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.
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.
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.
Liver disease is a leading cause of mortality in the United States. Tissue engineering and regenerative medicine (TE&RM) approaches to treating liver disease have the potential to provide temporary support with biohybrid artificial liver-assist devices or long-term therapy by replacing the diseased liver with functional tissue-engineered constructs that utilize a combination of cells, scaffolds and bioactive factors. A rate-limiting step for TE&RM technologies has been the loss of hepatocyte-specific functions in vitro after hepatocytes are isolated from their highly specialized in vivo microenvironment. 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 is predicated upon the hypothesis that the substrate upon which hepatocytes are seeded is a critical determinant of cell phenotype and function. Biologic scaffolds composed of extracellular matrix (ECM) derived from mammalian organs have been used to maintain cell phenotype in vitro. Two studies were performed to evaluate the ability of ECM scaffolds to maintain primary human hepatocyte (PHH) phenotype in vitro. The first study evaluated the effect of ECM scaffolds derived from porcine liver (PLECM) and human liver (HLECM) upon maintainence of PHH specific functions in vitro. Cytochrome P450 activity and albumin secretion were used as indicators of hepatocyte functionality. No differences in hepatoycte-specific functions were measured when comparing PHH cultured with PLECM and HLECM. From a clinical perspective, the results are appealing because of the limited availability of human liver tissue compared to porcine liver tissue for the manufacture of ECM scaffolds.The second study cultured PHH between two layers of PLECM and compared with Matrigel double gel cultures and adsorbed type-1 collagen. Albumin secretion, hepatic transport activity and ammonia metabolism were used as markers of hepatocyte functionality. PHH cultured between two layers of porcine liver ECM had significantly higher levels of albumin secretion, hepatic transport activity, and ammonia metabolism compared to PHH cultured on collagen.
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 hepatocyte-specific 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. 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.
