Collagen Microstructure Research Articles

Polarized reflectance from articular cartilage depends upon superficial zone collagen network microstructure.

Polarized reflectance from articular cartilage involves light scattering dependent on surface features, sub-surface optical properties, and collagen birefringence. To understand how surface roughness, zonal collagen microstructure, and chondrocyte organization contribute to polarized reflectance signals, experiments were conducted on bovine cartilage explants and osteochondral cores to compare polarized reflectance texture with split lines and relate these signals to cartilage zonal features and chondrocyte distribution. Texture parameter sensitivity to articular surface damage was determined from polarized reflectance maps and optimized to detect surface damage. Results indicate that polarized reflectance texture predominantly derives from the superficial zone collagen network, while the parameter average value also depends on surface roughness and total cartilage thickness.

Polarization-sensitive optical coherence tomography with a conical beam scan for the investigation of birefringence and collagen alignment in the human cervix.

By measuring the phase retardance of a cervical extracellular matrix, our in-house polarization-sensitive optical coherence tomography (PS-OCT) was shown to be capable of (1) mapping the distribution of collagen fibers in the non-gravid cervix, (2) accurately determining birefringence, and (3) measuring the distinctive depolarization of the cervical tissue. A conical beam scan strategy was also employed to explore the 3D orientation of the collagen fibers in the cervix by interrogating the samples with an incident light at 45° and successive azimuthal rotations of 0-360°. Our results confirmed previous observations by X-ray diffraction, suggesting that in the non-gravid human cervix collagen fibers adjacent to the endocervical canal and in the outermost areas tend to arrange in a longitudinal fashion whereas in the middle area they are oriented circumferentially. PS-OCT can assess the microstructure of the human cervical collagen in vitro and holds the potential to help us better understand cervical remodeling prior to birth pending the development of an in vivo probe.

Image-based Characterization of 3D Collagen Networks and the Effect of Embedded Cells.

Collagen microstructure is closely related to the mechanical properties of tissues and affects cell migration through the extracellular matrix. To study these structures, three-dimensional (3D) in vitro collagen-based gels are often used, attempting to mimic the natural environment of cells. Some key parameters of the microstructure of these gels are fiber orientation, fiber length, or pore size, which define the mechanical properties of the network and therefore condition cell behavior. In the present study, an automated tool to reconstruct 3D collagen networks is used to extract the aforementioned parameters of gels of different collagen concentration and determine how their microstructure is affected by the presence of cells. Two different experiments are presented to test the functionality of the method: first, collagen gels are embedded within a microfluidic device and collagen fibers are imaged by using confocal fluorescence microscopy; second, collagen gels are directly polymerized in a cell culture dish and collagen fibers are imaged by confocal reflection microscopy. Finally, we investigate and compare the collagen microstructure far from and in the vicinities of MDA-MB 23 cells, finding that cell activity during migration was able to strongly modify the orientation of the collagen fibers and the porosity-related values.

Tissue Level Mechanical Properties and Extracellular Matrix Investigation of the Bovine Jugular Venous Valve Tissue.

Jugular venous valve incompetence has no long-term remedy and symptoms of transient global amnesia and/or intracranial hypertension continue to discomfort patients. During this study, we interrogate the synergy of the collagen and elastin microstructure that compose the bi-layer extracellular matrix (ECM) of the jugular venous valve. In this study, we investigate the jugular venous valve and relate it to tissue-level mechanical properties, fibril orientation and fibril composition to improve fundamental knowledge of the jugular venous valves toward the development of bioprosthetic venous valve replacements. Steps include: (1) multi loading biaxial mechanical tests; (2) isolation of the elastin microstructure; (3) imaging of the elastin microstructure; and (4) imaging of the collagen microstructure, including an experimental analysis of crimp. Results from this study show that, during a 3:1 loading ratio (circumferential direction: 900 mN and radial direction: 300 mN), elastin may have the ability to contribute to the circumferential mechanical properties at low strains, for example, shifting the inflection point toward lower strains in comparison to other loading ratios. After isolating the elastin microstructure, light microscopy revealed that the overall elastin orients in the radial direction while forming a crosslinked mesh. Collagen fibers were found undulated, aligning in parallel with neighboring fibers and orienting in the circumferential direction with an interquartile range of −10.38° to 7.58° from the circumferential axis (n = 20). Collagen crimp wavelength and amplitude was found to be 38.46 ± 8.06 µm and 4.51 ± 1.65 µm, respectively (n = 87). Analyzing collagen crimp shows that crimp permits about 12% true strain circumferentially, while straightening of the overall fibers accounts for more. To the best of the authors’ knowledge, this is the first study of the jugular venous valve linking the composition and orientation of the ECM to its mechanical properties and this study will aid in forming a structure-based constitutive model.

High-speed collagen fiber modeling and orientation quantification for optical coherence tomography imaging.

Quantifying collagen fiber architecture has clinical and scientific relevance across a variety of tissue types and adds functionality to otherwise largely qualitative imaging modalities. Optical coherence tomography (OCT) is uniquely suited for this task due to its ability to capture the collagen microstructure over larger fields of view than traditional microscopy. Existing image processing techniques for quantifying fiber architecture, while accurate and effective, are very slow for processing large datasets and tend to lack structural specificity. We describe here a computationally efficient method for quantifying and visualizing collagen fiber organization. The algorithm is demonstrated on swine atria, bovine anterior cruciate ligament, and human cervical tissue samples. Additionally, we show an improved performance for images with crimped fiber textures and low signal to noise when compared to similar methods.

Abstract P6-09-12: Using multiphoton laser scanning microscopy to assess neoadjuvant therapy outcome in core needle biopsies: A novel methodology

Abstract Background: Over-expression of Human Epidermal Growth Factor receptor-2 (HER2) in breast cancer is associated with an aggressive clinical course and poor prognosis. Targeting HER2 over-expression has been shown to be a remarkably effective therapeutic modality in the metastatic, adjuvant and neoadjuvant setting and the pathologic response to neoadjuvant treatment in HER2-positive breast cancer has been shown to be an excellent surrogate for a good outcome. The stromal tumor microenvironment is implicated in fostering tumor growth, facilitating cell migration and ultimately resulting in metastatic disease. Specifically, the collagenous extracellular matrix, which includes fibrillar collagen, has been suggested to play a role in the migration of malignant breast epithelial cells within the surrounding stroma. We have developed a novel methodology which uses an intrinsic optical signature to quantitatively evaluate fibrillar collagen (Burke et al. BMC Cancer 15 (2015): 929). Here, we evaluate the ability of this quantitative methodology to predict the pathologic response after neoadjuvant HER2-targeted treatment as assessed by the Residual Cancer Burden score/class (RCB). This quantitative evaluation in pre-treatment biopsies is then correlated with the pathologic response to treatment in the post-therapy resection. Material and Methods: Clinical pathologic variables for 29 cases of HER2-positive breast cancer that had undergone neoadjuvant chemotherapy plus HER2-targeted therapy were retrieved from the medical record database at URMC, including the post-treatment RCB score and ER/PR/HER2 status. Second harmonic generation (SHG) is an intrinsic optical signal produced by fibrillar collagen. To quantify collagen microstructure in the pre-treatment core biopsy, we used SHG imaging to determine the average forward to backward-light scattering ratio (F/B). The F/B ratio is sensitive to structural properties of collagen fibers. Results: Logistic regression was used to assess the association between F/B and the binary response variable RCB class (0/1 or 2/3). A likelihood ratio test was used to calculate the p-value to test whether the regression coefficient for F/B was zero (i.e. no effect) in the tumor-stromal interface. The average F/B ratio at the leading edge of the tumor stratified by RCB class is shown in Table 1. When evaluated in the bulk of the tumor tissue, F/B was not correlated with RCB status; however, when evaluated at the leading edge of the tumor stromal interface, F/B was significantly correlated with RCB status (p=0.035). Table 1:RCB class and average F/BRCB class (n)Average F/B ± SEM0/1 (19)16.95 ± 1.062/3 (10)12.32 ± 1.84 Conclusions: We have previously shown that the measurement of F/B in the primary tumor after resection is an independent prognostic indicator of metastasis-free survival in breast cancer. Our results in the current study furthers these observations and suggests that the evaluations of the microstructure of collagen fibers by F/B measurement from the pre-treatment biopsy, specifically at the leading edge of the tumor-stroma interface, may be useful for predicting pathologic response to trastuzumab-based neoadjuvant therapy. Further studies in a larger patient cohort are warranted. Citation Format: Desa DE, Turner BM, Buscaglia B, Hill RL, Strawderman RL, Hicks DG, Brown EB. Using multiphoton laser scanning microscopy to assess neoadjuvant therapy outcome in core needle biopsies: A novel methodology [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-09-12.

Quantification of collagen fiber structure using second harmonic generation imaging and two-dimensional discrete Fourier transform analysis: Application to the human optic nerve head.

Second harmonic generation (SHG) microscopy is widely used to image collagen fiber microarchitecture due to its high spatial resolution, optical sectioning capabilities and relatively nondestructive sample preparation. Quantification of SHG images requires sensitive methods to capture fiber alignment. This article presents a two-dimensional discrete Fourier transform (DFT)-based method for collagen fiber structure analysis from SHG images. The method includes integrated periodicity plus smooth image decomposition for correction of DFT edge discontinuity artefact, avoiding the loss of peripheral image data encountered with more commonly used windowing methods. Outputted parameters are as follows: the collagen fiber orientation distribution, aligned collagen content and the degree of collagen fiber dispersion along the principal orientation. We demonstrate its application to determine collagen microstructure in the human optic nerve head, showing its capability to accurately capture characteristic structural features including radial fiber alignment in the innermost layers of the bounding sclera and a circumferential collagen ring in the mid-stromal tissue. Higher spatial resolution rendering of individual lamina cribrosa beams within the nerve head is also demonstrated. Validation of the method is provided in the form of correlative results from wide-angle X-ray scattering and application of the presented method to other fibrous tissues.

Mechanical Characterization and Constitutive Modeling of Rabbit Aortas in Health and Diabetes

Diabetes is a major risk factor to cause macrovascular diseases and plays a pivotal role in aortic wall remodeling. However, the effects of diabetes on elastic properties of aortas remain largely unknown. Thirty adult rabbits (1.6-2.2 kg) were collected and the type I diabetic rabbit model was induced by injection of alloxan. A total of 15 control and 15 diabetic rabbit (abdominal) aortas were harvested. Uniaxial and biaxial tensile tests were performed to measure ultimate tensile strength and to characterize biaxial mechanical behaviors of the aortas. A material model was fitted to the biaxial experimental data to obtain constitutive parameters. Histological and mass fraction analyses were performed to investigate the underlying microstructure and dry weight percentages of elastin and collagen in the control and the diabetic aortas. No statistically significant difference was found in ultimate tensile strength between the control and the diabetic aortas. Regarding biaxial mechanical responses, the diabetic aortas exhibited significantly lower extensibility and significantly higher tissue stiffness than the control aortas. Notably, tissue stiffening occurred in both circumferential and axial directions for the diabetic aortas; however, mechanical anisotropy does not change significantly. The material model was able to fit biaxial experimental data very well. Histology showed that a number of isolated foam cells were embedded in the diabetic aortas and hyperplasia of collagen was identified. The dry weight percentages of collagen within the diabetic aortas increased significantly as compared to the control aortas, whereas no significant change was found for that of elastin. The results suggest that the diabetes impairs elastic properties and alters microstructure of the aortas and consequently, these changes may further contribute to complex aortic wall remodeling.

Mammary fibroblasts remodel fibrillar collagen microstructure in a biomimetic nanocomposite hydrogel

Architecture and microstructure of type I collagen fibers constitute central regulators of tumor invasion with aligned fibers providing a route for migration of stromal and cancer cells. Several different aspects of fibrillar collagen, such as stiffness, density, thickness, and pore size, may regulate migration of cancer cells, but determining effects of any one parameter requires clear decoupling of physical properties of collagen networks. The objective of this work is to develop and apply an in vitro three-dimensional (3D) tumor-extra cellular matrix (ECM) model with tunable physical parameters to define how stromal fibroblasts modulate collagen microstructure to control migration of breast cancer cells. We incorporated two different types of polyhedral oligomeric silsesquioxane (POSS) nano-molecules into a collagen/alginate matrix to induce different mechanisms of gelling. The resultant biomimetic, nanocomposite hydrogels show different collagen fibrillar microstructures while maintaining constant overall matrix stiffness, density, and porosimetry. Spheroids of human mammary fibroblasts embedded in these 3D matrices remodel the collagen network to varying extents based on differences in underlying matrix microstructures. The remodeled collagen matrix shows oriented, thicker fibrillar tracks, facilitating invasion of tumor cells. By decoupling effects of matrix stiffness and architecture, our nanocomposite hydrogels serve as robust platforms to investigate how biophysical properties of tumor environments control key processes regulating tumor progression in breast cancer and other malignancies. Statement of SignificanceOur manuscript demonstrates a new type of nanocomposite hydrogel with two different gelling mechanisms, produced by incorporating two types of polyhedral oligomeric silsesquioxane (POSS) nano-molecules into a collagen/alginate matrix. The resultant biomimetic hydrogels show different fibrillar collagen microstructures while maintaining constant overall matrix stiffness, density, and porosimetry. These gels allow us to uncouple effects of matrix stiffness versus architecture on migration and invasion of breast cancer cells and stromal fibroblasts. Upon embedding spheroids of human mammary fibroblasts (HMFs) and dissociated 231 breast cancer cells, we showed that HMFs remodeled the collagen network to differing extents dependent on starting matrix microstructures in each hydrogel. The remodeled collagen matrix showed aligned collagen fibers perpendicular to the surface of a spheroid with migrating HMFs following these fibers as occurs in tumors in vivo. To our knowledge, this is the first study showing significant different fibrillar collagen microstructures with constant collagen density and gel stiffness. This study establishes a new type of nanocomposite 3D hydrogels for studies of biophysical and cellular interactions in engineered tumor environments.

Effect of cryopreservation of teeth on the structural integrity of dentin

The autotransplantation of teeth after cryopreservation has become an increasingly viable method for whole tooth replacement. While the immediate success rates are quite high, damage introduced by cryopreservation within the dentin or enamel could be detrimental to the durability of these teeth. Objectiveto determine whether cryopreservation alters the microstructure of dentin or causes a reduction of its resistance to mechanical failures. MethodsThird molars were obtained from young donors (18≤age≤30yrs) and subjected to a cryopreservation protocol involving storage for 10days in cryoprotectant solution at −196°C. After treatment, the mid-coronal dentin was characterized in terms of its elastic modulus, strength and fatigue behavior. Scanning electron microscopy and Raman spectroscopy were used to evaluate the microstructure and integrity of collagen after cryopreservation. ResultsThere was no significant difference in the elastic modulus or flexural strength between dentin from the cryopreserved and non-cryopreserved (control) teeth. However, the cryopreservation treatment caused a significant decrease in the fatigue strength of dentin with respect to the controls, with average reduction of nearly 20%. While there were no differences apparent in the collagen matrix or fracture surfaces between the cryopreserved and control groups, the microstructure of dentin from the cryopreserved teeth exhibited unique features and damage that appear to have caused the decrease in durability. SignificanceAutotransplantation of cryopreserved teeth may be a viable option for whole tooth restorations, but hidden damage within the dentin could render these teeth more susceptible to mechanical failures by fatigue and fracture.

Types of advanced optical microscopy techniques for breast cancer research: a review.

A cancerous cell is characterized by morphological and metabolic changes which are the key features of carcinogenesis. Adenosine triphosphate (ATP) in cancer cells is primarily produced by aerobic glycolysis rather than oxidative phosphorylation. In normal cellular metabolism, nicotinamide adenine dinucleotide (NADH) is considered as a principle electron donor and flavin adenine dinucleotide (FAD) as an electron acceptor. During metabolism in a cancerous cell, a net increase in NADH is found as the pathway switched from oxidative phosphorylation to aerobic glycolysis. Often during initiation and progression of cancer, the developmental regulation of extracellular matrix (ECM) is restricted and becomes disorganized. Tumor cell behavior is regulated by the ECM in the tumor micro environment. Collagen, which forms the scaffold of tumor micro-environment also influences its behavior. Advanced optical microscopy techniques are useful for determining the metabolic characteristics of cancerous, normal cells and tissues. They can be used to identify the collagen microstructure and the function of NADH, FAD, and lipids in living system. In this review article, various optical microscopy techniques applied for breast cancer research are discussed including fluorescence, confocal, second harmonic generation (SHG), coherent anti-Stokes Raman scattering (CARS), and fluorescence lifetime imaging (FLIM).

Mueller polarimetric imaging for characterizing the collagen microstructures of breast cancer tissues in different genotypes

Abstract Mueller polarimetric imaging has been proven a valid method for detecting tissue in biomedical field. In the present study, we applied the Mueller matrix microscopy to characterize the microstructures of breast cancer tissues in different genotypes. The experimental results demonstrated that Mueller matrix parameters were effective in distinguishing the genotypes of breast cancer, especially for triple-negative type. Then, we conducted quantitative analysis of Mueller matrix and immunohistochemistry (IHC) experiments. On that basis, we verified the relationship between collagen structures and polarization properties as well as the remodeling of collagen structures in breast cancer, which could provide new evidence for using high invasiveness and metastatic dissemination as triple-negative breast cancer. In comparison with traditional methods, Mueller polarimetry is independent of complex tumor marks. Besides, it is quick, sensitive and non-stained. Thus, it has a potential in the application for clinical diagnosis and is expected as a new method for researching the pathological process of breast cancer.

Directing fibroblast self-assembly to fabricate highly-aligned, collagen-rich matrices

Extracellular matrix composition and organization play a crucial role in numerous biological processes ranging from cell migration, differentiation, survival and metastasis. Consequently, there have been significant efforts towards the development of biomaterials and in vitro models that recapitulate the complexity of native tissue architecture. Here, we demonstrate an approach to fabricating highly aligned cell-derived tissue constructs via the self-assembly of human dermal fibroblasts. By optimizing mold geometry, cell seeding density, and media composition we can direct human dermal fibroblasts to adhere to one another around a non-adhesive agarose peg to facilitate the development of cell-mediated circumferential tension. By removing serum and adding ascorbic acid and l-proline, we tempered fibroblast contractility to enable the formation of stable tissue constructs. Similarly, we show that the alignment of cells and the ECM they synthesize can be modulated by changes to seeding density and that constructs seeded with the lowest number of cells have the highest degree of fibrillar collagen alignment. Finally, we show that this highly aligned, tissue engineered construct can be decellularized and that when re-seeded with fibroblasts, it provides instructive cues which enable cells to adhere to and align in the direction of the remaining collagen fiber network. Statement of SignificanceCell and extracellular matrix organization is directly related to biological function including cell signaling and tissue mechanics. Changes to this organization are often associated with injury or disease. The majority of in vitro tissue engineering models investigating cell and matrix organization rely on the addition of stress-shielding exogenous proteins and polymers and, or the application of external forces to promote alignment. Here we present a completely cell-based approach that relies on the development of cell-mediated tension to direct anisotropic cellular alignment and matrix synthesis using human dermal fibroblasts. A major challenge with this approach is excessive cellular contractility that results in necking and failure of the tissue construct. While other groups have tried to overcome this challenge by simply adding more cells, here we show that matrix alignment is inversely related to cell seeding density. To engineer tissue constructs with the highest degree of alignment, we optimized media components to reduce cellular contractility and promote collagen synthesis such that fibroblast toroids remained stable for at least 28 days in culture. We subsequently showed that these collagen-rich tissue constructs could be decellularized while maintaining their collagen microstructure and that cells adhered to and responded to the decellularized cell-derived matrix by aligning and elongating along the collagen fibers. The complexity of cell-derived matrices has been shown to better recapitulate in vivo tissue architecture and composition. This study provides a straight-forward approach to fabricating instructive cell-derived matrices with a high degree of uniaxial alignment generated purely by cell-mediated tension.

Fibroblast-seeded collagen gels in response to dynamic equibiaxial mechanical stimuli: A biomechanical study

The remodeling of fibroblast-seeded collagen gels in response to dynamic mechanical stimuli was investigated by using a newly developed biaxial culture system capable of cyclically stretching planar soft tissues. Fibroblast-seeded collagen gels were subjected to three distinct dynamic mechanical conditions for six days: Cyclic Equibiaxial Stretching at two constant strain magnitudes (CES-7% and CES-20%), and Cyclic Equibiaxial Stretching with incrementally Increasing stain magnitude (ICES, 7% → 15% → 20% each for two days). The frequency of cyclic stretching was set at 1 Hz. At the end of culture, mechanical properties of the gels were examined by biaxial mechanical testing and checked again upon the removal of seeded cells. Collagen microstructure within the gels was illustrated by multiphoton microscopy. The mRNA levels of collagen type I and type III and fibronectin in the cells were examined by reverse transcription PCR. The protein expression of α-smooth muscle actin was detected by immunohistochemistry. We found that the gels cultured under cyclic stretching were stiffer than those cultured under static stretching. Particularly, the stiffness appeared to be significantly enhanced when the ICES was employed. The enhancement of mechanical properties by cyclic stretching appeared to persist upon cell removal, suggesting an irreversible remodeling of extracellular matrix. Second harmonic generation images showed that collagen fibers became thicker and more compact in the gels cultured under cyclic stretching, which may explain the mechanical findings. The mRNA expression of collagen type I in the cells of the ICES was significantly greater than that of the other groups except for the CES-20%. This study suggests that when cyclic stretching is to be used in engineering soft tissues, incrementally increasing strain magnitude may prove useful in the development of the tissue.

Hierarchical Characterization and Nanomechanical Assessment of Biomimetic Scaffolds Mimicking Lamellar Bone via Atomic Force Microscopy Cantilever-Based Nanoindentation.

The hierarchical structure of bone and intrinsic material properties of its two primary constituents, carbonated apatite and fibrillar collagen, when being synergistically organized into an interpenetrating hard-soft composite, contribute to its excellent mechanical properties. Lamellar bone is the predominant structural motif in mammalian hard tissues; therefore, we believe the fabrication of a collagen/apatite composite with a hierarchical structure that emulates bone, consisting of a dense lamellar microstructure and a mineralized collagen fibril nanostructure, is an important first step toward the goal of regenerative bone tissue engineering. In this work, we exploit the liquid crystalline properties of collagen to fabricate dense matrices that assemble with cholesteric organization. The matrices were crosslinked via carbodiimide chemistry to improve mechanical properties, and are subsequently mineralized via the polymer-induced liquid-precursor (PILP) process to promote intrafibrillar mineralization. Neither the crosslinking procedure nor the mineralization affected the cholesteric collagen microstructures; notably, there was a positive trend toward higher stiffness with increasing crosslink density when measured by cantilever-based atomic force microscopy (AFM) nanoindentation. In the dry state, the average moduli of moderately (X51; 4.8 ± 4.3 GPa) and highly (X76; 7.8 ± 6.7 GPa) crosslinked PILP-mineralized liquid crystalline collagen (LCC) scaffolds were higher than the average modulus of bovine bone (5.5 ± 5.6 GPa).

Mechanical response of human subclavian and iliac arteries to extension, inflation and torsion

Peripheral vascular trauma due to injuries of the upper and lower limbs are life-threatening, and their treatment require rapid diagnosis and highly-qualified surgical procedures. Experienced surgeons have recognized that subclavian arteries, affected by injuries of the upper limbs, require a more careful handling due to fragility than common iliac arteries, which are may be affected by injures of the lower limbs. We investigated these two artery types with comparable diameter to evaluate the differences in the biomechanical properties between subclavian and iliac arteries. Human subclavian and common iliac arteries of 14 donors either from the right or the left side (age: 63 yrs, SD: 19,9 female and 5 male) were investigated. Extension-inflation-torsion experiments at different axial strains (0–20%), transmural pressures (0–200 mmHg) and torsion (±25°) on preconditioned arterial tubes were performed. Residual stresses in both circumferential and axial direction were determined. Additionally, the microstructure of the tissues was determined via second-harmonic generation imaging and by histological investigations. At physiological conditions (pi=13.3 kPa, λz=1.1) common iliac arteries revealed higher Cauchy stresses in circumferential and axial directions but a more compliant response in the circumferential direction than subclavian arteries. Both arteries showed distinct stiffer behavior in circumferential than in axial direction. Circumferential stiffness of common iliac arteries at physiological conditions increased significantly with aging (r=-0.67,p=0.02). The median inversion stretches, where the axial force is basically independent of the transmural pressure, were determined to be 1.05 for subclavian arteries and 1.11 for common iliac arteries. Both arteries exhibited increased torsional stiffness, when either axial prestretch or inflation pressure was increased. Residual stresses in the circumferential direction were significantly lower for subclavian arteries than for common iliac arteries at measurements after 30 min (p=0.05) and 16hrs (p=0.01). Investigations of the collagen microstructure revealed different collagen fiber orientations and dispersions in subclavian and iliac arteries. The difference in the collagen microstructure revealed further that the adventitia seems to contribute significantly to the passive mechanical response of the tested arteries at physiological loadings. Histological investigations indicated pronounced thickened intimal layers in subclavian and common iliac arteries, with a thickness comparable to the adventitial layer. In conclusion, we obtained biomechanical differences between subclavian and common iliac arteries, which possibly resulted from their different mechanical loadings/environments and respective in vivo movements caused by their anatomical locations. The biomechanical differences explored in this study are well reflected by the microstructure of the collagen and the histology of the investigated arteries, and the results can improve trauma patient care and endovascular implant design. Statement of SignificanceDuring surgical interventions surgeons experienced that subclavian arteries (SAs) supplying the upper extremities, appear more fragile and prone to damage during surgical repair than common iliac arteries (CIAs), supplying the lower extremities. To investigate this difference in a systematic way the aim of this study was to compare the biomechanical properties of these two arteries from the same donors in terms of geometry, extension-inflation-torsion behavior, residual stresses, microstructure, and histology. In regard to cardiovascular medicine the material behavior of aged human arteries is of crucial interest. Moreover, the investigation of SA is important as it can help to improve surgical procedures at this challenging location. Over the long-term it might well be of value in the construction of artificial arteries for substituting native arteries. In addition, the analysis of mechanical stresses can improve design and material choice for endovascular implants to optimize long-term implant function.

Diffusion and Binding of Laponite Clay Nanoparticles into Collagen Fibers for the Formation of Leather Matrix.

Understanding accessibilityand interactions of clay nanoparticles with collagen fibers is an important fundamental issue for the conversion of collagen to leather matrix. In this study, we have investigated the diffusion and binding of Laponite into the collagen fiber network. Our results indicate that the diffusion behaviors of Laponite into the collagen exhibit the Langmuir adsorption, verifying its affinity for collagen. The introduction of Laponite leads to a shift in the isoelectric point of collagen from ∼6.8 to ∼4.5, indicating the ionic bonding between the positively charged amino groups of the collagen and negatively charged Laponite under the tanning conditions. Fluorescence microscopy, atomic force microscopy, field-emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, and wide-angle X-ray diffraction analyses reveal that Laponite nanoparticles can penetrate into collagen microstructure and evenly distributed onto collagen fibrils, not altering native D-periodic banding patterns of collagen fibrils. Attenuated total reflectance-Fourier transform infrared and Raman spectroscopy detections further demonstrate the presence of noncovalent interactions, namely, ionic and hydrogen bonding, between Laponite and collagen. These findings provide a theoretical basis for the use of Laponite as an emerging tanning agent in leather manufacture.

Optical discrimination of type I and type III collagen through second order susceptibility imaging

Second order susceptibility (SOS) microscopy is used as a contrast mechanism for distinguishing type I and III collagen microstructure in tissues. We found that the SOS tensor elements for type III collagen are χzzz/χzxx = 1.20 ± 0.14 and χxzx/χzxx = 0.49 ± 0.15 which are different from the type I collagen values of χzzz/χzxx = 1.44 ± 0.15 and χxzx/χzxx = 0.58 ± 0.14. In addition, relative proportion of collagen type was determined by analyzing the histogram of SOS ratio. Through the use of a Gaussian mixture model, the ratio of collagen I and III was determined. We found that in rat skin, type III collagen is present in rat skin in an average ratio of 0.36 ± 0.07. This result shows that SOS microscopy can be applied to in vivo label-free studies to investigate the interaction of types I and III collagen molecules and related diseases.

Optimization of collagen-elastin-like polypeptide composite tissue engineering scaffolds using response surface methodology.

The ability of a tissue-engineered scaffold to regenerate functional tissues depends on its mechanical and biochemical properties. Though the commonly used collagen scaffolds have good biochemical properties, they fail due to their poor mechanical and physical properties. We have reinforced the collagen matrix with elastin-like polypeptide (ELP) to improve the mechanical and physical properties and optimized the composite composition using a novel statistical method of response surface methodology (RSM). RSM used a central composite design to correlate the 2 input factor variables (collagen and ELP concentrations) and 3 output objectives (tensile strength, elastic modulus, and toughness) using a second order polynomial equation. Upon uniaxial tensile testing and subsequent RSM optimization, a composite prepared using 6 mg/mL collagen and 18 mg/mL ELP was identified as having an optimal combination of all the three tensile properties. Physical properties of the 6:18 mg/mL composite versus the 6:0 mg/mL collagen-only hydrogel characterized by swelling ratio, differential scanning calorimetry, and FTIR spectroscopy revealed that the addition of ELP reduced the residual water content in the composites and provided evidence of the presence of collagen-ELP interactions. Scanning electron microscopy images of the collagen-only hydrogel showed porous fibrillar and dense afibrillar collagenous microstructure, but the collagen-ELP composite showed a dense collagenous microstructure with characteristic ELP aggregates. We surmise that because of the low water content and dense microstructure, the 6:18 mg/mL collagen-ELP composite had improved mechanical properties. Taken together, the composites prepared in this research can form good quality, rigid porous structures required for tissue engineering applications.

Characterization of the collagen microstructural organization of human cervical tissue.

The cervix shortens and softens as its collagen microstructure remodels in preparation for birth. Altered cervical tissue collagen microstructure can contribute to a mechanically weak cervix and premature cervical dilation and delivery. To investigate the local microstructural changes associated with anatomic location and pregnancy, we used second-harmonic generation microscopy to quantify the orientation and spatial distribution of collagen throughout cervical tissue from 4 pregnant and 14 non-pregnant women. Across patients, the alignment and concentration of collagen within the cervix was more variable near the internal os and less variable near the external os. Across anatomic locations, the spatial distribution of collagen within a radial zone adjacent to the inner canal of the cervix was more homogeneous than that of a region comprising the middle and outer radial zones. Two regions with different collagen distribution characteristics were found. The anterior and posterior sections in the outer radial zone were characterized by greater spatial heterogeneity of collagen than that of the rest of the sections. Our findings suggest that the microstructural alignment and distribution of collagen varies with anatomic location within the human cervix. These observed differences in collagen microstructural alignment may reflect local anatomic differences in cervical mechanical loading and function. Our study deepens the understanding of specific microstructural cervical changes in pregnancy and informs investigations of potential mechanisms for normal and premature cervical remodeling.