Smooth muscle cell growth in photopolymerized hydrogels with cell adhesive and proteolytically degradable domains: synthetic ECM analogs for tissue engineering
Introduction
The goal of the current project was the development of synthetic polymers that can mimic several of the properties of the natural extracellular matrix (ECM), such as biospecific cell adhesion, degradation by proteolytic processes involved in cell migration and tissue remodeling, and the ability to control cellular functions such as ECM synthesis. These materials are photopolymerizable derivatives of polyethylene glycol (PEG) that form hydrogel materials. PEG was chosen for this application due to its hydrophilicity, biocompatibility, and intrinsic resistance to protein adsorption and cell adhesion [1], [2]. This resistance to protein adsorption and cell adhesion makes the base material essentially a blank slate, devoid of biological interactions, upon which the desired biofunctionality can be built. Previous work has shown that aqueous solutions of acrylated PEG derivatives can be photopolymerized in direct contact with cells and tissues without deleterious effects [3], [4]. For tissue engineering purposes, cells can be suspended in the aqueous polymer solution; after photopolymerization, cells will be homogeneously seeded throughout the hydrogel scaffold. Photopolymerization can be carried out ex vivo or in situ.
These materials can be rendered bioactive by inclusion of peptides or polysaccharides in the polymer backbone or by grafting peptides, proteins, or polysaccharides into the hydrogel network during the photopolymerization process. In order to create a material that might be degraded by tissue formation processes, we have chosen to target degradation to proteolytic enzymes involved in cell migration and tissue remodeling [5], [6], [7], [8]. Proteases, such as the matrix metalloproteases, are crucial in the cell migration process, as they allow cells to clear a pathway through the dense matrix [9]. We have previously shown that PEG-based hydrogels with peptides in their backbone that are degradation substrates for particular enzymes, such as collagenase or plasmin, can undergo proteolytic degradation [10]. This degradation scheme should match the rate of material resorption to the rate of tissue formation. In order to achieve biospecific cell adhesion to these materials, we have grafted cell adhesion peptides into the hydrogel structure in a manner similar to that used by Hern and Hubbell [11], who demonstrated peptide concentration-dependent adhesion of fibroblasts to the surface of PEG diacrylate hydrogels grafted with RGD peptides. The PEG hydrogels offer an advantage over most other scaffold materials in that they are intrinsically resistant to cell adhesion [1], [2]. This allows the cell-material interactions to be limited to the adhesive ligands provided. If cell selective ligands, such as the REDV peptide that has been shown to be endothelial cell specific [12], are incorporated into a cell non-adhesive scaffold, the resultant scaffold material should allow adhesion of only the desired cell type. We have also previously shown that growth factors, such as transforming growth factor-beta1, can be covalently grafted into these photopolymerized hydrogel scaffolds and maintain their activity. Cells grown in scaffolds with immobilized transforming growth factor-beta exhibited dramatically increased synthesis of collagen and improved mechanical properties of the resultant engineered tissues, even compared to cells in similar scaffolds but with the growth factor soluble in the culture media [13]. The hydrogels used as tissue engineering scaffolds can be formed from blends of several of these bioactive PEG derivatives in order to create a matrix substitute with the desired biological activities for a given application.
Section snippets
Materials and methods
Chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated.
Proteolytic degradation of hydrogel scaffolds
In order to link scaffold degradation to tissue formation, scaffolds were synthesized that could be degraded by proteolytic enzymes secreted by cells during migration. These scaffolds were made by incorporating proteolytically degradable peptide sequences into the backbone of a PEG diacrylate derivative as an ABA block copolymer (A=PEG monoacrylate, B=peptide). The peptide sequences examined were LGPA, targeted for degradation by collagenase [5], [6], and a 9-mer of alanine, targeted for
Discussion
Materials used as scaffolds to support and guide tissue formation must meet certain basic criteria; they must be biocompatible, must support cell growth, must be bioresorbable, must provide the mechanical support necessary to maintain the tissue form, and should be easily processed. A number of synthetic biodegradable polymers, such as poly(lactic-co-glycolic acid), largely meet these requirements [18], [19], [20]. Ideally, however, a scaffold material should also have a number of biological
Acknowledgments
Funding for this work was provided by the NIH Heart, Lung, and Blood Institute (R01 HL60485) and the Theodore N. Law Foundation.
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