Collagen Anisotropy Research Articles

An innovative 4D printing approach for fabrication of anisotropic collagen scaffolds

Collagen anisotropy is known to provide the essential topographical cues to guide tissue-specific cell function. Recent work has shown that extrusion-based printing using collagenous inks yield 3D scaffolds with high geometric precision and print fidelity. However, these scaffolds lack collagen anisotropy. In this study, extrusion-based 3D printing was combined with a magnetic alignment approach in an innovative 4D printing scheme to generate 3D collagen scaffolds with high degree of collagen anisotropy. Specifically, the 4D printing process parameters-collagen (Col):xanthan gum (XG) ratio (Col:XG; 1:1, 4:1, 9:1 v/v), streptavidin-coated magnetic particle concentration (SMP; 0, 0.2, 0.4 mg ml-1), and print flow speed (2, 3 mm s-1)-were modulated and the effects of these parameters on rheological properties, print fidelity, and collagen alignment were assessed. Further, the effects of collagen anisotropy on human mesenchymal stem cell (hMSC) morphology, orientation, metabolic activity, and ligamentous differentiation were investigated. Results showed that increasing the XG composition (Col:XG 1:1) enhanced ink viscosity and yielded scaffolds with good print fidelity but poor collagen alignment. On the other hand, use of inks with lower XG composition (Col:XG 4:1 and 9:1) together with 0.4 mg ml-1SMP concentration yielded scaffolds with high degree of collagen alignment albeit with suboptimal print fidelity. Modulating the print flow speed conditions (2 mm s-1) with 4:1 Col:XG inks and 0.4 mg ml-1SMP resulted in improved print fidelity of the collagen scaffolds while retaining high level of collagen anisotropy. Cell studies revealed hMSCs orient uniformly on aligned collagen scaffolds. More importantly, collagen anisotropy was found to trigger tendon or ligament-like differentiation of hMSCs. Together, these results suggest that 4D printing is a viable strategy to generate anisotropic collagen scaffolds with significant potential for use in tendon and ligament tissue engineering applications.

Abstract PO3-07-01: Multiscale Computational Analysis of Patient-Matched Multi-Modal Imaging of Breast Tissue Microenvironment

Abstract Introduction: Mammographic breast density is a strong quantitative cancer risk factor, but its link to underlying tissue and cellular level changes are weakly understood. Understanding the complex restructuring changes in the breast tumor microenvironment and its impact on breast density is integral to improving risk assessment and treatment strategies. A novel approach was developed to stratify mammographic dense breast tissue into areas of active dense tissue and passive dense tissue. Active dense tissue is structurally reorganizing and links to cancer dynamics, whereas passive dense mammographic tissue remains organized.1,2 A complementary wavelet-based analysis technique to measure the multiscale anisotropy of collagen fibers from Second-Harmonic Generation (SHG) imaging demonstrated clinical cancer diagnostic potential in mouse carcinoma and human pancreatic cancer.3,4 This tool is now leveraged for a multi-modal study in which patient-matched H&E tissue biopsies are imaged using both bright-field microscopy and SHG imaging to compare with the patient’s corresponding mammograms. Stratifying the mammographic density into active and passive dense areas, a correlation between biopsy tissue anisotropy and mammographic density subtypes can be calculated to help understand how tumor microenvironment changes affect a patient’s mammographic breast tissue composition. Methods: SHG and brightfield-imaged biopsy slides from 10 patients (five malignant, five benign) collected from Maine Medical Center (Maine, US) were analyzed using the 2D Wavelet Transform Modulus Maxima (WTMM) Anisotropy Method generating a large-scale and a small-scale anisotropy factor as well as a scale-combined anisotropy calculated from their large- and small-scale anisotropy separation. A multi-modal anisotropy score was calculated by adding the imaged area’s H&E combined anisotropy score with its respective SHG combined anisotropy score. This analysis was done on eight randomly selected ductal areas for each imaging modality on a slide and correlated with the measured areas of density subtypes from the whole mammographic view. Linear regression was used for determining correlation between H&E and SHG anisotropy factors as well as with active and passive mammographic density subtype measurements. Results: The malignant patients’ small-scale H&E anisotropy factor positively correlated with their small scale SHG anisotropy factor (R2=0.571). No correlation was found for the benign patients (R2=0.011). The strongest correlations were found between benign patients’ mammographic passive (positive correlation) and active (negative correlation) dense tissues vs the H&E anisotropy scale-combined factor (R2=0.858 and R2=0.961, respectively). The largest malignant patient correlation was a negative relationship between SHG anisotropy scale-combined factor and overall mammographic density (inclusive of both active and passive dense tissue) (R2=0.891). The multi-modal anisotropy score correlated with overall mammographic density for both benign (positive correlation, R2=0.698) and malignant patients (negative correlation, R2=0.773). Combining the multi-modal anisotropy factor from SHG and H&E with the active dense tissue measurement using K-Means clustering, 5/5 malignant and 4/5 benign patients were correctly classified. Conclusion: Density in mammograms correlates with underlying quantitative changes in a patient’s tissue and collagen anisotropy. Combining both H&E and SHG analysis on tissue biopsy with active and passive density metrics from computational mammography leads to the best predictive potential than any one method on their own.

Spatial Configurations of 3D Extracellular Matrix Collagen Density and Anisotropy Simultaneously Guide Angiogenesis.

Extracellular matrix (ECM) collagen density and fibril anisotropy are thought to affect the development of new vasculatures during pathologic and homeostatic angiogenesis. Computational simulation is emerging as a tool to investigate the role of matrix structural configurations on cell guidance. However, prior computational models have only considered the orientation of collagen as a model input. Recent experimental evidence indicates that cell guidance is simultaneously influenced by the direction and intensity of alignment (i.e., degree of anisotropy) as well as the local collagen density. The objective of this study was to explore the role of ECM collagen anisotropy and density during sprouting angiogenesis through simulation in the AngioFE and FEBio modeling frameworks. AngioFE is a plugin for FEBio (Finite Elements for Biomechanics) that simulates cell-matrix interactions during sprouting angiogenesis. We extended AngioFE to represent ECM collagen as deformable 3D ellipsoidal fibril distributions (EFDs). The rate and direction of microvessel growth were modified to depend simultaneously on the ECM collagen anisotropy (orientation and degree of anisotropy) and density. The sensitivity of growing neovessels to these stimuli was adjusted so that AngioFE could reproduce the growth and guidance observed in experiments where microvessels were cultured in collagen gels of varying anisotropy and density. We then compared outcomes from simulations using EFDs to simulations that used AngioFE's prior vector field representation of collagen anisotropy. We found that EFD simulations were more accurate than vector field simulations in predicting experimentally observed microvessel guidance. Predictive simulations demonstrated the ability of anisotropy gradients to recruit microvessels across short and long distances relevant to wound healing. Further, simulations predicted that collagen alignment could enable microvessels to overcome dense tissue interfaces such as tumor-associated collagen structures (TACS) found in desmoplasia and tumor-stroma interfaces. This approach can be generalized to other mechanobiological relationships during cell guidance phenomena in computational settings.

T 2 orientation anisotropy mapping of articular cartilage using qMRI

Objective. To provide orientation-independent MR parameters potentially sensitive to articular cartilage degeneration by measuring isotropic and anisotropic components of T 2 relaxation, as well as 3D fiber orientation angle and anisotropy via multi-orientation MR scans. Approach. Seven bovine osteochondral plugs were scanned with a high angular resolution of thirty-seven orientations spanning 180° at 9.4 T. The obtained data was fitted to the magic angle model of anisotropic T 2 relaxation to produce pixel-wise maps of the parameters of interest. Quantitative Polarized Light Microscopy (qPLM) was used as a reference method for the anisotropy and fiber orientation. Main results. The number of scanned orientations was found to be sufficient for estimating both fiber orientation and anisotropy maps. The relaxation anisotropy maps demonstrated a high correspondence with qPLM reference measurements of the collagen anisotropy of the samples. The scans also enabled calculating orientation-independent T 2 maps. Little spatial variation was observed in the isotropic component of T 2 while the anisotropic component was much faster in the deep radial zone of cartilage. The estimated fiber orientation spanned the expected 0°–90° in samples that had a sufficiently thick superficial layer. The orientation-independent magnetic resonance imaging (MRI) measures can potentially reflect the true properties of articular cartilage more precisely and robustly. Significance. The methods presented in this study will likely improve the specificity of cartilage qMRI by allowing the assessment of the physical properties such as orientation and anisotropy of collagen fibers in articular cartilage.

Probing the alignment-dependent mechanical behaviors and time-evolutional aligning process of collagen scaffolds.

Efficiently manipulating and reproducing collagen (COL) alignment in vitro remains challenging because many of the fundamental mechanisms underlying and guiding the alignment process are not known. We reconcile experiments and coarse-grained molecular dynamics simulations to investigate the mechanical behaviors of a growing COL scaffold and assay how changes in fiber alignment and various cross-linking densities impact their alignment dynamics under shear flow. We find higher cross-link densities and alignment levels significantly enhance the apparent tensile/shear moduli and strength of a bulk COL system, suggesting potential measures to facilitate the design of stronger COL based materials. Since fibril alignment plays a key factor in scaffold mechanics, we next investigate the molecular mechanism behind fibril alignment with Couette flow by computationally investigating the effects of COL's structural properties such as chain lengths, number of chains, tethering conditions, and initial COL conformations on the COL's final alignment level. Our computations suggest that longer chain lengths, more chains, greater amounts of tethering, and initial anisotropic COL conformations benefit the final alignment, but the effect of chain lengths may be more dominant over other factors. These results provide important parameters for consideration in manufacturing COL-based scaffolds where alignment and cross-linking are necessary for regulating performance.

Effects of the lower energy and pulse stacking in carbon dioxide laser skin treatment: an objective analysis using second harmonic generation

ABSTRACTPurposeTo evaluate the effect of fractional carbon dioxide (CO2) laser treatment using lower power associated with pulse stacking within collagen fibers, using second harmonic generation microscopy and computerized image analysis.MethodsTwenty male Wistar rats aging eight weeks were used. Each treatment area received a single-pass CO2 fractional laser with different parameters. The 20 animals were divided into two groups and euthanized after 30 and 60 days. Second harmonic generation images were obtained and program ImageJ was utilized to evaluate the collagen organization within all areas. Collagen anisotropy, entropy and optical density were quantified.ResultsIncreased anisotropy over time was observed in all four areas, but only reached statistical significance (p = 0.0305) when the mildest parameters were used (area four). Entropy decreased over time in all areas, but without significance(p = 0.1779) in area four. Density showed an overtime increase only in area four, but no statistical significance was reached (p = 0.6534).ConclusionsWhen combined, the results obtained in this study regarding anisotropy, entropy and density tend to demonstrate that it is possible to achieve collagen remodeling with the use of lower power levels associated with stacked pulses.

Biochemical predictors of cartilaginous tissue metabolic disorders for early osteoarthrosis evidence diagnostics.

The complete laboratory and clinical instrumental examination was conducted, it included serum COMP test, circadian excretion of type II collagen C-terminal telopeptides Urine CartiLaps (СТХ II) and Т2 relaxometry in 29 patients of both sexes of the main group with early (0-I) X-ray osteoarthrosis stages, 30 subjects of comparison group with no X-ray osteoarthrosis evidences aged 44.7±5.9 years and 25 healthy subjects aged 26.3±2.6 years of the control group. The increase (р<0,05) of COMP and Urine CartiLaps levels as well as the increase of Т2 relaxation signal was found at early osteoarthrosis evidences. It was proven that there was (р<0.01) a connection (R=0.8) between COMP and Urine CTX II levels as well as (р<0.05) results of Т2 relaxometry (R=0.8). It was proven that collagen anisotropy and formation of chondromalacia areas as Т2 relaxometry showed in patients with early OA evidences were connected with accumulation of serum COMP and increase of type II collagen circadian renal excretion. The combination of laboratory and radiological methods of articular hyaline cartilage assessment may be used for finding early osteoarthrosis stages.

Abstract 1984: Anisotropy of collagen I matrix influences therapeutic response and secretome of Luminal A cells

Abstract High degree of collagen I fiber orientation has been associated with guided tumor cell invasion, but little is known about its impact in therapeutic response. Anisotropic substrates have been shown to influence the phenotype of stem cells which suggests that it can influence tumor cell behavior beyond cell migration. We hypothesized that the anisotropy of collagen I matrix impacts therapeutic response by modulating the secretome of breast cancer cells and this effect is mediated by integrin signaling. To test our hypothesis, the cell secretome, response to hormone ablation therapy and integrin inhibitors were evaluated in breast tumor cells cultured in collagen type 1 substrates of defined orientation. The orientation of electrospun collagen I fibrils was regulated by adjusting the syringe flow rate and collector velocity to obtain random (&amp;lt;70%) or aligned (&amp;gt;80%) oriented fibrils. Average porosity (45± 3.4 %), matrix stiffness (0.8 ±0.24 MPa) and fiber diameter (1.6 ± 0.86μm) were kept constant across collagen substrates. Estrogen-dependent Luminal A cells (MCF-7 and T-47D) cells were cultured on collagen substrates in a hormone-deprived environment for 3-6 days. Polycaprolactone (PCL) fibrous substrates of aligned and random orientation were used as negative fibrous controls for integrin signaling. Evaluation of cell growth showed that a fibrillar structure in combination with a high degree of fibril orientation significantly increased cell proliferation (&amp;gt;75%) as compared to gelatin-coated tissue culture plastic (TCP). Collagen fibers significantly supported hormone-independent tumor growth at levels equivalent to or above those observed in the estrogen-treated control. Increased proliferation levels were supported on collagen fibrous substrates treated with Tamoxifen and Fulvestrant, and this effect was enhanced in aligned collagen I. Inhibition of α2β1 integrin resulted in enhanced proliferation rates in random fibers and suppression of cell growth in aligned fibers. PCL aligned substrates where not sufficient to support hormone-independent cell growth. MCF-7 cells on collagen substrates significantly stimulated the growth (&amp;gt;7 fold) of adjacent cells on TCP in a dose-response manner as follows: aligned &amp;gt; random &amp;gt; gelatin. Expression levels of 1000 factors were quantified in conditioned medium to identify differences in the secretome signatures associated with cell-matrix signaling. MCF-7 cells cultured on collagen fibrous substrates displayed secretome signatures that were different across all substrates. Fourteen factors were identified to be upregulated in fibrous substrates by more than ten folds including apoptotic regulators RalA and BCL-2. Our results show for the first time the influence of collagen I fibril orientation in supporting hormone-independent growth and as a novel regulator therapeutic response in luminal A cells. Citation Format: Ana M. Reyes, Jorge Almodovar, Elaine Alarid, Maribella Domenech. Anisotropy of collagen I matrix influences therapeutic response and secretome of Luminal A cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1984.

Cellular and collagen reference values of gingival and periodontal ligament tissues in rats: a pilot study.

Reference data are lacking on the periodontal ligament and the gingival tissue of the rat model, which would be useful for studies of new medical or biomaterial periodontal treatments. The objective of the current study was to propose cellular and collagen reference values of gingival and periodontal ligament tissues in rat, using a simple and reliable quantitative method after decalcification. Mandibular samples of ten adult Sprague-Dawley rats were used. Mild decalcification was carried out using ethylenediaminetetraacetic acid (EDTA) to preserve the morphology of tissues. Half of the samples were decalcified and the other half were not. The gingiva and the periodontal ligament were analyzed. Descriptive histology and computer-assisted image analysis were performed. The data showed that qualitatively, cellular and extracellular matrix morphologies were well preserved compared to non-decalcified periodontal soft tissue biopsies. Histomorphometrically, constitutive cellularity and the total amount of native collagen, collagen directionality and collagen anisotropy in both experimental conditions did not significantly differ. Taken together, these results suggested that EDTA decalcification did not negatively affect the studied endpoints. Moreover, this mild decalcification method allowed in situ maintenance of the periodontal soft and hard tissue integrity. The structural and compositional computerized assessment performed in the healthy periodontal soft tissue could provide reference values that will be required for future assessment on the effects of pathological, reparative and regenerative processes in rat periodontal soft tissues.

Use of Mueller matrix colposcopy in the characterization of cervical collagen anisotropy.

Annually, about 15 million preterm infants are born in the world. Of these, due to complications resulting from their premature birth, about 1 million would die before the age of five. Since the high incidence of preterm birth (PTB) is partially due to the lack of effective diagnostic modalities, methodologies are needed to determine risk of PTB. We propose a noninvasive tool based on polarized light imaging aimed at measuring the organization of collagen in the cervix. Cervical collagen has been shown to remodel with the approach of parturition. We used a full-field Mueller matrix polarimetric colposcope to assess and compare cervical collagen content and structure in nonpregnant and pregnant women in vivo. Local collagen directional azimuth was used and a total of eight cervices were imaged.

Increased stiffness of collagen fibrils following cyclic tensile loading.

Alterations in mechanical loading can induce growth and remodeling in soft connective tissues. Numerous studies have measured changes in the collagen structure and mechanical properties of cellularized native and engineered tissues in response to cyclic mechanical loading. However, a recent experimental study demonstrated that cyclic loading also caused significant stiffening and strengthening of acellular collagen constructs. In this work, we developed an anisotropic hyperelastic model of the collagen constructs to investigate whether the measured changes in the tissue-level properties can be attributed to changes in the anisotropic collagen structure or mechanical properties of the collagen fibrils. The model parameters describing the elastic properties, damage properties, and morphology of the fibril were fit to the stress-stretch response measured for the constructs subjected to different preconditioning strains and cycles. The results showed that the changes in the collagen anisotropy measured in experiments were insufficient to explain the increase in the stiffness and strength of the collagen constructs with cyclic loading and that the increase in the strength of the collagen constructs may be attributed mainly to the increase in the effective stiffness of the fibrils. These findings suggest that mechanical loading can induce changes in the stiffness and failure properties of the collagen fibril network through passive chemomechanical processes in addition to active cellular processes.

Soy Protein/Cellulose Nanofiber Scaffolds Mimicking Skin Extracellular Matrix for Enhanced Wound Healing.

Historically, soy protein and extracts have been used extensively in foods due to their high protein and mineral content. More recently, soy protein has received attention for a variety of its potential health benefits, including enhanced skin regeneration. It has been reported that soy protein possesses bioactive molecules similar to extracellular matrix (ECM) proteins and estrogen. In wound healing, oral and topical soy has been heralded as a safe and cost-effective alternative to animal protein and endogenous estrogen. However, engineering soy protein-based fibrous dressings, while recapitulating ECM microenvironment and maintaining a moist environment, remains a challenge. Here, the development of an entirely plant-based nanofibrous dressing comprised of cellulose acetate (CA) and soy protein hydrolysate (SPH) using rotary jet spinning is described. The spun nanofibers successfully mimic physicochemical properties of the native skin ECM and exhibit a high water retaining capability. In vitro, CA/SPH nanofibers promote fibroblast proliferation, migration, infiltration, and integrin β1 expression. In vivo, CA/SPH scaffolds accelerate re-epithelialization and epidermal thinning as well as reduce scar formation and collagen anisotropy in a similar fashion to other fibrous scaffolds, but without the use of animal proteins or synthetic polymers. These results affirm the potential of CA/SPH nanofibers as a novel wound dressing.

Use of Mueller matrix polarimetry and optical coherence tomography in the characterization of cervical collagen anisotropy.

Preterm birth (PTB) presents a serious medical health concern throughout the world. There is a high incidence of PTB in both developed and developing countries ranging from 11% to 15%, respectively. Recent research has shown that cervical collagen orientation and distribution changes during pregnancy may be useful in predicting PTB. Polarization imaging is an effective means to measure optical anisotropy in birefringent materials, such as the cervix's extracellular matrix. Noninvasive, full-field Mueller matrix polarimetry (MMP) imaging methodologies, and optical coherence tomography (OCT) imaging were used to assess cervical collagen content and structure in nonpregnant porcine cervices. We demonstrate that the highly ordered structure of the nonpregnant porcine cervix can be observed with MMP. Furthermore, when utilized ex vivo, OCT and MMP yield very similar results with a mean error of 3.46% between the two modalities.

Improved quantification of collagen anisotropy with polarization-resolved second harmonic generation microscopy.

Imaging tissue samples by polarization-resolved second harmonic generation microscopy provides both qualitative and quantitative insights into collagen organization in a label-free manner. Polarization-resolved second harmonic generation microscopy goes beyond simple intensity-based imaging by adding the laser beam polarization component and applying different quantitative metrics such as the anisotropy factor. It thus provides valuable information on collagen arrangement not available with intensity measurements alone. Current established approaches are limited to calculating the anisotropy factor for only a particular laser beam polarization and no general guidelines on how to select the best laser beam polarization have yet been defined. Here, we introduce a novel methodology for selecting the optimal laser beam polarization for characterizing tissues using the anisotropy in the purpose of identifying cancer signatures. We show that the anisotropy factor exhibits a similar laser beam polarization dependence to the second harmonic intensity and we combine it with the collagen orientation index computed by Fast Fourier Transform analysis of the recorded images to establish a framework for choosing the laser beam polarization that is optimal for an accurate interpretation of polarization-resolved second harmonic generation microscopy images and anisotropy maps, and hence a better differentiation between healthy and dysplastic areas. SHG image of skin tissue (a) and a selected area of interest for which we compute the SHG intensity (b) and anisotropy factor (c) dependence on the laser beam polarization and also the FFT spectrum (d) to evaluate the collagen orientation index.

Depth-dependent changes in collagen organization in the human peripapillary sclera.

PurposeThe collagen structure of the human peripapillary sclera plays a significant role in determining optic nerve head (ONH) biomechanics, and is therefore of interest in the study of glaucoma. The aim of the current work was to map the anisotropic collagen structure of the normal human peripapillary sclera as a function of tissue depth.MethodsWide-angle x-ray scattering was used to quantify collagen fibril orientation at 0.5mm intervals across six 150μm-thick serial sections through the peripapillary sclera of eight normal European-derived human eyes. Two structural parameters were measured: 1) the relative number of fibrils preferentially aligned at a given angle within the tissue plane, 2) the degree of collagen alignment (anisotropy).ResultsThe inner-most one-third of the peripapillary scleral stroma (nearest to the choroid) was characterised by collagen fibrils either randomly arranged or preferentially aligned radially with respect to the ONH. In contrast, the outer two-thirds of the tissue was dominated by a circumferential arrangement of collagen encircling the ONH. In all tissue regions the degree of collagen anisotropy peaked in the mid-stroma and progressively decreased towards the tissue surfaces, with the largest depth variations occurring in the inferior-nasal quadrant, and the smallest occurring in the superior-nasal quadrant.ConclusionsSignificant, region-specific variations in collagen structure are present in the human peripapillary sclera as a function of depth. In normal eyes, the circumferential collagen fibril architecture is most prominent in the outer two-thirds of the stroma, possibly as a mechanical adaption to more effectively support the lamina cribrosa at the level of its insertion into the scleral canal wall.

Biomechanical model of the human cornea: Considering shear stiffness and regional variation of collagen anisotropy and density

A numerical model based on continuum mechanics theory has been developed which represents the 3D anisotropic behaviour of the corneal stroma. Experimental data has been gathered from a number of previous studies to provide the basis and calibration parameters for the numerical modelling. The resulting model introduces numerical representation of collagen fibril density and its related regional variation, interlamellar cohesion and age-related stiffening in an anisotropic model of the human cornea. Further, the model incorporates previous modelling developments including representation of lamellae anisotropy and stiffness of the underlying matrix. Wide angle X-ray scattering has provided measured data which quantifies relative fibril anisotropy in the 2D domain. Accurate numerical description of material response to deformation is essential to providing representative simulations of corneal behaviour. Representing experimentally obtained 2D anisotropy and regional density variation in the 3D domain is an essential component of this accuracy. The constitutive model was incorporated into finite element analysis. Combining with inverse analysis, the model was calibrated to an extensive experimental database of ex vivo corneal inflation tests and ex vivo corneal shear tests. This model represents stiffness of the underlying matrix which is 2–3 orders of magnitude than the mechanical response representing the collagen fibrils in the lamellae. The presented model, along with its age dependent material coefficients, allows finite element modelling for an individual patient with material stiffness approximated based on their age. This has great potential to be used in both daily clinical practice for the planning and optimisation of corrective procedures and in pre-clinical optimisation of diagnostic procedures.

Changes in scleral collagen organization in murine chronic experimental glaucoma.

The organization of scleral collagen helps to determine the eye's biomechanical response to intraocular pressure (IOP), and may therefore be important in glaucoma. This study provides a quantitative assessment of changes in scleral collagen fibril organization in bead-induced murine experimental glaucoma. Wide-angle X-ray scattering was used to study the effect of bead-induced glaucoma on posterior scleral collagen organization in one eye of 12 CD1 mice, with untreated fellow eyes serving as controls. Three collagen parameters were measured: the local preferred fibril directions, the degree of collagen anisotropy, and the total fibrillar collagen content. The mouse sclera featured a largely circumferential orientation of fibrillar collagen with respect to the optic nerve head canal. Localized alteration to fibril orientations was evident in the inferior peripapillary sclera of bead-treated eyes. Collagen anisotropy was significantly (P<0.05) reduced in bead-treated eyes in the superior peripapillary (Treated: 43±8%; 49±6%) and midposterior (Treated: 39±4%; 43±4%) sclera, and in the peripapillary region overall (Treated: 43±6%; 47±3%). No significant differences in total collagen content were found between groups. Spatial changes in collagen fibril anisotropy occur in the posterior sclera of mice with bead-induced chronic IOP elevation and axonal damage. These results support the idea that dynamic changes in scleral form and structure play a role in the development of experimental glaucoma in mice, and potentially in human glaucoma.

Magnetic resonance mapping of the rim of articular cartilage defects of the patella

Elevated T2-values of articular cartilage are associated with an increase in cartilage water that results from a damaged collagen matrix, and provide a marker for cartilage damage. We used T2 mapping to analyse the rim of cartilage defects that appeared to be intact on the morphological sequences, to determine whether there are early biochemical changes already present. We calculated T2 values for the rim of cartilage defects in 25 patients and compared these values to another area of control cartilage in these patients. A highly significant increase in T2 values of the deep, superficial, and global layer of the rim versus the control cartilage was measured. ANOVA showed a significant correlation of the defect levels with the T2 values of the deep and global zone of the adjacent cartilage tissue, but not with the superficial zone. Although cartilage appears to be intact on morphological sequences, T2 mapping can show a loss of structural anisotropy of collagen and the associated increase in cartilage water that indicates the destruction of the adjacent cartilage. Preoperative information about the degree of damage of the collagen matrix will support decision making for cartilage repair.

STRESS-FIBER REMODELING IN 3D: ‘CONTACT GUIDANCE VS STRETCH AVOIDANCE’

Abstract When engineering heart valves in vitro, matrix anisotropy is considered important for long-term in vivo functionality. However, it is not fully understood how to guide, maintain and control matrix anisotropy. Experiments suggest that collagen anisotropy is affected by actin-mediated cell traction and associated cellular orientation. Although cellular orientation in 2D can be manipulated via imposed uniaxial cyclic stretch, 3D data are lacking. We questioned how cyclic stretch influences actin and collagen orientation in 3D constructs. A novel micro-tissue platform system was designed, able to dynamically and biochemically load small-scale cell-populated fibrous tissues. Flexible membranes of Bioflex culture plates were provided with a rectangular array silicone posts. These silicone posts constrained a contracting gel mixture of human vena saphena cells (HVSC), collagen type I and matrigel. The constrained tissues were subjected to pure uniaxial cyclic stretch (10%, 0.5Hz, in the presence or absence of agents) on the Flexcell system. F-actin was taken as a measure for the cell traction direction, and the F-actin orientation was quantified throughout the complete tissue thickness (~ 300m) using fiber-tracking software, and was fitted using a bi-model distribution function. Uniaxial cyclic stretching for 3 days (preceded by 3 days of static constraint) resulted in stress-fibers that were oriented perpendicular to the stretching direction only at tissue surfaces, as generally observed in 2D. Strikingly, however, in the tissue core F-actin (and cell) and collagen orientation was biaxial. Immediate cyclic stretching, starting before polymerization of the collagen matrix, resulted in a strong stretch avoidance throughout the tissue, of both F-actin and collagen. We systematically investigated the effect of biochemical treatment, including MMP1 to perturb matrix integrity, MMP-1 + ROCK-inhibitor to counteract the possible MMP1-induced response, as well the effect of a lower and a higher initial collagen density. Experimental data suggests that F-actin stress-fibers avoid cyclic stretch in 3D, unless collagen contact guidance dictates otherwise.

Anisotropic Collagen Fibrillogenesis Within Microfabricated Scaffolds: Implications For Biomimetic Tissue Engineering

Anisotropic collagen fibrillogenesis is demonstrated within the pores of an accordion-like honeycomb poly(glycerol sebacate) tissue engineering scaffold. Confocal reflectance microscopy and image analysis demonstrate increased fibril distribution order, fibril density, and alignment in accordion-like honeycomb pores compared with collagen gelled unconstrained. Finite element modeling predicts how collagen gel and scaffold mechanics couple in matching native heart muscle stiffness and anisotropy.