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

ABSTRACT: 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.

SUBMITTER: Miller JS

PROVIDER: S-EPMC2837100 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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

Miller Jordan S JS Shen Colette J CJ Legant Wesley R WR Baranski Jan D JD Blakely Brandon L BL Chen Christopher S CS

Biomaterials 20100206 13

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 i ...[more]

PMID: 20138664

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Project description:Hydrogel scaffolds are used in biomedicine to study cell differentiation and tissue evolution, where it is critical to control the delivery of chemical cues both spatially and temporally. While large molecules can be physically entrapped in a hydrogel, moderate molecular weight therapeutics must be tethered to the hydrogel network through a labile linkage to allow controlled release. We synthesized and characterized a library of polymerizable ortho-nitrobenzyl (o-NB) macromers with different functionalities at the benzylic position (alcohol, amine, BOC-amine, halide, acrylate, carboxylic acid, activated disulfide, N-hydroxysuccinyl ester, biotin). This library of polymerizable macromers containing o-NB groups should allow direct conjugation of nearly any type of therapeutic agent and its subsequent controlled photorelease from a hydrogel network. As proof-of-concept, we incorporated the N-hydroxysuccinyl ester macromer into hydrogels and then reacted phenylalanine with the NHS ester. Upon exposure to light (? = 365 nm; 10 mW/cm(2), 10 min), 81.3% of the phenylalanine was released from the gel. Utilizing the photodegradable macromer incorporating an activated disulfide, we conjugated a cell-adhesive peptide (GCGYGRGDSPG), a protein that exhibits enzymatic activity (bovine serum albumin (BSA)), and a growth factor (transforming growth factor-?1 (TGF-?1)) into hydrogels, controlled their release with light (? = 365 nm; 10 mW/cm(2), 0-20 min), and verified the bioactivity of the photoreleased molecules. The photoreleasable peptide allows real-time control over cell adhesion. BSA maintains full enzymatic activity upon sequestration and release from the hydrogel. Photoreleased TGF-?1 is able to induce chondrogenic differentiation of human mesenchymal stem cells comparable to native TGF-?1. Through this approach, we have demonstrated that photodegradable tethers can be used to sequester peptides and proteins into hydrogel depots and release them in an externally controlled, predictable manner without compromising biological function.

Project description:Synthetic hydrogels with well-defined mechanical properties have become invaluable tools for probing cell response to extracellular cues including matrix stiffness and integrin binding. These synthetic matrices are often decorated with either proteins or integrin-binding peptides to promote cell adhesion and to direct or probe cell behavior. For example, both collagen I-functionalized polyacrylamide and peptide-functionalized poly(ethylene glycol) hydrogels have been instrumental in elucidating the role of the elasticity or 'stiffness' of the matrix in promoting fibroblast activation in wound healing and fibrosis. However, the two methods of promoting integrin binding are not often directly compared in the same system, partly owing to differences in material designs, despite the potential differences in the way cells interact with whole proteins and protein mimetic peptides. We hypothesized that such a comparison could provide insight into the ways integrin binding affects fibroblast activation within commonly utilized in vitro cell culture models, and more broadly, to inform the design of materials to modulate fibroblast activation in studies of wound healing and disease. To enable this comparison, we developed a method to conjugate whole proteins to step-growth poly(ethylene glycol) (PEG) hydrogels and investigated fibroblast response to protein-peptide pairs: fibronectin and PHSRN(G)10RGDS or collagen I and (POG)3POGFOGER(POG)4, which are important in matrix remodeling and relevant to fibroblast activation. With this approach, we observed that human pulmonary fibroblasts adopted a similar morphology on fibronectin and PHSRN(G)10RGDS, although with a slight increase in the percentage of alpha smooth muscle actin (αSMA) expressing cells on PHSRN(G)10RGDS. Interestingly, we observed that fibroblasts formed activated clusters on the collagen mimic (POG)3POGFOGER(POG)4 while exhibiting less activation on collagen I. This cell activation and clustering is reminiscent of fibroblast foci that are observed in lung fibrosis, suggesting the relevance of these well-defined polymer-peptide hydrogels for investigating fibrosis and decoupling biochemical and biophysical cues.

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

Publications

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

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