Solomon R. Eisenberg

Cardiac muscle tissue engineering: toward an in vitro model for electrophysiological studies

The objective of this study was to establish a three-dimensional (3-D) in vitro model system of cardiac muscle for electrophysiological studies. Primary neonatal rat ventricular cells containing lower or higher fractions of cardiac myocytes were cultured on polymeric scaffolds in bioreactors to form regular or enriched cardiac muscle constructs, respectively. After 1 wk, all constructs contained a peripheral tissue-like region (50-70 micrometer thick) in which differentiated cardiac myocytes were organized in multiple layers in a 3-D configuration. Indexes of cell size (protein/DNA) and metabolic activity (tetrazolium conversion/DNA) were similar for constructs and neonatal rat ventricles. Electrophysiological studies conducted using a linear array of extracellular electrodes showed that the peripheral region of constructs exhibited relatively homogeneous electrical properties and sustained macroscopically continuous impulse propagation on a centimeter-size scale. Electrophysiological properties of enriched constructs were superior to those of regular constructs but inferior to those of native ventricles. These results demonstrate that 3-D cardiac muscle constructs can be engineered with cardiac-specific structural and electrophysiological properties and used for in vitro impulse propagation studies.The objective of this study was to establish a three-dimensional (3-D) in vitro model system of cardiac muscle for electrophysiological studies. Primary neonatal rat ventricular cells containing lower or higher fractions of cardiac myocytes were cultured on polymeric scaffolds in bioreactors to form regular or enriched cardiac muscle constructs, respectively. After 1 wk, all constructs contained a peripheral tissue-like region (50-70 μm thick) in which differentiated cardiac myocytes were organized in multiple layers in a 3-D configuration. Indexes of cell size (protein/DNA) and metabolic activity (tetrazolium conversion/DNA) were similar for constructs and neonatal rat ventricles. Electrophysiological studies conducted using a linear array of extracellular electrodes showed that the peripheral region of constructs exhibited relatively homogeneous electrical properties and sustained macroscopically continuous impulse propagation on a centimeter-size scale. Electrophysiological properties of enriched constructs were superior to those of regular constructs but inferior to those of native ventricles. These results demonstrate that 3-D cardiac muscle constructs can be engineered with cardiac-specific structural and electrophysiological properties and used for in vitro impulse propagation studies.

Confined and unconfined stress relaxation of cartilage: appropriateness of a transversely isotropic analysis

Previous studies have shown that stress relaxation behavior of calf ulnar growth plate and chondroepiphysis cartilage can be described by a linear transverse isotropic biphasic model. The model provides a good fit to the observed unconfined compression transients when the out-of-plane Poissons ratio is set to zero. This assumption is based on the observation that the equilibrium stress in the axial direction (deltaz) is the same in confined and unconfined compression, which implies that the radial stress deltar = 0 in confined compression. In our study, we further investigated the ability of the transversely isotropic model to describe confined and unconfined stress relaxation behavior of calf cartilage. A series of confined and unconfined stress relaxation tests were performed on calf articular cartilage (4.5 mm diameter, approximately 3.3 mm height) in a displacement-controlled compression apparatus capable of measuring delta(z) and delta(r). In equilibrium, delta(r) > 0 and delta(z) in confined compression was greater than in unconfined compression. Transient data at each strain were fitted by the linear transversely isotropic biphasic model and the material parameters were estimated. Although the model could provide good fits to the unconfined transients, the estimated parameters overpredicted the measured delta(r). Conversely, if the model was constrained to match equilibrium delta(r), the fits were poor. These findings suggest that the linear transversely isotropic biphasic model could not simultaneously describe the observed stress relaxation and equilibrium behavior of calf cartilage.

Cultivation in rotating bioreactors promotes maintenance of cardiac myocyte electrophysiology and molecular properties.

We tested the hypothesis that cardiomyocytes maintained their phenotype better if cultured as three-dimensional tissue constructs than if cultured as confluent monolayers. Neonatal rat cardiomyocytes were cultured on biomaterial scaffolds in rotating bioreactors for 1 week, and resulting tissue constructs were compared with confluent monolayers and slices of native ventricular tissue with respect to proteins involved in cell metabolism (creatine kinase isoform MM), contractile function (sarcomeric myosin heavy chain), and intercellular communication (connexin 43), as well as action potential characteristics (e.g., membrane resting potential, maximum depolarization slope, and action potential duration), and macroscopic electrophysiological properties (maximum capture rate). The molecular and electrophysiological properties of cardiomyocytes cultured in tissue constructs, although inferior to those of native neonatal ventricles, were superior to those of the same cells cultured as monolayers. Construct levels of creatine kinase, myosin heavy chain, and connexin 43 were 40-60% as high as ventricle levels, whereas monolayer levels of the same proteins were only 11-20% as high. Construct action potential durations were 1.8-fold higher than those in ventricles, whereas monolayer action potential durations were 2.4-fold higher. Pharmacological studies using 4-aminopyridine showed that prolonged action potential duration and reduced maximum capture rate in tissue constructs as compared with native ventricles could be explained by decreased transient outward potassium current.

A three-dimensional finite element method for computing magnetically induced currents in tissues

Time-varying magnetic fields used both in nerve stimulation and in magnetic resonance imaging induce electric fields and currents in conducting tissues. Knowledge of the spatial distributions of these induced electric fields and currents in the tissues is very limited because of the complex geometry and inhomogeneous, anisotropic conductivities of the tissues, as well as the spatial nonuniformity of the applied magnetic fields. In this paper, we present a finite element solution method that can be used to compute the induced electric field and current density distributions in tissues when the time rate of change of the applied magnetic field is low enough that the propagation time and magnetic diffusion time in the conductive tissues are negligible, and when the conduction current in the tissues is substantially larger than the displacement current. This finite element implementation is tested for some simple conductive models with both spatially uniform and nonuniform magnetic fields. Our solutions for a homogeneous isotropic conductive slab and a homogeneous anisotropic conductive slab exposed to a uniform magnetic field are in good agreement with analytical results. The finite element approach enables us to include conductive inhomogeneity and anisotropy. I allows us to closely model the complex geometry of the tissues. Therefore, it is well suited for realistic models of the conductive anatomy of biological tissues. >

Compressive behavior of articular cartilage is not completely explained by proteoglycan osmotic pressure

It has been hypothesized that applied mechanical or osmotic loads which decrease cartilage volume by 5% or more are sufficient to relieve all collagen tensile forces, and that further changes in the applied load are completely supported by changes in proteoglycan osmotic pressure. In this view, cartilage should behave mechanically like a concentrated solution of proteoglycans. We tested this hypothesis by measuring the equilibrium axial and radial stresses in bovine articular cartilage during uniaxial confined compression. If the hypothesis is correct, the observed changes in the radial and axial stresses in confined compression should be equal for compression greater than 5%. However, the observed change in axial stress was always substantially greater than the change in radial stress over the range of strains (5-26%) and saline concentrations (0.05-0.15 M) tested. This indicates that the mechanical behavior of cartilage in confined compression cannot solely be explained by changes in proteoglycan osmotic pressure even for strains as large as 26%. A linear isotropic model was found to describe the observed equilibrium behavior adequately. In addition, the inferred shear modulus was found to be independent of saline concentration and similar to measurements by others of the flow-independent shear modulus. Our results have implications regarding the relative contribution of the proteoglycans and collagen to the mechanical properties of the tissue in compression, and suggest that tensile forces in the collagen network may play an important role in determining tissue behavior in confined compression even for relatively large volume changes.

Effect of paddle placement and size on defibrillation current distribution: a three-dimensional finite element model

A realistic three-dimensional finite-element model (FEM) of the conductive anatomy of a canine thorax was constructed for use in the study of transthoracic electrical defibrillation. The model was constructed from a series of 21 cross-sectional computed tomography (CT) scans of a 14.5 kg beagle, each separated by 0.82 cm. The electrical conductive properties of eight distinct tissues were incorporated, including the anisotropic properties of skeletal muscle. Current density distributions were obtained for six paddle pairings and two paddle sizes. A quantitative basis for comparing the resulting distributions was formulated. The results suggest that placing one or both of the paddles near the heart delivers a higher fraction of current to the heart. However, such placements also produce a less uniform myocardial current density distribution and thus have a higher potential for causing damage. Some paddle positions produced myocardial current densities close to the threshold for damage in successful defibrillations. The results indicate that 12 cm paddles may offer modest advantages over 8 cm paddles in clinical defibrillation.<<ETX>>

Effects of the local mechanical environment on vertebrate tissue differentiation during repair: does repair recapitulate development?

SUMMARY The local mechanical environment is a crucial factor in determining cell and tissue differentiation during vertebrate skeletal development and repair. Unlike the basic response of bone to mechanical load, as described in Wolffs law, the mechanobiological relationship between the local mechanical environment and tissue differentiation influences everything from tissue type and molecular architecture to the formation of complex joints. This study tests the hypothesis that precisely controlled mechanical loading can regulate gene expression, tissue differentiation and tissue architecture in the adult skeleton and that precise manipulation of the defects local mechanical environment can initiate a limited recapitulation of joint tissue development. We generated tissue type predictions using finite element models (FEMs) interpreted by published mechanobiological fate maps of tissue differentiation. The experiment included a custom-designed external fixator capable of introducing daily bending, shear or a combination of bending and shear load regimens to induce precisely controlled mechanical conditions within healing femoral defects. Tissue types and ratios were characterized using histomorphometrics and molecular markers. Tissue molecular architecture was quantified using polarized light and Fourier transforms, while immunological staining and in situ hybridization were used to characterize gene expression. The finite element models predicted the differentiation of cartilage within the defects and that substantial fibrous tissues would develop along the extreme excursion peripheries in the bending group. The three experimentally induced loading regimens produced contiguous cartilage bands across all experimental defects, inhibiting bony healing. Histomorphometric analysis of the ratios of cartilage to bone in the experimental groups were not significantly different from those for the knee joint, and Fourier transform analysis determined significantly different collagen fibril angle specializations within superficial, intermediate and deep layers of all experimental cartilages (P<0.0001), approximating those for articular cartilage. All stimulations resulted in the expression of collagen type II, while the bending stimulation also resulted in the expression of the joint-determining gene GDF-5. These findings indicate that the local mechanical environment is an important regulator of gene expression, tissue differentiation and tissue architecture.

Induction of a neoarthrosis by precisely controlled motion in an experimental mid-femoral defect

Bone regeneration during fracture healing has been demonstrated repeatedly, yet the regeneration of articular cartilage and joints has not yet been achieved. It has been recognized however that the mechanical environment during fracture healing can be correlated to the contributions of either the endochondral or intramembranous processes of bone formation, and to resultant tissue architecture. Using this information, the goal of this study was to test the hypothesis that induced motion can directly regulate osteogenic and chondrogenic tissue formation in a rat mid‐femoral bone defect and thereby influence the anatomical result. Sixteen male Sprague Dawley rats (400 ± 20 g) underwent production of a mid‐diaphyseal, non‐critical sized 3.0 mm segmental femoral defect with rigid external fixation using a custom designed four pin fixator. One group of eight animals represented the controls and underwent surgery and constant rigid fixation. In the treatment group the custom external fixator was used to introduce daily interfragmentary bending strain in the eight treatment animals (12°s angular excursion), with a hypothetical symmetrical bending load centered within the gap. The eight animals in the treatment group received motion at 1.0 Hz, for 10 min a day, with a 3 days on, one day off loading protocol for the first two weeks, and 2 days on, one day off for the remaining three weeks. Data collection included histological and immunohistological identification of tissue types, and mean collagen fiber angles and angular conformity between individual fibers in superficial, intermediate, and deep zones within the cartilage. These parameters were compared between the treatment group, rat knee articular cartilage, and the control group as a structural outcome assessment. After 35 days the control animals demonstrated varying degrees of osseous union of the defect with some animals showing partial union. In every individual within the mechanical treatment group the defect completely failed to unite. Bony arcades developed in the experimental group, capping the termini of the bone segments on both sides of the defect in four out of six animals completing the study. These new structures were typically covered with cartilage, as identified by specific histological staining for Type II collagen and proteoglycans. The distribution of collagen within analogous superficial, intermediate, and deep zones of the newly formed cartilage tissue demonstrated preferred fiber angles consistent with those seen in articular cartilage. Although not resulting in complete joint development, these neoarthroses show that the induced motion selectively controlled the formation of cartilage and bone during fracture repair, and that it can be specifically directed. They further demonstrate that the spatial organization of molecular components within the newly formed tissue, at both microanatomical and gross levels, are influenced by their local mechanical environment, confirming previous theoretical models.

A microstructural model of elastostatic properties of articular cartilage in confined compression.

A microstructural model of cartilage was developed to investigate the relative contribution of tissue matrix components to its elastostatic properties. Cartilage was depicted as a tensed collagen lattice pressurized by the Donnan osmotic swelling pressure of proteoglycans. As a first step in modeling the collagen lattice, two-dimensional networks of tensed, elastic, interconnected cables were studied as conceptual models. The models were subjected to the boundary conditions of confined compression and stress-strain curves and elastic moduli were obtained as a function of a two-dimensional equivalent of swelling pressure. Model predictions were compared to equilibrium confined compression moduli of calf cartilage obtained at different bath concentrations ranging from 0.01 to 0.50 M NaCl. It was found that a triangular cable network provided the most consistent correspondence to the experimental data. The model showed that the cartilage collagen network remained tensed under large confined compression strains and could therefore support shear stress. The model also predicted that the elastic moduli increased with increasing swelling pressure in a manner qualitatively similar to experimental observations. Although the model did not preclude potential contributions of other tissue components and mechanisms, the consistency of model predictions with experimental observations suggests that the cartilage collagen network, prestressed by proteoglycan swelling pressure, plays an important role in supporting compression.

Experimental validation of the influence of white matter anisotropy on the intracranial EEG forward solution

Forward solutions with different levels of complexity are employed for localization of current generators, which are responsible for the electric and magnetic fields measured from the human brain. The influence of brain anisotropy on the forward solution is poorly understood. The goal of this study is to validate an anisotropic model for the intracranial electric forward solution by comparing with the directly measured ‘gold standard’. Dipolar sources are created at known locations in the brain and intracranial electroencephalogram (EEG) is recorded simultaneously. Isotropic models with increasing level of complexity are generated along with anisotropic models based on Diffusion tensor imaging (DTI). A Finite Element Method based forward solution is calculated and validated using the measured data. Major findings are (1) An anisotropic model with a linear scaling between the eigenvalues of the electrical conductivity tensor and water self-diffusion tensor in brain tissue is validated. The greatest improvement was obtained when the stimulation site is close to a region of high anisotropy. The model with a global anisotropic ratio of 10:1 between the eigenvalues (parallel: tangential to the fiber direction) has the worst performance of all the anisotropic models. (2) Inclusion of cerebrospinal fluid as well as brain anisotropy in the forward model is necessary for an accurate description of the electric field inside the skull. The results indicate that an anisotropic model based on the DTI can be constructed non-invasively and shows an improved performance when compared to the isotropic models for the calculation of the intracranial EEG forward solution.