesis and transparency in healing cornea.

Tenomodulin knockout mice exhibit worse late healing outcomes with augmented trauma-induced heterotopic ossification of Achilles tendon.

Heterotopic ossification (HO) represents a common problem after tendon injury with no effective treatment yet being developed. Tenomodulin (Tnmd), the best-known mature marker for tendon lineage cells, has important effects in tendon tissue aging and function. We have reported that loss of Tnmd leads to inferior early tendon repair characterized by fibrovascular scaring and therefore hypothesized that its lack will persistently cause deficient repair during later stages. Tnmd knockout (Tnmd-/-) and wild-type (WT) animals were subjected to complete Achilles tendon surgical transection followed by end-to-end suture. Lineage tracing revealed a reduction in tendon-lineage cells marked by ScleraxisGFP, but an increase in alpha smooth muscle actin myofibroblasts in Tnmd-/- tendon scars. At the proliferative stage, more pro-inflammatory M1 macrophages and larger collagen II cartilaginous template were detected in this group. At the remodeling stage, histological scoring revealed lower repair quality in the injured Tnmd-/- tendons, which was coupled with higher HO quantified by micro-CT. Tendon biomechanical properties were compromised in both groups upon injury, however we identified an abnormal stiffening of non-injured Tnmd-/- tendons, which possessed higher static and dynamic E-moduli. Pathologically thicker and abnormally shaped collagen fibrils were observed by TEM in Tnmd-/- tendons and this, together with augmented HO, resulted in diminished running capacity of Tnmd-/- mice. These novel findings demonstrate that Tnmd plays a protecting role against trauma-induced endochondral HO and can inspire the generation of novel therapeutics to accelerate repair.

Developing exquisite collagen fibrillar assemblies in the presence of keratin nanoparticles for improved cellular affinity.

Recently, the collagen-keratin (CK) composites have received much attention for the purpose of biomedical applications due to the intrinsic biocompatibility and biodegradability of these two proteins. However, few studies have reported the CK composites developed by the self-assembly approach and the influence of the keratin on the collagen self-assembly in vitro was still unknown. In this study, the keratin nanoparticles (KNPs) were successfully prepared by the reduction method, and we focused on investigating the effect of the varying concentrations of KNPs on the mechanism of the fibrillogenesis process of collagen. The intermolecular interaction between the two proteins revealed by the ultraviolet spectroscopy, Fourier transform-infrared (FT-IR) spectroscopy and circular dichromatic (CD) spectroscopy showed that KNPs would interact with the collagen, and keratin significantly influenced the hydrogen bonding interaction existed in collagen molecules. The SEM images exhibited the formation of exquisite fibrillar networks after incorporating the KNPs into collagen, and it was conspicuous that the KNPs could uniformly distribute on the surface of collagen fibrils via electrostatic interaction, for both of the two proteins possessed many charged moieties. In addition, the AFM images confirmed the presence of the characteristic D-periodicity of collagen fibrils, indicating that the introduction of KNPs did not disrupt the self-assembly nature of the native collagen. The cell adhesion, proliferation and migration experiments on the CK fibrils were also performed in this study. The results demonstrated that the CK composites showed a better cellular affinity compared with the collagen, thus it might be a promising candidate for the biomedical applications.

Quantitative three-dimensional collagen orientation analysis of human meniscus posterior horn in health and osteoarthritis using micro-computed tomography.

OBJECTIVE: Knee osteoarthritis (OA) is associated with meniscal degeneration that may involve disorganization of the meniscal collagen fiber network. Our aims were to quantitatively analyze the microstructural organization of human meniscus samples in 3D using micro-computed tomography (µCT), and to compare the local microstructural organization between OA and donor samples. METHOD: We collected posterior horns of both medial and lateral human menisci from 10 end-stage medial compartment knee OA patients undergoing total knee replacement (medial & lateral OA) and 10 deceased donors without knee OA (medial & lateral donor). Posterior horns were dissected and fixed in formalin, dehydrated in ascending ethanol concentrations, treated with hexamethyldisilazane (HMDS), and imaged with µCT. We performed local orientation analysis of collagenous microstructure in 3D by calculating structure tensors from greyscale gradients within selected integration window to determine the polar angle for each voxel. RESULTS: In donor samples, meniscus bundles were aligned circumferentially around the inner border of meniscus. In medial OA menisci, the organized structure of collagen network was lost, and main orientation was shifted away from the circumferential alignment. Quantitatively, medial OA menisci had the lowest mean orientation angle compared to all groups, -24° (95%CI -31 to -18) vs medial donor and -25° (95%CI -34 to -15) vs lateral OA. CONCLUSIONS: HMDS-based µCT imaging enabled quantitative analysis of meniscal collagen fiber bundles and their orientations in 3D. In human medial OA menisci, the collagen disorganization was profound with overall lower orientation angles, suggesting collagenous microstructure disorganization as an important part of meniscus degradation.

Correlation of light microscopic findings with transmission electron microscopy within a vascular occlusion device.

Host response to an implanted biomaterial is a complex process involving microscopic changes in extracellular matrix (ECM) composition. Reliable pathology analysis is imperative for accurate assessment of the tissue response to an implanted device. Plastic histology is commonly used for histology evaluation of medical devices to assess the device-tissue interface; however, this technique is prone to variable staining that can confound histology interpretation. Appropriately, we propose using transmission electron microscopy (TEM) to confirm histologic ECM findings in order to provide sufficient host-response data. Tissue response to an absorbable shape memory polymer intravascular occlusion device with a nitinol wire backbone was evaluated. Representative plastic-embedded, micro-ground sections from 30-day, 60-day, and 90-day timepoints were analyzed. ECM regions were selected, and ultrathin sections were created for TEM evaluation. Histological changes in ECM composition were compared for light microscopy (LM) and TEM findings; specifically, TEM fibrillary patterns for collagen and fibrin were used to confirm LM results. Throughout this study, LM reveals inconsistent staining in plastic-embedded sections. TEM, on the other hand, provides clear insight into the tissue response by morphologically discerning distinct fibrillary patterns within ECM structures; loose to dense collagen surrounds the implant as fibrin degrades, demonstrating progression of postimplant ECM maturation. Moreover, TEM serves as a definitive method for confirming tissue substrate morphology when LM findings prove ambiguous.

Intratumoral heterogeneity of second-harmonic generation scattering from tumor collagen and its effects on metastatic risk prediction.

BACKGROUND: Metastases are the leading cause of breast cancer-related deaths. The tumor microenvironment impacts cancer progression and metastatic ability. Fibrillar collagen, a major extracellular matrix component, can be studied using the light scattering phenomenon known as second-harmonic generation (SHG). The ratio of forward- to backward-scattered SHG photons (F/B) is sensitive to collagen fiber internal structure and has been shown to be an independent prognostic indicator of metastasis-free survival time (MFS). Here we assess the effects of heterogeneity in the tumor matrix on the possible use of F/B as a prognostic tool. METHODS: SHG imaging was performed on sectioned primary tumor excisions from 95 untreated, estrogen receptor-positive, lymph node negative invasive ductal carcinoma patients. We identified two distinct regions whose collagen displayed different average F/B values, indicative of spatial heterogeneity: the cellular tumor bulk and surrounding tumor-stroma interface. To evaluate the impact of heterogeneity on F/B's prognostic ability, we performed SHG imaging in the tumor bulk and tumor-stroma interface, calculated a 21-gene recurrence score (surrogate for OncotypeDX®, or S-ODX) for each patient and evaluated their combined prognostic ability. RESULTS: We found that F/B measured in tumor-stroma interface, but not tumor bulk, is prognostic of MFS using three methods to select pixels for analysis: an intensity threshold selected by a blinded observer, a histogram-based thresholding method, and an adaptive thresholding method. Using both regression trees and Random Survival Forests for MFS outcome, we obtained data-driven prediction rules that show F/B from tumor-stroma interface, but not tumor bulk, and S-ODX both contribute to predicting MFS in this patient cohort. We also separated patients into low-intermediate (S-ODX < 26) and high risk (S-ODX ≥26) groups. In the low-intermediate risk group, comprised of patients not typically recommended for adjuvant chemotherapy, we find that F/B from the tumor-stroma interface is prognostic of MFS and can identify a patient cohort with poor outcomes. CONCLUSIONS: These data demonstrate that intratumoral heterogeneity in F/B values can play an important role in its possible use as a prognostic marker, and that F/B from tumor-stroma interface of primary tumor excisions may provide useful information to stratify patients by metastatic risk.

Collagen XI regulates the acquisition of collagen fibril structure, organization and functional properties in tendon.

Collagen XI is a fibril-forming collagen that regulates collagen fibrillogenesis. Collagen XI is normally associated with collagen II-containing tissues such as cartilage, but it also is expressed broadly during development in collagen I-containing tissues, including tendons. The goals of this study are to define the roles of collagen XI in regulation of tendon fibrillar structure and the relationship to function. A conditional Col11a1-null mouse model was created to permit the spatial and temporal manipulation of Col11a1 expression. We hypothesize that collagen XI functions to regulate fibril assembly, organization and, therefore, tendon function. Previous work using cho mice with ablated Col11a1 alleles supported roles for collagen XI in tendon fibril assembly. Homozygous cho/cho mice have a perinatal lethal phenotype that limited the studies. To circumvent this, a conditional Col11a1flox/flox mouse model was created where exon 3 was flanked with loxP sites. Breeding with Scleraxis-Cre (Scx-Cre) mice yielded a tendon-specific Col11a1-null mouse line, Col11a1Δten/Δten. Col11a1flox/flox mice had no phenotype compared to wild type C57BL/6 mice and other control mice, e.g., Col11a1flox/flox and Scx-Cre. Col11a1flox/flox mice expressed Col11a1 mRNA at levels comparable to wild type and Scx-Cre mice. In contrast, in Col11a1Δten/Δten mice, Col11a1 mRNA expression decreased to baseline in flexor digitorum longus tendons (FDL). Collagen XI protein expression was absent in Col11a1Δten/Δten FDLs, and at ~50% in Col11a1+/Δten compared to controls. Phenotypically, Col11a1Δten/Δten mice had significantly decreased body weights (p < 0.001), grip strengths (p < 0.001), and with age developed gait impairment becoming hypomobile. In the absence of Col11a1, the tendon collagen fibrillar matrix was abnormal when analyzed using transmission electron microscopy. Reducing Col11a1 and, therefore collagen XI content, resulted in abnormal fibril structure, loss of normal fibril diameter control with a significant shift to small diameters and disrupted parallel alignment of fibrils. These alterations in matrix structure were observed in developing (day 4), maturing (day 30) and mature (day 60) mice. Altering the time of knockdown using inducible I-Col11a1-/- mice indicated that the primary regulatory foci for collagen XI was in development. In mature Col11a1Δten/Δten FDLs a significant decrease in the biomechanical properties was observed. The decrease in maximum stress and modulus suggest that fundamental differences in the material properties in the absence of Col11a1 expression underlie the mechanical deficiencies. These data demonstrate an essential role for collagen XI in regulation of tendon fibril assembly and organization occurring primarily during development.

Fibrillogenesis of acrylic acid-grafted-collagen without self-assembly property inspired by the hybrid fibrils of xenogeneic collagen.

Along with advancements in both protein and chemistry science, the chemical modification of proteins is attracting more and more attention. More specifically, the attachment of polymers or reactive moieties into collagen offers a method to add novel functions to this protein. However, the fibrillogenesis of the modified collagen with high grafting density cannot always be achieved. Here, inspired by the hybrid fibrils of xenogeneic collagen, fibrillogenesis of acrylic acid-grafted-collagen (AAc-g-Col) without self-assembly property was achieved by the induction of natural collagen (Col). The step-by-step co-assembly process of AAc-g-Col and Col was confirmed by turbidity assay. The formation of Col/AAc-g-Col hybrid fibrils was verified by TEM since the acryloyl groups of the hybrid fibrils were labelled using HS-AuNPs based on the Michael addition. Moreover, rheology, SEM, and MTT assays revealed that the fibrillary structures and biocompatibility of the Col/AAc-g-Col hydrogel were comparable to that of the Col hydrogel, although they presented a lower viscoelasticity.

Non-disruptive collagen characterization in clinical histopathology using cross-modality image synthesis.

The importance of fibrillar collagen topology and organization in disease progression and prognostication in different types of cancer has been characterized extensively in many research studies. These explorations have either used specialized imaging approaches, such as specific stains (e.g., picrosirius red), or advanced and costly imaging modalities (e.g., second harmonic generation imaging (SHG)) that are not currently in the clinical workflow. To facilitate the analysis of stromal biomarkers in clinical workflows, it would be ideal to have technical approaches that can characterize fibrillar collagen on standard H&E stained slides produced during routine diagnostic work. Here, we present a machine learning-based stromal collagen image synthesis algorithm that can be incorporated into existing H&E-based histopathology workflow. Specifically, this solution applies a convolutional neural network (CNN) directly onto clinically standard H&E bright field images to extract information about collagen fiber arrangement and alignment, without requiring additional specialized imaging stains, systems or equipment.

Extracellular matrix-mimetic composite hydrogels of cross-linked hyaluronan and fibrillar collagen with tunable properties and ultrastructure.

A platform of enzymatically-crosslinked Collagen/Tyramine hyaluronan derivative (Col/HA-Tyr) hydrogels with tunable compositions and gelation conditions was developed to evaluate the impact of the preparation conditions on their physical, chemical and biological properties. At low HA-Tyr content, hydrogels exhibited a fibrillar structure, with lower mechanical properties compared to pure Col hydrogels. At high HA-Tyr and Horse Radish Peroxydase (HRP) content, a microfibrillar network was formed beside the banded Col fibrils and a synergistic effect of the hybrid structure on mechanical properties was observed. These hydrogels were highly resistant against enzymatic degradation while keeping a high degree of hydration. Unlike HA-Tyr hydrogels, encapsulation of human dermal fibroblasts within Col/HA-Tyr hydrogels allowed for high cell viability. These results showed that high HA-Tyr and HRP concentrations are required to positively impact the physical properties of hydrogels while preserving collagen fibrils. Those Col/HA-Tyr hydrogels appear promising for novel tissue engineering applications following a biomimetic approach.

The Effect of the Wooden Breast Fibrotic Myopathy in Broilers on Fibrillar Collagen Organization and Decorin-Collagen Binding.

The wooden breast myopathy is identified by the palpation of a rigid pectoralis major muscle and results in myofiber necrosis and fibrosis in fast-growing, meat-type broilers. The fibrosis in wooden breast-affected muscle is characterized by the replacement of myofibers with extracellular matrix proteins, especially fibril-forming collagens. Studies have shown differences in collagen organization in fast-growing broiler lines, with tightly packed and highly aligned collagen organizations having a higher phenotypic incidence of wooden breast. The objective of the current study was to analyze collagen fibril organization further in two fast-growing broiler lines (Lines A and B) with incidence of wooden breast compared with a slower growing broiler Line C with no phenotypically detectable wooden breast. The small leucine-rich proteoglycan decorin was also studied for its interaction with collagen by immunogold detection. Decorin binds to fibrillar collagens and organizes their alignment and crosslinking, both of which will affect collagen functional properties. Key findings from the study showed that collagen shifts to larger diameter collagen fibril bundles with the wooden breast myopathy. Specifically, broilers affected with wooden breast from Line A had a more dramatic shift toward larger collagen fibril bundles compared with those affected from Line B. Wooden breast-affected Line A had collagen fibril bundles up to 8.4 µm, whereas Line B maximum size was 5.1 µm. Although decorin-collagen binding was not different overall in the wooden breast myopathy or broiler line, for small-diameter collagen fibril bundles, wooden breast-affected Line A had more decorin-collagen binding than wooden breast-affected Line B. Taken together, these data provide further evidence that multiple fibrotic myopathies are likely in fast-growing meat-type broilers.

Efecto de la miopatía fibrótica de pechuga de madera en pollos de engorde en la organización del colágeno fibrilar y en la unión entre decorina y colágeno. La miopatía de madera de la pechuga se identifica por la palpación de un músculo pectoralis major rígido y da como resultado necrosis y fibrosis de fibras musculares en pollos de engorde de rápido crecimiento. La fibrosis en el músculo afectado por pechuga de madera se caracteriza por la sustitución de las fibras musculares con proteínas de la matriz extracelular, especialmente colágeno que forma fibrillas. Los estudios han demostrado diferencias en la organización del colágeno en las líneas de pollos de engorde de rápido crecimiento, con organizaciones de colágeno altamente alineadas y altamente empacadas que tienen una mayor incidencia fenotípica para la pechuga de madera. El objetivo del presente estudio fue analizar la organización de las fibrillas de colágeno en dos líneas de pollos de engorde de rápido crecimiento (Líneas A y B) con una incidencia de pechos de madera en comparación con la Línea C de pollos de engorde de crecimiento más lento, sin pecho de madera detectable fenotípicamente. El proteoglicano pequeño decorina rica en leucina también se estudió por su interacción con el colágeno mediante detección por el método inmunogold. La decorina se une a los colágenos fibrilares y organiza su alineación y sus enlaces cruzados, los cuales afectarán las propiedades funcionales del colágeno. Los hallazgos más importantes del estudio demostraron que el colágeno se organiza en fibrillas de mayor diámetro en la miopatía de pechuga de madera. Específicamente, los pollos de engorde afectados con la pechuga de madera de la Línea A mostraron una tendencia mayor para mostrar paquetes de fibrillas de colágeno más grandes en comparación con los afectados de la Línea B. La Línea A afectada por la pechuga de madera tuvo paquetes de fibrillas de colágeno de hasta 8.4 µm, mientras que el tamaño máximo de la Línea B fue de 5.1 µm. Aunque en general, el enlace entre la decorina y colágeno no fue diferente en la miopatía de madera de la pechuga o en la línea de pollos de engorde con haces de fibrillas de colágeno de diámetro menor, la línea A afectada por pechuga de madera tuvo más uniones de decorina-colágeno en comparación con la línea B afectada por pechuga de madera. En general, los datos proporcionan evidencia adicional de que es más probable la presentación de miopatías fibróticas múltiples en pollos de engorde de rápido crecimiento.

Defective Fibrillar Collagen Organization by Fibroblasts Contributes to Airway Remodeling in Asthma.

Rationale: Histologic stains have been used as the gold standard to visualize extracellular matrix (ECM) changes associated with airway remodeling in asthma, yet they provide no information on the biochemical and structural characteristics of the ECM, which are vital to understanding alterations in tissue function.Objectives: To demonstrate the use of nonlinear optical microscopy (NLOM) and texture analysis algorithms to image fibrillar collagen (second harmonic generation) and elastin (two-photon excited autofluorescence), to obtain biochemical and structural information on the remodeled ECM environment in asthma.Methods: Nontransplantable donor lungs from donors with asthma (n = 13) and control (n = 12) donors were used for the assessment of airway collagen and elastin fibers by NLOM, and extraction of lung fibroblasts for in vitro experiments.Measurements and Main Results: Fibrillar collagen is not only increased but also highly disorganized and fragmented within large and small asthmatic airways compared with control subjects, using NLOM imaging. Furthermore, such structural alterations are present in pediatric and adult donors with asthma, irrespective of fatal disease. In vitro studies demonstrated that asthmatic airway fibroblasts are deficient in their packaging of fibrillar collagen-I and express less decorin, important for collagen fibril packaging. Packaging of collagen fibrils was found to be more disorganized in asthmatic airways compared with control subjects, using transmission electron microscopy.Conclusions: NLOM imaging enabled the structural assessment of the ECM, and the data suggest that airway remodeling in asthma involves the progressive accumulation of disorganized fibrillar collagen by airway fibroblasts. This study highlights the future potential clinical application of NLOM to assess airway remodeling in vivo.

Chronic inflammation deteriorates structure and function of collagen fibril in rat temporomandibular joint disc.

Collagen is the building component of temporomandibular joint (TMJ) discs and is often affected by inflammation in temporomandibular disorders. The macromechanical properties of collagen are deteriorated by chronic inflammation. However, the mechanism by which inflammation influences disc function remains unknown. The relationship between the ultrastructure and nanomechanical properties of collagen in inflamed discs should be clarified. Seven-week-old female Sprague-Dawley rats were randomly divided into two groups. Chronic TMJ inflammation was induced by intra-articular injection of complete Freund's adjuvant, and samples were harvested after 5 weeks. Picrosirius staining revealed multiple colours under polarized light, which represented alternative collagen bundles in inflamed discs. Using atomic force microscopy scanning, the magnitude of Young's modulus was reduced significantly accompanied with disordered collagen fibril arrangement with porous architecture of inflamed discs. Transmission electron microscopy scanning revealed a non-uniform distribution of collagen fibres, and oversized collagen fibrils were observed in inflamed discs. Fourier transform infrared microspectroscopy revealed a decrease in 1 338 cm-1/amide II area ratio of collagen in different regions. The peak positions of amide I and amide II bands were altered in inflamed discs, indicating collagen unfolding. Our results suggest that sustained inflammation deteriorates collagen structures, resulting in the deterioration of the ultrastructure and nanomechanical properties of rat TMJ discs.

Collagen fibrillar structures in vocal fold scarring and repair using stem cell therapy: a detailed histological, immunohistochemical and atomic force microscopy study.

Regenerative medicine opens new opportunities in the repair of cicatricial lesions of the vocal folds. Here, we present a thorough morphological study, with the focus on the collagen structures in the mucosa of the vocal folds, dedicated to the effects of stem cells on the vocal folds repair after cicatricial lesions. We used a conventional experimental model of a mature scar of the rabbit vocal folds, which was surgically excised with a simultaneous implantation of autologous bone marrow-derived mesenchymal stem cells (MSC) into the defect. The restoration of the vocal folds was studied 3 months postimplantation of stem cells and 6 months after the first surgery. The collagen structure assessment included histology, immunohistochemistry and atomic force microscopy (AFM) studies. According to the data of optical microscopy and AFM, as well as to immunohistochemical analysis, MSC implantation into the vocal fold defect leads not only to the general reduction of scarring, normal ratio of collagens type I and type III, but also to a more complete restoration of architecture and ultrastructure of collagen fibres in the mucosa, as compared to the control. The collagen structures in the scar tissue in the vocal folds with implanted MSC are more similar to those in the normal mucosa of the vocal folds than to those of the untreated scars. AFM has proven to be an instrumental technique in the assessment of the ultrastructure restoration in such studies. LAY DESCRIPTION: Regenerative medicine opens new opportunities in the repair of the vocal fold scars. Because collagen is a main component in the vocal fold mucosa responsible for the scar formation and repair, we focus on the collagen structures in the mucosa of the vocal folds, using a thorough morphological study based on histology and atomic force microscopy (AFM). Atomic force microscopy is a scanning microscopic technique which allows revealing the internal structure of a tissue with a resolution up to nanometres. We used a conventional experimental model of a mature scar of the rabbit vocal folds, surgically excised and treated with a mesenchymal stem cells transplant. Our morphological study, primarily AFM, explicitly shows that the collagen structures in the scarred vocal folds almost completely restore after the stem cell treatment. Thus, the modern microscopic methods, and especially AFM are instrumental tools for monitoring the repair of the vocal folds scars.

Tensile behavior and structural characterization of pig dermis.

Skin, the outermost layer of the body, fulfills a broad range of functions, protecting internal organs from damage and infection, while regulating the body's temperature and water content via the exchange of heat and fluids. It must be able to withstand and recover from extensive deformation and damage that can occur during growth, movement, and potential injuries. A detailed investigation of the evolution of the collagen architecture of the dermis as a function of deformation is conducted, which reveals new aspects that help us to understand the mechanical response of skin. Juvenile pig is used as a model material because of its similarity to human skin. The dermis is found to have a tridimensional woven structure of collagen fibers, which evolves with deformation. After failure, we observe that the fibers have straightened and aligned in the direction of tension. The effects of strain-rate change, cyclic loading, stress relaxation, and orientation are quantitatively established. Digital image correlation techniques are implemented to quantify skin's anisotropy; measurements of the Poisson ratio are reported. This is coupled with transmission electron microscopy which enables obtaining quantitative strain parameters evaluated through the orientation and curvature of the collagen fibers and their changes, for the first time in all three dimensions of the tissue. A model experiment using braided human hair in tension exhibits a similar J-curve response to skin, and we propose that this fiber configuration is at least partially responsible for the monotonic increase of the tangent modulus of skin with strain. The obtained results are intended to serve as a basis for structurally-based models of skin. STATEMENT OF SIGNIFICANCE: Our study reveals a new aspect of the dermis: it is comprised of a tridimensional woven structure of collagen fibers, which evolves with deformation. This is enabled by primarily two techniques, transmission electron microscopy on three perpendicular planes and confocal images with second harmonic generation fluorescence of collagen, captured at different intervals of depth. After failure, the fibers have straightened and aligned in the direction of tension. Digital image correlation techniques are implemented to quantify skin's anisotropy; measurements of the Poisson ratio are reported. A model experiment using braided human hair in tension exhibits a similar J-curve response to skin, and we propose that this fiber configuration is at least partially responsible for the monotonic increase of the tangent modulus of skin with strain.

A novel approach to enhance the spinnability of collagen fibers by graft polymerization.

Collagen is an important natural biopolymer that cannot be electrospun easily due to the lost properties occurs in the associated degrading chains while dissolving and spinning. Grafting polymerization of methyl methacrylate-co-Ethyl Acrylate was applied to modify the surface of acid soluble collagen (ASC). The branched copolymer on the surface of collagen significantly influenced the initial viscosity. Since chain entanglement is crucial for fiber formation during electrospinning, the dependency of entanglement concentration on branch densities possessing the approximate same viscosity was investigated; in which the mean fiber diameters of all considered samples remained broadly constant. Increasing the number of branching onto ASC chains significantly decreased the deteriorative impact of the electrospinning conditions. It has also increased the stability of the collagen-based fibers under high humidity conditions. The short chain branched ASC-g-P(MMA-co-EA) can effectively influence the thermal stability of electrospun collagen fibers while the long chain branched ASC-g-P(MMA-co-EA) can provide a higher chain entanglement density leading to the more fiber uniformity.

Bi-layered silane-TiO2/collagen coating to control biodegradation and biointegration of Mg alloys.

Magnesium alloys have shown high potential as biodegradable implants for bone repair applications. However, their fast degradation in physiological media demands tuning their corrosion rate to accompany the natural tissue healing processes. Here, a new bi-layered silane-TiO2/collagen coating efficient in stabilizing and biofunctionalizing the surface of AZ31 and ZE41 Mg alloys is presented. Corrosion tests performed in cell culture medium over 7 weeks showed that the bi-layered coating promotes the formation of a stable layer of Mg(OH)2/MgCO3/CaCO3 that provides effective protection to the alloys at advanced immersion stages. The intrinsic reactivity of each alloy plus formation of transitory calcium phosphate phases, resulted in distinct corrosion behavior in the short term. Cell experiments showed that the bi-layered coating improved osteoblasts and fibroblasts proliferation compared to bare and silane-TiO2-coated alloys. Different responses in terms of cell adhesion could be related to the intrinsic corrosion rate of each alloy and some toxicity from the alloying elements. The results evidenced the complex interplay between alloy nature, coating-alloy combination and cell type. The silane-TiO2/collagen coating showed to be a promising strategy to improve cell response and viability and to control degradation rate of Mg alloys in the long term.

Biomimetic intrafibrillar mineralized collagen promotes bone regeneration via activation of the Wnt signaling pathway.

PURPOSE: The purpose of this study was to assess the effects of biomimetic intrafibrillar mineralized collagen (IMC) bone scaffold materials on bone regeneration and the underlying biological mechanisms. MATERIALS AND METHODS: A critical-sized bone defect in the rat femur was created; then IMC, extrafibrillar mineralized collagen, and nano-hydroxyapatite bone scaffold materials were grafted into the defect. Ten weeks after implantation, micro-computed tomography and histology were applied to evaluate the bone regeneration. Furthermore, microarray technology was applied for transcriptional profile analysis at two postoperative time points (7 and 14 days). Subsequently, the critical genes involved in bone regeneration identified by transcriptional analysis were verified both in vivo through immunohistochemical analysis and in vitro by quantitative real-time transcription polymerase chain reaction evaluation. RESULTS: Significantly increased new bone formation was found in the IMC group based on micro-computed tomography and histological evaluation (P<0.05). Transcriptional analysis revealed that the early process of IMC-guided bone regeneration involves the overexpression of genes mainly associated with inflammation, immune response, skeletal development, angiogenesis, neurogenesis, and the Wnt signaling pathway. The roles of the Wnt signaling pathway-related factors Wnt5a, ß-catenin, and Axin2 were further confirmed both in vivo and in vitro. CONCLUSION: The IMC bone scaffold materials significantly enhanced bone regeneration via activation of the Wnt signaling pathway.

Evaluation of dermal collagen stained with picrosirius red and examined under polarized light microscopy.

The special picrosirius red staining highlights the natural birefringence of collagen fibers when exposed to polarized light. The results from birefringence allow to evaluate the organization of the collagen fibers in the tissues. The authors intend to elucidate all steps to obtain and capture images of histological sections stained with picrosirius red and evaluated under polarized light microscopy, as well as possible artefacts that may occur.