No abstract

Tissue engineering and regenerative medicine (TE&RM) approaches to treating liver disease have the potential to provide temporary support with biohybrid-liver-assist devices or long-term therapy by replacing the diseased liver with functional constructs. A rate-limiting step for TE&RM strategies has been the loss of hepatocytespecific functions after hepatocytes are isolated from their highly specialized in vivo microenvironment and placed in in vitro culture systems. The identification of a biologic substrate that can maintain a functional hepatocyte differentiation profile during in vitro culture would advance potential TE&RM therapeutic strategies. The present study compared two different biologic substrates for their ability to support human hepatocyte function in vitro: porcine-liver-derived extracellular matrix (PLECM) or Matrigel TM . Because Matrigel has been shown to be the most useful matrix for static, traditional hepatocyte culture, we directly compared PLECM with Matrigel in each experiment. Albumin secretion, hepatic transport activity, and ammonia metabolism were used to determine hepatocyte function. Hepatocytes cultured between two layers of PLECM or Matrigel showed equally high levels of albumin expression and secretion, ammonia metabolism, and hepatic transporter expression and function. We conclude that like Matrigel, PLECM represents a favorable substrate for in vitro culture of human hepatocytes.

Sinusoidal endothelial cells (SECs) are notoriously difficult to culture in vitro. SECs represent a highly specialized endothelial cell (EC) population, and traditional methods of SEC isolation from the liver initiate a process of SEC dedifferentiation. Acellular extracellular matrix (ECM) scaffolds were investigated in a physiologically relevant in vitro culture model for their ability to maintain SEC phenotype. The cell culture model used SECs only or a coculture of SECs with hepatocytes on ECM substrates derived from the liver (L-ECM), bladder (UBM-ECM), or small intestine submucosa (SIS-ECM). The effect of the ECM substrate upon SEC dedifferentiation was evaluated using scanning electron microscopy (SEM) and confocal microscopy. When SECs alone were cultured on uncoated glass slides, collagen I, UBM-ECM, or SIS-ECM, SECs showed signs of dedifferentiation after 1 day. In contrast, SECs alone cultured on L-ECM maintained their differentiated phenotype for at least 3 days, indicated by the presence of many fenestrations on SEC surface, expression of anti-rat hepatic sinusoidal endothelial cells mouse IgG MoAb (SE-1), and lack of expression of CD31. When SECs were cocultured with hepatocytes on any of the ECM scaffolds, the SECs maintained a near-normal fenestrated phenotype for at least 1 day. However, SEM revealed that the shape, size, frequency, and organization of the fenestrations varied greatly depending on ECM source. At all time points, SECs cocultured with hepatocytes on L-ECM maintained the greatest degree of differentiation. The present study demonstrated that the acellular ECM scaffold derived from the liver maintained SEC differentiation in culture longer than any of the tested substrate materials. The replacement of complex tissues and 3-dimensional organs may require specialized scaffolds to support multiple, functional cell phenotypes.

Hepatocyte transplantation to treat liver disease is largely limited by the availability of useful cells. Amniotic epithelial cells (hAECs) from term human placenta express surface markers and genes characteristic of embryonic stem cells and have the ability to differentiate into all three germ layers, including tissues of endodermal origin (i.e. liver). Thus, hAECs could provide a source of stem cell-derived hepatocytes for transplantation. We investigated the differentiation of hAECs in vitro and after transplantation into the liver of SCID/Beige mice. Moreover, we tested the ability of rat amniotic epithelial cells (rAECs) to replicate and differentiate upon transplantation into a syngenic model of liver repopulation. In vitro results indicate that the presence of extracellular matrix proteins together with a cocktail of growth factors, cytokines and hormones are required for differentiation of hAECs into hepatocyte-like cells. Differentiated hAECs expressed hepatocyte markers at levels comparable to those of fetal hepatocytes. They were able to metabolize ammonia, testosterone and 17α-hydroxyprogesterone caproate, and expressed inducible fetal cytochromes. After transplantation into the liver of Retrorsine (RS) treated SCID/beige mice, naïve hAECs differentiated into hepatocyte-like cells which expressed mature liver genes such as cytochromes, plasma proteins, transporters and other hepatic enzymes at levels equal to adult liver tissue. When transplanted in a syngenic animal pretreated with RS, rAECs were able to engraft and generate a progeny of cells with morphology and protein expression typical of mature hepatocytes. Conclusion amniotic epithelial cells possess the ability to differentiate into cells with characteristics of functional hepatocytes, in vitro and in vivo, thus representing a useful and non controversial source of cells for transplantation.

Utilization of novel biologically-derived biomaterials in bioprosthetic heart valves (BHV) requires robust constitutive models to predict the mechanical behavior under generalized loading states. Thus, it is necessary to perform rigorous experimentation involving all functional deformations to obtain both the form and material constants of a strain-energy density function. In this study, we generated a comprehensive experimental biaxial mechanical dataset that included high in-plane shear stresses using glutaraldehyde treated bovine pericardium (GLBP) as the representative BHV biomaterial. Compared to our previous study (Sacks, JBME, v.121, pp. 551-555, 1999), GLBP demonstrated a substantially different response under high shear strains. This finding was underscored by the inability of the standard Fung model, applied successfully in our previous GLBP study, to fit the high-shear data. To develop an appropriate constitutive model, we utilized an interpolation technique for the pseudo-elastic response to guide modification of the final model form. An eight parameter modified Fung model utilizing additional quartic terms was developed, which fitted the complete dataset well. Model parameters were also constrained to satisfy physical plausibility of the strain energy function. The results of this study underscore the limited predictive ability of current soft tissue models, and the need to collect experimental data for soft tissue simulations over the complete functional range.

In the present study, the effects of initial collagen fiber orientation on the medium-term (up to 50 Â 10 6 cycles) fatigue response of heart valve soft tissue biomaterials was investigated. Glutaraldehyde treated bovine pericardium (GLBP), preselected for uniform structure and collagen fiber orientation, was used as the representative heart valve biomaterial. Using specialized instrumentation, GLBP specimens were subjected to cyclic tensile loading to maximum stress levels of 500 6 50 kPa at a frequency of 22 Hz. Two sample groups were examined, one with the preferred collagen fiber direction parallel (PD) and perpendicular (XD) to the direction of applied strain. The primary findings indicated that GLBP fatigue response was highly sensitive to the direction of loading with respect to fiber orientation. Specifically, when loading perpendicular to the preferred collagen fiber orientation, fiber reorientation is the dominant mechanism. In contrast, when loaded parallel to the preferred fiber direction a reduction in both collagen fiber crimp and fiber reorientation occurred. Moreover, alterations in the degree and direction of mechanical anisotropy can be inducted by cyclic loading when specimens are loaded perpendicular to the preferred fiber direction. Fourier Transform Infrared Spectroscopy (FT-IR) results indicate that molecular-level damage to collagen occurs in both groups after only 20 Â 10 6 cycles. Taken as a whole, the results of this study suggest that initial collagen orientation plays a critical role in bioprosthetic heart valve biomaterial fatigue response.

DBP polyurethane possesses physical (water absorption) and biomechanical properties comparable to unmodified polyurethane and can resist intrinsic heart-valve leaflet calcification in blood-stream implants.

Transplantation of functional adrenal cortex cells could reduce morbidity and increase the quality of life of patients with adrenal insufficiency. Our aim was to determine whether adrenal extracellular matrix (ECM) scaffolds promote adrenocortical cell endocrine function and proliferation in vitro. We seeded decellularized porcine adrenal ECM with primary human fetal adrenocortical (HFA) cells. Adrenocortical function was quantified by cortisol secretion of HFA-ECM constructs after stimulation with adrenocorticotropic hormone. Proliferation was assessed by adenosine triphosphate assay. HFA-ECM construct morphology was evaluated by immunofluorescence microscopy and scanning electron microscopy. Adrenal HFA-ECM constructs coated with laminin were compared to uncoated constructs. Laminin coating did not significantly affect HFA morphology, proliferation, or function. We demonstrated HFA cell attachment to adrenal ECM scaffolds. Cortisol production and HFA cell proliferation were significantly increased in HFA-ECM constructs compared to controls (p < 0.05), and cortisol secretion rate per cell is comparable to that of human adult and fetal explants. We conclude that adrenal ECM supports endocrine function and proliferation of adrenocortical cells in vitro. Adrenal ECM scaffolds may form the basis for biocompatible tissue-engineered adrenal replacements.