Fabrication of dense anisotropic collagen scaffolds using biaxial compression.

We developed a new method to manufacture dense, aligned, and porous collagen scaffolds using biaxial plastic compression of type I collagen gels. Using a novel compression apparatus that constricts like an iris diaphragm, low density collagen gels were compressed to yield a permanently densified, highly aligned collagen material. Micro-porosity scaffolds were created using hydrophilic elastomer porogens that can be selectively removed following biaxial compression, with porosity modulated by using different porogen concentrations. The resulting scaffolds exhibit collagen densities that are similar to native connective tissues (∼10% collagen by weight), pronounced collagen alignment across multiple length scales, and an interconnected network of pores, making them highly relevant for use in tissue culture, the study of physiologically relevant cell-matrix interactions, and tissue engineering applications. The scaffolds exhibited highly anisotropic material behavior, with the modulus of the scaffolds in the fiber direction over 100 times greater than the modulus in the transverse direction. Adipose-derived mesenchymal stem cells were seeded onto the biaxially compressed scaffolds with minimal cell death over seven days of culture, along with cell proliferation and migration into the pore spaces. This fabrication method provides new capabilities to manufacture structurally and mechanically relevant cytocompatible scaffolds that will enable more physiologically relevant cell culture studies. Further improvement of manufacturing techniques has the potential to produce engineered scaffolds for direct replacement of dense connective tissues such as meniscus and annulus fibrosus. STATEMENT OF SIGNIFICANCE: In vitro studies of cell-matrix interactions and the engineering of replacement materials for collagenous connective tissues require biocompatible scaffolds that replicate the high collagen density (15-25%/wt), aligned fibrillar organization, and anisotropic mechanical properties of native tissues. However, methods for creating scaffolds with these characteristics are currently lacking. We developed a new apparatus and method to create high density, aligned, and porous collagen scaffolds using a biaxial compression with porogens technique. These scaffolds have a highly directional structure and mechanical properties, with the tensile strength and modulus up to 100 times greater in the direction of alignment. We also demonstrated that the scaffolds are a suitable material for cell culture, promoting cell adhesion, viability, and an aligned cell morphology comparable to the cell morphology observed in native aligned tissues.

Molecular level detection and localization of mechanical damage in collagen enabled by collagen hybridizing peptides.

Mechanical injury to connective tissue causes changes in collagen structure and material behaviour, but the role and mechanisms of molecular damage have not been established. In the case of mechanical subfailure damage, no apparent macroscale damage can be detected, yet this damage initiates and potentiates in pathological processes. Here, we utilize collagen hybridizing peptide (CHP), which binds unfolded collagen by triple helix formation, to detect molecular level subfailure damage to collagen in mechanically stretched rat tail tendon fascicle. Our results directly reveal that collagen triple helix unfolding occurs during tensile loading of collagenous tissues and thus is an important damage mechanism. Steered molecular dynamics simulations suggest that a likely mechanism for triple helix unfolding is intermolecular shearing of collagen α-chains. Our results elucidate a probable molecular failure mechanism associated with subfailure injuries, and demonstrate the potential of CHP targeting for diagnosis, treatment and monitoring of tissue disease and injury.

Nanoscale Imaging of Collagen Gels with Focused Ion Beam Milling and Scanning Electron Microscopy.

In vitro polymerized type I collagen hydrogels have been used extensively as a model system for three-dimensional (3D) cell and tissue culture, studies of fibrillogenesis, and investigation of multiscale force transmission within connective tissues. The nanoscale organization of collagen fibrils plays an essential role in the mechanics of these gels and emergent cellular behavior in culture, yet quantifying 3D structure with nanoscale resolution to fully characterize fibril organization remains a significant technical challenge. In this study, we demonstrate that a new imaging modality, focused ion beam scanning electron microscopy (FIB-SEM), can be used to generate 3D image datasets for visualizing and quantifying complex nanoscale organization and morphometry in collagen gels. We polymerized gels at a number of concentrations and conditions commonly used for in vitro models, stained and embedded the samples, and performed FIB-SEM imaging. The resulting image data had a voxel size of 25 nm, which is the highest resolution 3D data of a collagen fibril network ever obtained for collagen gels. This resolution was essential for discerning individual fibrils, fibril paths, and their branching and grouping. The resulting volumetric images revealed that polymerization conditions have a significant impact on the complex fibril morphology of the gels. We segmented the fibril network and demonstrated that individual collagen fibrils can be tracked in 3D space, providing quantitative analysis of network descriptors such as fibril diameter distribution, length, branch points, and fibril aggregations. FIB-SEM 3D reconstructions showed considerably less lateral grouping and overlap of fibrils than standard 2D SEM images, likely due to artifacts in SEM introduced by dehydration. This study demonstrates the utility of FIB-SEM for 3D imaging of collagen gels and quantitative analysis of 3D fibril networks. We anticipate that the method will see application in future studies of structure-function relationships in collagen gels as well as native collagenous tissues.

Continuum description of the Poisson's ratio of ligament and tendon under finite deformation.

Ligaments and tendons undergo volume loss when stretched along the primary fiber axis, which is evident by the large, strain-dependent Poisson's ratios measured during quasi-static tensile tests. Continuum constitutive models that have been used to describe ligament material behavior generally assume incompressibility, which does not reflect the volumetric material behavior seen experimentally. We developed a strain energy equation that describes large, strain dependent Poisson's ratios and nonlinear, transversely isotropic behavior using a novel method to numerically enforce the desired volumetric behavior. The Cauchy stress and spatial elasticity tensors for this strain energy equation were derived and implemented in the FEBio finite element software (www.febio.org). As part of this objective, we derived the Cauchy stress and spatial elasticity tensors for a compressible transversely isotropic material, which to our knowledge have not appeared previously in the literature. Elastic simulations demonstrated that the model predicted the nonlinear, upwardly concave uniaxial stress-strain behavior while also predicting a strain-dependent Poisson's ratio. Biphasic simulations of stress relaxation predicted a large outward fluid flux and substantial relaxation of the peak stress. Thus, the results of this study demonstrate that the viscoelastic behavior of ligaments and tendons can be predicted by modeling fluid movement when combined with a large Poisson's ratio. Further, the constitutive framework provides the means for accurate simulations of ligament volumetric material behavior without the need to resort to micromechanical or homogenization methods, thus facilitating its use in large scale, whole joint models.

A nitinol based flexor tendon fixation device: gapping and tensile strength measurements in cadaver flexor tendon.

In this study, a new nitinol based fixation device was investigated for use in repairing severed digital flexor tendons. The device, composed of superelastic nitinol, is tubular in shape with inward facing tines for gripping tissue. Its cellular structure was designed such that it has a large effective Poisson's ratio, which facilitates a "finger trap" effect. This allows for reduced tendon compression during a resting state (to permit vascular perfusion) and increased compression during loading (to drive the tines into the tissue for gripping). To test the feasibility of using this device for flexor tendon repair, it was tested on cadaver flexor digitorum profundus tendons. The tendons were excised, cut in the region corresponding to a zone II laceration, and repaired using the device. The device was easy to install and did not prevent the tendon from bending. Constant strain rate tensile testing revealed a mean tensile strength of 57.6 ± 7.7 N, with a force of 53.2 ± 7.8 N at a 2 mm gap. This exceeds the suggested primary repair strength of 45 N, which has been proposed as the necessary strength for enabling early mobilization. Although considerable future studies will be needed to determine the suitability of the new repair device for clinical use, this study demonstrates the feasibility of utilizing a tubular, nitinol repair device for flexor tendon fixation.

Tendon fascicles exhibit a linear correlation between Poisson's ratio and force during uniaxial stress relaxation.

The underlying mechanisms for the viscoelastic behavior of tendon and ligament tissue are poorly understood. It has been suggested that both a flow-dependent and flow-independent mechanism may contribute at different structural levels. We hypothesized that the stress relaxation response of a single tendon fascicle is consistent with the flow-dependent mechanism described by the biphasic theory (Armstrong et al., 1984, "An Analysis of the Unconfined Compression of Articular Cartilage," ASME J. Biomech. Eng., 106, pp. 165-173). To test this hypothesis, force, lateral strain, and Poisson's ratio were measured as a function of time during stress relaxation testing of six rat tail tendon fascicles from a Sprague Dawley rat. As predicted by biphasic theory, the lateral strain and Poisson's ratio were time dependent, a large estimated volume loss was seen at equilibrium and there was a linear correlation between the force and Poisson's ratio during stress relaxation. These results suggest that the fluid dependent mechanism described by biphasic theory may explain some or all of the apparent viscoelastic behavior of single tendon fascicles.

Effects of decorin proteoglycan on fibrillogenesis, ultrastructure, and mechanics of type I collagen gels.

The proteoglycan decorin is known to affect both the fibrillogenesis and the resulting ultrastructure of in vitro polymerized collagen gels. However, little is known about its effects on mechanical properties. In this study, 3D collagen gels were polymerized into tensile test specimens in the presence of decorin proteoglycan, decorin core protein, or dermatan sulfate (DS). Collagen fibrillogenesis, ultrastructure, and mechanical properties were then quantified using a turbidity assay, 2 forms of microscopy (SEM and confocal), and tensile testing. The presence of decorin proteoglycan or core protein decreased the rate and ultimate turbidity during fibrillogenesis and decreased the number of fibril aggregates (fibers) compared to control gels. The addition of decorin and core protein increased the linear modulus by a factor of 2 compared to controls, while the addition of DS reduced the linear modulus by a factor of 3. Adding decorin after fibrillogenesis had no effect, suggesting that decorin must be present during fibrillogenesis to increase the mechanical properties of the resulting gels. These results show that the inclusion of decorin proteoglycan during fibrillogenesis of type I collagen increases the modulus and tensile strength of resulting collagen gels. The increase in mechanical properties when polymerization occurs in the presence of the decorin proteoglycan is due to a reduction in the aggregation of fibrils into larger order structures such as fibers and fiber bundles.

Three-dimensional quantification of femoral head shape in controls and patients with cam-type femoroacetabular impingement.

An objective measurement technique to quantify 3D femoral head shape was developed and applied to normal subjects and patients with cam-type femoroacetabular impingement (FAI). 3D reconstructions were made from high-resolution CT images of 15 cam and 15 control femurs. Femoral heads were fit to ideal geometries consisting of rotational conchoids and spheres. Geometric similarity between native femoral heads and ideal shapes was quantified. The maximum distance native femoral heads protruded above ideal shapes and the protrusion area were measured. Conchoids provided a significantly better fit to native femoral head geometry than spheres for both groups. Cam-type FAI femurs had significantly greater maximum deviations (4.99 ± 0.39 mm and 4.08 ± 0.37 mm) than controls (2.41 ± 0.31 mm and 1.75 ± 0.30 mm) when fit to spheres or conchoids, respectively. The area of native femoral heads protruding above ideal shapes was significantly larger in controls when a lower threshold of 0.1 mm (for spheres) and 0.01 mm (for conchoids) was used to define a protrusion. The 3D measurement technique described herein could supplement measurements of radiographs in the diagnosis of cam-type FAI. Deviations up to 2.5 mm from ideal shapes can be expected in normal femurs while deviations of 4-5 mm are characteristic of cam-type FAI.

Micromechanical model of a surrogate for collagenous soft tissues: development, validation and analysis of mesoscale size effects.

Aligned, collagenous tissues such as tendons and ligaments are composed primarily of water and type I collagen, organized hierarchically into nanoscale fibrils, microscale fibers and mesoscale fascicles. Force transfer across scales is complex and poorly understood. Since innervation, the vasculature, damage mechanisms and mechanotransduction occur at the microscale and mesoscale, understanding multiscale interactions is of high importance. This study used a physical model in combination with a computational model to isolate and examine the mechanisms of force transfer between scales. A collagen-based surrogate served as the physical model. The surrogate consisted of extruded collagen fibers embedded within a collagen gel matrix. A micromechanical finite element model of the surrogate was validated using tensile test data that were recorded using a custom tensile testing device mounted on a confocal microscope. Results demonstrated that the experimentally measured macroscale strain was not representative of the microscale strain, which was highly inhomogeneous. The micromechanical model, in combination with a macroscopic continuum model, revealed that the microscale inhomogeneity resulted from size effects in the presence of a constrained boundary. A sensitivity study indicated that significant scale effects would be present over a range of physiologically relevant inter-fiber spacing values and matrix material properties. The results indicate that the traditional continuum assumption is not valid for describing the macroscale behavior of the surrogate and that boundary-induced size effects are present.

Micromechanical models of helical superstructures in ligament and tendon fibers predict large Poisson's ratios.

Experimental measurements of the Poisson's ratio in tendon and ligament tissue greatly exceed the isotropic limit of 0.5. This is indicative of volume loss during tensile loading. The microstructural origin of the large Poisson's ratios is unknown. It was hypothesized that a helical organization of fibrils within a fiber would result in a large Poisson's ratio in ligaments and tendons, and that this helical organization would be compatible with the crimped nature of these tissues, thus modeling their classic nonlinear stress-strain behavior. Micromechanical finite element models were constructed to represent crimped fibers with a super-helical organization, composed of fibrils embedded within a matrix material. A homogenization procedure was performed to determine both the effective Poisson's ratio and the Poisson function. The results showed that helical fibril organization within a crimped fiber was capable of simultaneously predicting large Poisson's ratios and the nonlinear stress-strain behavior seen experimentally. Parametric studies revealed that the predicted Poisson's ratio was strongly dependent on the helical pitch, crimp angle and the material coefficients. The results indicated that, for physiologically relevant parameters, the models were capable of predicting the large Poisson's ratios seen experimentally. It was concluded that helical organization within a crimped fiber can produce both the characteristic nonlinear stress-strain behavior and large Poisson's ratios, while fiber crimp alone could only account for the nonlinear stress-strain behavior.