Collagen Fiber Angle Research Articles

Effects of Age and Multiple Vaginal Births on Lower Urinary Tract Structure and Function in Nonhuman Primates.

The relative roles of urinary sphincter damage, aging, and childbirth in stress urinary incontinence (SUI), have not been established. This study was performed to elucidate the roles of these factors. The study included: (1) 8 female cynomolgus monkeys (17-19 years of age and 7-8 vaginal births each); (2) six 5-yearold nulliparous monkeys with surgically created chronic urinary sphincter dysfunction; and (3) six 5-year-old, nulliparous, nosurgery controls. Sedated abdominal leak point pressure (ALPP) and maximum urethral sphincter pressures (MUP) were measured. Sphincters, bladders, and pelvic support muscles were quantified for collagen content. Additionally, bladders were analyzed for collagen fiber thickness, length, and angle using CT-FIRE analysis of Picrosirius red-stained tissues. Resting MUP values were similar in the controls and older multiparous monkeys (P>0.05). However, aging and multiple births reduced pudendal nerve-stimulated increases in MUP (P<0.05 vs. controls). ALPP values were lower in the older multiparous versus younger groups of monkeys (P<0.05). Sphincter collagen content was greater, and muscle content less, in the injury model (P<0.05 vs. controls). However, these measures were not affected by age and childbirth (P>0.05 vs. young groups). Bladder collagen content was greater, and muscle content less, in the old multiparous monkeys (P<0.05 vs. younger groups). Additionally, collagen fibers were thicker and more angular in the bladders of the older multiparous monkeys than in the other nonhuman primate groups (P<0.05). Pelvic support muscles had higher collagen and lower muscle content in the older multiparous monkeys than in the younger groups of monkeys (P<0.05). SUI, associated with aging and multiple childbirths, appeared to be more strongly associated with bladder dysfunction, reduced pelvic muscle support, and the compensatory response to neural stimulation than with selective urinary sphincter dysfunction.

Real-time polarization microscopy of fibrillar collagen in histopathology

Over the past two decades, fibrillar collagen reorganization parameters such as the amount of collagen deposition, fiber angle and alignment have been widely explored in numerous studies. These parameters are now widely accepted as stromal biomarkers and linked to disease progression and survival time in several cancer types. Despite all these advances, there has not been a significant effort to make it possible for clinicians to explore these biomarkers without adding steps to the clinical workflow or by requiring high-cost imaging systems. In this paper, we evaluate previously described polychromatic polarization microscope (PPM) to visualize collagen fibers with an optically generated color representation of fiber orientation and alignment when inspecting the sample by a regular microscope with minor modifications. This system does not require stained slides, but is compatible with histological stains such as H&E. Consequently, it can be easily accommodated as part of regular pathology review of tissue slides, while providing clinically useful insight into stromal composition.

Osteoarthritic meniscal entheses exhibit altered collagen fiber orientation

ABSTRACT Purpose/Aim The knee menisci are vital for maintaining the stability of the joint, allowing for force distribution, and protecting the underlying articular cartilage during loading. Each meniscus is attached to the underlying bone via two ligamentous entheses composed of collagen fibers that are continuous throughout all four zones of the attachment: ligament, uncalcified fibrocartilage, calcified fibrocartilage, and subchondral bone. The collagen fibers of the meniscal entheses are important for proper functionality of the entheses, particularly in preventing meniscal extrusion which is a common hallmark of osteoarthritis. The goal of this work was to assess changes in collagen fiber orientation present in osteoarthritic knee joints. Materials and Methods Entheses were harvested from patients undergoing total knee arthroplasties and prepared histological sections were stained with picrosirius red to identify collagen fiber angle and fiber deviation. Results In the calcified fibrocartilage the collagen fibers of the lateral anterior enthesis inserted at significantly (p < 0.1) shallower angles, and the fiber deviation was significantly (p < 0.1) less compared to the lateral posterior enthesis. These differences in the calcified fibrocartilage may occur as an adaptation to loading regimes of the osteoarthritic joint. When compared to the collagen fiber orientation of healthy entheses, collagen fibers in osteoarthritic tissue inserted at shallower insertion angles and demonstrated higher levels of deviation. Conclusions Changes to meniscal enthesis collagen fiber orientation with end stage osteoarthritis could offer an explanation for the change in functionality of diseased tissue and may contribute to meniscal extrusion and ultimately the degeneration of articular cartilage.

Collagen Organization in Relation to Ductal Carcinoma In Situ Pathology and Outcomes.

There is widespread interest in discriminating indolent from aggressive ductal carcinoma in situ (DCIS). We sought to evaluate collagen organization in the DCIS tumor microenvironment in relation to pathologic characteristics and patient outcomes. We retrieved fixed tissue specimens for 90 DCIS cases within the population-based Vermont DCIS Cohort. We imaged collagen fibers within 75 μm of the tumor/stromal boundary on hematoxylin and eosin-stained slides using multiphoton microscopy with second-harmonic generation. Automated software quantified collagen fiber length, width, straightness, density, alignment, and angle to the tumor/stroma boundary. Factor analysis identified linear combinations of collagen fiber features representing composite attributes of collagen organization. Multiple collagen features were associated with DCIS grade, necrosis pattern, or periductal fibrosis (P < 0.05). After adjusting for treatments and nuclear grade, risk of recurrence (defined as any second breast cancer diagnosis) was lower among cases with greater collagen fiber width [hazard ratio (HR), 0.57 per one standard deviation increase; 95% confidence interval (CI), 0.39-0.84] and fiber density (HR, 0.60; 95% CI, 0.42-0.85), whereas risk was elevated among DCIS cases with higher fiber straightness (HR, 1.47; 95% CI, 1.05-2.06) and distance to the nearest two fibers (HR, 1.47; 95% CI, 1.06-2.02). Fiber length, alignment, and fiber angle were not associated with recurrence (P > 0.05). Five composite factors were identified, accounting for 72.4% of the total variability among fibers; three were inversely associated with recurrence (HRs ranging from 0.60 to 0.67; P ≤ 0.01). Multiple aspects of collagen organization around DCIS lesions are associated with recurrence risk. Collagen organization should be considered in the development of prognostic DCIS biomarker signatures.

T2* and quantitative susceptibility mapping in an equine model of post-traumatic osteoarthritis: assessment of mechanical and structural properties of articular cartilage

To investigate the potential of quantitative susceptibility mapping (QSM) and T2* relaxation time mapping to determine mechanical and structural properties of articular cartilage via univariate and multivariate analysis. Samples were obtained from a cartilage repair study, in which surgically induced full-thickness chondral defects in the stifle joints of seven Shetland ponies caused post-traumatic osteoarthritis (14 samples). Control samples were collected from non-operated joints of three animals (6 samples). Magnetic resonance imaging (MRI) was performed at 9.4T, using a 3-D multi-echo gradient echo sequence. Biomechanical testing, digital densitometry (DD) and polarized light microscopy (PLM) were utilized as reference methods. To compare MRI parameters with reference parameters (equilibrium and dynamic moduli, proteoglycan content, collagen fiber angle and -anisotropy), depth-wise profiles of MRI parameters were acquired at the biomechanical testing locations. Partial least squares regression (PLSR) and Spearman's rank correlation were utilized in data analysis. PLSR indicated a moderate-to-strong correlation (ρ=0.49-0.66) and a moderate correlation (ρ=0.41-0.55) between the reference values and T2* relaxation time and QSM profiles, respectively (excluding superficial-only results). PLSR correlations were noticeably higher than direct correlations between bulk MRI and reference parameters. 3-D parametric surface maps revealed spatial variations in the MRI parameters between experimental and control groups. Quantitative parameters from 3-D multi-echo gradient echo MRI can be utilized to predict the properties of articular cartilage. With PLSR, especially the T2* relaxation time profile appeared to correlate with the properties of cartilage. Furthermore, the results suggest that degeneration affects the QSM-contrast in the cartilage. However, this change in contrast is not easy to quantify.

An investigation on mechanical property differences of arteries based on structure and morphology analysis of collagen fibers

ObjectiveThe structure and morphology of collagen fibers greatly influence the mechanical property of arteries. Cyosection immunofluorescence represents a powerful technique for the direct observation of the structure and morphology of the collagen fibers. The objective of this work is to investigate the mechanical property variation of arteries between elderly and young rats, and to get insight of the mechanical property differences based on the structure and morphology of the collagen fibers using cyosection immunofluorescence method.MethodFemoral, iliac, carotid and abdominal arteries were obtained from 4 pairs of healthy Sprague Dawley rats with the maturity of 8 weeks (young) and 8 months (elderly). The mechanical properties of the obtained arteries were investigated based on the cross‐section morphology. The structure and morphology of the collagen fibers were evidently observed using cyosection immunofluorescence method.ResultsThe abdominal artery segments exhibited greater orientation difference between elderly and young groups compared with those of carotid and femoral arteries. It was observed that elderly rats showed thicker and higher content ratio collagen fibers of abdominal aorta compared with young group using cyosection immunofluorescence method. In addition, the abdominal aorta collagen fiber angle frequency distribution in the median layer exhibited a broad peak (young group: 0°–30°; elderly group: 17°–49°, P value&lt;0.05), while it showed more uniform distribution ranging from −90° to 90° in femoral and carotid arteries.ConclusionsThe abdominal artery in elderly rats shows distinguished variation in the mechanical property compared with young rats in terms of the angle distribution and fiber width than those of femoral artery, carotid artery, and iliac artery. Consequently, this finding reveals the morphological growth and aging laws of arteries to understand microstructure‐based constitutive model in the vascular remodeling and disease.Support or Funding InformationThis research is supported in part by the Shenzhen Science and Technology Innovation Institution (China) Grant JCYJ20160427170536358 (Y Huo).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

GAG content, fiber stiffness, and fiber angle affect swelling-based residual stress in the intact annulus fibrosus.

Biological tissues with a high glycosaminoglycan (GAG) content have an excellent ability to swell by absorbing water molecules from the surrounding environment. Our recent work showed that anisotropy in tissue swelling depends on the fiber-network architecture, including fiber angle, fiber stiffness, and lamellae structure. However, that work did not evaluate the effect of in situ boundary conditions, such as the kidney-bean shape of the annulus fibrosus (AF), on swelling behavior. The biochemical composition of intact AF is inhomogeneous with respect to GAG composition, collagen fiber angle, and fiber stiffness. Moreover, the GAG content in the inner AF decreases significantly with degeneration. In this study, we investigated the role of GAG content, fiber angle, and fiber stiffness in AF swelling and residual strain development using a finite element model based on a human lumbar disk. Our results showed that the annular ring structure had a great impact on swelling by developing region-dependent compressive stress/stretch in the inner layers and tensile stress/stretch in the outer AF. Swelling-based residual stretch was comparable to experimentally measured values, suggesting an important role of tissue swelling in maintaining residual stresses. Moreover, GAG loss in the inner AF, as observed with degeneration, decreased circumferential-direction stress by over 65%. Homogeneous distributions of fiber angle and stiffness overestimated or underestimated AF swelling behavior, such as swelling ratio, circumferential/axial stretch, and fiber stretch/reorientation. These findings demonstrate the need to include native fiber architecture in finite element models, to accurately predict tissue failure, as well as to cultivate engineered disks.

Skin stiffness in ray-finned fishes: Contrasting material properties between species and body regions.

The skin of aquatic vertebrates surrounds all the mechanical lineages of the body and must, therefore, play an important role in locomotion. A cross-woven collagenous dermal design has converged across several clades of vertebrates. Despite this intriguing pattern, the biomechanical role of skin in swimming fishes remains largely unknown. A direct force transmission role for fish skin has been proposed, a hypothesis that is supported by the arrangement of the connective tissues linking the skin to the axial musculature. To evaluate this direct force-transmission hypothesis, we undertook hundreds of uniaxial tensile tests on skin samples from coho salmon (Oncorhynchus kisutch), Florida pompano (Trachinotus carolinus), and red snapper (Lutjanus campechanus). To do this, we developed highly precise, low-cost, custom-built material testing units. To augment our data, we also assembled a data set of skin stiffness of four additional species of actinopterygians fishes from previously published studies. We found that stiffness varies significantly between species and that the skin of our study species was increasingly stiff along a rostrocaudal gradient. Placing our results in the context of the limited body of previous work, we found that species with lower skin stiffness exhibit shorter propulsive wavelengths and low thrust production at the caudal fin and species with higher skin stiffness possess longer propulsive wavelengths and high thrust production at the caudal fin. In addition, we found that mean collagen fiber angle was close to 50° and that fiber angle was lower in posterior samples than in anterior and midlateral samples. Taken as a whole, our mechanical and morphological results support the hypothesis that the skin functions as an important direct force-transmission device in actinopterygians whereby muscular force generated in anterior myotomes is transmitted to the posterior of the body through the increasingly stiff skin.

Mechanical properties of striped bass fish skin: Evidence of an exotendon function of the stratum compactum

Teleost fish skin is a multifunctional natural material with high penetration resistance owing to specialized puncture mechanisms of both the individual scale and the intact scaled integument. In this paper, we explore the possible additional role of the skin in fish undulatory locomotion by examining the structural and mechanical properties of the dermal stratum (s.) compactum layer of striped bass (Morone saxatilis) skin. The structure, mechanical response and function of s. compactum was investigated by combining several methods: optical microscopy and histology, tensile tests on descaled skin specimens in different anatomical locations and orientations, puncture tests, and flexural tests on whole fish with disruption of the s. compactum. Local histological features of the s. compactum, such as collagen fiber angle and degree of crimping, were shown to explain corresponding patterns determined for the tensile properties of the skin along the long axis of the fish, including changes in stiffness, strength and locking strain at stiffening. The fish bending tests demonstrated a tendon-like response of the whole fish and a significant contribution of the s. compactum to the flexural stiffness of the fish. Collectively, the findings show that the s. compactum is a strong tissue with a tendon-like nonlinear response, and which provides an appreciable mechanical protection against sharp puncture and lacerations. Our results also support the theory of an exotendon function of the s. compactum in teleost fish skin. These findings may inspire the design of new multifunctional protective and locomotory systems for a variety of engineering applications.

Diet Alters Age‐related Remodeling of Aortic Extracellular Matrix in Mice Susceptible to Atherosclerosis

Atherosclerosis is a chronic inflammatory disease of the arterial wall, constituting a significant source of morbidity and mortality in the United States. In many arteries, remodeling of the collagenous framework occurs as a result of atheromatous plaque development in the intima of the arterial wall, termed atherogenesis. A major challenge in understanding how collagen contributes to the functional properties of the arterial wall is identifying how vascular cells respond to mechanical loading by restructuring the surrounding extracellular matrix. Therefore, the objective of this study is to evaluate collagen architecture to understand the remodeling capabilities of the aorta during aging with or without atherosclerotic lesion development. Groups of 5 apolipoprotein E (apoE) knockout (KO) mice were fed a chow diet for 6 weeks or an additional 6 months. To promote atherosclerotic lesion formation within the aorta, 5 mice were fed a high-fat Western diet (42 kcal% fat) for an additional 6 months. The apoE KO mice are susceptible to atherosclerosis due to a genetic defect in their ability to clear circulating plasma lipoproteins. Aortic diameters were measured in vivo prior to euthanasia using small animal ultrasound (Vevo 770). Following the duration of each diet, mice were euthanized at 6 weeks old or ~7.5 months old. Segments from non-lesioned areas of the thoracic and abdominal aorta were imaged using second harmonic generation (SHG) microscopy. Optical sectioning with SHG microscopy permits visualization of collagen fibers throughout the vessel wall thickness without staining. The images of the collagen fibers were used to determine angle measurements with the circumferential (0°) and axial directions (±90°) serving as the reference points. Image analysis was performed using Continuity 6.4b software. A multiple regression analysis was performed in R to identify variables (mouse age, diet, anatomical location, through-thickness position) having a significant effect on mean fiber angle. Our data indicate that the aortas of 6-week old apoE KO mice have a mean absolute collagen fiber angle of 35.17±1.89° but that fibers become more circumferentially aligned at 12.52° after 6 months of Western diet feeding. In contrast, collagen fiber average absolute angle undergoes a significant increase in apoE KO mice fed a chow diet for 6 months, with fibers becoming more axially aligned, at 61.61°. Six-month-old mice on Western diet demonstrate increased gain in body weight compared to 6-month-old mice on chow diet (31.05±4.21g vs 24.53±2.99g p<0.05), which further supports differences in these mice independent of aging. Body weight for 6-week-old mice was 16.45±2.55g. However, aortic diameters were not significantly different between mice on chow diet for 6 months vs. those on Western diet. We conclude that the aorta, while showing an equivalent increase in collagen content during aging, displays distinct remodeling responses in the presence of atherogenic stimuli, even in non-lesioned areas. Acknowledgements: Special thanks to Dr. Bruce Gao and Zhonghai Wang at Clemson University for use of the SHG microscope. Support or Funding Information This work was supported by National Science Foundation grants EPS-0903795 and CMMI-1200358, and National Institute of Health grants P30 GM103336-01A1 and R01 CA154731. Average collagen fiber angle diverges with aging in apoE KO mice on chow diet vs. Western diet. Compared to 6-week old apoE KO mice, multiple regression analysis showed a significant difference in average absolute fiber angle in response to age and high-fat diet. On average, absolute fiber angle was increased by 26.44±2.67° in older mice on same diet and reduced 22.35±2.71° in mice fed a Western diet for 6 months. Collagen content (% area) for each of the three treatment groups obtained from Picrosirius Red staining. Collagen content increases significantly with age from 6 weeks to 7.5 months (P<0.05), but is not altered by Western diet. CD = chow diet; WD = Western diet

Theory of MRI contrast in the annulus fibrosus of the intervertebral disc.

Here we develop a three-dimensional analytic model for MR image contrast of collagen lamellae in the annulus fibrosus of the intervertebral disc of the spine, based on the dependence of the MRI signal on collagen fiber orientation. High-resolution MRI scans were performed at 1.5 and 7T on intact whole disc specimens from ovine, bovine, and human spines. An analytic model that approximates the three-dimensional curvature of the disc lamellae was developed to explain inter-lamellar contrast and intensity variations in the annulus. The model is based on the known anisotropic dipolar relaxation of water in tissues with ordered collagen. Simulated MRI data were generated that reproduced many features of the actual MRI data. The calculated inter-lamellar image contrast demonstrated a strong dependence on the collagen fiber angle and on the circumferential location within the annulus. This analytic model may be useful for interpreting MR images of the disc and for predicting experimental conditions that will optimize MR image contrast in the annulus fibrosus.

Comparison of Aortic Collagen Fiber Angle Distribution in Mouse Models of Atherosclerosis Using Second-Harmonic Generation (SHG) Microscopy.

Characterization of collagen fiber angle distribution throughout the blood vessel wall provides insight into the mechanical behavior of healthy and diseased arteries and their capacity to remodel. Atherosclerotic plaque contributes to the overall mechanical behavior, yet little is known experimentally about how collagen fiber orientation is influenced by atherogenesis. We hypothesized that atherosclerotic lesion development, and the factors contributing to lesion development, leads to a shift in collagen fiber angles within the aorta. Second-harmonic generation microscopy was used to visualize the three-dimensional organization of collagen throughout the aortic wall and to examine structural differences in mice maintained on high-fat Western diet versus age-matched chow diet mice in a model of atherosclerosis. Image analysis was performed on thoracic and abdominal sections of the aorta from each mouse to determine fiber orientation, with the circumferential (0°) and blood flow directions (axial ±90°) as the two reference points. All measurements were used in a multiple regression analysis to determine the factors having a significant influence on mean collagen fiber angle. We found that mean absolute angle of collagen fibers is 43° lower in Western diet mice compared with chow diet mice. Mice on a chow diet have a mean collagen fiber angle of ±63°, whereas mice on a Western diet have a more circumferential fiber orientation (~20°). This apparent shift in absolute angle coincides with the development of extensive aortic atherosclerosis, suggesting that atherosclerotic factors contribute to collagen fiber angle orientation.

Moderately Degenerated Human Intervertebral Disks Exhibit a Less Geometrically Specific Collagen Fiber Orientation Distribution.

Study Design Collagen fiber orientation analysis in moderately degenerated human cadaveric annulus fibrosus (AF) tissue samples.Objective Little is known about the changes in tissue architecture during early degeneration of intervertebral disks (IVDs). As collagen organization strongly affects the disk function, the objective of this study was to quantify the AF collagen orientation and its spatial distribution in moderately degenerated IVDs (Pfirrmann grade III).Methods AF tissue samples were dissected from four circumferential (anterior, left and right lateral, and posterior) and two radial (outer and inner) locations. Cryosections were imaged using Second Harmonic Generation microscopy, and the collagen fiber orientations per location were determined utilizing a fiber-tracking image analysis algorithm. Also, the proportionality between the fibers aligned in the primary direction versus other oriented fibers was determined.Results Mean collagen fiber angles ranged between 21 and 31 degrees for outer and 15 to 19 degrees for inner AF samples. Mean collagen orientations at circumferential locations were only significantly different from each other at inner anterior and lateral location. Similarly, fiber angles between the outer and inner AF were not significantly different except at the posterior location. Fiber orientation proportionality did not show large variations. Except for a significant difference in outer AF proportionality between posterior and lateral positions, no other differences were observed.Conclusion The results of this study provide the first quantitative evidence that the collagen fiber orientation of moderately degenerated disks exhibits a spatial rather than homogeneous distribution and typical collagen orientation gradients characterizing healthy IVDs are only partially retained.

Diet‐induced Vascular Remodeling Produces a Shift in Collagen Fiber Angle Distribution in a Mouse Model of Atherosclerosis

Atherosclerosis is a chronic inflammatory disease characterized by accumulation of lipids in the artery wall. Clinical complications include plaque rupture, in which thrombosis of the artery can lead to myocardial infarction. In response to plaque development, arteries undergo long-lasting changes in size/composition, termed vascular remodeling. The study objective is to measure collagen fiber angle distribution to understand how the aortic matrix remodels in response to altered mechanical loading resulting from plaque formation. To promote aortic plaque formation, apolipoprotein E (apoE) knockout (KO) and apoE matrix metalloproteinase-12 (MMP12) DKO mice were fed a Western diet for 6 months. Control mice were fed a chow diet. Segments of the thoracic and abdominal aorta were optically sectioned using second harmonic generation (SHG) microscopy to visualize collagen fibers and analyzed using Continuity 6. Multivariate statistical analysis was performed in R to identify variables (mouse strain, diet, aortic position, relative depth in wall) having a significant effect on absolute fiber angle. Our data indicate that the aortas of both apoE KO and apoE MMP12 DKO mice fed Western diet for 6 months undergo a significant shift in collagen fiber angle distribution. This shift coincides with the development of extensive aortic atherosclerosis, suggesting that changes in collagen fiber angle may be part of the vascular remodeling process. We thank Dr. Bruce Gao for use of the SHG microscope. This work was funded by NSF EPS-0903795 and CMMI-1200358.

Abstract P1-06-06: Alteration of stromal collagen fiber orientation in DCIS

Abstract Approximately 20% of new diagnoses of breast cancer are ductal carcinoma in situ (DCIS), a non-invasive form of breast cancer. Treatment decision-making for DCIS is challenging since current predictors of disease-free survival are limited, so that most women are presented with options for surgery, radiation and tamoxifen - all options with consequences for quality of life. Prior studies of prognostic factors for DCIS have focused on morphologic, genetic, and protein expression patterns of the DCIS cells. However, laboratory evidence suggests that the tumor microenvironment may play a key role in tumor invasion and progression. Collagen is the most abundant component of the stroma surrounding the breast ducts in which cancers develop. We previously observed that, in invasive breast cancer, tumors with greater numbers of collagen fibers aligned perpendicularly from the tumor were more likely to predict poor survival than tumors with collagen fibers in primarily parallel patterns near the tumor boundary (Conklin Am J Pathol 2011). To improve our ability to predict breast cancer outcomes in women with DCIS, we examined the alignment of collagen adjacent to ducts affected by DCIS to test whether alignment patterns were similar to patterns observed in tissue labeled as “normal” from biopsy and surgical sections. We evaluated collagen alignment in 255 Wisconsin women diagnosed with DCIS in 1997-2000 and followed for a median of 11.2 years (range 1-15). Stromal collagen alignment was evaluated from routine H&amp;E tissue slides prepared at the time of diagnosis using second harmonic generation (SHG) microscopy, a label-free multiphoton laser scanning technique that selectively images collagen. SHG images were acquired and evaluated for 3-5 regions on each DCIS and normal slide for each patient; the angles of collagen fibers with respect to the DCIS lesion/stroma boundary were calculated using customized imaging software. Data for the distribution of angles were compared for normal ducts and DCIS lesions using compositional data analysis with the number of fibers totaled according to 5-angle bins (1-5, 6-10, 11-15, …, 86-90 degrees). Repeated measures linear regression models were fit to log-transformed ratios of binned counts as a function of tissue type. Dependence among repeated counts within a single region was modeled using an unstructured variance-covariance matrix. Dependence among measurements within a single subject was modeled using a compound symmetry correlation structure. Overall, the distribution of collagen fiber angles from DCIS lesions differed significantly (P = 0.0002) from the distribution of collagen fibers surrounding normal ducts. Collagen fibers surrounding DCIS lesions were 11-18% more likely to orient at 75-90 degrees relative to the lesion boundary than fibers surrounding normal ducts; fibers were more similarly aligned in both DCIS lesions and normal ducts at other smaller angles. These results underscore the relevance of the tumor microenvironment, in particular the arrangement of the collagen fiber matrix. Planned data analysis will next examine whether collagen fiber alignment patterns differ between DCIS patients who did and did not experience a second breast cancer diagnosis over the course of follow-up. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-06-06.

Three-Dimensional Distribution of Transverse Collagen Fibers in the Anterior Human Corneal Stroma

Recent investigations of human corneal structure and biomechanics have shown that stromal collagen fibers (lamellae) are organized into a complex, highly intertwined three-dimensional meshwork of transverse oriented fibers that increases stromal stiffness and controls corneal shape. The purpose of this study was to characterize the three-dimensional distribution of transverse collagen fibers along the major meridians of the cornea using an automated method to rapidly quantify the collagen fibers' angular orientation. Three eyes from three donors were perfusion-fixed under pressure, excised, and cut into four quadrants. Quadrants were physically sectioned using a vibratome and scanned using nonlinear optical high-resolution macroscopy. Planes were analyzed numerically using software to identify collagen fiber angles relative to the corneal surface, stromal depth, and radial position within the anterior 250 μm of the stroma. The range of fiber angles and the fiber percentage having an angular displacement greater than ±3.5° relative to the corneal surface ("transverse fibers") was highest in the anterior stroma and decreased with depth. Numerical analysis showed no significant differences in fiber angles and transverse fibers between quadrants, meridians, and radial position. These results match our previous observation of a depth-dependent gradient in stromal collagen interconnectivity in the central cornea, and show that this gradient extends from the central cornea to the limbus. The lack of a preferred distribution of angled fibers with regard to corneal quadrant or radial position likely serves to evenly distribute loads and to avoid the formation of areas of stress concentration.

Biaxial Tensile Testing and Constitutive Modeling of Human Supraspinatus Tendon

The heterogeneous composition and mechanical properties of the supraspinatus tendon offer an opportunity for studying the structure-function relationships of fibrous musculoskeletal connective tissues. Previous uniaxial testing has demonstrated a correlation between the collagen fiber angle distribution and tendon mechanics in response to tensile loading both parallel and transverse to the tendon longitudinal axis. However, the planar mechanics of the supraspinatus tendon may be more appropriately characterized through biaxial tensile testing, which avoids the limitation of nonphysiologic traction-free boundary conditions present during uniaxial testing. Combined with a structural constitutive model, biaxial testing can help identify the specific structural mechanisms underlying the tendon's two-dimensional mechanical behavior. Therefore, the objective of this study was to evaluate the contribution of collagen fiber organization to the planar tensile mechanics of the human supraspinatus tendon by fitting biaxial tensile data with a structural constitutive model that incorporates a sample-specific angular distribution of nonlinear fibers. Regional samples were tested under several biaxial boundary conditions while simultaneously measuring the collagen fiber orientations via polarized light imaging. The histograms of fiber angles were fit with a von Mises probability distribution and input into a hyperelastic constitutive model incorporating the contributions of the uncrimped fibers. Samples with a wide fiber angle distribution produced greater transverse stresses than more highly aligned samples. The structural model fit the longitudinal stresses well (median R(2) ≥ 0.96) and was validated by successfully predicting the stress response to a mechanical protocol not used for parameter estimation. The transverse stresses were fit less well with greater errors observed for less aligned samples. Sensitivity analyses and relatively affine fiber kinematics suggest that these errors are not due to inaccuracies in measuring the collagen fiber organization. More likely, additional strain energy terms representing fiber-fiber interactions are necessary to provide a closer approximation of the transverse stresses. Nevertheless, this approach demonstrated that the longitudinal tensile mechanics of the supraspinatus tendon are primarily dependent on the moduli, crimp, and angular distribution of its collagen fibers. These results add to the existing knowledge of structure-function relationships in fibrous musculoskeletal tissue, which is valuable for understanding the etiology of degenerative disease, developing effective tissue engineering design strategies, and predicting outcomes of tissue repair.

Collagen morphology in human meniscal attachments: A SEM study

Qualitative analysis of meniscal attachments from five human knees was completed using scanning electron microscopy (SEM). In addition, quantitative analysis to determine the collagen crimping angle and length in each attachment was done. Morphological differences were revealed between the distinct zones of the attachments from the meniscus transition to the bony insertion. Collagen fibers near to the meniscus appeared inhomogeneous in a radial cross-section view. The sheath surrounding the fibers seemed loose compared with the membrane wrapping around the fibers in the menisci. The midsubstance of human meniscal attachments was composed of collagen fibers running parallel to the longitudinal axis, with a few fibers running obliquely, and others transversely. The bony insertion showed that the crimping pattern vanishes as the collagen fibers approach the fibrocartilagenous enthesis. There were no differences between attachments for crimping angle or length. Collagen crimping angles for all attachments were similar with values of approximately 22°. Crimp length values tended to be smaller for the medial attachments (MA: 4.76 ± 1.95 μm; MP: 3.72 ± 2.31 μm) and higher for the lateral (LA: 6.49 ± 2.34 μm, LP: 6.91 ± 2.29 μm). SEM was demonstrated to be an effective method for revealing the morphology of fibrous connective tissue. The data of collagen fiber length and angle found in this study will allow for better development of microstructural models of meniscal attachments. This study will help to better understand the relation between the morphology and the architecture of collagen and the mechanical behavior of meniscal attachments.

Residual stresses in soft tissue as a consequence of growth and remodeling: application to an arterial geometry

Abstract We develop a thermodynamically consistent model for growth and remodeling in elastic arteries. The model is specialized to a cylindrical geometry, strain energy of the Holzapfel–Gasser–Ogden type and remodeling of the collagen fiber angle. A numerical method for calculating the evolution of the adaptation process is developed. For a particular choice of the thermodynamic forces of growth and remodeling (configurational forces), it is shown that an almost homogeneous transmural axial and tangential stress distribution is obtained. Residual stresses develop during this adaption process and these resemble what is found in experiments and by parameter identification methods.

A strain energy function for arteries accounting for wall composition and structure

Identification of a Strain Energy Function (SEF) is used when describing the complex mechanical properties of soft biological tissues such as the arterial wall. Classic SEFs, such as the one proposed by Chuong and Fung (J. Biomech. Eng. 105(3) (1983) 268), have been mostly phenomenological and neglect the particularities of the wall structure. A more structural model was proposed by Holzapfel et al. (J. Elasticity 61 (2000) 1–48.) when they included the characteristic angle at which the collagen fibers are helically wrapped, resulting in an excellent SEF for applications such as finite element modeling. We have expanded upon the idea of structural SEFs by including not only the wavy nature of the collagen but also the fraction of both elastin and collagen contained in the media, which can be determined by histology. The waviness of the collagen is assumed to be distributed log-logistically. In order to evaluate this novel SEF, we have used it to fit experimental data from inflation–extension tests performed on rat carotids. We have compared the results of the fit to the SEFs of Choung and Fung and Holzapfel et al. The novel SEF is found to behave similarly to that of Holzapfel et al., both succeed in describing the typical S-shaped pressure-radius curves with comparable quality of fit. The parameters of the novel SEF obtained from the fitting, bearing the physical meaning of the elastic modulus of collagen, the elastic modulus of elastin, the collagen waviness, and the collagen fiber angle, were compared to experimental data and discussed.