Introduction

Cell-based therapies, combined with tissue engineering, have the potential to bring regenerative medicine to the clinic. A major challenge is the development of selective substrates that are permissive and instructive for the growth of complex functional tissue in vitro. The requirements for selective cell attachment, survival, migration, growth, and differentiation will require an integrative understanding and application of cellular and molecular responses to the chemical, mechanical, and topological properties of substrates.

A wide range of natural and synthetically designed materials have been employed to culture and expand mammalian cells within tissue culture substrates, bioreactors, and implantable scaffolds. Despite their reduced bioactivity, synthetic biomaterials offer advantages over natural substrates, including reduced risk of immunological response and disease transmission and controlled properties. For example, the ability to manipulate structure through chemistry and fabrication provides opportunities to engineer specific properties, including biodegradation, fouling, drug elution, and biofunctionalization, which are desirable for introducing tissue-specific targeting and tissue regeneration and remodeling. The characterization of newer generation cell-interactive materials requires complex considerations and is further hampered by the lack of integrated data on extracellular matrix and protein conditioning, cell adhesion, motility, apoptosis, and differentiation processes, as well as the mechanisms of outside-in signaling on biomaterials. While rapid methods for quantifying protein adsorption have been developed (1,2,3,4), the characterization of cell-biomaterial interactions at the cellular and subcellular level remains time-consuming and expensive (5). A need exists for qualitative and quantitative tools that can rapidly assess cellular response to biomaterial substrates.

Green fluorescent protein (GFP) technology has revolutionized the detection and analysis of dynamic structural and biochemical changes in living cells (6,7). We have developed genetically engineered cell lines expressing a series of GFP fusion genes that visually report on a variety of physiological properties in living cells. The present study describes a new way to utilize these GFP fusion genes as visual live reporters for quantitative characterization of cell-biomaterials interaction. Using quantitative image analysis, the reporter information can be further dissected to yield molecular level metrology of the cell-material interactions.

Materials and Methods

Polymer Synthesis and Fabrication

Biomaterials used in this study were from the family of tyrosine-derived polycarbonates synthesized utilizing previous published procedures (8,9,10,11,12). Polymers derived from desaminotyrosyl tyrosine alkyl ester (DTR) monomers were named poly(DTR carbonate)s with R representing ethyl (E) or octyl (O). Copolymers of x mole% desaminotyrosyl tyrosine alkyl ester with y mole% desaminotyrosyl tyrosine with a free acid (DT) and z% polyethylene glycol (PEG) blocks of 1000 MW were identified as poly(x%DTE-co-y%DT-co-z%-PEG₁₀₀₀ carbonate). Finally, copolymers of x mole% iodinated desaminotyrosyl tyrosine alkyl ester with y mole% iodinated desaminotyrosyl tyrosine (DT) and z% PEG blocks of 1000 MW were identified as poly(x%I₂DTE-co-y%I₂DTE-co-z%-PEG₁₀₀₀ carbonate) (Figure 1). This library of polymers was used to evaluate the sensitivity of cell reporters to changes in substrate hydrophobicity (hydrophobicity is increased with length of alkyl chain; thus, DTO is more hydrophobic than DTE) (13,14); stiffness (increased with iodination; decreased with PEG incorporation) (11,12), charge (incorporation of DT adds negative charge at physiologic pH) (8,15,16), and protein-repulsive character (increased PEG levels lower protein adsorption) (17,18). Two-dimensional polymer films were spincoated (Photo Resist Spinner; Headway Research, Garland, TX, USA) onto 15-mm glass coverslips with 100 µL of a 0.2-µm pore polytetrafluoroethylene (PTFE)-filtered, 1% polymer solution in 98.5% methylene chloride/1.5% methanol (v/v). All polymer surfaces were sterilized by exposure to UV.

Figure 1. Structure of tyrosine-derived polycarbonates. (Click to enlarge)

Cell Culture and Transfection

GFP-based plasmids were obtained from academic laboratories (19,20) (see Acknowledgments) and commercial sources (Clontech, Mountain View, CA, USA) (Table 1) and were targeted to specific organelles through the addition of a short localization signal [such as GFP-farnesylation (f)] and to specific cytoskeletal proteins (such as GFP-actin) (see Table 1). Several model cell lines were employed, including CHO-K1 (ATCC, Manassas, VA, USA) and Saos-2 (a gift from David Denhardt; Rutgers University). The cells were propagated in F12H (Invitrogen, Carlsbad, CA, USA) supplemented with glutamine, penicillin-streptomycin, and 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA). Cells were transfected with Lipofectamine™ supplemented with PLUS™ reagent (Invitrogen), and stable lines were selected using 0.5 mg/mL (Saos-2) or 1 mg/mL (CHO-K1) G-418 (Sigma-Aldrich). Utilizing G-418 selection, cell populations with as high as 80% of total cells expressing the fluorescent reporters were achieved. Where indicated, cells were fixed via treatment with a 4% paraformaldehyde solution for 15 min at room temperature.