Elsevier

Biomaterials

Volume 26, Issue 16, June 2005, Pages 3227-3234
Biomaterials

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

https://doi.org/10.1016/j.biomaterials.2004.09.021Get rights and content

Abstract

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 ∼41% and migration by ∼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.

Introduction

Polyethylene glycol (PEG) diacrylate hydrogel scaffolds may be useful for optimizing engineered tissue formation. These hydrogel scaffolds are highly resistant to protein adsorption due to PEG's hydrophilicity, yet provide the opportunity to incorporate bioactive factors [1], [2], [3], [4]. This combination of protein-resistant scaffold and incorporated biofunctionality makes it possible to control the identity and concentration of bioactive factors presented to cells [1], [2]. For example, fibroblasts, osteoblasts, and vascular smooth muscle cells (SMCs) have all been shown to adhere to and grow on PEG-based hydrogel scaffolds when these scaffolds were modified with adhesion peptides [5], [6], [7], [8]. In fact, the incorporation of adhesion peptides was required for cells to interact at any significant level with hydrogel scaffolds, while cell attachment and spreading was shown to vary depending on the bulk peptide concentration [5], [6], [7], [8]. Thus, these scaffolds provide a blank slate to which cell adhesion peptides can be added in a controlled fashion to enable cell interaction. However, many of these studies have simply focused on studying cell interactions with hydrogels modified with the RGD adhesion peptide. Additional signals that may include a combination of cell adhesion peptides and growth factors will be necessary to elicit cell behavior that is conducive to the formation of engineered tissues [9], [10].

Previous studies have demonstrated that it is possible to covalently immobilize growth factors with retained effects on proliferation and extracellular matrix production. Kuhl and Griffith-Cima tethered epidermal growth factor (EGF) to glass slides via a PEG-based polymer chain and showed that immobilized EGF stimulated DNA synthesis and morphological changes in rat hepatocytes [11]. These alterations were comparable to those observed in the presence of soluble EGF. Similarly, transforming growth factor-β2 (TGF-β2) can retain its effect on collagen tissue deposition when conjugated to collagen via a PEG-based chain [12]. Thus, conjugation to a polymer chain does not impede the interaction between cell receptor binding sites and the growth factors, EGF and TGF-β2, making it possible to control the growth factor's localized concentration.

This technology for covalently immobilizing growth factors can be readily applied to PEG hydrogel scaffolds to influence cell behavior. For example, EGF and TGF-β have been tethered to PEG chains by reaction with an N-hydroxysuccinimidyl ester of PEG monoacrylate [13], [14]. These PEG-modified growth factors can then be immobilized to PEG hydrogel scaffolds during photopolymerization. The immobilized EGF retained its mitogenic activity and also promoted cell migration on hydrogel surfaces containing both EGF and the RGD adhesion peptide [13]. The TGF-β-modified scaffolds promoted collagen production by vascular SMCs seeded within the hydrogel scaffolds, and the resultant engineered tissues had improved mechanical properties compared to those formed with scaffolds modified with only RGD [14]. From these two examples, it is clear that growth factor immobilization in the PEG diacrylate hydrogel system is feasible and can help to influence cell behavior within the scaffold to optimize tissue formation.

In this study, hydrogel scaffolds were modified with the growth factor, basic fibroblast growth factor (bFGF), to stimulate two important aspects of tissue formation—proliferation and migration. bFGF was selected because it is a potent mitogen and chemotactic agent for vascular SMCs [15], [16]. bFGF is also normally found in the vascular environment as it is associated with the extracellular matrix [15], [16]. Thus, this growth factor may play an important role in developing engineered tissues that more closely mimic the structural and functional properties of native blood vessels. Furthermore, we have developed methods that allow immobilization of the growth factor in a concentration gradient. This should mimic the presentation of bioactive factors found in vivo to elicit enhanced and directional cell migration, which may be useful for optimizing tissue formation. The ability to create a stable, immobilized gradient with a known concentration profile of a growth factor should improve our understanding of cellular responses to gradients. Such materials should also allow guidance of cell migration in many tissue-engineering applications.

Section snippets

Cell maintenance

Human aortic smooth muscle cells (HASMCs) were obtained from Cell Applications (San Diego, CA). They were maintained at 37 °C/5% CO2 on Dulbecco's modified Eagle's medium (DMEM) (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; BioWhittaker, Walkersville, MD), 2mM L-glutamine (Sigma, St. Louis, MO), 1000 U/L penicillin (Sigma, St. Louis, MO), and 100 mg/L streptomycin (Sigma, St. Louis, MO). For the gradient bFGF studies, SMCs were maintained on SMC Growth Medium (Cell

Characterization of PEG-conjugated bFGF

Western analysis demonstrated that bFGF was successfully conjugated to PEG. Visual inspection of the gel showed that only a small percentage of the growth factor remained unmodified, while the majority of the growth factor showed an increase in molecular weight corresponding to the attachment of PEG chains. Multiple PEG chains attached to bFGF, resulting in a product with a range of molecular weights (MW of unmodified bFGF=17.5 kDa). Thus, bFGF was conjugated to PEG via the reaction between

Discussion

PEG-conjugated bFGF was investigated for its effect on vascular SMC behavior when covalently incorporated within PEG-based hydrogel scaffolds. bFGF is a potent mitogenic and chemotactic agent present throughout the vascular environment and as a result may be useful for developing an engineered blood vessel. In PEG-based hydrogels, covalently immobilized bFGF has the potential to elicit cell behavior that is conducive to the formation of engineered tissues, while providing the opportunity to

Acknowledgments

The authors would like to thank the NIH for financial support. Technical assistance provided by Dr. Wafa Elbjeirami, Andre Gobin, and James Moon is greatly appreciated.

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