Effect of Electrospun Fiber Mat Thickness and Support Method on Cell Morphology.

Electrospun fiber mats (EFMs) are highly versatile biomaterials used in a myriad of biomedical applications. Whereas some facets of EFMs are well studied and can be highly tuned (e.g., pore size, fiber diameter, etc.), other features are under characterized. For example, although substrate mechanics have been explored by several groups, most studies rely on Young's modulus alone as a characterization variable. The influence of fiber mat thickness and the effect of supports are variables that are often not considered when evaluating cell-mechanical response. To assay the role of these features in EFM scaffold design and to improve understanding of scaffold mechanical properties, we designed EFM scaffolds with varying thickness (50-200 µm) and supporting methodologies. EFM scaffolds were comprised of polycaprolactone and were either electrospun directly onto a support, suspended across an annulus (3 or 10 mm inner diameter), or "tension-released" and then suspended across an annulus. Then, single cell spreading (i.e., Feret diameter) was measured in the presence of these different features. Cells were sensitive to EFM thickness and suspended gap diameter. Overall, cell spreading was greatest for 50 µm thick EFMs suspended over a 3 mm gap, which was the smallest thickness and gap investigated. These results are counterintuitive to conventional understanding in mechanobiology, which suggests that stiffer materials, such as thicker, supported EFMs, should elicit greater cell polarization. Additional experiments with 50 µm thick EFMs on polystyrene and polydimethylsiloxane (PDMS) supports demonstrated that cells can "feel" the support underlying the EFM if it is rigid, similar to previous results in hydrogels. These results also suggest that EFM curvature may play a role in cell response, separate from Young's modulus, possibly because of internal tension generated. These parameters are not often considered in EFM design and could improve scaffold performance and ultimately patient outcomes.

Beyond Linear Elastic Modulus: Viscoelastic Models for Brain and Brain Mimetic Hydrogels.

With their high degree of specificity and investigator control, in vitro disease models provide a natural complement to in vivo models. Especially in organs such as the brain, where anatomical limitations make in vivo experiments challenging, in vitro models have been increasingly used to mimic disease pathology. However, brain mimetic models may not fully replicate the mechanical environment in vivo, which has been shown to influence a variety of cell behaviors. Specifically, many disease models consider only the linear elastic modulus of brain, which describes the stiffness of a material with the assumption that mechanical behavior is independent of loading rate. Here, we characterized porcine brain tissue using a modified stress relaxation test, and across a panel of viscoelastic models, showed that stiffness depends on loading rate. As such, the linear elastic modulus does not accurately reflect the viscoelastic properties of native brain. Among viscoelastic models, the Maxwell model was selected for further analysis because of its simplicity and excellent curve fit (R2 = 0.99 ± 0.0006). Thus, mechanical response of native brain and hydrogel mimetic models was analyzed using the Maxwell model and the linear elastic model to evaluate the effects of strain rate, time post mortem, region, tissue type (i.e., bulk brain vs white matter), and in brain mimetic models, hydrogel composition, on observed mechanical properties. In comparing the Maxwell and linear elastic models, linear elastic modulus is consistently lower than the Maxwell elastic modulus across all brain regions. Additionally, the Maxwell model is sensitive to changes in viscosity and small changes in elasticity, demonstrating improved fidelity. These findings demonstrate the insufficiency of linear elastic modulus as a primary mechanical characterization for brain mimetic materials and provide quantitative information toward the future design of materials that more closely mimic mechanical features of brain.

Exploring the mechanical behavior of degrading swine neural tissue at low strain rates via the fractional Zener constitutive model.

The ability of the fractional Zener constitutive model to predict the behavior of postmortem swine brain tissue was examined in this work. Understanding tissue behavior attributed to degradation is invaluable in many fields such as the forensic sciences or cases where only cadaveric tissue is available. To understand how material properties change with postmortem age, the fractional Zener model was considered as it includes parameters to describe brain stiffness and also the parameter α, which quantifies the viscoelasticity of a material. The relationship between the viscoelasticity described by α and tissue degradation was examined by fitting the model to data collected in a previous study (Bentil, 2013). This previous study subjected swine neural tissue to in vitro unconfined compression tests using four postmortem age groups (<6h, 24h, 3 days, and 1 week). All samples were compressed to a strain level of 10% using two compressive rates: 1mm/min and 5mm/min. Statistical analysis was used as a tool to study the influence of the fractional Zener constants on factors such as tissue degradation and compressive rate. Application of the fractional Zener constitutive model to the experimental data showed that swine neural tissue becomes less stiff with increased postmortem age. The fractional Zener model was also able to capture the nonlinear viscoelastic features of the brain tissue at low strain rates. The results showed that the parameter α was better correlated with compressive rate than with postmortem age.

Sequential biomechanics of the human upper thoracic spine and pectoral girdle.

Thoracic spine flexibility affects head motion, which is critical to control in motor vehicle crashes given the frequency and severity of head injuries. The objective of this study is to investigate the dynamic response of the human upper thoracic region. An original experimental/analytical approach, Isolated Segment Manipulation (ISM), is introduced to quantify the intact upper thoracic spine-pectoral girdle (UTS-PG) dynamic response of six adult post-mortem human subjects (PMHS). A continuous series of small displacement, frontal perturbations were applied to the human UTS-PG using fifteen combinations of speed and constraint per PMHS. The non-parametric response of the T1-T6 lumped mass segment was obtained using a system identification technique. A parametric mass-damper-spring model was used to fit the non-parametric system response. Mechanical parameters of the upper thoracic spine were determined from the experimental model and analyzed in each speed/constraint configuration. The natural frequencies of the UTS-PG were 22.9 ± 7.1 rad/sec (shear, n=58), 32.1 ± 7.4 rad/sec (axial, n=58), and 27.8 ± 7.7 rad/sec (rotation, n=65). The damping ratios were 0.25 ± 0.20 (shear), 0.42 ± 0.24 (axial), and 0.58± 0.32 (rotation). N-way analysis of variance (Type III constrained sum of squares, no interaction effects) revealed that the relative effects of test speed, pectoral girdle constraint, and PMHS anthropometry on the UTS-PG dynamic properties varied per property and direction. While more work is needed to verify accuracy in realistic crash scenarios, the UTS-PG model system dynamic properties could eventually aid in developing integrated anthropomorphic test device (ATD) thoracic spine and shoulder components to provide improved head kinematics and belt interaction.

Inherent interfacial mechanical gradients in 3D hydrogels influence tumor cell behaviors.

Cells sense and respond to the rigidity of their microenvironment by altering their morphology and migration behavior. To examine this response, hydrogels with a range of moduli or mechanical gradients have been developed. Here, we show that edge effects inherent in hydrogels supported on rigid substrates also influence cell behavior. A Matrigel hydrogel was supported on a rigid glass substrate, an interface which computational techniques revealed to yield relative stiffening close to the rigid substrate support. To explore the influence of these gradients in 3D, hydrogels of varying Matrigel content were synthesized and the morphology, spreading, actin organization, and migration of glioblastoma multiforme (GBM) tumor cells were examined at the lowest (<50 µm) and highest (>500 µm) gel positions. GBMs adopted bipolar morphologies, displayed actin stress fiber formation, and evidenced fast, mesenchymal migration close to the substrate, whereas away from the interface, they adopted more rounded or ellipsoid morphologies, displayed poor actin architecture, and evidenced slow migration with some amoeboid characteristics. Mechanical gradients produced via edge effects could be observed with other hydrogels and substrates and permit observation of responses to multiple mechanical environments in a single hydrogel. Thus, hydrogel-support edge effects could be used to explore mechanosensitivity in a single 3D hydrogel system and should be considered in 3D hydrogel cell culture systems.

Dynamic properties of the upper thoracic spine-pectoral girdle (UTS-PG) system and corresponding kinematics in PMHS sled tests.

Anthropomorphic test devices (ATDs) should accurately depict head kinematics in crash tests, and thoracic spine properties have been demonstrated to affect those kinematics. To investigate the relationships between thoracic spine system dynamics and upper thoracic kinematics in crash-level scenarios, three adult post-mortem human subjects (PMHS) were tested in both Isolated Segment Manipulation (ISM) and sled configurations. In frontal sled tests, the T6-T8 vertebrae of the PMHS were coupled through a novel fixation technique to a rigid seat to directly measure thoracic spine loading. Mid-thoracic spine and belt loads along with head, spine, and pectoral girdle (PG) displacements were measured in 12 sled tests conducted with the three PMHS (3-pt lap-shoulder belted/unbelted at velocities from 3.8 - 7.0 m/s applied directly through T6-T8). The sled pulse, ISM- derived characteristic properties of that PMHS, and externally applied forces due to head-neck inertia and shoulder belt constraint were used to predict kinematic time histories of the T1-T6 spine segment. The experimental impulse applied to the upper thorax was normalized to be consistent with a T6 force/sled acceleration sinusoidal profile, and the result was an improvement in the prediction of T3 X-axis displacements with ISM properties. Differences between experimental and model-predicted displacement-time history increases were quantified with respect to speed. These discrepancies were attributed to the lack of rotational inertia of the head-neck late in the event as well as restricted kyphosis and viscoelasticity of spine constitutive structures through costovertebral interactions and mid-spine fixation. The results indicate that system dynamic properties from sub-injurious ISM testing could be useful for characterizing forward trajectories of the upper thoracic spine in higher energy crash simulations, leading to improved biofidelity for both ATDs and finite element models.

Constitutive modeling of rate-dependent stress-strain behavior of human liver in blunt impact loading.

An understanding of the mechanical deformation behavior of the liver under high strain rate loading conditions could aid in the development of vehicle safety measures to reduce the occurrence of blunt liver injury. The purpose of this study was to develop a constitutive model of the stress-strain behavior of the human liver in blunt impact loading. Experimental stress and strain data was obtained from impact tests of 12 unembalmed human livers using a drop tower technique. A constitutive model previously developed for finite strain behavior of amorphous polymers was adapted to model the observed liver behavior. The elements of the model include a nonlinear spring in parallel with a linear spring and nonlinear dashpot. The model captures three features of liver stress-strain behavior in impact loading: (1) relatively stiff initial modulus, (2) rate-dependent yield or rollover to viscous "flow" behavior, and (3) strain hardening at large strains. Six material properties were used to define the constitutive model. This study represents a novel application of polymer mechanics concepts to understand the rate-dependent large strain behavior of human liver tissue under high strain rate loading. Applications of this research include finite element simulations of injury-producing liver or abdominal impact events.

Mechanical characterization of electrospun polycaprolactone (PCL): a potential scaffold for tissue engineering.

This paper investigates the mechanical behavior of electrospun polycaprolactone (PCL) under tensile loading. PCL in bulk form degrades slowly and is biocompatible, two properties that make it a viable option for tissue engineering applications in biomedicine. Of particular interest is the use of electrospun PCL tubes as scaffolds for tissue engineered blood vessel implants. Stress relaxation and tensile tests have been conducted with specimens at room temperature (21 degrees C) and 37 degrees C. Additionally, to probe the effects of moisture on mechanical behavior, specimens were tested either dry (in air) or submerged in water. In general, the electrospun PCL was found to exhibit rate dependence, as well as some dependence on the test temperature and on whether the sample was wet or dry. Two different models were investigated to describe the experimentally observed material behavior. The models used were Fung's theory of quasilinear viscoelasticity (QLV) and the eight-chain model developed for rubber elastomers by Arruda and Boyce (1993, "A Three-Dimensional Constitutive Model for the Large Stretch Behavior of Rubber Elastic Materials," J. Mech. Phys. Solids, 41(2), pp. 389-412). The implementation and fitting results, as well as the advantages and disadvantages of each model, are presented. In general, it was found that the QLV theory provided a better fit.

Using pressure to predict liver injury risk from blunt impact.

Liver trauma research suggests that rapidly increasing internal pressure plays a role in causing blunt liver injury. Knowledge of the relationship between pressure and the likelihood of liver injury could be used to enhance the design of crash test dummies. The objectives of this study were (1) to characterize the relationship between impact-induced pressures and blunt liver injury in an experimental model to impacts of ex vivo organs; and (2) to compare human liver vascular pressure and tissue pressure in the parenchyma with other biomechanical variables as predictors of liver injury risk. Test specimens were 14 ex vivo human livers. Specimens were perfused with normal saline solution at physiological pressures, and a drop tower applied blunt impact at varying energies. Impact-induced pressures were measured by transducers inserted into the hepatic veins and the parenchyma (caudate lobe) of ex vivo specimens. Experimentally induced liver injuries were consistent with those documented in the Crash Injury Research and Engineering Network (CIREN) database. Binary logistic regression analysis demonstrated that injury predictors associated with tissue pressure measured in the parenchyma were the best indicators of serious liver injury risk. The best injury predictor overall was the product of the peak rate of tissue pressure increase and the peak tissue pressure, P T max * P T max (pseudo-R2 = .82, p = .001). A burst injury mechanism directly related to hydrostatic pressure is postulated for the ex vivo liver loaded dynamically in a drop test experiment.