Degradable hydrogels derived from PEG-diacrylamide for hepatic tissue engineering

Abstract

Engineered tissue constructs have the potential to augment or replace whole organ transplantation for the treatment of liver failure. Poly(ethylene glycol) (PEG)-based systems are particularly promising for the construction of engineered liver tissue due to their biocompatibility and amenability to modular addition of bioactive factors. To date, primary hepatocytes have been successfully encapsulated in non-degradable hydrogels based on PEG-diacrylate (PEGDA). In this study, we describe a hydrogel system based on PEG-diacrylamide (PEGDAAm) containing matrix-metalloproteinase sensitive (MMP-sensitive) peptide in the hydrogel backbone that is suitable for hepatocyte culture both in vitro and after implantation. By replacing hydrolytically unstable esters in PEGDA with amides in PEGDAAm, resultant hydrogels resisted non-specific hydrolysis, while still allowing for MMP-mediated hydrogel degradation. Optimization of polymerization conditions, hepatocellular density, and multicellular tissue composition modulated both the magnitude and longevity of hepatic function in vitro. Importantly, hepatic PEGDAAm-based tissues survived and functioned for over 3 weeks after implantation ectopically in the intraperitoneal (IP) space of nude mice. Together, these studies suggest that MMP-sensitive PEGDAAm-based hydrogels may be a useful material system for applications in tissue engineering and regenerative medicine. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 3331-3338, 2015.

Keywords: PEGDAAm; liver; matrix metalloproteinase; polyethylene glycol diacrylamide; tissue engineering.

Figure 1

(A) Synthesis of polyethylene glycol diacrylamide (PEGDAAm) from PEG. Reaction of PEG under anhydrous conditions with mesyl chloride yields PEG‐dimesylate, which is aminated under aqueous conditions to yield PEG‐diamine. Reaction of PEG‐diamine with acryloyl chloride in the presence of DIPEA yields PEG diacrylamide (PEGDAAm). (B) Synthesis of MMP‐sensitive PEGDAAm is effected in aqueous conditions (100 mM sodium borate, pH 9.0) by reacting the MMP‐sensitive peptide CGPQGIWGQGCR with PEGDAAm. (C) Schematic of hydrogel mesh network resulting from photopolymerization of MMP‐sensitive PEGDAAm from (B) with cell‐adhesive peptides conjugated to PEG and tethered as pendant chains (see text for details). (D) Reaction of MMP‐degradable peptides with a 1.6 molar excess of PEGDAAm resulted in ∼85% conjugation (sum of medium and high molecular weights). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 2

(A) MMP‐sensitive hydrogels were polymerized at 40% w/w and then swollen to equilibrium over 24 h (n = 3). (B) Degradation of MMP‐sensitive PEGDAAm hydrogels in collagenase (0.2 mg mL⁻¹ with 0.2 mg mL⁻¹ NaN₃). (C) Assessment of the stability of MMP‐sensitive PEGDAAm hydrogels in 0.1 N NaOH compared to MMP‐sensitive PEGDA‐based hydrogels.²⁵ Bars indicate standard deviation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 3

Cellular concentration (A) impacts hepatic tissue function, as measured by albumin production (B) and urea synthesis (C) (**p < 0.01, *p < 0.5, 5% PEGDAAm, 10 mmol RGDS. UV 10 mW cm⁻² for 210 s).

Figure 4

UV intensity and exposure duration (A) for material polymerization modulate albumin secretion (B) and urea synthesis (C) of encapsulated hepatocytes. (**p < 0.01, 5% PEGDDAAm, 10 mmol RGDS, 8 × 10⁶ hepatocytes/mL).

Figure 5

(A) Inclusion of LECs in hepatic hydrogels results in interconnected cellular network (green, calcein; red, ethidium homodimer, 5% PEGDAAm, 10 mmol RGDS. UV 10 mW cm⁻² for 210 s, +LEC 6 × 10⁶ heps/mL) and prolongs hydrogel lifetime (B) and hepatic tissue function (C). Hepatic hydrogels with LECs contain cytokeratin‐positive hepatocytes (green) after 3 weeks in culture (D). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 6

PEGDAAm‐based hepatic tissues survived and function after implantation in vivo to 2 weeks. (5% PEGDAAm, 10 mmol RGDS). UV 10 mW cm⁻² for 210 s, 8 × 10⁶ Hep/J2 +LEC 6 × 10⁶ heps/mL in all animals w/cells. Sham animals had blank MMP‐degradable PEGDAAm hydrogels. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]