Constitutive modelling of abdominal organs

Abdominal organs are very susceptible to trauma. In order to protect them properly against car crash and other impact consequences, we need to be able to simulate the abdominal organ deformation. Such simulation should account for proper stress-strain relation as well as stress dependence on strain rate. As the step in this direction, this paper presents three-dimensional, non-linear, viscoelastic constitutive models for liver and kidney tissue. The models have been constructed basing on in vivo experiments conducted in Highway Safety Research Institute and the Medical Centre of The University of Michigan (Melvin et al., 1973). The proposed models are valid for compressive nominal strains up to 35% and fast (impact) strain rates between 0.2 and 22.5 s(-1). Similar models can find applications in computer and robot assisted surgery, e.g. the realistic simulation of surgical procedures (including virtual reality) and non-rigid registration.     

Viscoelastic properties of single attached cells under compression

The viscoelastic properties of single, attached C2C12 myoblasts were measured using a recently developed cell loading device. The device allows global compression of an attached cell, while simultaneously measuring the associated forces. The viscoelastic properties were examined by performing a series of dynamic experiments over two frequency decades (0.1-10 Hz) and at a range of axial strains (approximately 10-40%). Confocal laser scanning microscopy was used to visualize the cell during these experiments. To analyze the experimentally obtained force-deformation curves, a nonlinear viscoelastic model was developed. The nonlinear viscoelastic model was able to describe the complete series of dynamic experiments using only a single set of parameters, yielding an elastic modulus of 2120 +/- 900 Pa for the elastic spring, an elastic modulus of 1960 +/- 1350 for the nonlinear spring, and a relaxation time constant of 0.3 +/- 0.12 s. To our knowledge, it is the first time that the global viscoelastic properties of attached cells have been quantified over such a wide range of strains. Furthermore, the experiments were performed under optimal environmental conditions and the results are, therefore, believed to reflect the viscoelastic mechanical behavior of cells, such as would be present in vivo.     

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.     

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.     

A new apparatus and surgical technique for the dual perfusion of human tumor xenografts in situ in nude rats

We present a new perfusion system and surgical technique for simultaneous perfusion of 2 tissue-isolated human cancer xenografts in nude rats by using donor blood that preserves a continuous flow. Adult, athymic nude rats (Hsd:RH-Foxn1(rnu)) were implanted with HeLa human cervical or HT29 colon adenocarcinomas and grown as tissue-isolated xenografts. When tumors reached an estimated weight of 5 to 6 g, rats were prepared for perfusion with donor blood and arteriovenous measurements. The surgical procedure required approximately 20 min to complete for each tumor, and tumors were perfused for a period of 150 min. Results showed that tumor venous blood flow, glucose uptake, lactic acid release, O(2) uptake and CO(2) production, uptake of total fatty acid and linoleic acid and conversion to the mitogen 13-HODE, cAMP levels, and activation of several marker kinases were all well within the normal physiologic, metabolic, and signaling parameters characteristic of individually perfused xenografts. This new perfusion system and technique reduced procedure time by more than 50%. These findings demonstrate that 2 human tumors can be perfused simultaneously in situ or ex vivo by using either rodent or human blood and suggest that the system may also be adapted for use in the dual perfusion of other organs. Advantages of this dual perfusion technique include decreased anesthesia time, decreased surgical manipulation, and increased efficiency, thereby potentially reducing the numbers of laboratory animals required for scientific investigations.     

Preparation of "tissue-isolated" rat tumors for perfusion: a new surgical technique that preserves continuous blood flow

A surgical technique for preparing "tissue-isolated" rat tumors for perfusion was developed that ensures continuous blood flow to the tissue. The tumor may be perfused in situ or ex vivo. Tumor venous blood flow, tumor glucose and lactic acid metabolism, as well as host animal respiration and core temperature, were unchanged during the procedure. Following addition of 14C-D-Glucose to the tumor afferent blood, all radioactivity appeared in the tumor venous blood. None appeared in the host systemic blood, indicating complete separation of tumor and host vasculature during in situ perfusion.     

Design of an ex vivo culture system to investigate the effects of shear stress on cardiovascular tissue

Mechanical forces are known to affect the biomechanical properties of native and engineered cardiovascular tissue. In particular, shear stress that results from the relative motion of heart valve leaflets with respect to the blood flow is one important component of their mechanical environment in vivo. Although different types of bioreactors have been designed to subject cells to shear stress, devices to expose biological tissue are few. In an effort to address this issue, the aim of this study was to design an ex vivo tissue culture system to characterize the biological response of heart valve leaflets subjected to a well-defined steady or time-varying shear stress environment. The novel apparatus was designed based on a cone-and-plate viscometer. The device characteristics were defined to limit the secondary flow effects inherent to this particular geometry. The determination of the operating conditions producing the desired shear stress profile was streamlined using a computational fluid dynamic (CFD) model validated with laser Doppler velocimetry. The novel ex vivo tissue culture system was validated in terms of its capability to reproduce a desired cone rotation and to maintain sterile conditions. The CFD results demonstrated that a cone angle of 0.5 deg, a cone radius of 40 mm, and a gap of 0.2 mm between the cone apex and the plate could limit radial secondary flow effects. The novel cone-and-plate permits to expose nine tissue specimens to an identical shear stress waveform. The whole setup is capable of accommodating four cone-and-plate systems, thus concomitantly subjecting 36 tissue samples to desired shear stress condition. The innovative design enables the tissue specimens to be flush mounted in the plate in order to limit flow perturbations caused by the tissue thickness. The device is capable of producing shear stress rates of up to 650 dyn cm(-2) s(-1) (i.e., maximum shear stress rate experienced by the ventricular surface of an aortic valve leaflet) and was shown to maintain tissue under sterile conditions for 120 h. The novel ex vivo tissue culture system constitutes a valuable tool toward elucidating heart valve mechanobiology. Ultimately, this knowledge will permit the production of functional tissue engineered heart valves, and a better understanding of heart valve biology and disease progression.     

Scale-dependent mechanical properties of native and decellularized liver tissue

Decellularization, a technique used in liver regenerative medicine, is the removal of all the cellular components from a tissue or organ, leaving behind an intact structure of extracellular matrix. The biomechanical properties of this novel scaffold material are currently unknown and are important due to the mechanosensitivity of liver cells. Characterizing this material is important for bioengineering liver tissue from this decellularized scaffold as well as creating new 3-dimensional mimetic structures of liver extracellular matrix. This study set out to characterize the biomechanical properties of perfused liver tissue in its native and decellularized states on both a macro- and nano-scale. Poroviscoelastic finite element models were then used to extract the fluid and solid mechanical properties from the experimental data. Tissue-level spherical indentation-relaxation tests were performed on 5 native livers and 8 decellularized livers at two indentation rates and at multiple perfusion rates. Cellular-level spherical nanoindentation was performed on 2 native livers and 1 decellularized liver. Tissue-level results found native liver tissue to possess a long-term Young’s modulus of 10.5 kPa and decellularized tissue a modulus of 1.18 kPa. Cellular-level testing found native tissue to have a long-term Young’s modulus of 4.40 kPa and decellularized tissue to have a modulus of 0.91 kPa. These results are important for regenerative medicine and tissue engineering where cellular response is dependent on the mechanical properties of the engineered scaffold.     

Biochemical and ultrastructural evaluation of isolated rat liver systems perfused with a hemoglobin-free medium

The viability of hemoglobin-free perfused rat liver was examined with respect to several liver functions and to the intactness of subcellular structures under electron microscopic observation. Provided that rat livers were perfused with the oxygenated buffer solution at a flow rate between 3 and 3.5 ml/min per g of liver, all the biochemical parameters measured in the perfused liver system, i.e. the rates of glucose, pyruvate, and lactate production, the rate of oxygen consumption and the tissue contents of adenine nucleotides, were similar to those observed with perfusion systems containing erythrocytes or albumin. The perfused liver showed a sensitive response to norepinephrine, involving a reduction of pyridine nucleotides and enhancements of glucose production and oxygen consumption. On electron microscopic examination, changes in hepatic-structure indicative of hypoxic injury particularly vacuolar degeneration and mitochondrial swelling, were not detected in the liver after 70 min of perfusion; the fact that the fine structure of the hepatocyte was preserved in all parts of the organ confirmed that the supply of oxygen to the perfused liver was sufficient under the conditions employed. From viewpoint of the generally accepted criteria for the viability of perfused liver, therefore, the results confirmed that the perfusion of liver with a hemoglobin- and albumin-free medium is a convenient and reliable tool for biochemical investigation of the reactions occurring in whole liver.     

Particle deposition in arteries ex vivo: effects of pressure,flow, and waveform

The goal of this study was to quantify the effect of hemodynamic pressure, flow and waveform perturbations on the deposition of protein-sized particles in porcine carotid arteries ex vivo. An ex vivo perfusion system was used to control the pressure and flow environment for excised arterial tissue. Confocal laser microscopy images revealed that 200 nm particles were deposited intimally and that more spheres were evident along vessels perfused under oscillatory waveform conditions than all others. Under all pressure, flow and waveform conditions, particles were excluded from the media and adventitia of the vessel wall. The steady flow data support the use of Darcy's Law with pressure-dependent hydraulic permeability to model arterial tissue.     

Permeability characteristics of isolated perfused rat lungs

We examined the factors that influence the permeability characteristics of isolated perfused rat lungs and compared the ex vivo permeability-surface area product (PS) with that obtained in vivo. In lungs perfused for 20 min with homologous blood or a physiological salt solution (PSS) containing 4 g/100 ml albumin, mean PS values, obtained by the single-sample method of Kern et al. [Am. J. Physiol. 245 (Heart Circ. Physiol. 14): H229-H236, 1983], were 9.9 +/- 0.6 (SE) and 6.8 +/- 0.3 cm3.min-1.g wet lung-1.10(-2), respectively. These values were similar to lung PS obtained in intact rats (7.7 +/- 0.4 cm3.min-1.g wet lung-1.10(-2). In perfused lungs, PS values were influenced by the perfusate albumin concentration, the length of perfusion time, and the degree of vascular recruitment. Twenty minutes after lung isolation, PS was 126% higher in lungs perfused with albumin-free PSS containing Ficoll than in lungs perfused with albumin-PSS. Moreover, PS in Ficoll-PSS-perfused lungs increased even higher after 2 h of perfusion, and this time-dependent increase in PS was attenuated by addition of 0.1 g/100 ml albumin to the perfusate. Two hours of ex vivo ventilation with hypoxic (0 or 3% 0(2)) or hyperoxic (95% 0(2)) gas mixture did not affect PS values in perfused lungs. However, PS was elevated in lungs perfused ex vivo with protamine, which causes endothelial cell injury, or in lungs from rats exposed in vivo to human recombinant tumor necrosis factor.(ABSTRACT TRUNCATED AT 250 WORDS)     

24-h Langendorff-perfused neonatal rat heart used to study the impact of adenoviral gene transfer

The human genome project has increased the demand for simple experimental systems that allow the impact of gene manipulations to be studied under controlled ex vivo conditions. We hypothesized that, in contrast to adult hearts, neonatal hearts allow long-term perfusion and efficient gene transfer ex vivo. A Langendorff perfusion system was modified to allow perfusion for >24 h with particular emphasis on uncompromised contractile activity, sterility, online measurement of force of contraction, inotropic response to beta-adrenergic stimulation, and efficient gene transfer. The hearts were perfused with serum-free medium (DMEM + medium 199, 4 + 1) supplemented with hydrocortisone, triiodothyronine, ascorbic acid, insulin, pyruvate, l-carnitine, creatine, taurine, l-glutamine, mannitol, and antibiotics recirculating (500 ml/2 hearts) at 1 ml/min. Hearts from 2 day-old rats beat constantly at 135-155 beats/min and developed active force of 1-2 mN. During 24 h of perfusion, twitch tension increased to approximately 165% of initial values (P < 0.05), whereas the inotropic response to isoprenaline remained constant. A decrease in total protein content of 10% and histological examination indicated moderate edema, but actin and calsequestrin concentration remained unchanged and perfusion pressure remained constant at 7-11 mmHg. Perfusion with a LacZ-encoding adenovirus at 3 x 108 active virus particles yielded homogeneous transfection of approximately 80% throughout the heart and did not affect heart rate, force of contraction, or response to isoprenaline compared with uninfected controls (n = 7 each). Taken together, the 24-h Langendorff-perfused neonatal rat heart is a relatively simple, inexpensive, and robust new heart model that appears feasible as a test bed for functional genomics.     

Modes of endocytosis of latex particles in sinusoidal endothelial and Kupffer cells of normal and perfused rat liver

The endocytosis of latex particles (0.33, 0.46 and 0.80 micron in diameter) in the sinusoidal endothelial and Kupffer cells of the rat liver was studied electron microscopically. When the liver was perfused with serum-free oxygenated Krebs Ringer bicarbonate, latex particles of all three sizes were taken up by the endothelial cells. After a 10-min perfusion, particles were incorporated by the luminal cell surface of the perikarya or of the thick portion of the endothelial cells. A large patch of bristle coat was surrounding the ingested particle. The number of ingested particles in the endothelial cells, however, was much less than in the Kupffer cells. In in vivo experiments, no endocytosis of the latex particles was observed in the endothelial cells. In the Kupffer cells, particles were engulfed by the ruffled membranes or sank into the cytoplasm without a large patch of the bristle coat both in the perfusion system and in vivo. These observations show that at least 0.80 micron latex particles are taken up by the bristle-coated membranes in the sinusoidal endothelial cells of the perfused liver. The endocytic mechanism for latex particles in the endothelial cells is different from that of the Kupffer cells.     

Influence of oxygen uptake through the liver surface on the metabolism of ex vivo perfused liver during hypoxia

The low quality of transplants having undergone hypoxic injury can lead to postoperative complications. The aim of the present research is to estimate, by means of mathematical modeling, how the process of oxygen uptake through the liver surface influences the metabolism of ex vivo perfused liver under hypoxia. The value of oxygen uptake through the surface was established to depend on the degree of oxygenation of the perfusion medium. A decrease in the oxygenation of the perfusion medium resulted in a decreased oxygen uptake through the liver surface. Stoichiometric modeling of the liver metabolism shows that upon the decreased oxygenation of the perfusion medium more energy is required for the process of oxygen uptake through the surface even at a lower level as compared to the normal oxygen supply. The application of the Pareto optimality allows estimating the optimum distribution of the energy resources in liver under ex vivo conditions. Both upon the normal and decreased oxygenation of the perfusion medium, the phenomenon of “free competition” for the resource was observed, with the energy being optimally distributed among all the metabolic fluxes. Moreover, this energy is also spent on the accompanying processes, e.g. for the transport of interstitial fluid.     

Primary cultures of rat hepatocytes in hollow fiber chambers

Summary Hepatocyte culture may represent an alternative to the use of animals to study drug detoxification by the liver. An ideal in vitro system should closely mimic the in vivo environment by providing continuous media perfusion and oxygenation, and should facilitate sampling of cells and culture media. To meet these criteria, a hollow fiber bioreactor seeded with isolated rat hepatocytes was developed and tested by measuring the formation of three products of the oxidative metabolism of diazepam and the glucuronidation of phenolsulfonphthalein (PSP). To compare the performance of conventional monolayer culture to that of the bioreactor system, diazepam metabolism was studied for 45 days in both systems. The oxygen dependency of diazepam metabolism was evaluated by perfusing the bioreactor in an oxygen-rich atmosphere (30%). Total diazepam metabolism was twofold higher in the O₂-rich perfused hollow fiber cultures than in the cultures perfused under normal conditions, reflecting an increase in temazepam and oxazepam production. Diazepam detoxification activity was significantly enhanced by oxygen (P≤0.001) over the life of the perfused cultures. PSP metabolism was similar in all three culture systems. By Day 10, diazepam metabolism in the oxygenated bioreactor system was 44% of the in vivo activity of rat hepatocytes. This activity dropped to 30% by Day 25 of culture. These results justify the use of perfused culture systems for in vitro detoxification studies as an alternative to animal use and emphasize the capacity of a culture device perfused under O₂-enriched conditions to maintain long-term P450 activity of rat hepatocytes.     

Identifying a minimal rheological configuration: a tool for effective and efficient constitutive modeling of soft tissues

We describe a modeling methodology intended as a preliminary step in the identification of appropriate constitutive frameworks for the time-dependent response of biological tissues. The modeling approach comprises a customizable rheological network of viscous and elastic elements governed by user-defined 1D constitutive relationships. The model parameters are identified by iterative nonlinear optimization, minimizing the error between experimental and model-predicted structural (load-displacement) tissue response under a specific mode of deformation. We demonstrate the use of this methodology by determining the minimal rheological arrangement, constitutive relationships, and model parameters for the structural response of various soft tissues, including ex vivo perfused porcine liver in indentation, ex vivo porcine brain cortical tissue in indentation, and ex vivo human cervical tissue in unconfined compression. Our results indicate that the identified rheological configurations provide good agreement with experimental data, including multiple constant strain rate load/unload tests and stress relaxation tests. Our experience suggests that the described modeling framework is an efficient tool for exploring a wide array of constitutive relationships and rheological arrangements, which can subsequently serve as a basis for 3D constitutive model development and finite-element implementations. The proposed approach can also be employed as a self-contained tool to obtain simplified 1D phenomenological models of the structural response of biological tissue to single-axis manipulations for applications in haptic technologies.     

Effects of forskolin on contractile responses and protein phosphorylation in the isolated perfused rat heart.

Continuous perfusion of rat hearts with concentrations of forskolin between 0.1 and 12 microM resulted in transient increases in tension after 45 s, followed by a return to the control value after 5 min. In contrast, the content of cyclic AMP increased linearly with time over this period, reaching values up to 35 times control after 5 min. Increases in contractile force, intracellular cyclic AMP concentration and the proportion of phosphorylase in the a form were dependent on the concentration of forskolin when measured 45 s and 120 s after initiation of perfusion. In hearts perfused for 45 s with various concentrations of forskolin, the measured cyclic AMP-dependent protein kinase activity ratio and phosphorylase a content for a given measured intracellular cyclic AMP concentration were both much less than the corresponding values in hearts perfused for 30 s with various concentrations of isoprenaline. The phosphorylation of the contractile proteins troponin-I and C-protein also showed a concentration-dependent increase in hearts perfused with forskolin. There was a strong correlation between the cyclic AMP-dependent protein kinase activity ratios and the phosphorylation of the contractile proteins under all perfusion conditions. These results suggest that cyclic AMP is compartmented in perfused rat heart, and that much of the cyclic AMP produced in response to forskolin is unavailable to activate cyclic AMP-dependent protein kinase.     

Rheology of the vitreous gel: effects of macromolecule organization on the viscoelastic properties

The macromolecular organization of vitreous gel is responsible for its viscoelastic properties. Knowledge of this correlation enables us to relate the physical properties of vitreous to its pathology, as well as optimize surgical procedures such as vitrectomy. Herein, we studied the rheological properties (e.g. dynamic deformation, shear stress-strain flow, and creep compliance) of porcine vitreous humor using a stressed-control shear rheometer. All experiments were performed in a closed environment with the temperature set to that of the human body (i.e. 37°C) to mimic in-vivo conditions. We modeled the creep deformation using the two-element retardation spectrum model. By associating each element of the model to an individual biopolymeric system in the vitreous gel, a distinct response to the applied stress was observed from each component. We hypothesized that the first viscoelastic response with the short time scale (~1 s) is associated with the collagen structure, while the second viscoelastic response with longer time scale (~100 s) is related to the microfibrilis and hyaluronan network. Consequently, we were able to differentiate the role of each main component from the overall viscoelastic properties.     

Large deformation shear properties of liver tissue

Characterization of the mechanical properties of soft biological tissues is important for establishing the mechanical tolerances of the tissues, and for input to computational models. In this work, the viscoelastic properties of bovine liver tissue in shear loading have been measured using relaxation and constant shear rate loading. The tissue is nonlinearly viscoelastic for strains greater than 0.2%, has a yield strain of approximately 10, and shows moderate strain-rate sensitivity. The response can be modelled using a nonlinear viscoelastic differential model previously developed for brain tissue.     

Stimulation of hepatic glycogenolysis by phorbol 12-myristate 13-acetate.

In isolated perfused rat livers, infusion of phorbol 12-myristate 13-acetate (PMA) (150 nM) resulted in a 3-fold stimulation of the rate of glucose production. This response was maximal at a perfusate PMA concentration of 150 nM, and was significantly diminished at higher concentrations of PMA (e.g. 300 nM). Stimulation of glycogenolysis by PMA was greatly decreased in livers perfused with Ca2+-free medium. PMA infusion into livers perfused in the absence of Ca2+ did not result in Ca2+ efflux from the livers. Additionally, in hepatocytes isolated from livers of fed rats, neither PMA nor 1-oleoyl-2-acetyl-rac-glycerol stimulated the rate of glucose production. Although indomethacin has been demonstrated to block PMA-stimulated hepatic glycogenolysis [Garcia-Sainz & Hernandez-Sotomayor (1985) Biochem. Biophys. Res. Commun. 132, 204-209], infusion of PMA into perfused rat livers did not alter the rates of production of either prostaglandin E2 or 6-oxo-prostaglandin F1 alpha in the livers. These data, along with the observed increases in the perfusion pressure and decrease in O2 consumption in isolated perfused livers suggest that phorbol-ester-stimulated glycogenolysis is not a consequence of a direct effect of phorbol ester on liver parenchymal cells.