Effects of collagen fiber orientation on the response of biologically derived soft tissue biomaterials to cyclic loading

In the present study, the effects of initial collagen fiber orientation on the medium-term (up to 50 x 10(6) cycles) fatigue response of heart valve soft tissue biomaterials was investigated. Glutaraldehyde treated bovine pericardium (GLBP), preselected for uniform structure and collagen fiber orientation, was used as the representative heart valve biomaterial. Using specialized instrumentation, GLBP specimens were subjected to cyclic tensile loading to maximum stress levels of 500 +/- 50 kPa at a frequency of 22 Hz. Two sample groups were examined, one with the preferred collagen fiber direction parallel (PD) and perpendicular (XD) to the direction of applied strain. The primary findings indicated that GLBP fatigue response was highly sensitive to the direction of loading with respect to fiber orientation. Specifically, when loading perpendicular to the preferred collagen fiber orientation, fiber reorientation is the dominant mechanism. In contrast, when loaded parallel to the preferred fiber direction a reduction in both collagen fiber crimp and fiber reorientation occurred. Moreover, alterations in the degree and direction of mechanical anisotropy can be inducted by cyclic loading when specimens are loaded perpendicular to the preferred fiber direction. Fourier Transform Infrared Spectroscopy (FT-IR) results indicate that molecular-level damage to collagen occurs in both groups after only 20 x 10(6) cycles. Taken as a whole, the results of this study suggest that initial collagen orientation plays a critical role in bioprosthetic heart valve biomaterial fatigue response.     

Quantification of the Temporal Evolution of Collagen Orientation in Mechanically Conditioned Engineered Cardiovascular Tissues

Load-bearing soft tissues predominantly consist of collagen and exhibit anisotropic, non-linear visco-elastic behavior, coupled to the organization of the collagen fibers. Mimicking native mechanical behavior forms a major goal in cardiovascular tissue engineering. Engineered tissues often lack properly organized collagen and consequently do not meet in vivo mechanical demands. To improve collagen architecture and mechanical properties, mechanical stimulation of the tissue during in vitro tissue growth is crucial. This study describes the evolution of collagen fiber orientation with culture time in engineered tissue constructs in response to mechanical loading. To achieve this, a novel technique for the quantification of collagen fiber orientation is used, based on 3D vital imaging using multiphoton microscopy combined with image analysis. The engineered tissue constructs consisted of cell-seeded biodegradable rectangular scaffolds, which were either constrained or intermittently strained in longitudinal direction. Collagen fiber orientation analyses revealed that mechanical loading induced collagen alignment. The alignment shifted from oblique at the surface of the construct towards parallel to the straining direction in deeper tissue layers. Most importantly, intermittent straining improved and accelerated the alignment of the collagen fibers, as compared to constraining the constructs. Both the method and the results are relevant to create and monitor load-bearing tissues with an organized anisotropic collagen network. The work was performed at the Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands.     

Collagen fiber alignment and biaxial mechanical behavior of porcine urinary bladder derived extracellular matrix

The collagen fiber alignment and biomechanical behavior of naturally occurring extracellular matrix (ECM) scaffolds are important considerations for the design of medical devices from these materials. Both should be considered in order to produce a device to meet tissue specific mechanical requirements (e.g., tendon vs. urinary bladder), and could ultimately affect the remodeling response in vivo. The present study evaluated the collagen fiber alignment and biaxial mechanical behavior of ECM scaffold material harvested from porcine urinary bladder tunica mucosa and basement membrane (together referred to as urinary bladder matrix (UBM)) and ECM harvested from urinary bladder submucosa (UBS). Since the preparation of UBM allows for control of the direction of delamination, the effect of the delamination method on the mechanical behavior of UBM was determined by delaminating the submucosa and other abluminal layers by scraping along the longitudinal axis of the bladder (apex to neck) (UBML) or along the circumferential direction (UBMC). The processing of UBS does not allow for similar directional control. UBML and UBS had similar collagen fiber distributions, with a preferred collagen fiber alignment along the longitudinal direction. UBMC showed a more homogenous collagen fiber orientation. All samples showed a stiffer mechanical behavior in the longitudinal direction. Despite similar collagen fiber distributions, UBML and UBS showed quite different mechanical behavior for the applied loading patterns with UBS showing a much more pronounced toe region. The mechanical behavior for UBMC in both directions was similar to the mechanical behavior of UBML. There are distinct differences in the mechanical behavior of different layers of ECM from the porcine urinary bladder, and the processing methods can substantially alter the mechanical behavior observed.     

Cyclic strain anisotropy regulates valvular interstitial cell phenotype and tissue remodeling in three-dimensional culture

Many planar connective tissues exhibit complex anisotropic matrix fiber arrangements that are critical to their biomechanical function. This organized structure is created and modified by resident fibroblasts in response to mechanical forces in their environment. The directionality of applied strain fields changes dramatically during development, aging, and disease, but the specific effect of strain direction on matrix remodeling is less clear. Current mechanobiological inquiry of planar tissues is limited to equibiaxial or uniaxial stretch, which inadequately simulates many in vivo environments. In this study, we implement a novel bioreactor system to demonstrate the unique effect of controlled anisotropic strain on fibroblast behavior in three-dimensional (3-D) engineered tissue environments, using aortic valve interstitial fibroblast cells as a model system. Cell seeded 3-D collagen hydrogels were subjected to cyclic anisotropic strain profiles maintained at constant areal strain magnitude for up to 96 h at 1 Hz. Increasing anisotropy of biaxial strain resulted in increased cellular orientation and collagen fiber alignment along the principal directions of strain and cell orientation was found to precede fiber reorganization. Cellular proliferation and apoptosis were both significantly enhanced under increasing biaxial strain anisotropy (P<0.05). While cyclic strain reduced both vimentin and alpha-smooth muscle actin compared to unstrained controls, vimentin and alpha-smooth muscle actin expression increased with strain anisotropy and correlated with direction (P<0.05). Collectively, these results suggest that strain field anisotropy is an independent regulator of fibroblast cell phenotype, turnover, and matrix reorganization, which may inform normal and pathological remodeling in soft tissues.     

Molecular orientation of collagen in intact planar connective tissues under biaxial stretch

Understanding of the mechanical behavior of collagenous tissues at different size scales is necessary to understand their physiological function as well as to guide their use as heterograft biomaterials. We conducted a first investigation of the kinematics of collagen at the molecular and fiber levels under biaxial stretch in an intact planar collagenous tissue. A synchrotron small angle X-ray scattering (SAXS) technique combined with a custom biaxial stretching apparatus was used. Collagen fiber behavior under biaxial stretch was then studied with the same specimens using small angle light scattering (SALS) under identical biaxial stretch states. Both native and glutaraldehyde modified bovine pericardium were investigated to explore the effects of chemical modification to collagen. Results indicated that collagen fiber and molecular orientation did not change under equibiaxial strain, but were observed to profoundly change under uniaxial stretch. Interestingly, collagen molecular strain initiated only after approximately 15% global tissue strain, potentially due to fiber-level reorganization occurring prior to collagen molecule loading. Glutaraldehyde treatment also did not affect collagen molecular strain behavior, indicating that chemical fixation does not alter intrinsic collagen molecular stiffness. No detectable changes in the angular distribution and D-period strain were found after 80 min of stress relaxation. It can be speculated that other mechanisms may be responsible for the reduction in stress with time under biaxial stretch. The results of this first study suggest that collagen fiber/molecular kinematics under biaxial stretch are more complex than under uniaxial deformation, and warrant future studies.     

Orientation of human glioblastoma cells embedded in type I collagen,caused by exposure to a 10 T static magnetic field

We investigated the preferred orientation of human glioblastoma cells (A172) following exposure to static magnetic fields (SMF) at 10 Tesla in the presence or absence of collagen. A172 cells embedded in collagen gel were oriented perpendicular to the direction of the SMF. A172 cells cultured in the absence of collagen did not exhibit any specific orientation pattern after 7 days of exposure to the SMF. Thus we succeeded in evoking the magnetic orientation of human glioblastoma cells by exposure to the SMF. Our results suggest that the orientation of glioblastoma cell processes may be due to the arrangement of microtubules under the influence of magnetically oriented collagen fiber.     

Response of heterograft heart valve biomaterials to moderate cyclic loading

We have recently demonstrated that noncalcific tissue damage can lead to significant collagen degradation in clinically explanted bioprosthetic heart valves (BHVs). In the present study we quantified the early response of glutaraldehyde treated bovine pericardium (GLBP) to cyclic tensile loading to begin to elucidate the mechanisms of noncalcific tissue degeneration in BHV biomaterials. GLBP specimens were cycled at 30 Hz to a maximum uniaxial strain of 16% (corresponding to approximately 1-MPa peak stress), with the loading direction parallel to the preferred collagen fiber (PD) direction. After 30 x 10(6) cycles, specimens were subjected to biaxial mechanical testing, then cycled until 65 x 10(6) cycles. The results indicated a permanent change in the unloaded tissue dimensions of +7.1% strain in the PD direction and -7.7% strain in the cross fiber direction (XD) after 65 x 10(6) cycles and an increase of the collagen crimp period from 40.6 to 45.2 microm by 65 x 10(6) cycles (p = 0.05). Fourier transform IR spectroscopy analysis indicated that cyclic fatigue of GLBP leads to both collagen conformational changes and early denaturation. Furthermore, no significant changes in areal strain were found under 1-MPa equibiaxial stress, indicating that cyclic loading changed the collagen fiber orientation but not the overall tissue compliance. These observations suggest that while deterioration of collagen begins immediately, fiber straightening and reorientation dominates the changes in the mechanical behavior up to 65 x 10(6) cycles. The present study underscores the complexity of the response of biologically derived biomaterials to cyclic mechanical loading. Improved understanding of these phenomena can potentially guide the development of novel chemical treatment methods that seek to improve BHV durability by minimizing these degenerative processes.     

Morphology,proliferation, alignment,and new collagen synthesis of mesenchymal stem cells on a microgrooved collagen membrane

The topographic cues of the extracellular matrix may have significant effects upon cellular behavior, such as adhesion, spreading, migration, proliferation, differentiation, and in particular, morphology and orientation. In this study, we examined the effects of microgrooved collagen membrane (MCM) on mesenchymal stem cell (MSC) behavior. The MCM (9 μm in periodicity, and 1–2 μm in depth) was fabricated on an untreated (nonpolar) and smooth polystyrene substrate, based on the absorption and self-assembly properties of collagen on the polystyrene substrate. Methyl thiazolyl tetrazolium assay revealed that cell proliferation on the MCM was enhanced compared with the smooth collagen membrane at day 2. Qualitative observation of MSC behavior using confocal laser scanning microscopy and scanning electron microscopy showed that MSCs grew with a highly elongated morphology and were aligned strictly along the direction of the microgrooves. Additionally, scanning electron microscopy revealed the oriented cells produced a collagenous matrix on the MCM that had a preferential orientation, whereas the collagenous matrix produced by randomly oriented MSCs on the smooth collagen membrane was disorganized. Future studies should investigate the fabrication of oriented topographical substrates, based on the natural biomaterial collagen, to guide cell alignment and oriented growth along definite directions. These substrates may help produce aligned collagenous matrices that could have good potential for the production of tissue substitutes.     

Biaxial mechanical evaluation of cholecyst-derived extracellular matrix: a weakly anisotropic potential tissue engineered biomaterial

A new acellular, natural, biodegradable matrix has been discovered in the cholecyst-derived extracellular matrix (CEM). This matrix is rich in collagen and contains several other macromolecules useful in tissue remodeling. In this study, the principal material axes, collagen fiber orientations, and biaxial mechanical properties in a physiological loading regime were characterized. Fiber direction was determined by polarized light microscopy, and the principal axes and degree of anisotropy were determined mechanically. Macroscopic equibiaxial strain tests were then conducted on preconditioned specimens. While 13% of the area of CEM contains collagen fibers oriented between 50 degrees and 60 degrees from the neck-fundus axis, the principal material axis was oriented 63 degrees +/- 13.7 degrees , with an aspect ratio of 0.11 +/- 0.06, indicating a weak anisotropy in that direction. Under biaxial loading, CEM exhibited a large toe region followed by an exponential rise in stress in both principal and perpendicular axis directions, similar to other materials currently under research. There was no significant difference between the biaxial stress-strain profile and the burst stress-strain profile. The results demonstrate that CEM is weakly anisotropic and it has the ability to support large strains across a physiological loading regime.     

The effects of collagen fiber orientation on the flexural properties of pericardial heterograft biomaterials

Improving cardiac valve bioprostheses (BHV) utilizing heterograft biomaterials requires a better understanding of their mechanical behavior. Flexure is a major mode of deformation for BHV leaflets during valve operation, inducing more complex deformation patterns within the tissue compared to tensile loads. In this study, we investigated the relation between collagen fiber preferred direction and the resulting flexural properties of native and glutaraldehyde-treated bovine pericardium. 20 mm x 4 mm strips were cut from the presorted sheets of bovine pericardium and divided into four groups: two directions of collagen fiber orientation in two groups of native and chemically treated specimens. Specimens were flexed in two different directions using a three-point bending technique (ASAIO J. 45(1999)59) and their flexural mechanical response compared. Results indicated that: (1) the relationship between the applied flexing moment and change of curvature of specimens was non-linear in both native and chemically fixed groups, (2) there were no directional differences in flexural properties when the bovine pericardium is flexed towards either the epi-pericardial or visceral surfaces in both native and chemically fixed specimens, (3) native and chemically fixed bovine pericardium were stiffer when flexed perpendicular to local preferred collagen fiber direction, and (4) chemical fixation increased the flexural rigidity of bovine pericardium. Results of this study indicate that the flexural properties of bovine pericardium are dominated by inter-fiber cross-links as opposed to the stiffness of the collagen fibers themselves. These findings can be used to guide the development of novel chemical treatment methods that seek to optimize biomechanical properties of heterograft biomaterials.     

Collagen-agarose co-gels as a model for collagen-matrix interaction in soft tissues subjected to indentation

The mechanical properties of soft tissues depend on the collagen fiber network and the surrounding non-fibrillar matrix. The mechanical role of non-fibrillar material remains poorly understood. Our recent study (Lake and Barocas, Ann Biomed Eng 2011) introduced collagen-agarose co-gels as a simple experimental model system to evaluate the mechanical contribution of non-fibrillar matrix, and evaluated co-gel properties in uniaxial tension. In this study, we utilized similar co-gels to examine collagen-matrix interaction in tissues subjected to incremental stress-relaxation indentation tests. Mechanical testing was performed using two orthogonal custom test devices, and polarized light imaging was used to quantify 3D collagen fiber kinematics under load. The addition of agarose led to concentration-dependent changes in the time-dependent mechanical response and magnitude/spread of collagen fiber reorganization of tissue analogs. Specifically, peak/relaxed loads increased, and relaxation rate decreased, with increasing agarose concentration. In addition, increasing agarose content led to larger magnitude changes in orientation direction and alignment strength that were more localized near the indenter. Results suggest that non-fibrillar material significantly contributes to the behavior of co-gels in indentation, likely by reducing permeability and resisting volume change, thereby providing insight into the properties of artificial and native tissues subjected to non-tensile loading.     

Distribution map of collagen fiber orientation in a whole calf skin

It is important to obtain information about the collagen fiber orientation in biological fibrous tissues such as human and animal skins because the collagen fiber orientation in the skins may closely relate to the motional functions of the body. However, no reports are yet available on the distribution of collagen fiber orientation in a whole skin. The microwave method recently developed by the author was applied for giving the distribution of collagen fiber orientation. A map representing the distribution of collagen fiber orientation was obtained for a leather sheet prepared from the entire skin of a calf. It was found that the direction of collagen fiber orientation was, on an average, parallel to the calf's spinal column and limbs, corresponding to the direction perpendicular to that of skin motions due to eating and walking, and that the degree of orientation was large in the parts where the skin motions were marked. These findings suggest that the distribution map of fiber orientation in calf leather significantly affects the motional functions of its different parts and also hopefully gives useful information for performing an appropriate transplant of skins. Anat Rec 254:147–152, 1999. © 1999 Wiley‐Liss, Inc.     

Mechanical evaluation and design of a multilayered collagenous repair biomaterial

One method of fabricating implantable biomaterials is to utilize biologically derived, chemically modified tissues to form constructs that are both biocompatible and remodelable. Rigorous mechanical characterization is a necessary component in material evaluation to ensure that the constructs will withstand in vivo loading. In this study we performed an in-depth biaxial mechanical and quantitative structural analysis of GraftPatch (GP), a biomaterial constructed by assembling chemically treated layers of porcine small intestinal submucosa (SIS). The mechanical behavior of GP was compared to both native SIS and to glutaraldehyde-treated bovine pericardium (GLBP) as a reference biomaterial. Under biaxial loading, GP was found to be stiffer than native SIS and mechanically anisotropic, with the preferred fiber direction demonstrating greater stiffness. Quantitative structural analysis using small-angle light scattering indicated a uniform fiber structure similar to GLBP and SIS. To enable test-protocol-independent quantitative comparisons, the biaxial mechanical data were fit to an orthotropic constitutive model, which indicated a similar degree of mechanical anisotropy between the three groups. We also demonstrate how the constitutive model can be used to design layered biocomposite materials that can undergo large deformations.     

Human root dentin: structural anisotropy and Vickers microhardness isotropy

The demanding mechanical functions and the variable structure of dentin make it an invaluable material for studying the structure-mechanical function relations of a mineralized collagen-containing tissue. The mineralized collagen fibril axes in human root dentin are mainly located on the incremental plane. Within this plane there is a preferred orientation in the general root-crown direction. The apatite crystals are aligned in three dimensions within an individual collagen fibril, but this orientation does not necessarily extend to the neighboring fibrils. Crystals are also present as aggregates without any preferred orientation. The structure is therefore clearly anisotropic with respect to the collagen fibril orientation, but less so with respect to overall crystal orientation. Vickers microhardness measurements of the root dentin are essentially the same on the three orthogonal planes with respect to the incremental plane. Knoop microhardness measurements are also the same on all three orthogonal planes when the major diagonal is aligned perpendicular to the collagen fibril axis preferred orientation direction. In-plane variations of up to 20% are observed in the orthogonal direction. The material is thus isotropic in the three main directions with respect to Vickers microhardness, but anisotropic in structure. This paradoxical situation is attributed mainly to the variable modes of crystal organization.     

Effects of cell seeding and cyclic stretch on the fiber remodeling in an extracellular matrix-derived bioscaffold

The porcine small intestine submucosa, an extracellular matrix-derived bioscaffold (ECM-SIS), has been successfully used to enhance the healing of ligaments and tendons. Since the collagen fibers of ECM-SIS have an orientation of +/- 30 degrees , its application in improving the healing of the parallel-fibered ligament and tendon may not be optimal. Therefore, the objective was to improve the collagen fiber alignment of ECM-SIS in vitro with fibroblast seeding and cyclic stretch. The hypothesis was that with the synergistic effects of cell seeding and mechanical stimuli, the collagen fibers in the ECM-SIS can be remodeled and aligned, making it an improved bioscaffold with enhanced conductive properties. Three experimental groups were established: group I (n = 14), ECM-SIS was seeded with fibroblasts and cyclically stretched; group II (n = 13), ECM-SIS was seeded with fibroblasts but not cyclically stretched; and group III (n = 8), ECM-SIS was not seeded with fibroblasts but cyclically stretched. After 5 days' experiments, the scaffolds from all the three groups (n = 9 for group I; n = 8 for groups II and III) were processed for quantification of the collagen fiber orientation with a small-angle light scattering (SALS) system. For groups I and II, in which the scaffolds were seeded with fibroblasts, the cell morphology and orientation and newly produced collagen fibrils were examined with confocal fluorescent microscopy (n = 3/group) and transmission electronic microscopy (n = 2/group). The results revealed that the collagen fiber orientation in group I was more aligned closer to the stretching direction when compared to the other two groups. The mean angle decreased from 25.3 degrees to 7.1 degrees (p < 0.05), and the associated angular dispersion was also reduced (37.4 degrees vs. 18.5 degrees , p < 0.05). In contrast, groups II and III demonstrated minimal changes. The cells in group I were more aligned in the stretching direction than those in group II. Newly produced collagen fibrils could be observed along the cells in both groups I and II. This study demonstrated that a combination of fibroblast seeding and cyclic stretch could remodel and align the collagen fiber orientation in ECM-SIS bioscaffolds. The better-aligned ECM-SIS has the prospect of eliciting improved effects on enhancing the healing of ligaments and tendons.     

Sequential Multimodal Microscopic Imaging and Biaxial Mechanical Testing of Living Multicomponent Tissue Constructs

Understanding relationships between mechanical stimuli and cellular responses require measurements of evolving tissue structure and mechanical properties. We developed a 3D tissue bioreactor that couples to both the stage of a custom multimodal microscopy system and a biaxial mechanical testing platform. Time dependent changes in microstructure and mechanical properties of fibroblast seeded cruciform fibrin gels were investigated while cultured under either anchored (1.0:1.0 stretch ratio) or strip biaxial (1.0:1.1) conditions. A multimodal nonlinear optical microscopy-optical coherence microscopy (NLOM-OCM) system was used to delineate noninvasively the relative spatial distributions of original fibrin, deposited collagen, and fibroblasts during month long culture. Serial in-culture mechanical testing was also performed to track the evolution of bulk mechanical properties under sterile conditions. Over the month long time course, seeded cells and deposited collagen were randomly distributed in equibiaxially anchored constructs, but exhibited preferential alignment parallel to the direction of the 10% stretch in constructs cultured under strip biaxial stretch. Surprisingly, both anchored and strip biaxial stretched constructs exhibited isotropic mechanical properties (including progressively increasing stiffness) despite developing a very different collagen microstructural organization. In summary, our biaxial bioreactor system integrating both NLOM-OCM and mechanical testing provided complementary information on microstructural organization and mechanical properties and, thus, may enable greater fundamental understanding of relationships between engineered soft tissue mechanics and mechanobiology.     

Three-dimensional fibril-reinforced finite element model of articular cartilage

Collagen fiber orientations in articular cartilage are tissue depth-dependent and joint site-specific. A realistic three-dimensional (3D) fiber orientation has not been implemented in modeling fluid flow-dependent response of articular cartilage; thus the detailed mechanical role of the collagen network may have not been fully understood. In the present study, a previously developed fibril-reinforced model of articular cartilage was extended to account for the 3D fiber orientation. A numerical procedure for the material model was incorporated into the finite element code ABAQUS using the “user material” option. Unconfined compression and indentation testing was evaluated. For indentation testing, we considered a mechanical contact between a solid indenter and a medial femoral condyle, assuming fiber orientations in the surface layer to follow the split-line pattern. The numerical results from the 3D modeling for unconfined compression seemed reasonably to deviate from that of axisymmetric modeling. Significant fiber orientation dependence was observed in the displacement, fluid pressure and velocity for the cases of moderate strain-rates, or during early relaxation. The influence of fiber orientation diminished at static and instantaneous compressions.     

Tissue-engineered valves with commissural alignment

We present a novel approach to producing bioartificial valves using the tissue-equivalent method of entrapping cells within a biopolymer gel and using a mold design that presents appropriate mechanical constraints to the cell-induced gel compaction to yield both the fibril alignment and the geometry of a native valve. Bileaflet valves were fabricated from bovine collagen and neonatal human dermal fibroblasts as proof of principle. The resultant valves possessed both commissure-tocommissure alignment of collagen fibers in the leaflets and circumferential alignment in the root. While this alignment was manifested in planar biaxial tensile mechanical properties, histology of the leaflets revealed an aligned collagen matrix but lacking other extracellular matrix (ECM) components present in the native valve. The apparent lack of ECM production by the fibroblasts after contracting and aligning the collagen fibrils is consistent with peak loads during biaxial testing being only approximately 10% of native leaflet values and a 0:1 coupling index that was only approximately 50% of native leaflet values despite exhibiting comparable values for the anisotropy index.     

Mechanical and structural contribution of non-fibrillar matrix in uniaxial tension: a collagen-agarose co-gel model

The mechanical role of non-fibrillar matrix and the nature of its interaction with the collagen network in soft tissues remain poorly understood, in part because of the lack of a simple experimental model system to quantify these interactions. This study’s objective was to examine mechanical and structural properties of collagen-agarose co-gels, utilized as a simplified model system, to understand better the relationships between the collagen network and non-fibrillar matrix. We hypothesized that the presence of agarose would have a pronounced effect on microstructural reorganization and mechanical behavior. Samples fabricated from gel solutions containing 1.0 mg/mL collagen and 0, 0.125, or 0.25% w/v agarose were evaluated via scanning electron microscopy, incremental tensile stress-relaxation tests, and polarized light imaging. While the incorporation of agarose did not dramatically alter collagen network morphology, agarose led to concentration-dependent changes in mechanical and structural properties. Specifically, resistance of co-gels to volume change corresponded with differences in fiber reorientation and elastic/viscoelastic mechanics. Results demonstrate strong relationships between tissue properties and offer insight into behavior of tissues of varying Poisson’s ratio and fiber kinematics. Results also suggest that non-fibrillar material may have significant effects on properties of artificial and native tissues even in tension, which is generally assumed to be collagen dominated.     

Strain-induced Collagen Organization at the Micro-level in Fibrin-based Engineered Tissue Constructs

Full understanding of strain-induced collagen organization in complex tissue geometries to create tissues with predefined collagen architecture has not been achieved. This is mainly due to our limited knowledge of collagen remodeling in developing tissues. Here we investigate strain-induced collagen (re)organization in fibrin based engineered tissues using nondestructive time-lapse imaging. The tissues start from a biaxially constrained myofibroblast-populated fibrin gel and are used to study: (A) remodeling from a static equi-biaxial loading condition to static uniaxial loading; and (B) remodeling of a biaxially constrained tissue under uniaxial cyclic straining before and after a change in strain direction. Under static conditions, collagen oriented parallel to the direction of strain, whereas under cyclic conditions the orientation in the constrained tissue was perpendicular to the direction of strain. It is concluded that due to the biaxial constraints the uniaxially, cyclically strained cells can exert forces in two directions and strain shield themselves. A subsequent change in the direction of cyclic straining resulted in a rapid reorientation of collagen at the tissue surface. Reorientation was significantly slower in deeper tissue layers, where tissue remodeling was dominated by contact guidance provided by the endogenous matrix. These findings emphasize the relevance of achieving a functional collagen organization right from the start of tissue culture.