Fabrication of 3D hepatic tissues by additive photopatterning of cellular hydrogels.

We have fabricated a hepatic tissue construct using a multilayer photopatterning platform for embedding cells in hydrogels of complex architecture. We first explored the potential of established hepatocyte culture models to stabilize isolated hepatocytes for photoencapsulation (e.g., double gel, Matrigel, cocultivation with nonparenchymal cells). Using photopolymerizable PEG hydrogels, we then tailored both the chemistry and architecture of the hydrogels to further support hepatocyte survival and liver-specific function. Specifically, we incorporated adhesive peptides to ligate key integrins on these adhesion-dependent cells. To identify the appropriate peptides for incorporation, the integrin expression of cultured hepatocytes was monitored by flow cytometry and their functional role in cell adhesion was assessed on full-length extracellular matrix (ECM) molecules and their adhesive peptide domains. In addition, we modified the hydrogel architecture to minimize barriers to nutrient transport for these highly metabolic cells. Viability of encapsulated cells was improved in photopatterned hydrogels with structural features of 500 microm in width over unpatterned, bulk hydrogels. Based on these findings, we fabricated a multilayer photopatterned PEG hydrogel structure containing the adhesive RGD peptide sequence to ligate the alpha5beta1 integrin of cocultured hepatocytes. Three-dimensional photopatterned constructs were visualized by digital volumetric imaging and cultured in a continuous flow bioreactor for 12 d where they performed favorably in comparison to unpatterned, unperfused constructs. These studies will have impact in the field of liver biology as well as provide enabling tools for tissue engineering of other organs.

Covalent immobilization of RGDS on hydrogel surfaces to direct cell alignment and migration.

This study extends the capability for directing cell behavior using PEG-based hydrogels in tissue-engineering applications to include control over the spatial distribution of the adhesive peptide, RGDS. A continuous linear gradient was formed by simultaneously using a gradient maker to combine precursor solutions and using photopolymerization to lock the RGDS gradient in place. Hydrogels containing entrapped gradients of bovine serum albumin (BSA) were characterized using Coomassie brilliant blue stain, which indicated that BSA concentration increases along the hydrogel's length and that the steepness of the gradient's slope can be varied by changing the relative BSA concentrations in the precursor solutions. Human dermal fibroblasts responded to covalently immobilized RGDS gradients by changing their morphology to align in the direction of increasing RGDS concentration. After 24 h, approximately 46% of fibroblasts were aligned with the RGDS-gradient axis. This proportion of cells further increased to approximately 53% (p < 0.05) and approximately 58% after 48 and 96 h, respectively. Also, fibroblasts migrated differentially depending on the concentration of RGDS. Fibroblasts migrated approximately 48% further going up the concentration gradient (0 to 6 micromol/ml PEG-RGDS) than going down the concentration gradient. Migration up the concentration gradient was also approximately 33% greater than migration on control surfaces with a constant concentration of RGDS (2 micromol/ml), while migration down the gradient was reduced approximately 12% relative to the control surface. In addition, directed migration was further enhanced by increasing the RGDS gradient's slope. This hydrogel system is expected to be useful for directing cell migration to enhance the formation of engineered tissues.

Covalently immobilized gradients of bFGF on hydrogel scaffolds for directed cell migration.

Basic fibroblast growth factor (bFGF) was immobilized to hydrogel scaffolds with retention of mitogenic and chemotactic activity. The bFGF was functionalized in order to incorporate it covalently within polyethylene glycol (PEG) hydrogel scaffolds by reaction with acryloyl-PEG-NHS. Hydrogels were formed by exposing aqueous solutions of PEG diacrylate, acryloyl-PEG-RGDS, and acryloyl-PEG-bFGF to long-wavelength ultraviolet light in the presence of a photoinitiator. These bFGF-modified hydrogels with RGD adhesion sites were evaluated for their effect on vascular smooth muscle cell (SMC) behavior, increasing SMC proliferation by approximately 41% and migration by approximately 15%. A covalently immobilized bFGF gradient was formed using a gradient maker to pour the hydrogel precursor solutions and then photopolymerizing to lock in the concentration gradient. Silver staining was used to detect the bFGF gradient, which increased linearly along the hydrogel's length. Cells were observed to align on hydrogels modified with a bFGF gradient in the direction of increasing tethered bFGF concentration as early as 24 h after seeding. SMCs also migrated differentially, up the concentration gradient, on bFGF-gradient hydrogels compared to control hydrogels with and without a constant bFGF concentration. These hydrogel scaffolds may be useful for studying protein gradient effects on cell behavior and for directing cell migration in tissue-engineering applications.