Collagen Fiber Microstructure Research Articles

Quantification of age-related changes in the structure and mechanical function of skin with multiscale imaging.

The mechanical properties of skin change during aging but the relationships between structure and mechanical function remain poorly understood. Previous work has shown that young skin exhibits a substantial decrease in tissue volume, a large macro-scale Poisson's ratio, and an increase in micro-scale collagen fiber alignment during mechanical stretch. In this study, label-free multiphoton microscopy was used to quantify how the microstructure and fiber kinematics of aged mouse skin affect its mechanical function. In an unloaded state, aged skin was found to have less collagen alignment and more non-enzymatic collagen fiber crosslinks. Skin samples were then loaded in uniaxial tension and aged skin exhibited a lower mechanical stiffness compared to young skin. Aged tissue also demonstrated less volume reduction and a lower macro-scale Poisson's ratio at 10% uniaxial strain, but not at 20% strain. The magnitude of 3D fiber realignment in the direction of loading was not different between age groups, and the amount of realignment in young and aged skin was less than expected based on theoretical fiber kinematics affine to the local deformation. These findings provide key insights on how the collagen fiber microstructure changes with age, and how those changes affect the mechanical function of skin, findings which may help guide wound healing or anti-aging treatments.

In vivo imaging of bone collagen dynamics in zebrafish

Type I collagen plays a pivotal role in shaping bone morphology and determining its physical properties by serving as a template for ossification. Nevertheless, the mechanisms underlying bone collagen formation, particularly the principles governing its orientation, remain unknown owing to the lack of a method that enables continuous in vivo observations. To address this challenge, we constructed a method to visualize bone collagen by tagging with green fluorescent protein (GFP) in zebrafish and observed the interactions between osteoblasts and collagen fibers during bone formation in vivo. When collagen type I alpha 2 chain (Col1a2)-GFP was expressed under the control of the osteoblast-specific promoters osx or osc in zebrafish, bone collagen was observed clearly enough to identify its localization, whereas collagen from other organs was not. Therefore, we determined that this method was of sufficient quality for the detailed in vivo observation of bone collagen. Next, bone collagen in the scales, fin rays, and opercular bones of zebrafish was observed in detail, when bone formation is more active. High-magnification imaging showed that Col1a2-GFP can visualize collagen sufficiently to analyze the collagen fiber orientation and microstructure of bones.Furthermore, by simultaneously observation of bone collagen and osteoblasts, we successfully observed dynamic changes in the morphology and position of osteoblasts from the early stages of bone formation. It was also found that the localization pattern and orientation of bone collagen significantly differed depending on the choice of the expression promoter. Both promoters (osx and osc) used in this study are osteoblast-specific, but their Col1a2-GFP localizing regions within the bone were exclusive, with osx region localizing mainly to the outer edge of the bone and osc region localizing to the central area of the bone. This suggests the existence of distinct osteoblast subpopulations with different gene expression profiles, each of which may play a unique role in osteogenesis.These findings would contribute to a better understanding of the mechanisms governing bone collagen formation by osteoblasts.

Fiber orientation and crimp level might control the auxetic effect of biological tissues

We propose an analytical micromechanical model for studying the lamellar-composite-like structure of fibrous soft tissue. The tissue under consideration is made up of several lamellae, and is designed to resemble the annulus fibrosus (AF) tissue or media layer of arterial tissue, for example. The collagen fibers are arranged in parallel in each lamella and the fiber orientation differs from one lamella to its neighbors. The parallel fibers in each lamella of AF tissue, for example, have been observed to have a crimped microstructure. The proposed model incorporates this quality, considering fiber waviness as a sinusoidal shape and taking into account the fiber dispersion in different layers, where both fiber and matrix are considered as solid phases. We find that collagen-fiber waviness and layer orientation have a significant influence on Poisson’s ratio. The effective Poisson’s ratio predicted by the proposed model demonstrates that the crimped collagen fiber microstructure might weaken the auxetic effect of fibrous soft tissue, which might explain why, as the literature suggests, the auxetic behavior is more difficult to observe than large Poisson’s ratios. As opposed to the many studies that use the well-known hyperelastic fiber-based constitutive model, in which out-of-plane expansion is often observed, the present work explains the auxetic response found in modeling and in experimental data from the perspective of collagen fiber microstructure.

A direct fiber approach to model sclera collagen architecture and biomechanics

Sclera collagen fiber microstructure and mechanical behavior are central to eye physiology and pathology. They are also complex, and are therefore often studied using modeling. Most models of sclera, however, have been built within a conventional continuum framework. In this framework, collagen fibers are incorporated as statistical distributions of fiber characteristics such as the orientation of a family of fibers. The conventional continuum approach, while proven successful for describing the macroscale behavior of the sclera, does not account for the sclera fibers are long, interwoven and interact with one another. Hence, by not considering these potentially crucial characteristics, the conventional approach has only a limited ability to capture and describe sclera structure and mechanics at smaller, fiber-level, scales. Recent advances in the tools for characterizing sclera microarchitecture and mechanics bring to the forefront the need to develop more advanced modeling techniques that can incorporate and take advantage of the newly available highly detailed information. Our goal was to create a new computational modeling approach that can represent the sclera fibrous microstructure more accurately than with the conventional continuum approach, while still capturing its macroscale behavior. In this manuscript we introduce the new modeling approach, that we call direct fiber modeling, in which the collagen architecture is built explicitly by long, continuous, interwoven fibers. The fibers are embedded in a continuum matrix representing the non-fibrous tissue components. We demonstrate the approach by doing direct fiber modeling of a rectangular patch of posterior sclera. The model integrated fiber orientations obtained by polarized light microscopy from coronal and sagittal cryosections of pig and sheep. The fibers were modeled using a Mooney-Rivlin model, and the matrix using a Neo-Hookean model. The fiber parameters were determined by inversely matching experimental equi-biaxial tensile data from the literature. After reconstruction, the direct fiber model orientations agreed well with the microscopy data both in the coronal plane (adjusted R2 = 0.8234) and in the sagittal plane (adjusted R2 = 0.8495) of the sclera. With the estimated fiber properties (C10 = 5746.9 MPa; C01 = −5002.6 MPa, matrix shear modulus 200 kPa), the model's stress-strain curves simultaneously fit the experimental data in radial and circumferential directions (adjusted R2's 0.9971 and 0.9508, respectively). The estimated fiber elastic modulus at 2.16% strain was 5.45 GPa, in reasonable agreement with the literature. During stretch, the model exhibited stresses and strains at sub-fiber level, with interactions among individual fibers which are not accounted for by the conventional continuum methods. Our results demonstrate that direct fiber models can simultaneously describe the macroscale mechanics and microarchitecture of the sclera, and therefore that the approach can provide unique insight into tissue behavior questions inaccessible with continuum approaches.

Development and mechanical characterization of decellularized scaffolds for an active aortic graft.

Decellularized porcine aortas are proposed as scaffolds for revolutionary active aortic grafts. A change in the static and dynamic mechanical properties, associated with the microstructure of elastin and collagen fibers, corresponds to alteration in the cyclic expansion and perfusion, in addition to possible graft damage. Therefore, the present study thoroughly investigates the mechanical response of the decellularized scaffolds of human and porcine origin to static and dynamic mechanical loads. The responses of the native human and porcine aortas are also compared; this is unavailable in the literature. Because the aorta is subjected to pulsatile blood pressure, dynamical responses to cyclic loads and their associated viscoelastic properties are particularly relevant for advanced graft design. In parallel, this study examines the microstructure of the decellularized aorta. The resulting data are compared to the analogous data obtained for the native human and porcine tissues. The results indicate that by using an optimized decellularization protocol - based on sodium dodecyl sulfate (SDS) and DNase - that minimizes mechanical and structural changes of the tissue, layered scaffolds with static and dynamic properties very similar to natural human aortas are obtained. In particular, a decellularized porcine aorta is non-inferior to a decellularized human aorta. STATEMENT OF SIGNIFICANCE: About 55,000 patients undergo abdominal aortic aneurysm repair annually in the USA. The currently implanted grafts present a large mechanical mismatch with the native tissue. This increases the pulsatile nature of the blood flow with negative consequences to the organ perfusion. For this reason, biomimetic and mechanically compatible grafts for aortic repair are urgently needed and they can be obtained through tissue engineering. In this study, scaffolds from porcine and human aortas are obtained from an optimized decellularization protocol. They are accurately compared to the native tissue and present the ideal static and dynamic mechanical properties for developing innovative aortic grafts.

Using Diffusion Tensor Imaging to Explore the Changes in the Microstructure of Canine Vocal Fold Scar Tissue

To apply diffusion tensor imaging (DTI) in measurement of the diffusion characteristics of water molecules in vocal fold scar tissue, combined with the analysis of textural characteristics of collagen fibers in the cover layer of the vocal folds to explore the feasibility of DTI in the qualitative and quantitative diagnosis of vocal fold scars and the evaluation of microstructural changes of vocal fold scar tissue. A unilateral injury was created using micro-cup forceps in the left vocal fold of six beagles. The contralateral normal vocal fold was used as a self-control. Five months postinjury, the larynges were excised and placed into a magnetic resonance imaging (MRI) system (9.4T BioSpec MRI, Bruker, German) for scanning and extraction of the diffusion parameters, fractional anisotropy (FA) and tensor trace in the anterior, middle, and posterior portions of the vocal fold cover layer. These parameters were then analyzed for statistical significance between the scarred vocal fold and the normal vocal fold. After MRI scanning, the tissue of the vocal folds was divided into anterior, middle, and posterior parts for sectioning and staining with hematoxylin and eosin, and samples were subsequently digitally scanned for texture analysis. The irregularity parameters, energy, contrast, correlation, and homogeneity, of collagen fibers of the vocal folds and the mean gray value of collagen fibers were calculated by the gray-level co-occurrence matrix (GLCM) texture analysis method. The differences in the mean value of the two sides of the vocal fold were compared. In addition, Pearson correlation analysis was performed between DTI parameters and irregularity parameters. The FA of the left vocal fold cover layer was significantly lower compared to the self-control group (P=0.0366), and the tensor trace value on the left vocal fold cover layer was significantly higher compared to the self-control group (P=0.0353). The FA was significantly higher in the anterior part of the right vocal fold cover layer compared to the middle and posterior parts of the same side (P=0.0352), and the tensor trace was significantly lower in the anterior part of the right vocal fold cover layer compared to the middle and posterior parts of the same side (P=0.0298). There were no significant differences in FA and tensor trace between the middle and posterior parts of the vocal fold cover layer. The mean gray value of the left vocal folds cover layer was significantly smaller than the right vocal fold cover layer (P=0.0219), the energy of the left vocal fold cover layer was significantly smaller than that of the right vocal fold cover layer (P < 0.0001), the contrast of the left vocal folds cover layer was significantly larger than that of the right vocal fold cover layer (P=0.0002), the correlation of the left vocal folds cover layer was significantly smaller than the right vocal fold cover layer (P=0.0002), and the homogeneity of the left vocal folds cover layer was significantly smaller than the right vocal fold cover layer (P=0.0003). Pearson correlation analysis yielded values of r=0.926, P=0.000 between the FA and mean gray value; r=-0.918, P=0.000 between FA and energy; r=-0.924, P=0.000 between the FA and homogeneity, r=-0.949, P=0.000 between tensor trace and mean gray value; r=0.893, P=0.000 between the tensor trace and energy; and r=0.929, P=0.000 between the tensor trace and homogeneity. FA and tensor trace can be used as effective parameters to reflect microstructural changes in vocal fold scars. DTI is an objective and quantitative method of analyzing vocal fold scarring, and it noninvasively evaluates the microstructure of vocal fold collagen fibers.

Biomechanical changes in myopic sclera correlate with underlying changes in microstructure

Myopia alters the microstructural and biomechanical properties of the posterior sclera, which is characterized as a layered structure with potentially different inter-layer collagen fibril characteristics. Scanning acoustic microscopy (SAM) has been used to investigate how the micron-scale bulk mechanical properties of the posterior sclera are affected by myopia. Other investigators have employed second harmonic generation (SHG) imaging to characterize the collagen microstructure of tissues. In the present study, SAM and SHG imaging were used to investigate the existence of biomechanically-distinct scleral layers and identify relationships between mechanical properties and tissue microstructure in myopic guinea pig (GP) eyes. Diffusers were worn over the right eyes of six, 1-week-old GPs for one week to induce unilateral form-deprivation myopia. GPs were euthanized, enucleated, and eyes were cryosectioned. Twelve-micron-thick adjacent vertical cryosections were scanned with SAM or SHG. SAM maps of bulk modulus, mass density, and acoustic attenuation were estimated. A fiber-extraction algorithm applied to SHG images estimated collagen fiber length, width, straightness, alignment, and number density. Results revealed that the posterior sclera may exhibit biomechanically distinct layers that are affected differently in myopia. Specifically, a layered structure was observed in the mechanical-parameter maps of control eyes that was less apparent in myopic eyes. Collagen fibers in myopic eyes had smaller diameters and were more aligned. Myopia-associated biomechanical changes were most significant in the outermost and innermost scleral layers. SAM-measured mechanical parameters were correlated with collagen fiber microstructure, particularly fiber length, alignment, and number density, which may imply the biomechanical parameters estimated from SAM measurements are related to tissue microstructure. Interestingly, some changes were greatest in more-peripheral regions, suggesting interventions to strengthen the sclera may be effective away from the optic nerve and efficacy may be achieved best when intervention is applied to the outermost layer.

Role of fibulin-5 insufficiency and prolapse progression on murine vaginal biomechanical function

The vagina plays a critical role in supporting the pelvic organs and loss of support leads to pelvic organ prolapse. It is unknown what microstructural changes influence prolapse progression nor how decreased elastic fibers contributes to vaginal remodeling and smooth muscle contractility. The objective for this study was to evaluate the effect of fibulin-5 haploinsufficiency, and deficiency with progressive prolapse on the biaxial contractile and biomechanical function of the murine vagina. Vaginas from wildtype (n = 13), haploinsufficient (n = 13), and deficient mice with grade 1 (n = 9) and grade 2 or 3 (n = 9) prolapse were explanted for biaxial contractile and biomechanical testing. Multiaxial histology (n = 3/group) evaluated elastic and collagen fiber microstructure. Western blotting quantified protein expression (n = 6/group). A one-way ANOVA or Kruskal–Wallis test evaluated statistical significance. Pearson’s or Spearman’s test determined correlations with prolapse grade. Axial contractility decreased with fibulin-5 deficiency and POP (p < 0.001), negatively correlated with prolapse grade (ρ = − 0.80; p < 0.001), and positively correlated with muscularis elastin area fraction (ρ = − 0.78; p = 0.004). Circumferential (ρ = 0.71; p < 0.001) and axial (ρ = 0.69; p < 0.001) vaginal wall stresses positively correlated with prolapse grade. These findings demonstrated that fibulin-5 deficiency and prolapse progression decreased vaginal contractility and increased vaginal wall stress. Future work is needed to better understand the processes that contribute to prolapse progression in order to guide diagnostic, preventative, and treatment strategies.

Constitutive Modeling of Corneal Tissue: Influence of Three-Dimensional Collagen Fiber Microstructure.

The cornea, the transparent tissue in the front of the eye, along with the sclera, plays a vital role in protecting the inner structures of the eyeball. The precise shape and mechanical strength of this tissue are mostly determined by the unique microstructure of its extracellular matrix. A clear picture of the 3D arrangement of collagen fibrils within the corneal extracellular matrix has recently been obtained from the secondary harmonic generation images. However, this important information about the through-thickness distribution of collagen fibrils was seldom taken into account in the constitutive modeling of the corneal behavior. This work creates a generalized structure tensor (GST) model to investigate the mechanical influence of collagen fibril through-thickness distribution. It then uses numerical simulations of the corneal mechanical response in inflation experiments to assess the efficacy of the proposed model. A parametric study is also done to investigate the influence of model parameters on numerical predictions. Finally, a brief comparison between the performance of this new constitutive model and a recent angular integration (AI) model from the literature is given.

Ex-vivo quantification of ovine pia arachnoid complex biomechanical properties under uniaxial tension

BackgroundThe pia arachnoid complex (PAC) is a cerebrospinal fluid-filled tissue conglomerate that surrounds the brain and spinal cord. Pia mater adheres directly to the surface of the brain while the arachnoid mater adheres to the deep surface of the dura mater. Collagen fibers, known as subarachnoid trabeculae (SAT) fibers, and microvascular structure lie intermediately to the pia and arachnoid meninges. Due to its structural role, alterations to the biomechanical properties of the PAC may change surface stress loading in traumatic brain injury (TBI) caused by sub-concussive hits. The aim of this study was to quantify the mechanical and morphological properties of ovine PAC.MethodsOvine brain samples (n = 10) were removed from the skull and tissue was harvested within 30 min post-mortem. To access the PAC, ovine skulls were split medially from the occipital region down the nasal bone on the superior and inferior aspects of the skull. A template was used to remove arachnoid samples from the left and right sides of the frontal and occipital regions of the brain. 10 ex-vivo samples were tested with uniaxial tension at 2 mm s−1, average strain rate of 0.59 s−1, until failure at < 5 h post extraction. The force and displacement data were acquired at 100 Hz. PAC tissue collagen fiber microstructure was characterized using second-harmonic generation (SHG) imaging on a subset of n = 4 stained tissue samples. To differentiate transverse blood vessels from SAT by visualization of cell nuclei and endothelial cells, samples were stained with DAPI and anti-von Willebrand Factor, respectively. The Mooney-Rivlin model for average stress–strain curve fit was used to model PAC material properties.ResultsThe elastic modulus, ultimate stress, and ultimate strain were found to be 7.7 ± 3.0, 2.7 ± 0.76 MPa, and 0.60 ± 0.13, respectively. No statistical significance was found across brain dissection locations in terms of biomechanical properties. SHG images were post-processed to obtain average SAT fiber intersection density, concentration, porosity, tortuosity, segment length, orientation, radial counts, and diameter as 0.23, 26.14, 73.86%, 1.07 ± 0.28, 17.33 ± 15.25 µm, 84.66 ± 49.18°, 8.15%, 3.46 ± 1.62 µm, respectively.ConclusionFor the sizes, strain, and strain rates tested, our results suggest that ovine PAC mechanical behavior is isotropic, and that the Mooney-Rivlin model is an appropriate curve-fitting constitutive equation for obtaining material parameters of PAC tissues.

A Pilot Study on Linking Tissue Mechanics with Load-Dependent Collagen Microstructures in Porcine Tricuspid Valve Leaflets.

The tricuspid valve (TV) is composed of three leaflets that coapt during systole to prevent deoxygenated blood from re-entering the right atrium. The connection between the TV leaflets’ microstructure and the tissue-level mechanical responses has yet to be fully understood in the TV biomechanics society. This pilot study sought to examine the load-dependent collagen fiber architecture of the three TV leaflets, by employing a multiscale, combined experimental approach that utilizes tissue-level biaxial mechanical characterizations, micro-level collagen fiber quantification, and histological analysis. Our results showed that the three TV leaflets displayed greater extensibility in the tissues’ radial direction than in the circumferential direction, consistently under different applied biaxial tensions. Additionally, collagen fibers reoriented towards the direction of the larger applied load, with the largest changes in the alignment of the collagen fibers under radially-dominant loading. Moreover, collagen fibers in the belly region of the TV leaflets were found to experience greater reorientations compared to the tissue region closer to the TV annulus. Furthermore, histological examinations of the TV leaflets displayed significant regional variation in constituent mass fraction, highlighting the heterogeneous collagen microstructure. The combined experimental approach presented in this work enables the connection of tissue mechanics, collagen fiber microstructure, and morphology for the TV leaflets. This experimental methodology also provides a new research platform for future developments, such as multiscale models for the TVs, and the design of bioprosthetic heart valves that could better mimic the mechanical, microstructural, and morphological characteristics of the native tricuspid valve leaflets.

Acoustic Fabrication of Collagen-Fibronectin Composite Gels Accelerates Microtissue Formation.

Ultrasound can influence biological systems through several distinct acoustic mechanisms that can be manipulated by varying reaction conditions and acoustic exposure parameters. We recently reported a new ultrasound-based fabrication technology that exploits the ability of ultrasound to generate localized mechanical forces and thermal effects to control collagen fiber microstructure non-invasively. Exposing solutions of type I collagen to ultrasound during the period of microfibril assembly produced changes in collagen fiber structure and alignment, and increased the biological activity of the resultant collagen hydrogels. In the extracellular matrix, interactions between fibronectin and collagen fibrils influence the biological activity of both proteins. Thus, in the present study, we examined how addition of fibronectin to collagen solutions prior to ultrasound exposure affects protein organization and the biological activity of the composite hydrogels. Results indicate that ultrasound can alter the distribution of fibronectin within 3D hydrogels via thermal and non-thermal mechanisms to produce composite hydrogels that support accelerated microtissue formation. The use of acoustic energy to drive changes in protein conformation to functionalize biomaterials has much potential as a unique, non-invasive technology for tissue engineering and regenerative medicine.

Acoustic modification of collagen hydrogels facilitates cellular remodeling.

Developing tunable biomaterials that have the capacity to recreate the physical and biochemical characteristics of native extracellular matrices (ECMs) with spatial fidelity is important for a variety of biomedical, biological, and clinical applications. Several factors have made the ECM protein, collagen I, an attractive biomaterial, including its ease of isolation, low antigenicity and toxicity, and biodegradability. However, current collagen gel formulations fail to recapitulate the range of collagen structures observed in native tissues, presenting a significant challenge in achieving the full potential of collagen-based biomaterials. Collagen fiber structure can be manipulated in vitro through mechanical forces, environmental factors, or thermal mechanisms. Here, we describe a new ultrasound-based fabrication technology that exploits the ability of ultrasound to generate localized mechanical forces to control the collagen fiber microstructure non-invasively. The results indicate that exposing soluble collagen to ultrasound (7.8 or 8.8 ​MHz; 3.2–10 ​W/cm2) during hydrogel formation leads to local variations in collagen fiber structure and organization that support increased levels of cell migration. Furthermore, multiphoton imaging revealed increased cell-mediated collagen remodeling of ultrasound-exposed but not sham-exposed hydrogels, including formation of multicellular aggregates, collagen fiber bundle contraction, and increased binding of collagen hybridizing peptides. Skin explant ​cultures obtained from diabetic mice showed similar enhancement of cell-mediated remodeling of ultrasound-exposed but not sham-exposed collagen hydrogels. Using the mechanical forces associated with ultrasound to induce local changes in collagen fibril structure and organization ​to functionalize native biomaterials is a promising non-invasive and non-toxic technology for tissue engineering and regenerative medicine.

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.

Thin Lamina Cribrosa Beams Have Different Collagen Microstructure Than Thick Beams.

PurposeTo compare the collagen microstructural crimp characteristics between thin and thick lamina cribrosa (LC) beams.MethodsSeven eyes from four sheep were fixed at 5 mm Hg IOP in 10% formalin. For each eye, one to three coronal cryosections through the LC were imaged with polarized light microscopy and analyzed to visualize the LC and determine collagen fiber microstructure. For every beam, we measured its width and three characteristics of the crimp of its collagen fibers: waviness, tortuosity, and amplitude. Linear mixed effects models were used to test whether crimp characteristics were associated with the LC beam width.ResultsFor each eye and over all the eyes, LC beam width was positively associated with crimp waviness and tortuosity, and negatively associated with crimp amplitude (P's < 0.0001). Thin beams, average width 13.11 μm, had average (SD) waviness, tortuosity, and amplitude of 0.27 (0.17) radians, 1.017 (0.028) and 1.88 (1.41) μm, respectively. For thick beams, average width 26.10 μm, these characteristics were 0.33 (0.18) radians, 1.025 (0.037) and 1.58 (1.36) μm, respectively.ConclusionsOur results suggest heterogeneity in LC beam mechanical properties. Thin beams were less wavy than their thicker counterparts, suggesting that thin beams may stiffen at lower IOP than thick beams. This difference may allow thin beams to support similar amounts of IOP-induced force as thicker beams, thus providing a similar level of structural support to the axons at physiologic IOP, despite the differences in width. Measurements of beam-level mechanical properties are needed to confirm these predictions.

Resliced image space construction for coronary artery collagen fibers.

Collagen fibers play an important role in the biomechanics of the blood vessel wall. The objective of this study was to determine the 3D microstructure of collagen fibers in the media and adventitia of coronary arteries. We present a novel optimal angle consistence algorithm to reform image slices in the visualization and analysis of 3D collagen images. 3D geometry was reconstructed from resliced image space where the 3D skeleton was extracted as the primary feature for accurate reconstruction of geometrical parameters. Collagen fibers (range 80–200) were reconstructed from the porcine coronary artery wall for the measurement of various morphological parameters. Collagen waviness and diameters were 1.37 ± 0.19 and 2.61 ± 0.89 μm, respectively. The biaxial distributions of orientation had two different peaks at 110.7 ± 25.2° and 18.4 ± 19.3°. Results for width, waviness, and orientation were found to be in good agreement with manual measurements. In addition to accurately measuring 2D features more efficiently than the manual approach, the present method produced 3D features that could not be measured in the 2D manual approach. These additional parameters included the tilt angle (5.10 ± 2.95°) and cross-sectional area (CSA; 5.98 ± 3.79 μm2) of collagen fibers. These 3D collagen reconstructions provide accurate and reliable microstructure for biomechanical modeling of vessel wall mechanics.

Consistent trilayer biomechanical modeling of aortic valve leaflet tissue

Aortic valve tissue exhibits highly nonlinear, anisotropic, and heterogeneous material behavior due to its complex microstructure. A thorough understanding of these characteristics permits us to develop numerical models that can shed insight on the function of the aortic valve in health and disease. Herein, we take a closer look at consistently capturing the observed physical response of aortic valve tissue in a continuum mechanics framework. Such a treatment is the first step in developing comprehensive multiscale and multiphysics models.We highlight two important aspects of aortic valve tissue behavior: the role of the collagen fiber microstructure and the native prestressing. We propose a model that captures these two features as well as the heterogeneous layer-scale topology of the tissue. We find the model can reproduce the experimentally observed multiscale mechanical behavior in a manner that provides intuition on the underlying mechanics.

大動脈中膜内エラスチン・コラーゲン線維の加圧時の微細構造変化

大動脈中膜内エラスチン・コラーゲン線維の加圧時の微細構造変化

Multiphoton microscopy observations of 3D elastin and collagen fiber microstructure changes during pressurization in aortic media.

Elastin and collagen fibers play important roles in the mechanical properties of aortic media. Because knowledge of local fiber structures is required for detailed analysis of blood vessel wall mechanics, we investigated 3D microstructures of elastin and collagen fibers in thoracic aortas and monitored changes during pressurization. Using multiphoton microscopy, autofluorescence images from elastin and second harmonic generation signals from collagen were acquired in media from rabbit thoracic aortas that were stretched biaxially to restore physiological dimensions. Both elastin and collagen fibers were observed in all longitudinal-circumferential plane images, whereas alternate bright and dark layers were observed along the radial direction and were recognized as elastic laminas (ELs) and smooth muscle-rich layers (SMLs), respectively. Elastin and collagen fibers are mainly oriented in the circumferential direction, and waviness of collagen fibers was significantly higher than that of elastin fibers. Collagen fibers were more undulated in longitudinal than in radial direction, whereas undulation of elastin fibers was equibiaxial. Changes in waviness of collagen fibers during pressurization were then evaluated using 2-dimensional fast Fourier transform in mouse aortas, and indices of waviness of collagen fibers decreased with increases in intraluminal pressure. These indices also showed that collagen fibers in SMLs became straight at lower intraluminal pressures than those in EL, indicating that SMLs stretched more than ELs. These results indicate that deformation of the aorta due to pressurization is complicated because of the heterogeneity of tissue layers and differences in elastic properties of ELs, SMLs, and surrounding collagen and elastin.

Inverted orientation improves decellularization of whole porcine hearts.

The use of decellularized tissue as a suitable scaffold for engineered tissue has emerged over the past decade as one of the most promising biofabrication platforms. The decellularization process removes all native cells, leaving the natural biopolymers, extracellular matrix materials and native architecture intact. This manuscript describes heart orientation as important in optimizing decellularization efficiency and maintaining extracellular matrix integrity. To our knowledge, this is the first study to assess flow dynamics in a whole heart throughout the decellularization procedure. Our findings compared to currently published methods demonstrate that continuous complex real-time measurements and analyses are required to produce an optimal scaffold for cardiac regeneration.