Fibril Alignment Research Articles

Biomimetic Injectable 3D Hydrogels with Aligned Topography for Neural Tissue Engineering

Spinal cord trauma leads to destruction of the highly organized cytoarchitecture that carries information along the axis of the spinal column. Currently, there are no clinically accepted strategies that can help regenerate severed axons after spinal cord injury. Experimental neuroregenerative efforts include the creation of optimal biomaterials with aligned topography to support enhanced neuronal regeneration. Hydrogels are soft biomaterials with high water content that are widely used as scaffolds to interface with the central nervous system (CNS). Current available methods to create topography within the 3D amorphous hydrogels are typically complex. Here we examine a simple and reproducible method that results in consistently aligned fibrils within 3D matrices using thermally gelling biomimetic polymers that are compatible with neuronal cells. A collagen type I (Col)-based thermally gelling hydrogel system was used in combination with two other native extracellular matrix proteins: laminin I (LN), and hyaluronic acid (HA). Gelling kinetics for all gel types (Col, Col LN, Col HA) were examined, and we found that all three combinations of polymer formed consistent gels at 37°C. Col solution was faster to form gels (17 min), while Col LN and Col HA took longer (~22 minutes). A method of aspiration and ejection was used to produce Col-based hydrogels containing aligned fibrils. Gels were then examined using scanning electron microscopy (SEM). SEM images confirmed successful alignment in all gel types, and the size of fibers was consistent with reported values for collagen (~250 nm in diameter). We found that embryonic spinal cord neurons survive and produce processes aligned to collagen fibrils after 14 days in vitro. Next, we investigated the functionality of aligned and non-aligned Col hydrogels implanted acutely after a contusion type spinal cord injury to the thoracic spinal cord at T7/T8. Pan neuronal antibody-positive fibrils were found within all implants, aligned hydrogels supported neurite growth along the parallel direction of the implanted hydrogels. Our data indicate that thermally gelling biomimetic hydrogels can produce aligned matrices by a method of aspiration and ejection. The material composition and process of aligning hydrogels outlined here presents a novel platform for regenerative therapies for the CNS that is compatible with the survival and growth of neuronal cells in vitro and in vivo.

Shear-induced alignment of collagen fibrils using 3D cell printing for corneal stroma tissue engineering

The microenvironments of tissues or organs are complex architectures comprised of structural proteins including collagen. Particularly, the cornea is organized in a lattice pattern of collagen fibrils which play a significant role in its transparency. This paper introduces a transparent bioengineered corneal structure for transplantation. The structure is fabricated by inducing shear stress to a corneal stroma-derived decellularized extracellular matrix bioink based on a 3D cell printing technique. The printed structure recapitulates the native macrostructure of the cornea with aligned collagen fibrils which results in the construction of a highly matured and transparent cornea stroma analog. The level of shear stress, controlled by the various size of the printing nozzle, manipulates the arrangement of the fibrillar structure. With proper parameter selection, the printed cornea exhibits high cellular alignment capability, indicating a tissue-specific structural organization of collagen fibrils. In addition, this structural regulation enhances critical cellular events in the assembly of collagen over time. Interestingly, the collagen fibrils that remodeled along with the printing path create a lattice pattern similar to the structure of native human cornea after 4 weeks in vivo. Taken together, these results establish the possibilities and versatility of fabricating aligned collagen fibrils; this represents significant advances in corneal tissue engineering.

The matrix: building more bioactive extracellular matrices via tuning of substrate stiffness

Background & Aim Mesenchymal stromal cells (MSCs) have long been at the forefront of cellular therapy and regenerative medicine, and they are being explored in more than 700 NIH-funded trials for their therapeutic potential. The low frequency of MSCs in the human body, poor yield of MSCs from tissue biopsies, and high number of MSCs required for clinical applications demand that tissue-isolated MSCs undergo a lengthy expansion ex vivo, during which they exhibit senescence and become unsuitable for therapeutic applications. Decellularized extracellular matrices (dECM) present a novel solution to this issue, as MSCs grown on dECM maintain greater viability as compared to those grown on conventional tissue culture substrates. The effect of environmental parameters on extracellular matrix bioactivity requires further elucidation. Particularly, we will investigate the influence of substrate stiffness on the composition of MSC-derived ECM, to produce customized dECM which enhances cellular growth and functionality. This is achieved by culturing MSCs on hydrogels of varying stiffnesses, and assessing the bioactivity of the ECM produced during culture. Methods, Results & Conclusion Mechanical properties of tunable stiffness polyacrylamide gels were verified using an Instron Microtester. Cells used were isolated from the decidua basalis, and ECM was sourced from decidua basalis hTERT-transfected MSCs. ECM deposition was induced by addition of ascorbic acid. Instron measurements confirm the elastic moduli of fully-hydrated polyacrylamide gels to be 4 kPa, 10 kPa, and 40 kPa, tailored to mimic the stiffness of adipose tissue, skeletal muscle tissue, and collagenous bone tissue, respectively. MSCs exhibit a spindle-shaped morphology when cultured on the hydrogels. Confocal microscopy reveals microarchitectural differences between ECM deposited on softer vs. stiffer hydrogels. We have shown that MSCs can be grown on hydrogels of varying stiffnesses, and can be induced to laydown extracellular matrix. The functionalization of the gels with collagen I to aid cell adhesion has been confirmed with immunofluorescence. The deposition of ECM has also been confirmed with confocal microscopy, and the alignment of ECM fibrils has been shown to depend on substrate stiffness. Further work will involve proteomic characterization of the dECMs, and culturing MSCs on the dECMs to assess bioactivity. We expect that the substrate stiffness will modulate the ECM bioactivity, allowing customization of tailored dECM for MSC expansion.

Composite Gels Containing Whey Protein Fibrils and Bacterial Cellulose Microfibrils.

In this study, we investigated the gelation of WPI fibrils in the presence of bacterial cellulose (BC) microfibrils at pH 2 upon prolonged heating. Rheology and microstructure were investigated as a function of BC microfibril concentration. The presence of BC microfibrils did not influence the gelation dynamics and resulting overall structure of the WPI fibrillar gel. The storage modulus and loss modulus of the mixed WPI‐BC microfibril gels increased with increasing BC microfibril concentration, whereas the ratio between loss modulus and storage modulus remained constant. The WPI fibrils and BC microfibrils independently form two coexisting gel networks. Interestingly, near to the BC microfibrils more aligned WPI fibrils seemed to be formed, with individual WPI fibrils clearly distinguishable. The level of alignment of the WPI fibrils seemed to be dependent on the distance between BC microfibrils and WPI fibrils. This also is in line with our observation that with more BC microfibrils present, WPI fibrils are more aligned than in a WPI fibrillar gel without BC microfibrils. The large deformation response of the gels at different BC microfibril concentration and NaCl concentration is mainly influenced by the concentration of NaCl, which affects the WPI fibrillar gel structures, changing form linear fibrillar to a particulate gel. The WPI fibrillar gel yields the dominant contribution to the gel strength.

Adjusting the pore size of nano-cellulose aerogel by heat treatment in the gel stage

How to prepare a kind of porous material with the specific pore sizes according to the actual needs is always a tough problem. In this work, it has been found that it is an easy way to prepare nano-cellulose aerogels with adjustable pore size. The nano-cellulose (CNC) obtained by acid hydrolysis of microcrystalline cellulose (MCC). The prepared nano-cellulose wet gel was heat treated in an aqueous phase. Heat provides sufficient energy for further alignment of cellulose fibrils. Nano-cellulose aerogels are prepared by solvent exchange in tert-butanol and freeze drying. Solvent exchange in tert-butanol keeps the aerogel intact in a three-dimensional network structure. The characterization results show that the uniformity of the pores and the internal structure improved as the heat-treatment temperature increased. At the same time, the specific surface area and porosity of the aerogel increased, and the shrinkage decreased. This study suggests new possibilities for controlling the pore size of cellulose aerogels.

A Review of Structural and Biomechanical Changes in the Cornea in Aging, Disease, and Photochemical Crosslinking.

The study of corneal biomechanics is motivated by the tight relationship between biomechanical properties and visual function within the ocular system. For instance, variation in collagen fibril alignment and non-enzymatic crosslinks rank high among structural factors which give rise to the cornea's particular shape and ability to properly focus light. Gradation in these and other factors engender biomechanical changes which can be quantified by a wide variety of techniques. This review summarizes what is known about both the changes in corneal structure and associated changes in corneal biomechanical properties in aging, keratoconic, and photochemically crosslinked corneas. In addition, methods for measuring corneal biomechanics are discussed and the topics are related to both clinical studies and biomechanical modeling simulations.

Probing the importance of ECM alignment on cell migration

Screening for the influence of size, density and alignment of ECM fibrils in fibrous tissues shows that highly aligned adhesions are correlated with rapid migration.

Fabrication of juglone functionalized silver nanoparticle stabilized collagen scaffolds for pro-wound healing activities

The effective wound management strategies depends on identification and manipulation of the molecular defects in the pathophysiology of wound. Poor vascularization, protease susceptibility and microbial invasion at wound site affect the early wound closure. Hence, an efficient wound dressing material needs to promote angiogenesis, control proteolytic activity and microbial attack. The present study, describes designing and developing a novel wound dressing material by stabilization of collagen with juglone functionalized silver nanoparticle. Stabilization of collagen with juglone functionalized silver nanoparticles enhanced thermal properties, influenced the uniform alignment of collagen fibrils which enhanced collagen's ability to promote cell proliferation. FTIR and CD analyses revealed that juglone functionalized silver nanoparticles did not induced any structural changes in the collagen molecule. Juglone functionalized silver nanoparticles controlled the proteolytic activity in a spatio-temporal manner and elicited the angiogenic response by upregulation of cell adhesion molecules like β catenin and VE cadherin which had promoted the cell attachment and cell-cell contact. It had also promoted the expression of angiogenic signaling molecules like VEGF and VEGFR2. Further, the in vivo studies proved that the juglone functionalized silver nanoparticles had a potential role in rapid wound closure due to the cumulative property of juglone and silver nanoparticles.

Effect of collagen packing and moisture content on leather stiffness

Applications for skin derived collagen materials, such as leather and acellular dermal matrices, usually require both strength and flexibility. In general, both the tensile modulus (which has an impact on flexibility) and strength are known to increase with fiber alignment, in the tensile direction, for practically all collagen-based tissues. The structural basis for flexibility in leather was investigated and the moisture content was varied. Small angle X-ray scattering was used to determine collagen fibril orientation, elongation and lateral intermolecular spacing in leather conditioned by different controlled humidity environments. Flexibility was measured by a three point bending test. Leather was prepared by tanning under biaxial loading to create leather with increased fibril alignment and thus strength, but this treatment also increased the stiffness. As collagen aligns, it not only strengthens the material but it also stiffens because tensile loading is then applied along the covalent chain of the collagen molecules, rather than at an angle to it. Here it has been shown that with higher moisture content greater flexibility of the material develops as water absorption inside collagen fibrils produces a larger lateral spacing between collagen molecules. It is suggested that water provides a lubricating effect in collagen fibrils, enabling greater freedom of movement and therefore greater flexibility. When collagen molecules align in the strain direction during tanning, leather stiffens not only by the fiber alignment itself but also because collagen molecules pack closer together, reducing the ability of the molecules to move relative to each other.

Vibrational Circular Dichroism Sheds New Light on the Competitive Effects of Crowding and β-Synuclein on the Fibrillation Process of α-Synuclein.

The effects of crowding, using the crowding agent Ficoll 70, and the presence of β-synuclein on the fibrillation process of α-synuclein were studied by spectroscopic techniques, transmission electron microscopy, and thioflavin T assays. This combined approach, in which all techniques were applied to the same original sample, generated an unprecedented understanding of the effects of these modifying agents on the morphological properties of the fibrils. Separately, crowding gives rise to shorter mutually aligned fibrils, while β-synuclein leads to branched, short fibrils. The combination of both effects leads to short, branched, mutually aligned fibrils. Moreover, it is shown that the nondestructive technique of vibrational circular dichroism is extremely sensitive to the length and the higher-order morphology of the fibrils.

A novel method for continuous formation of cord-like collagen gels to fabricate durable fibers in which collagen fibrils are longitudinally aligned.

We developed a continuous formation method of cord-like collagen gels comprising fibrils preferentially aligned along the geometrical axes (CCGs). Collagen (2.5%) dissolved in a sodium phosphate buffer containing 280 mM of sodium chloride was introduced into a stainless cylinder (length 52 mm, diameter 2.0 mm) warmed at 38°C at a linear velocity of 2.5 mm/s. This process caused collagen fibril alignments under acute fibril formation in the cylinder, resulting in continuous formation of CCGs. Fibril formation rate, shear rate, and shear duration were substantial factors for successful CCG formation. Outstanding advantages of this method over conventional wet spinning include the capacity of this technique to form aligned fibrils in the entire gels and to control the diameter of cord-like gels over 1 mm. The air-drying of CCGs which were crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxy-succinimide produced dry collagen fibers with cross-sectional areas of 0.0123-0.135 mm2 . Upon the rewetting of the fibers, they failed at a stress of 54.5 ± 7.8 MPa, which is higher than the mean failure stress of anterior cruciate ligament tissue (13.3-37.8 MPa). These findings indicate that the CCG formation method enables the fabrication of collagen fibers which are potential components of collagen-based artificial tendons. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1011-1023, 2019.

Collagen Structure-Function Relationships from Solid-State NMR Spectroscopy.

The extracellular matrix of a tissue is as important to life as the cells within it. Its detailed molecular structure defines the environment of a tissue's cells and thus their properties, including differentiation and metabolic status. Collagen proteins are the major component of extracellular matrices. Self-assembled collagen fibrils provide both specific mechanical properties to handle external stresses on tissues and, at the molecular level, well-defined protein binding sites to interact with cells. How the cell-matrix interactions are maintained against the stresses on the tissue is an important and as yet unanswered question. Similarly, how collagen molecular and fibrillar structures change in aging and disease is a crucial open question. Solid-state NMR spectroscopy offers insight into collagen molecular conformation in intact in vivo and in vitro tissues, and in this Account we review how NMR spectroscopy is beginning to provide answers to these questions. In vivo 13C,15N labeling of the extracellular matrix has given insight into collagen molecular dynamics and generated multidimensional NMR "fingerprints" of collagen molecular structure that allow comparison of local collagen conformation between tissues. NMR studies have shown that charged collagen residues (Lys, Arg) adopt extended-side-chain conformations in the fibrillar structure to facilitate charge-charge interactions between neighboring collagen molecules, while hydrophobic residues (Leu, Ile) fold along the collagen molecular axis to minimize the hydrophobic area exposed to surrounding water. Detailed NMR and molecular modeling work has shown that the abundant Gly-Pro-Hyp (Hyp = hydroxyproline) triplets in collagen triple helices confer well-defined flexibility because the proline is conformationally metastable, in contrast to the expectation that these triplets confer structural rigidity. The alignment of the Gly-Pro-Hyp triplets within the fibril structure means that the Gly-Pro-Hyp molecular flexibility generates fibril flexibility. The fibrillar bands of Gly-Pro-Hyp are highly correlated with collagen ligand binding sites, leading to the hypothesis that the fibril alignment of Gly-Pro-Hyp triplets is essential to protect collagen-ligand binding against external stresses on the tissue. Non-enzymatic chemistry between collagen side-chain amine groups (Lys, Arg) and reducing sugars-glycation-is an important source of matrix structural change in aging and disease. Glycation leads to stiffening of collagen fibrils, which is widely speculated to be the result of intermolecular cross-linking. The chemistry of non-enzymatic glycation has been extensively detailed through NMR studies and has been shown to lead to side-chain modifications as the majority reaction products, rather than intermolecular cross-links, with resultant molecular misalignment in the fibrils. Thus, a picture is beginning to emerge in which collagen glycation causes stiffening through misalignment of collagen molecular flexible regions rather than intermolecular cross-linking, meaning that new thinking is needed on how to alleviate collagen structural changes in aging and disease.

Polymorphism in disease-related apolipoprotein C-II amyloid fibrils: a structural model for rod-like fibrils.

Human apolipoprotein (apo) C-II is one of several plasma apolipoproteins that form amyloid deposits in vivo and is an independent risk factor for cardiovascular disease. Lipid-free apoC-II readily self-assembles into twisted-ribbon amyloid fibrils but forms straight, rod-like amyloid fibrils in the presence of low concentrations of micellar phospholipids. Charge mutations exerted significantly different effects on rod-like fibril formation compared to their effects on twisted-ribbon fibril formation. For instance, the double mutant, K30D-D69K apoC-II, readily formed twisted-ribbon fibrils, while the rate of rod-like fibril formation in the presence of micellar phospholipid was negligible. Structural analysis of rod-like apoC-II fibrils, using hydrogen-deuterium exchange and NMR analysis showed exchange protection consistent with a core cross-β structure comprising the C-terminal 58-76 region. Molecular dynamics simulations of fibril arrangements for this region favoured a parallel cross-β structure. X-ray fibre diffraction data for aligned rod-like fibrils showed a major meridional spacing at 4.6 Å and equatorial spacings at 9.7, 23.8 and 46.6 Å. The latter two equatorial spacings are not observed for aligned twisted-ribbon fibrils and are predicted for a model involving two cross-β fibrils in an off-set antiparallel structure with four apoC-II units per rise of the β-sheet. This model is consistent with the mutational effects on rod-like apoC-II fibril formation. The lipid-dependent polymorphisms exhibited by apoC-II fibrils could determine the properties of apoC-II in renal amyloid deposits and their potential role in the development of cardiovascular disease.

Exploring the In Vivo Anti-Inflammatory Actions of Simvastatin-Loaded Porous Microspheres on Inflamed Tenocytes in a Collagenase-Induced Animal Model of Achilles Tendinitis.

Tendon rupture induces an inflammatory response characterized by release of pro-inflammatory cytokines and impaired tendon performance. This study sought to investigate the therapeutic effects of simvastatin-loaded porous microspheres (SIM/PMSs) on inflamed tenocytes in vitro and collagenase-induced Achilles tendinitis in vivo. The treatment of SIM/PMSs in lipopolysaccharide (LPS)-treated tenocytes reduced the mRNA expressions of pro-inflammatory cytokines (Matrix metalloproteinase-3 (MMP-3), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α)). In addition, the local injection of SIM/PMSs into the tendons of collagenase-induced Achilles tendinitis rat models suppressed pro-inflammatory cytokines (MMP-3, COX-2, IL-6, TNF-α, and MMP-13). This local treatment also upregulated anti-inflammatory cytokines (IL-4, IL-10, and IL-13). Furthermore, treatment with SIM/PMSs also improved the alignment of collagen fibrils and effectively prevented collagen disruption in a dose-dependent manner. Therefore, SIM/PMSs treatment resulted in an incremental increase in the collagen content, stiffness, and tensile strength in tendons. This study suggests that SIM/PMSs have great potential for tendon healing and restoration in Achilles tendinitis.

Impacts of maturation on the micromechanics of the meniscus extracellular matrix

To elucidate how maturation impacts the structure and mechanics of meniscus extracellular matrix (ECM) at the length scale of collagen fibrils and fibers, we tested the micromechanical properties of fetal and adult bovine menisci via atomic force microscopy (AFM)-nanoindentation. For circumferential fibers, we detected significant increase in the effective indentation modulus, Eind, with age. Such impact is in agreement with the increase in collagen fibril diameter and alignment during maturation, and is more pronounced in the outer zone, where collagen fibrils are more aligned and packed. Meanwhile, maturation also markedly increases the Eind of radial tie fibers, but not those of intact surface or superficial layer. These results provide new insights into the effect of maturation on the assembly of meniscus ECM, and enable the design of new meniscus repair strategies by modulating local ECM structure and mechanical behaviors.

An Agent-Based Discrete Collagen Fiber Network Model of Dynamic Traction Force-Induced Remodeling.

Microstructural properties of extracellular matrix (ECM) promote cell and tissue homeostasis as well as contribute to the formation and progression of disease. In order to understand how microstructural properties influence the mechanical properties and traction force-induced remodeling of ECM, we developed an agent-based model that incorporates repetitively applied traction force within a discrete fiber network. An important difference between our model and similar finite element models is that by implementing more biologically realistic dynamic traction, we can explore a greater range of matrix remodeling. Here, we validated our model by reproducing qualitative trends observed in three sets of experimental data reported by others: tensile and shear testing of cell-free collagen gels, collagen remodeling around a single isolated cell, and collagen remodeling between pairs of cells. In response to tensile and shear strain, simulated acellular networks with straight fibrils exhibited biphasic stress-strain curves indicative of strain-stiffening. When fibril curvature was introduced, stress-strain curves shifted to the right, delaying the onset of strain-stiffening. Our data support the notion that strain-stiffening might occur as individual fibrils successively align along the axis of strain and become engaged in tension. In simulations with a single, contractile cell, peak collagen displacement occurred closest to the cell and decreased with increasing distance. In simulations with two cells, compaction of collagen between cells appeared inversely related to the initial distance between cells. These results for cell-populated collagen networks match in vitro findings. A demonstrable benefit of modeling is that it allows for further analysis not feasible with experimentation. Within two-cell simulations, strain energy within the collagen network measured from the final state was relatively uniform around the outer surface of cells separated by 250 μm, but became increasingly nonuniform as the distance between cells decreased. For cells separated by 75 and 100 μm, strain energy peaked in the direction toward the other cell in the region in which fibrils become highly aligned and reached a minimum adjacent to this region, not on the opposite side of the cell as might be expected. This pattern of strain energy was partly attributable to the pattern of collagen compaction, but was still present when mapping strain energy divided by collagen density. Findings like these are of interest because fibril alignment, density, and strain energy may each contribute to contact guidance during tissue morphogenesis.

Artificially modified collagen fibril orientation affects leather tear strength.

Ovine leather has around half the tear strength of bovine leather and is therefore not suitable for high-value applications such as shoes. Tear strength has been correlated with the natural collagen fibril alignment (orientation index, OI). It is hypothesized that it could be possible to artificially increase the OI of the collagen fibrils and that an artificial increase in OI could increase tear strength. Ovine skins, after pickling and bating, were strained biaxially during chrome tanning. The strain ranged from 2 to 15% of the initial sample length, either uniformly in both directions by 10% or with 3% in one direction and 15% in the other. Once tanned, the leather tear strengths were measured and the collagen fibril orientation was measured using synchrotron-based small-angle X-ray scattering. The OI increased as a result of strain during tanning from 0.48 to 0.79 (P = 0.001) measured edge-on and the thickness-normalized tear strength increased from 27 to 43 N mm-1 (P < 0.001) after leather was strained 10% in two orthogonal directions. This is evidence to support a causal relationship between high OI (measured edge-on), highly influenced by thickness, and tear strength. It also provides a method to produce stronger leather. © 2017 Society of Chemical Industry.

Control of Cellulose Nano Fibril Alignment by Electric Field in Elongational Flow for High Strength Single Fiber Fabrication.

Control of Cellulose Nano Fibril Alignment by Electric Field in Elongational Flow for High Strength Single Fiber Fabrication.

Modulation of collagen population under honey assisted wound healing in diabetic rat model

Abstract Background Diabetic wound with impaired healing attributes owing to inappropriate synthesis and alignment of collagen fibrils with aberrant post translational modifications impose substantial economic burden. Honey: a recognized natural collagen pro-synthetic healing agent; stimulates rapid healing with its anti-inflammatory and anti-scarring approach. Present study investigates the effect of honey on collagen population in diabetic wound model. Methodology Full thickness wound was created on rat and divided into three groups: normal saline treated group (N), diabetic honey treated group (H), diabetic povidone iodine treated group (PI). Biopsies were collected from wound edges for collagen characterization. Main findings Histological attributes of collagen fibers in N and H group biopsy displayed proximity. Ultrastructure analysis using scanning electron microscope (SEM) revealed that the D-Spacing and collagen diameter under honey treatment were in close resemblance to normal skin collagen. FTIR spectra confirmed secondary structure of collagen with a greater peak height in PI group exhibiting excessive glycation than H group. Nano-indentation profiling showed decreased displacement in PI samples resulting from increased rigidity when compared to honey treated ones. Molecular expression of collagen I and III exhibited optimum ratio in H group similar to N group. Conclusions Based on these findings, it might be opined that honey predominantly helps in regulating synthesis, glycation and deposition followed by mechanical quality of collagen alike normal skin and its application also accelerated diabetic healing process.

Modeling the effect of collagen fibril alignment on ligament mechanical behavior.

Ligament mechanical behavior is primarily regulated by fibrous networks of type I collagen. Although these fibrous networks are typically highly aligned, healthy and injured ligament can also exhibit disorganized collagen architecture. The objective of this study was to determine whether variations in the collagen fibril network between neighboring ligaments can predict observed differences in mechanical behavior. Ligament specimens from two regions of bovine fetlock joints, which either exhibited highly aligned or disorganized collagen fibril networks, were mechanically tested in uniaxial tension. Confocal microscopy and FiberFit software were used to quantify the collagen fibril dispersion and mean fibril orientation in the mechanically tested specimens. These two structural parameters served as inputs into an established hyperelastic constitutive model that accounts for a continuous distribution of planar fibril orientations. The ability of the model to predict differences in the mechanical behavior between neighboring ligaments was tested by (1) curve fitting the model parameters to the stress response of the ligament with highly aligned fibrils and then (2) using this model to predict the stress response of the ligament with disorganized fibrils by only changing the parameter values for fibril dispersion and mean fibril orientation. This study found that when using parameter values for fibril dispersion and mean fibril orientation based on confocal imaging data, the model strongly predicted the average stress response of ligaments with disorganized fibrils ([Formula: see text]); however, the model only successfully predicted the individual stress response of ligaments with disorganized fibrils in half the specimens tested. Model predictions became worse when parameters for fibril dispersion and mean fibril orientation were not based on confocal imaging data. These findings emphasize the importance of collagen fibril alignment in ligament mechanics and help advance a mechanistic understanding of fibrillar networks in healthy and injured ligament.