Engineering hepatocellular morphogenesis and function via ligand-presenting hydrogels with graded mechanical compliance

Abstract

In order to evaluate the sensitivity of hepatocellular cultures to variations in both substrate stiffness and bioactive ligand presentation, hepatocytes were cultured on differentially compliant polyacrylamide gel discs functionalized with varying amounts of the ECM ligand, fibronectin (FN). Subconfluent cell cultures were established in a multiwell plate format enabling the systematic evaluation of cellular response to both underlying substrate rigidity and substrate ligand concentration. Hepatocellular morphogenesis, regulated by a combination of both ligand density and substrate compliance, resulted in a broad spectrum of patterns of cellular reorganization and assembly ranging from highly two-dimensionally spread cells to highly compact, three-dimensional spheroids. Cell compaction was promoted by increasing levels of substrate mechanical compliance and generally inhibited by increasing concentrations of substrate-bound FN. We identified regimes of substrate compliance in which cells are highly responsive or relatively insensitive to the level of substrate-based ligands. For example, while FN presentation did not have a large impact on cell morphogenesis for cultures on highly compliant gels (G' = 1.9 kPa), hepatocytes on "firm" substrates of intermediate compliance (G' = 5.6 kPa) exhibited approximately a 2-fold increase in cell area between the highest and lowest FN concentrations used in this study. Further, we show that increasing substrate compliance at constant ligand concentration results in increased levels of liver-specific albumin secretion while increasing levels of FN at constant substrate rigidity yield reduced liver-specific functional activity. These substrate-elicited differences in cell function also coincided with analogous changes in the transcript levels of metabolic, growth-related, and liver-specific gene markers. Notably, these results also demonstrated that "firm" gel substrates elicit the most hepatocyte functional sensitivity to substrate-based FN presentation. Overall, our findings indicate that hepatocellular responsiveness to ligand concentration can be acutely regulated by gradation of substrate compliance, suggesting that concerted biochemical and biophysical design strategies may be critical toward the fabrication of hepatospecific biomaterials that effectively support desired levels of liver-specific function.

Synthetic Materials in the Study of Cell Response to Substrate Rigidity

“…16 Califano and Reinhart-King 12 hypothesized that network formation by endothelial cells occurs when the combined effects of substrate mechanics and ligand density are optimized. This interplay between substrate biochemistry and mechanics has been noted in many cell types 6,38,59,62,67 and highlights the importance of using substrate systems that allow independent control of the two factors.…”

“…Higher collagen densities also corresponded to increased cytoskeletal organization and focal adhesion formation, especially on softer substrates. 38 Semler et al 67 saw an increase in cell area and proliferation and a decrease in albumin secretion and cytochrome p450 expression with increases in either ligand density or substrate stiffness. These shifts in cellular behavior with ligand density highlight the importance of carefully controlling substrate biochemistry, especially when comparing between distinct surfaces.…”

Tissue Cells Feel and Respond to the Stiffness of Their Substrate

“…Many emerging topics are not dealt with adequately in this brief review of substrate stiffness effects. These include in vitro models of fibrotic stiffening and related disease processes (82,83); perturbed secretion and uptake (84,85); 2D versus 3D responses (32,86); deformations of fibronectin and other matrix molecules (87); structure formation such as capillary development (15,88); deeper aspects of cell differentiation such as with stem cells (81); the relative sensitivity and contractility of some cells relative to others; and broader effects of matrix elasticity, as well as fluidity (i.e., matrix rheology), on cells in tissue development, remodeling, and regeneration. For the cell biologist, this review may suggest the need for a better understanding of mechanochemical pathways and the benefit of more biologically relevant elastic substrates than rigid coverslips and polystyrene for in vitro studies.…”

Polyelectrolyte Multilayer Nanofilms Used as Thin Materials for Cell Mechano‐Sensitivity Studies

“…The first strategy relies on varying the degree of ionic cross-linking between the polyanion and polycation (poly(allylamine hydrochloride)/polyacrylic) as the pH of the polyelectrolyte solutions is increased. [28,29] The second Collagen [12,[14][15][16] Poly-D-lysine [60] Fibronectin [16,50,61] PDMS Tensile tests 5 Á 10 4 -2 Á 10 6 Collagen [17] PEM layer [51] Fibronectin [18] vmIPN Shear rheometry 10 1 -10 4 RGD or RGE peptides [62] Biopolymeric gels Alginate Compression rheometry 10 1 -3 Á 10 3 RGD or RGE peptides [63] Oligopeptide [64] Agarose Shear rheometry 10 3 -4 Á 10 6 No coating [65] Collagen Tensile tests 5Á10 2 -2 Á 10 3 No coating [66] Chitosan gels Compression rheometry 10 3 -2 Á 10 4 No coating [67] Laminin [68] PEM films PLL/HA capped with PAH/PSS AFM indentations 10 3 -5 Á 10 5 No coating [30] PAH/PAA AFM indentations 2 Á 10 5 -10 10 Fibronectin [26] No coating or collagen [29] PLL/HA AFM indentations 10 3 -4 Á 10 5 No coating [40] relies on depositing an increasing number of stiff layers onto a soft base. [30] The third makes use of post-diffusion on the film of a cross-linker, converting ammonium and carboxylic groups into covalent amide bonds.…”