Spencer E. Szczesny

Interfibrillar shear stress is the loading mechanism of collagen fibrils in tendon

Despite the critical role tendons play in transmitting loads throughout the musculoskeletal system, little is known about the microstructural mechanisms underlying their mechanical function. Of particular interest is whether collagen fibrils in tendon fascicles bear load independently or if load is transferred between fibrils through interfibrillar shear forces. We conducted multiscale experimental testing and developed a microstructural shear lag model to explicitly test whether interfibrillar shear load transfer is indeed the fibrillar loading mechanism in tendon. Experimental correlations between fascicle macroscale mechanics and microscale interfibrillar sliding suggest that fibrils are discontinuous and share load. Moreover, for the first time, we demonstrate that a shear lag model can replicate the fascicle macroscale mechanics as well as predict the microscale fibrillar deformations. Since interfibrillar shear stress is the fundamental loading mechanism assumed in the model, this result provides strong evidence that load is transferred between fibrils in tendon and possibly other aligned collagenous tissues. Conclusively establishing this fibrillar loading mechanism and identifying the involved structural components should help develop repair strategies for tissue degeneration and guide the design of tissue engineered replacements.

Biaxial Tensile Testing and Constitutive Modeling of Human Supraspinatus Tendon

The heterogeneous composition, collagen fiber organization and mechanical properties of the supraspinatus tendon (SST) offer an opportunity for studying the structure-function relationships of fibrous musculoskeletal connective tissues. The objective of this study was to evaluate the contribution of collagen fiber organization to the planar tensile mechanics of the human SST. This was accomplished by fitting biaxial tensile data with a structural constitutive model that incorporates a sample-specific angular distribution of nonlinear fibers. Biaxial testing was employed to avoid the limitation of non-physiologic traction-free boundary conditions present during uniaxial testing. Samples were tested under a range of boundary conditions with simultaneous monitoring of collagen fiber orientation via polarized light imaging. The experimental data were input into a hyperelastic constitutive model incorporating the contributions of the uncrimped fibers. The model fit the longitudinal stresses well and was successfully validated. The transverse stresses were fit less well with greater errors observed for less aligned samples. Additional strain energy terms representing fiber-fiber interactions are likely necessary to provide closer approximation of the transverse stresses. This approach demonstrated that the longitudinal tensile mechanics of the SST are primarily dependent on the moduli, crimp, and angular distribution of its collagen fibers.

Biaxial mechanics and inter-lamellar shearing of stem-cell seeded electrospun angle-ply laminates for annulus fibrosus tissue engineering

The annulus fibrosus (AF) of the intervertebral disk plays a critical role in vertebral load transmission that is heavily dependent on the microscale structure and composition of the tissue. With degeneration, both structure and composition are compromised, resulting in a loss of AF mechanical function. Numerous tissue engineering strategies have addressed the issue of AF degeneration, but few have focused on recapitulation of AF microstructure and function. One approach that allows for generation of engineered AF with appropriate (+/−)30° lamellar microstructure is the use of aligned electrospun scaffolds seeded with mesenchymal stem cells (MSCs) and assembled into angle‐ply laminates (APL). Previous work indicates that opposing lamellar orientation is necessary for development of near native uniaxial tensile properties. However, most native AF tensile loads are applied biaxially, as the disk is subjected to multi‐axial loads and is constrained by its attachments to the vertebral bodies. Thus, the objective of this study was to evaluate the biaxial mechanical response of engineered AF bilayers, and to determine the importance of opposing lamellar structure under this loading regime. Opposing bilayers, which replicate native AF structure, showed a significantly higher modulus in both testing directions compared to parallel bilayers, and reached ∼60% of native AF biaxial properties. Associated with this increase in biaxial properties, significantly less shear, and significantly higher stretch in the fiber direction, was observed. These results provide additional insight into native tissue structure–function relationships, as well as new benchmarks for engineering functional AF tissue constructs.

Regional Variation in Human Supraspinatus Tendon Proteoglycans: Decorin, Biglycan, and Aggrecan

While tendons typically undergo primary tensile loading, the human supraspinatus tendon (SST) experiences substantial amounts of tension, compression, and shear in vivo. As a result, the functional roles of the extracellular matrix components, in particular the proteoglycans (PGs), are likely complex and important. The goal of this study was to determine the PG content in specific regions of the SST that exhibit differing mechanical function. The concentration of aggrecan, biglycan, and decorin was determined in six regions of the human SST using immunochemical techniques. We hypothesized that aggrecan concentrations would be highest in areas where the tendon likely experiences compression; biglycan levels would be highest in regions likely subjected to injury and/or active remodeling such as the anterior regions; decorin concentrations would be highest in regions of greatest tensile stiffness. Our results generally supported these hypotheses and demonstrated that aggrecan and biglycan share regional variability, with increased concentration in the anterior and posterior regions and smaller concentration in the medial regions. Decorin, however, was in high concentration throughout all regions. The data presented in this study represent the first regional measurements of PG in the SST. Together with our previous regional measurements of mechanical properties, these data can be used to evaluate SST structure–function relationships. With knowledge of the differences in specific PG content, their spatial variations in the SST, and their relationships to tendon mechanics, we can begin to associate defects in PG content with specific pathology, which may provide guidance for new therapeutic interventions.

Experimental and Computational Investigation of Altered Mechanical Properties in Myocardium after Hydrogel Injection

The material properties of myocardium are an important determinant of global left ventricular function. Myocardial infarction results in a series of maladaptive geometric alterations which lead to increased stress and risk of heart failure. In vivo studies have demonstrated that material injection can mitigate these changes. More importantly, the material properties of these injectates can be tuned to minimize wall thinning and ventricular dilation. The current investigation combines experimental data and finite element modeling to correlate how injectate mechanics and volume influence myocardial wall stress. Experimentally, mechanics were characterized with biaxial testing and injected hydrogel volumes were measured with magnetic resonance imaging. Injection of hyaluronic acid hydrogel increased the stiffness of the myocardium/hydrogel composite region in an anisotropic manner, significantly increasing the modulus in the longitudinal direction compared to control myocardium. Increased stiffness, in combination with increased volume from hydrogel injection, reduced the global average fiber stress by ~14% and the transmural average by ~26% in the simulations. Additionally, stiffening in an anisotropic manner enhanced the influence of hydrogel treatment in decreasing stress. Overall, this work provides insight on how injectable biomaterials can be used to attenuate wall stress and provides tools to further optimize material properties for therapeutic applications.

Incorporating plasticity of the interfibrillar matrix in shear lag models is necessary to replicate the multiscale mechanics of tendon fascicles.

Despite current knowledge of tendon structure, the fundamental deformation mechanisms underlying tendon mechanics and failure are unknown. We recently showed that a shear lag model, which explicitly assumed plastic interfibrillar load transfer between discontinuous fibrils, could explain the multiscale fascicle mechanics, suggesting that fascicle yielding is due to plastic deformation of the interfibrillar matrix. However, it is unclear whether alternative physical mechanisms, such as elastic interfibrillar deformation or fibril yielding, also contribute to fascicle mechanical behavior. The objective of the current work was to determine if plasticity of the interfibrillar matrix is uniquely capable of explaining the multiscale mechanics of tendon fascicles including the tissue post-yield behavior. This was examined by comparing the predictions of a continuous fibril model and three separate shear lag models incorporating an elastic, plastic, or elastoplastic interfibrillar matrix with multiscale experimental data. The predicted effects of fibril yielding on each of these models were also considered. The results demonstrated that neither the continuous fibril model nor the elastic shear lag model can successfully predict the experimental data, even if fibril yielding is included. Only the plastic or elastoplastic shear lag models were capable of reproducing the multiscale tendon fascicle mechanics. Differences between these two models were small, although the elastoplastic model did improve the fit of the experimental data at low applied tissue strains. These findings suggest that while interfibrillar elasticity contributes to the initial stress response, plastic deformation of the interfibrillar matrix is responsible for tendon fascicle post-yield behavior. This information sheds light on the physical processes underlying tendon failure, which is essential to improve our understanding of tissue pathology and guide the development of successful repair.

Quantification of Interfibrillar Shear Stress in Aligned Soft Collagenous Tissues via Notch Tension Testing.

The mechanical function of soft collagenous tissues is largely determined by their hierarchical organization of collagen molecules. While collagen fibrils are believed to be discontinuous and transfer load through shearing of the interfibrillar matrix, interfibrillar shear stresses have never been quantified. Scaling traditional shear testing procedures down to the fibrillar length scale is impractical and would introduce substantial artifacts. Here, through the use of a novel microscopic variation of notch tension testing, we explicitly demonstrate the existence of interfibrillar shear stresses within tendon fascicles and provide the first measurement of their magnitude. Axial stress gradients along the sample length generated by notch tension testing were measured and used to calculate a value of 32 kPa for the interfibrillar shear stress. This estimate is comparable to the interfibrillar shear stress predicted by previous multiscale modeling of tendon fascicles, which supports the hypothesis that fibrils are discontinuous and transmit load through interfibrillar shear. This information regarding the structure-function relationships of tendon and other soft collagenous tissues is necessary to identify potential causes for tissue impairment with degeneration and provide the foundation for developing regenerative repair strategies or engineering biomaterials for tissue replacement.

DTAF Dye Concentrations Commonly Used to Measure Microscale Deformations in Biological Tissues Alter Tissue Mechanics

Identification of the deformation mechanisms and specific components underlying the mechanical function of biological tissues requires mechanical testing at multiple levels within the tissue hierarchical structure. Dichlorotriazinylaminofluorescein (DTAF) is a fluorescent dye that is used to visualize microscale deformations of the extracellular matrix in soft collagenous tissues. However, the DTAF concentrations commonly employed in previous multiscale experiments (≥2000 µg/ml) may alter tissue mechanics. The objective of this study was to determine whether DTAF affects tendon fascicle mechanics and if a concentration threshold exists below which any observed effects are negligible. This information is valuable for guiding the continued use of this fluorescent dye in future experiments and for interpreting the results of previous work. Incremental strain testing demonstrated that high DTAF concentrations (≥100 µg/ml) increase the quasi-static modulus and yield strength of rat tail tendon fascicles while reducing their viscoelastic behavior. Subsequent multiscale testing and modeling suggests that these effects are due to a stiffening of the collagen fibrils and strengthening of the interfibrillar matrix. Despite these changes in tissue behavior, the fundamental deformation mechanisms underlying fascicle mechanics appear to remain intact, which suggests that conclusions from previous multiscale investigations of strain transfer are still valid. The effects of lower DTAF concentrations (≤10 µg/ml) on tendon mechanics were substantially smaller and potentially negligible; nevertheless, no concentration was found that did not at least slightly alter the tissue behavior. Therefore, future studies should either reduce DTAF concentrations as much as possible or use other dyes/techniques for measuring microscale deformations.

Altered lumbar spine structure, biochemistry and biomechanical properties in a canine model of mucopolysaccharidosis type VII

Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disorder characterized by a deficiency in β‐glucuronidase activity, leading to systemic accumulation of poorly degraded glycosaminoglycans (GAG). Along with other morbidities, MPS VII is associated with pediatric spinal deformity. The objective of this study was to examine potential associations between abnormal lumbar spine matrix structure and composition in MPS VII, and spine segment and tissue‐level mechanical properties, using a naturally occurring canine model with a similar clinical phenotype to the human form of the disorder. Segments from juvenile MPS VII and unaffected dogs were allocated to: radiography, gross morphology, histology, biochemistry, and mechanical testing. MPS VII spines had radiolucent lesions in the vertebral body epiphyses. Histologically, this corresponded to a GAG‐rich cartilaginous region in place of bone and elevated GAG staining was seen in the annulus fibrosus. Biochemically, MPS VII samples had elevated GAG in the outer annulus fibrosus and epiphyses, low calcium in the epiphyses, and high water content in all regions except the nucleus pulposus. MPS VII spine segments had higher range of motion and lower stiffness than controls. Endplate indentation stiffness and failure loads were significantly lower in MPS VII samples, while annulus fibrosus tensile mechanical properties were normal. Vertebral body lesions in MPS VII spines suggest a failure to convert cartilage to bone during development. Low stiffness in these regions likely contributes to mechanical weakness in motion segments and is a potential factor in the progression of spinal deformity.

In situ deformations in the immature brain during rapid rotations.

Head trauma is the leading cause of death and debilitating injury in children. Computational models are important tools used to understand head injury mechanisms but they must be validated with experimental data. In this communication we present in situ measurements of brain deformation during rapid, nonimpact head rotation in juvenile pigs of different ages. These data will be used to validate computational models identifying age-dependent thresholds of axonal injury. Fresh 5 days (n=3) and 4 weeks (n=2) old piglet heads were transected horizontally and secured in a container. The cut surface of each brain was marked and covered with a transparent, lubricated plate that allowed the brain to move freely in the plane of rotation. For each brain, a rapid (20-28 ms) 65 deg rotation was applied sequentially at 50 rad/s, 75 rad/s, and 75 rad/s. Each rotation was digitally captured at 2500 frames/s (480x320 pixels) and mark locations were tracked and used to compute strain using an in-house program in MATLAB. Peak values of principal strain (E(peak)) were significantly larger during deceleration than during acceleration of the head rotation (p<0.05), and doubled with a 50% increase in velocity. E(peak) was also significantly higher during the second 75 rad/s rotation than during the first 75 rad/s rotation (p<0.0001), suggesting structural alteration at 75 rad/s and the possibility that similar changes may have occurred at 50 rad/s. Analyzing only lower velocity (50 rad/s) rotations, E(peak) significantly increased with age (16.5% versus 12.4%, p<0.003), which was likely due to the larger brain mass and smaller viscoelastic modulus of the 4 weeks old pig brain compared with those of the 5 days old. Strain measurement error for the overall methodology was estimated to be 1%. Brain tissue strain during rapid, nonimpact head rotation in the juvenile pig varies significantly with age. The empirical data presented will be used to validate computational model predictions of brain motion under similar loading conditions and to assist in the development of age-specific thresholds for axonal injury. Future studies will examine the brain-skull displacement and will be used to validate brain-skull interactions in computational models.