Measuring collagen fibril diameter with differential interference contrast microscopy.

Collagen fibrils, linear arrangements of collagen monomers, 20-500 nm in diameter, comprising hundreds of molecules in their cross-section, are the fundamental structural unit in a variety of load-bearing tissues such as tendons, ligaments, skin, cornea, and bone. These fibrils often assemble into more complex structures, providing mechanical stability, strength, or toughness to the host tissue. Unfortunately, there is little information available on individual fibril dynamics, mechanics, growth, aggregation and remodeling because they are difficult to image using visible light as a probe. The principle quantity of interest is the fibril diameter, which is difficult to extract accurately, dynamically, in situ and non-destructively. An optical method, differential interference contrast (DIC) microscopy has been used to visualize dynamic structures that are as small as microtubules (25 nm diameter) and has been shown to be sensitive to the size of objects smaller than the wavelength of light. In this investigation, we take advantage of DIC microscopy's ability to report dimensions of nanometer scale objects to generate a curve that relates collagen diameter to DIC edge intensity shift (DIC-EIS). We further calibrate the curve using electron microscopy and demonstrate a linear correlation between fibril diameter and the DIC-EIS. Using a non-oil immersion, 40x objective (NA 0.6), collagen fibril diameters between ~100 nm to ~ 300 nm could be obtained with ±11 and ±4 nm accuracy for dehydrated and hydrated fibrils, respectively. This simple, nondestructive, label free method should advance our ability to directly examine fibril dynamics under experimental conditions that are physiologically relevant.

Tendon Extracellular Matrix Assembly, Maintenance and Dysregulation Throughout Life.

In his Lissner Award medal lecture in 2000, Stephen Cowin asked the question: "How is a tissue built?" It is not a new question, but it remains as relevant today as it did when it was asked 20 years ago. In fact, research on the organization and development of tissue structure has been a primary focus of tendon and ligament research for over two centuries. The tendon extracellular matrix (ECM) is critical to overall tissue function; it gives the tissue its unique mechanical properties, exhibiting complex non-linear responses, viscoelasticity and flow mechanisms, excellent energy storage and fatigue resistance. This matrix also creates a unique microenvironment for resident cells, allowing cells to maintain their phenotype and translate mechanical and chemical signals into biological responses. Importantly, this architecture is constantly remodeled by local cell populations in response to changing biochemical (systemic and local disease or injury) and mechanical (exercise, disuse, and overuse) stimuli. Here, we review the current understanding of matrix remodeling throughout life, focusing on formation and assembly during the postnatal period, maintenance and homeostasis during adulthood, and changes to homeostasis in natural aging. We also discuss advances in model systems and novel tools for studying collagen and non-collagenous matrix remodeling throughout life, and finally conclude by identifying key questions that have yet to be answered.

Quick-freeze/deep-etch electron microscopy visualization of the mouse posterior pole.

The mouse is one of the most commonly used mammalian systems to study human diseases. In particular it has been an invaluable tool to model a multitude of ocular pathologies affecting the posterior pole. The aim of this study was to create a comprehensive map of the ultrastructure of the mouse posterior pole using the quick-freeze/deep-etch method (QFDE). QFDE can produce detailed three-dimensional images of tissue structure and macromolecular moieties, without many of the artifacts introduced by structure-altering post-processing methods necessary to perform conventional transmission electron microscopy (cTEM). A total of 18 eyes from aged C57BL6/J mice were enucleated and the posterior poles were processed, either intact or with the retinal pigment epithelium (RPE) cell layer removed, for imaging by either QFDE or cTEM. QFDE images were correlated with cTEM cross-sections and en face images through the outer retina. Nicely preserved outer retinal architecture was observed with both methods, however, QFDE provided excellent high magnification imaging, with greater detail, of the apical, central, and basal planes of the RPE. Furthermore, key landmarks within Bruch's membrane, choriocapillaris, choroid and sclera were characterized and identified. In this study we developed methods for preparing the outer retina of the mouse for evaluation with QFDE and provide a map of the ultrastructure and cellular composition of the outer posterior pole. This technique should be applicable for morphological evaluation of mouse models, in which detailed visualization of subtle ocular structural changes is needed or in cases where post-processing methods introduce unacceptable artifacts.

Micromechanical Modeling Study of Mechanical Inhibition of Enzymatic Degradation of Collagen Tissues.

This study investigates how the collagen fiber structure influences the enzymatic degradation of collagen tissues. We developed a micromechanical model of a fibrous collagen tissue undergoing enzymatic degradation based on two central hypotheses. The collagen fibers are crimped in the undeformed configuration. Enzymatic degradation is an energy activated process and the activation energy is increased by the axial strain energy density of the fiber. We determined the intrinsic degradation rate and characteristic energy for mechanical inhibition from fibril-level degradation experiments and applied the parameters to predict the effect of the crimped fiber structure and fiber properties on the degradation of bovine cornea and pericardium tissues under controlled tension. We then applied the model to examine the effect of the tissue stress state on the rate of tissue degradation and the anisotropic fiber structures that developed from enzymatic degradation.

Differential contributions of conformation extension and domain unfolding to properties of fibronectin nanotextiles.

Fibronectin (FN) textiles are built as nanometer-thick fabrics. When uniaxially loaded, these fabrics exhibit a distinct threshold between elastic and plastic deformation with increasing stretch. Fabric mechanics are modeled using an eight-chain network and two-state model, revealing that elastic properties of FN depend on conformational extension of the protein and that plastic deformation depends on domain unfolding. Our results suggest how the molecular architecture of a molecule can be exploited for designer mechanical properties of a bulk material.

Mechanochemistry of collagen.

The collagen molecular family is the result of nearly one billion years of evolution. It is a unique family of proteins, the majority of which provide general mechanical support to biological tissues. Fibril forming collagens are the most abundant collagens in vertebrate animals and are generally found in positions that resist tensile loading. In animals, cells produce fibril-forming collagen molecules that self-assemble into larger structures known as collagen fibrils. Collagen fibrils are the fundamental, continuous, load-bearing elements in connective tissues, but are often further aggregated into larger load-bearing structures, fascicles in tendon, lamellae in cornea and in intervertebral disk. We know that failure to form fibrillar collagen is embryonic lethal, and excessive collagen formation/growth (fibrosis) or uncontrolled enzymatic remodeling (type II collagen: osteoarthritis) is pathological. Collagen is thus critical to vertebrate viability and instrumental in maintaining efficient mechanical structures. However, despite decades of research, our understanding of collagen matrix formation is not complete, and we know still less about the detailed mechanisms that drive collagen remodeling, growth, and pathology. In this perspective, we examine the known role of mechanical force on the formation and development of collagenous structure. We then discuss a mechanochemical mechanism that has the potential to unify our understanding of collagenous tissue assembly dynamics, which preferentially deposits and grows collagen fibrils directly in the path of mechanical force, where the energetics should be dissuasive and where collagen fibrils are most required. We term this mechanism: Mechanochemical force-structure causality. STATEMENT OF SIGNIFICANCE: Our mechanochemical-force structure causality postulate suggests that collagen molecules are components of mechanochemically-sensitive and dynamically-responsive fibrils. Collagen molecules assemble preferentially in the path of applied strain, can be grown in place by mechanical extension, and are retained in the path of force through strain-stabilization. The mechanisms that drive this behavior operate at the level of the molecules themselves and are encoded into the structure of the biomaterial. The concept might change our understanding of structure formation, enhance our ability to treat injuries, and accelerate the development of therapeutics to prevent pathologies such as fibrosis. We suggest that collagen is a mechanochemically responsive dynamic element designed to provide a substantial "material assist" in the construction of adaptive carriers of mechanical signals.

Traumatic brain injury and the pathways to cerebral tau accumulation.

Tau is a protein that has received national mainstream recognition for its potential negative impact to the brain. This review succinctly provides information on the structure of tau and its normal physiological functions, including in hibernation and changes throughout the estrus cycle. There are many pathways involved in phosphorylating tau including diabetes, stroke, Alzheimer's disease (AD), brain injury, aging, and drug use. The common mechanisms for these processes are put into context with changes observed in mild and repetitive mild traumatic brain injury (TBI). The phosphorylation of tau is a part of the progression to pathology, but the ability for tau to aggregate and propagate is also addressed. Summarizing both the functional and dysfunctional roles of tau can help advance our understanding of this complex protein, improve our care for individuals with a history of TBI, and lead to development of therapeutic interventions to prevent or reverse tau-mediated neurodegeneration.

Corneal biomechanics and biomaterials.

The ability to clearly observe one's environment in the visible spectrum provides a tremendous evolutionary advantage in most of the world's habitats. The complex optical processing system that has evolved in higher vertebrate animals gathers, focuses, detects, transduces, and interprets incoming visible light. The cornea resides at the front end of this imaging system, where it provides a clear optical aperture, substantial refractive power, and the structural stability required to protect the fragile intraocular components. Nature has resolved these simultaneous design requirements through an exceedingly clever manipulation of common extracellular-matrix structural materials (e.g., collagen and proteoglycans). In this review, we (a) examine the biophysical and optical roles of the cornea, (b) discuss increasingly popular approaches to altering its natural refractive properties with an emphasis on biomechanics, and (c) investigate the fast-rising science of corneal replacement via synthetic biomaterials. We close by considering relevant open problems that would benefit from the increased attention of bioengineers.

Disorganized collagen scaffold interferes with fibroblast mediated deposition of organized extracellular matrix in vitro.

Many tissue engineering applications require the remodeling of a degradable scaffold either in vitro or in situ. Although inefficient remodeling or failure to fully remodel the temporary matrix can result in a poor clinical outcome, very few investigations have examined in detail, the interaction of regenerative cells with temporary scaffoldings. In a recent series of investigations, randomly oriented collagen gels were directly implanted into human corneal pockets and followed for 24 months. The resulting remodeling response exhibited a high degree of variability which likely reflects differing regenerative/synthetic capacity across patients. Given this variability, we hypothesize that a disorganized, degradable provisional scaffold could be disruptive to a uniform, organized reconstruction of stromal matrix. In this investigation, two established corneal stroma tissue engineering culture systems (collagen scaffold-based and scaffold-free) were compared to determine if the presence of the disorganized collagen gel influenced matrix production and organizational control exerted by primary human corneal fibroblast cells (PHCFCs). PHCFCs were cultured on thin disorganized reconstituted collagen substrate (RCS--five donors: average age 34.4) or on a bare polycarbonate membrane (five donors: average age 32.4 controls). The organization and morphology of the two culture systems were compared over the long-term at 4, 8, and 11/12 weeks. Construct thickness and extracellular matrix organization/alignment was tracked optically with bright field and differential interference contrast (DIC) microscopy. The details of cell/matrix morphology and cell/matrix interaction were examined with standard transmission, cuprolinic blue and quick-freeze/deep-etch electron microscopy. Both the scaffold-free and the collagen-based scaffold cultures produced organized arrays of collagen fibrils. However, at all time points, the amount of organized cell-derived matrix in the scaffold-based constructs was significantly lower than that produced by scaffold-free constructs (controls). We also observed significant variability in the remodeling of RCS scaffold by PHCFCs. PHCFCs which penetrated the RCS scaffold did exert robust local control over secreted collagen but did not appear to globally reorganize the scaffold effectively in the time period of the study. Consistent with our hypothesis, the results demonstrate that the presence of the scaffold appears to interfere with the global organization of the cell-derived matrix. The production of highly organized local matrix by fibroblasts which penetrated the scaffold suggests that there is a mechanism which operates close to the cell membrane capable of controlling fibril organization. Nonetheless, the local control of the collagen alignment produced by cells within the scaffold was not continuous and did not result in overall global organization of the construct. Using a disorganized scaffold as a guide to produce highly organized tissue has the potential to delay the production of useful matrix or prevent uniform remodeling. The results of this study may shed light on the recent attempts to use disorganized collagenous matrix as a temporary corneal replacement in vivo which led to a variable remodeling response.

Lamina Cribrosa Thickening in Early Glaucoma Predicted by a Microstructure Motivated Growth and Remodeling Approach.

Glaucoma is among the leading causes of blindness worldwide. The ocular disease is characterized by irreversible damage of the retinal ganglion cell axons at the level of the lamina cribrosa (LC). The LC is a porous, connective tissue structure whose function is believed to provide mechanical support to the axons as they exit the eye on their path from the retina to the brain. Early experimental glaucoma studies have shown that the LC remodels into a thicker, more posterior structure which incorporates more connective tissue after intraocular pressure (IOP) elevation. The process by which this occurs is unknown. Here we present a microstructure motivated growth and remodeling (G&R) formulation to explore a potential mechanism of these structural changes. We hypothesize that the mechanical strain experienced by the collagen fibrils in the LC stimulates the G&R response at the micro-scale. The proposed G&R algorithm controls collagen fibril synthesis/degradation and adapts the residual strains between collagen fibrils and the surrounding tissue to achieve biomechanical homeostasis. The G&R algorithm was applied to a generic finite element model of the human eye subjected to normal and elevated IOP. The G&R simulation underscores the biomechanical need for a LC at normal IOP. The numerical results suggest that IOP elevation leads to LC thickening due to an increase in collagen fibril mass, which is in good agreement with experimental observations in early glaucoma monkey eyes. This is the first study to demonstrate that a biomechanically-driven G&R mechanism can lead to the LC thickening observed in early experimental glaucoma.

Development of Fluorescently Labeled, Functional Type I Collagen Molecules.

While de novo collagen fibril formation is well-studied, there are few investigations into the growth and remodeling of extant fibrils, where molecular collagen incorporation into and erosion from the fibril surface must delicately balance during fibril growth and remodeling. Observing molecule/fibril interactions is difficult, requiring the tracking of molecular dynamics while, at the same time, minimizing the effect of the observation on fibril structure and assembly. To address the observation-interference problem, exogenous collagen molecules are tagged with small fluorophores and the fibrillogenesis kinetics of labeled collagen molecules as well as the structure and network morphology of assembled fibrils are examined. While excessive labeling significantly disturbs fibrillogenesis kinetics and network morphology of assembled fibrils, adding less than ≈1.2 labels per collagen molecule preserves these characteristics. Applications of the functional, labeled collagen probe are demonstrated in both cellular and acellular systems. The functional, labeled collagen associates strongly with native fibrils and when added to an in vitro model of corneal stromal development at low concentration, the labeled collagen is incorporated into a fine extracellular matrix (ECM) network associated with the cells within 24 h.

Molecular mechanochemistry: low force switch slows enzymatic cleavage of human type I collagen monomer.

In vertebrate animals, fibrillar collagen accumulates, organizes, and persists in structures which resist mechanical force. This antidissipative behavior is possibly due to a mechanochemical force-switch which converts collagen from enzyme-susceptible to enzyme-resistant. Degradation experiments on native tissue and reconstituted fibrils suggest that collagen/enzyme kinetics favor the retention of loaded collagen. We used a massively parallel, single molecule, mechanochemical reaction assay to demonstrate that the effect is derivative of molecular mechanics. Tensile loads higher than 3 pN dramatically reduced (10×) the enzymatic degradation rate of recombinant human type I collagen monomers by Clostridium histolyticum compared to unloaded controls. Because bacterial collagenase accesses collagen at multiple sites and is an aggressive cleaver of the collagen triple helical domain, the results suggest that collagen molecular architecture is generally more stable when mechanically strained in tension. Thus the tensile mechanical state of collagen monomers is likely to be correlated to their longevity in tissues. Further, strain-actuated molecular stability of collagen may constitute the fundamental basis of a smart structural mechanism which enhances the ability of animals to place, retain, and load-optimize material in the path of mechanical forces.

Method for measurement of collagen monomer orientation in fluorescence microscopy.

SIGNIFICANCE: Collagen is the most abundant protein in vertebrates and is found in tissues that regularly experience tension, compression, and shear forces. However, the underlying mechanism of collagen fibril formation and remodeling is poorly understood. AIM: We explore how a collagen monomer is visualized using fluorescence microscopy and how its spatial orientation is determined. Defining the orientation of collagen monomers is not a trivial problem, as the monomer has a weak contrast and is relatively small. It is possible to attach fluorescence tags for contrast, but the size is still a problem for detecting orientation using fluorescence microscopy. APPROACH: We present two methods for detecting a monomer and classifying its orientation. A modified Gabor filter set and an automatic classifier trained by convolutional neural network based on a synthetic dataset were used. RESULTS: By evaluating the performance of these two approaches with synthetic and experimental data, our results show that it is possible to determine the location and orientation with an error of ∼37 deg of a single monomer with fluorescence microscopy. CONCLUSIONS: These findings can contribute to our understanding of collagen monomers interaction with collagen fibrils surface during fibril formation and remodeling.

Stiffening of the extracellular matrix is a sufficient condition for airway hyperreactivity.

The current therapeutic approach to asthma focuses exclusively on targeting inflammation and reducing airway smooth muscle force to prevent the recurrence of symptoms. However, even when inflammation is brought under control, airways in an asthmatic can still hyperconstrict when exposed to a low dose of agonist. This suggests that there are mechanisms at play that are likely triggered by inflammation and eventually become self-sustaining so that even when airway inflammation is brought back under control, these alternative mechanisms continue to drive airway hyperreactivity in asthmatics. In this study, we hypothesized that stiffening of the airway extracellular matrix is a core pathological change sufficient to support excessive bronchoconstriction even in the absence of inflammation. To test this hypothesis, we increased the stiffness of the airway extracellular matrix by photo-crosslinking collagen fibers within the airway wall of freshly dissected bovine rings using riboflavin (vitamin B2) and Ultraviolet-A radiation. In our experiments, collagen crosslinking led to a twofold increase in the stiffness of the airway extracellular matrix. This change was sufficient to cause airways to constrict to a greater degree, and at a faster rate when they were exposed to 10-5 M acetylcholine for 5 min. Our results show that stiffening of the extracellular matrix is sufficient to drive excessive airway constriction even in the absence of inflammatory signals.NEW & NOTEWORTHY Targeting inflammation is the central dogma on which current asthma therapy is based. Here, we show that a healthy airway can be made to constrict excessively and at a faster rate in response to the same stimulus by increasing the stiffness of the extracellular matrix, without the use of inflammatory agents. Our results provide an independent mechanism by which airway remodeling in asthma can sustain airway hyperreactivity even in the absence of inflammatory signals.

Probing collagen/enzyme mechanochemistry in native tissue with dynamic, enzyme-induced creep.

Mechanical strain or stretch of collagen has been shown to be protective of fibrils against both thermal and enzymatic degradation. The details of this mechanochemical relationship could change our understanding of load-bearing tissue formation, growth, maintenance, and disease in vertebrate animals. However, extracting a quantitative relationship between strain and the rate of enzymatic degradation is extremely difficult in bulk tissue due to confounding diffusion effects. In this investigation, we develop a dynamic, enzyme-induced creep assay and diffusion/reaction rate scaling arguments to extract a lower bound on the relationship between strain and the cutting rate of bacterial collagenase (BC) at low strains. The assay method permits continuous, forced probing of enzyme-induced strain which is very sensitive to degradation rate differences between specimens at low initial strain. The results, obtained on uniaxially loaded strips of bovine corneal tissue (0.1, 0.25, or 0.5 N), demonstrate that small differences in strain alter the enzymatic cutting rate of the BC substantially. It was estimated that a change in tissue elongation of only 1.5% (at approximately 5% strain) reduces the maximum cutting rate of the enzyme by more than half. Estimation of the average load per monomer in the tissue strips indicates that this protective "cutoff" occurs when the collagen monomers are transitioning from an entropic to an energetic mechanical regime. The continuous tracking of the enzymatic cleavage rate as a function of strain during the initial creep response indicates that the decrease in the cleavage rate of the BC is nonlinear (initially steep between 4.5 and 6.5% and then flattens out from 6.5 to 9.5%). The high sensitivity to strain at low strain implies that even lightly loaded collagenous tissue may exhibit significant strain protection. The dynamic, enzyme-induced creep assay described herein has the potential to permit the rapid characterization of collagen/enzyme mechanochemistry in many different tissue types.

LXRs regulate features of age-related macular degeneration and may be a potential therapeutic target.

Effective treatments and animal models for the most prevalent neurodegenerative form of blindness in elderly people, called age-related macular degeneration (AMD), are lacking. Genome-wide association studies have identified lipid metabolism and inflammation as AMD-associated pathogenic pathways. Given liver X receptors (LXRs), encoded by the nuclear receptor subfamily 1 group H members 2 and 3 (NR1H3 and NR1H2), are master regulators of these pathways, herein we investigated the role of LXR in human and mouse eyes as a function of age and disease and tested the therapeutic potential of targeting LXR. We identified immunopositive LXR fragments in human extracellular early dry AMD lesions and a decrease in LXR expression within the retinal pigment epithelium (RPE) as a function of age. Aged mice lacking LXR presented with isoform-dependent ocular pathologies. Specifically, loss of the Nr1h3 isoform resulted in pathobiologies aligned with AMD, supported by compromised visual function, accumulation of native and oxidized lipids in the outer retina, and upregulation of ocular inflammatory cytokines, while absence of Nr1h2 was associated with ocular lipoidal degeneration. LXR activation not only ameliorated lipid accumulation and oxidant-induced injury in RPE cells but also decreased ocular inflammatory markers and lipid deposition in a mouse model, thereby providing translational support for pursuing LXR-active pharmaceuticals as potential therapies for dry AMD.

Prelude to corneal tissue engineering - gaining control of collagen organization.

By most standard engineering practice principles, it is premature to credibly discuss the "engineering" of a human cornea. A professional design engineer would assert that we still do not know what a cornea is (and correctly so), therefore we cannot possibly build one. The proof resides in the fact that there are no clinically viable corneas based on classical tissue engineering methods available. This is possibly because tissue engineering in the classical sense (seeding a degradable scaffolding with a population synthetically active cells) does not produce conditions which support the generation of organized tissue. Alternative approaches to the problem are in their infancy and include the methods which attempt to recapitulate development or to produce corneal stromal analogs de novo which require minimal remodeling. Nonetheless, tissue engineering efforts, which have been focused on producing the fundamental functional component of a cornea (organized alternating arrays of collagen or "lamellae"), may have already provided valuable new insights and tools relevant to development, growth, remodeling and pathologies associated with connective tissue in general. This is because engineers ask a fundamentally different question (How can that be done?) than do biological scientists (How is that done?). The difference in inquiry has prompted us to closely examine (and to mimic) development as well as investigate collagen physicochemical behavior so that we may exert control over organization both in cell culture (in vitro) and on the benchtop (de novo). Our initial results indicate that reproducing corneal stroma-like local and long-range organization of collagen may be simpler than we anticipated while controlling spacing and fibril morphology remains difficult, but perhaps not impossible in the (reasonably) near term.

OPTIMIZING COLLAGEN TRANSPORT THROUGH TRACK-ETCHED NANOPORES.

Polymer transport through nanopores is a potentially powerful tool for separation and organization of molecules in biotechnology applications. Our goal is to produce aligned collagen fibrils by mimicking cell-mediated collagen assembly: driving collagen monomers in solution through the aligned nanopores in track-etched membranes followed by fibrillogenesis at the pore exit. We examined type I atelo-collagen monomer transport in neutral, cold solution through polycarbonate track-etched membranes comprising 80-nm-diameter, 6-µm-long pores at 2% areal fraction. Source concentrations of 1.0, 2.8 and 7.0 mg/ml and pressure differentials of 0, 10 and 20 inH(2)O were used. Membrane surfaces were hydrophilized via covalent poly(ethylene-glycol) binding to limit solute-membrane interaction. Collagen transport through the nanopores was a non-intuitive process due to the complex behavior of this associating molecule in semi-dilute solution. Nonetheless, a modified open pore model provided reasonable predictions of transport parameters. Transport rates were concentration- and pressure-dependent, with diffusivities across the membrane in semi-dilute solution two-fold those in dilute solution, possibly via cooperative diffusion or polymer entrainment. The most significant enhancement of collagen transport was accomplished by membrane hydrophilization. The highest concentration transported (5.99±2.58 mg/ml) with the highest monomer flux (2.60±0.49 ×10(3) molecules s(-1) pore(-1)) was observed using 2.8 mg collagen/ml, 10 inH(2)O and hydrophilic membranes.

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.