Lakiesha N. Williams

Myocardial Scaffold-based Cardiac Tissue Engineering: Application of Coordinated Mechanical and Electrical Stimulations

Recently, we developed an optimal decellularization protocol to generate 3D porcine myocardial scaffolds, which preserve the natural extracellular matrix structure, mechanical anisotropy, and vasculature templates and also show good cell recellularization and differentiation potential. In this study, a multistimulation bioreactor was built to provide coordinated mechanical and electrical stimulation for facilitating stem cell differentiation and cardiac construct development. The acellular myocardial scaffolds were seeded with mesenchymal stem cells (10(6) cells/mL) by needle injection and subjected to 5-azacytidine treatment (3 μmol/L, 24 h) and various bioreactor conditioning protocols. We found that after 2 days of culturing with mechanical (20% strain) and electrical stimulation (5 V, 1 Hz), high cell density and good cell viability were observed in the reseeded scaffold. Immunofluorescence staining demonstrated that the differentiated cells showed a cardiomyocyte-like phenotype by expressing sarcomeric α-actinin, myosin heavy chain, cardiac troponin T, connexin-43, and N-cadherin. Biaxial mechanical testing demonstrated that positive tissue remodeling took place after 2 days of bioreactor conditioning (20% strain + 5 V, 1 Hz); passive mechanical properties of the 2 day and 4 day tissue constructs were comparable to those of the tissue constructs produced by stirring reseeding followed by 2 weeks of static culturing, implying the effectiveness and efficiency of the coordinated simulations in promoting tissue remodeling. In short, the synergistic stimulations might be beneficial not only for the quality of cardiac construct development but also for patients by reducing the waiting time in future clinical scenarios.

The effects of water and microstructure on the mechanical properties of bighorn sheep (Ovis canadensis) horn keratin

The function of the bighorn sheep horn prompted quantification of the various parametric effects important to the microstructure and mechanical property relationships of this horn. These parameters included analysis of the stress-state dependence with the horn keratin tested under tension and compression, the anisotropy of the material structure and mechanical behavior, the spatial location along the horn, and the wet-dry horn behavior. The mechanical properties of interest were the elastic moduli, yield strength, ultimate strength, failure strain and hardness. The results showed that water has a more significant effect on the mechanical behavior of ram horn more than the anisotropy, location along the horn and the type of loading state. All of these parametric effects showed that the horn microstructure and mechanical properties were similar to those of long-fiber composites. In the ambient dry condition (10 wt.% water), the longitudinal elastic modulus, yield strength and failure strain were measured to be 4.0 G Pa, 62 MPa and 4%, respectively, and the transverse elastic modulus, yield strength and failure strain were 2.9 GPa, 37 MPa and 2%, respectively. In the wet condition (35 wt.% water), horn behaves more like an isotropic material; the elastic modulus, yield strength and failure strain were determined to be 0.6G Pa, 10 MPa and 60%, respectively.

A mechanistic study for strain rate sensitivity of rabbit patellar tendon

The ultrastructural mechanism for strain rate sensitivity of collagenous tissue has not been well studied at the collagen fibril level. Our objective is to reveal the mechanistic contribution of tendons key structural component to strain rate sensitivity. We have investigated the structure of the collagen fibril undergoing tension at different strain rates. Tendon fascicles were pulled and fixed within the linear region (12% local tissue strain) at multiple strain rates. Although samples were pulled to the same percent elongation, the fibrils were noticed to elongate differently, increasing with strain rate. For the 0.1, 10, and 70%/s strain rates, there were 1.84±3.6%, 5.5±1.9%, and 7.03±2.2% elongations (mean±S.D.), respectively. We concluded that the collagen fibrils underwent significantly greater recruitment (fibril strain relative to global tissue strain) at higher strain rates. A better understanding of tendon mechanisms at lower hierarchical levels would help establish a basis for future development of constitutive models and assist in tissue replacement design.

The anisotropic compressive mechanical properties of the rabbit patellar tendon.

In this study, we examine the transverse and longitudinal compressive mechanical behavior of the rabbit patellar tendon. The anisotropic compressive properties are of interest, because compression occurs where the tendon attaches to bone and where the tendon wraps around bone leading to the development of fibro-cartilaginous matrices. We quantified the time dependent viscoelastic and anisotropic behavior of the tendon under compression. For both orientations, sections of patellar tendon were drawn from mature male white New Zealand rabbits in preparation for testing. The tendons were sequentially compressed to 40% strain at strain rates of 0.1, 1 and 10% strain(s) using a computer-controlled stepper motor driven device under physiological conditions. Following monotonic loading, the tendons were subjected to stress relaxation. The tendon equilibrium compressive modulus was quantified to be 19.49+/-11.46 kPa for the transverse direction and 1.11+/-0.57 kPa for the longitudinal direction. The compressive modulus at applied strain rates of 0.1, 1 and 10% strain(s) in the transverse orientation were 13.48+/-2.31, 18.24+/-4.58 and 20.90+/-8.60 kPa, respectively. The compressive modulus at applied strain rates of 0.1, 1 and 10% strain/s in the longitudinal orientation were 0.19+/-0.11, 1.27+/-1.38 and 3.26+/-3.49 kPa, respectively. The modulus values were almost significantly different for the examination of the effect of orientation on the equilibrium modulus (p=0.054). Monotonic loading of the tendon showed visual differences of the strain rate dependency; however, no significant difference was shown in the statistical analysis of the effect of strain rate on compressive modulus. The statistical analysis of the effect of orientation on compressive modulus showed a significant difference. The difference shown in the orientation analysis validated the anisotropic nature of the tendon.

Anisotropic Compressive Properties of Passive Porcine Muscle Tissue

The body has approximately 434 muscles, which makes up 40-50% of the body by weight. Muscle is hierarchical in nature and organized in progressively larger units encased in connective tissue. Like many soft tissues, muscle has nonlinear visco-elastic behavior, but muscle also has unique characteristics of excitability and contractibility. Mechanical testing of muscle has been done for crash models, pressure sore models, back pain, and other disease models. The majority of previous biomechanical studies on muscle have been associated with tensile properties in the longitudinal direction as this is muscles primary mode of operation under normal physiological conditions. Injury conditions, particularly high rate injuries, can expose muscle to multiple stress states. Compressive stresses can lead to tissue damage, which may not be reversible. In this study, we evaluate the structure-property relationships of porcine muscle tissue under compression, in both the transverse and longitudinal orientations at 0.1 s-1, 0.01 s-1, or 0.001 s-1. Our results show an initial toe region followed by an increase in stress for muscle in both the longitudinal and transverse directions tested to 50% strain. Strain rate dependency was also observed with the higher strain rates showing significantly more stress at 50% strain. Muscle in the transverse orientation was significantly stiffer than in the longitudinal orientation indicating anisotropy. The mean area of fibers in the longitudinal orientation shows an increasing mean fiber area and a decreasing mean fiber area in the transverse orientation. Data obtained in this study can help provide insight on how muscle injuries are caused, ranging from low energy strains to high rate blast events, and can also be used in developing computational injury models.

Variation of diameter distribution, number density, and area fraction of fibrils within five areas of the rabbit patellar tendon

The purpose of this investigation is to show microstructural information at various regions within the rabbit patellar tendon. The properties of the rabbit patellar tendon are well documented mechanically, but detailed information at the microscopic level is not available. Increasing attention has been directed to soft tissue microscopy as the demand for development of biologically inspired materials increases. Microstructural examination of the tendon fibrils is performed to provide further insight into understanding of the structure to function relations within the rabbit patellar tendon. Limited studies on rabbit patellar tendon collagen fibrils at the microscopic level have been computed. Furthermore, evaluation of structure-function relations in multiple regions of any given specimen of a particular tissue type has not been conducted. In this study the number density, area fraction, and diameter distribution of collagen fibrils have been determined. Overall, this examination showed considerable variation within each section of the tendon. Correlating these structural results with mechanical tests of the tendon portions in the various regions could provide additional information on the mechanics of the rabbit tendon as well as insight into development of artificial tissue constructs.

Coupled experiment/finite element analysis on the mechanical response of porcine brain under high strain rates

This paper presents a coupled experimental/modeling study of the mechanical response of porcine brain under high strain rate loading conditions. Essentially, the stress wave propagation through the brain tissue is quantified. A Split-Hopkinson Pressure Bar (SPHB) apparatus, using a polycarbonate (viscoelastic) striker bar was employed for inducing compression waves for strain rates ranging from 50 to 750 s(-1). The experimental responses along with high speed video showed that the brain tissues response was nonlinear and inelastic. Also, Finite Element Analysis (FEA) of the SHPB tests revealed that the tissue underwent a non-uniform stress state during testing when glue is used to secure the specimen with the test fixture. This result renders erroneous the assumption of uniaxial loading. In this study, the uniaxial volume averaged stress-strain behavior was extracted from the FEA to help calibrate inelastic constitutive equations.

Geometric Effects on Stress Wave Propagation

The present study, through finite element simulations, shows the geometric effects of a bioinspired solid on pressure and impulse mitigation for an elastic, plastic, and viscoelastic material. Because of the bioinspired geometries, stress wave mitigation became apparent in a nonintuitive manner such that potential real-world applications in human protective gear designs are realizable. In nature, there are several toroidal designs that are employed for mitigating stress waves; examples include the hyoid bone on the back of a woodpeckers jaw that extends around the skull to its nose and a rams horn. This study evaluates four different geometries with the same length and same initial cross-sectional diameter at the impact location in three-dimensional finite element analyses. The geometries in increasing complexity were the following: (1) a round cylinder, (2) a round cylinder that was tapered to a point, (3) a round cylinder that was spiraled in a two dimensional plane, and (4) a round cylinder that was tapered and spiraled in a two-dimensional plane. The results show that the tapered spiral geometry mitigated the greatest amount of pressure and impulse (approximately 98% mitigation) when compared to the cylinder regardless of material type (elastic, plastic, and viscoelastic) and regardless of input pressure signature. The specimen taper effectively mitigated the stress wave as a result of uniaxial deformational processes and an induced shear that arose from its geometry. Due to the decreasing cross-sectional area arising from the taper, the local uniaxial and shear stresses increased along the specimen length. The spiral induced even greater shear stresses that help mitigate the stress wave and also induced transverse displacements at the tip such that minimal wave reflections occurred. This phenomenon arose although only longitudinal waves were introduced as the initial boundary condition (BC). In nature, when shearing occurs within or between materials (friction), dissipation usually results helping the mitigation of the stress wave and is illustrated in this study with the taper and spiral geometries. The combined taper and spiral optimized stress wave mitigation in terms of the pressure and impulse; thus providing insight into the rams horn design and woodpecker hyoid designs found in nature.

Nanomechanics of phospholipid bilayer failure under strip biaxial stretching using molecular dynamics

The current study presents a nanoscale in silico investigation of strain rate dependency of membrane (phospholipid bilayer) failure when placed under strip biaxial tension with two planar areas. The nanoscale simulations were conducted in the context of a multiscale modelling framework in which the macroscale damage (pore volume fraction) progression is delineated into pore nucleation (number density of pores), pore growth (size of pores), and pore coalescence (inverse of nearest neighbor distance) mechanisms. As such, the number density, area fraction, and nearest neighbor distances were quantified in association with the stress–strain behavior. Deformations of a 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer were performed using molecular dynamics to simulate mechanoporation of a neuronal cell membrane due to injury, which in turn can result in long-term detrimental effects that could ultimately lead to cell death. Structures with 72 and 144 phospholipids were subjected to strip biaxial tensile deformations at multiple strain rates. Formation of a water bridge through the phospholipid bilayer was the metric to indicate structural failure. Both the larger and smaller bilayers had similar behavior regarding pore nucleation and the strain rate effect on pore growth post water penetration. The applied strain rates, planar area, and cross-sectional area had no effect on the von Mises strains at which pores greater than 0.1 nm2 were detected (0.509 ± 7.8%) or the von Mises strain at failure (e failure = 0.68 ± 4.8%). Additionally, changes in bilayer planar and cross-sectional areas did not affect the stress response. However, as the strain rate increased from 2.0 × 108 s−1 to 1.0 × 109 s−1, the yield stress increased from 26.5 MPa to 66.7 MPa and the yield strain increased from 0.056 to 0.226.

Quantitative analysis of brain microstructure following mild blunt and blast trauma

We induced mild blunt and blast injuries in rats using a custom-built device and utilized in-house diffusion tensor imaging (DTI) software to reconstruct 3-D fiber tracts in brains before and after injury (1, 4, and 7 days). DTI measures such as fiber count, fiber length, and fractional anisotropy (FA) were selected to characterize axonal integrity. In-house image analysis software also showed changes in parameters including the area fraction (AF) and nearest neighbor distance (NND), which corresponded to variations in the microstructure of Hematoxylin and Eosin (H&E) brain sections. Both blunt and blast injuries produced lower fiber counts, but neither injury case significantly changed the fiber length. Compared to controls, blunt injury produced a lower FA, which may correspond to an early onset of diffuse axonal injury (DAI). However, blast injury generated a higher FA compared to controls. This increase in FA has been linked previously to various phenomena including edema, neuroplasticity, and even recovery. Subsequent image analysis revealed that both blunt and blast injuries produced a significantly higher AF and significantly lower NND, which correlated to voids formed by the reduced fluid retention within injured axons. In conclusion, DTI can detect subtle pathophysiological changes in axonal fiber structure after mild blunt and blast trauma. Our injury model and DTI method provide a practical basis for studying mild traumatic brain injury (mTBI) in a controllable manner and for tracking injury progression. Knowledge gained from our approach could lead to enhanced mTBI diagnoses, biofidelic constitutive brain models, and specialized pharmaceutical treatments.