A DNA-based molecular probe for optically reporting cellular traction forces.

We developed molecular tension probes (TPs) that report traction forces of adherent cells with high spatial resolution, can in principle be linked to virtually any surface, and obviate monitoring deformations of elastic substrates. TPs consist of DNA hairpins conjugated to fluorophore-quencher pairs that unfold and fluoresce when subjected to specific forces. We applied TPs to reveal that cellular traction forces are heterogeneous within focal adhesions and localized at their distal edges.

Measurement of mechanical tractions exerted by cells in three-dimensional matrices.

Quantitative measurements of cell-generated forces have heretofore required that cells be cultured on two-dimensional substrates. We describe a technique to quantitatively measure three-dimensional traction forces exerted by cells fully encapsulated in well-defined elastic hydrogel matrices. Using this approach we measured traction forces for several cell types in various contexts and revealed patterns of force generation attributable to morphologically distinct regions of cells as they extend into the surrounding matrix.

Degradable hydrogels derived from PEG-diacrylamide for hepatic tissue engineering.

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.

Bioactive hydrogels made from step-growth derived PEG-peptide macromers.

Synthetic hydrogels based on poly(ethylene glycol) (PEG) have been used as biomaterials for cell biology and tissue engineering investigations. Bioactive PEG-based gels have largely relied on heterobifunctional or multi-arm PEG precursors that can be difficult to synthesize and characterize or expensive to obtain. Here, we report an alternative strategy, which instead uses inexpensive and readily available PEG precursors to simplify reactant sourcing. This new approach provides a robust system in which to probe cellular interactions with the microenvironment. We used the step-growth polymerization of PEG diacrylate (PEGDA, 3400Da) with bis-cysteine matrix metalloproteinase (MMP)-sensitive peptides via Michael-type addition to form biodegradable photoactive macromers of the form acrylate-PEG-(peptide-PEG)(m)-acrylate. The molecular weight (MW) of these macromers is controlled by the stoichiometry of the reaction, with a high proportion of resultant macromer species greater than 500kDa. In addition, the polydispersity of these materials was nearly identical for three different MMP-sensitive peptide sequences subjected to the same reaction conditions. When photopolymerized into hydrogels, these high MW materials exhibit increased swelling and sensitivity to collagenase-mediated degradation as compared to previously published PEG hydrogel systems. Cell-adhesive acrylate-PEG-CGRGDS was synthesized similarly and its immobilization and stability in solid hydrogels was characterized with a modified Lowry assay. To illustrate the functional utility of this approach in a biological setting, we applied this system to develop materials that promote angiogenesis in an ex vivo aortic arch explant assay. We demonstrate the formation and invasion of new sprouts mediated by endothelial cells into the hydrogels from embedded embryonic chick aortic arches. Furthermore, we show that this capillary sprouting and three-dimensional migration of endothelial cells can be tuned by engineering the MMP-susceptibility of the hydrogels and the presence of functional immobilized adhesive ligands (CGRGDS vs. CGRGES peptide). The facile chemistry described and significant cellular responses observed suggest the usefulness of these materials in a variety of in vitro and ex vivo biologic investigations, and may aid in the design or refinement of material systems for a range of tissue engineering approaches.