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Micropatterning of proteins and mammalian cells on biomaterials ¹

Department of Chemical and Materials Engineering, University of Cincinnati, Cincinnati, Ohio, USA

²Correspondence: Department of Chemical and Materials Engineering, University of Cincinnati, Cincinnati, OH 45221, USA. E-mail: cho{at}alpha.che.uc.edu

SPECIFIC AIMS

Tissue engineering scaffolds with chemical and topographical features may potentially be used to exert precise control on the spatial organization and behavior of cells. A simple and general method was developed to fabricate micropatterned biomaterials that can be used to form protein arrays and also investigate the effects of chemical and topographical surface features on cell spreading and organization.

PRINCIPAL FINDINGS

1. A general method for forming micron-sized chemical and topographical features on biomaterials
Controlling spatial organization of cells is vital for engineering tissues that require precisely defined cellular architectures. For example, functional nerves or blood vessels form only when groups of cells are organized and aligned in very specific geometries. Among the many techniques that have been developed to fabricate micron-size patterns of protein to spatially control the adhesion of cells on substrates, self-assembled monolayers (SAMs) have taken the lead as the preferred method because of simplicity and high spatial resolutions. In this technique, complementary patterns of alkanethiols and oligo-ethylene glycol terminated alkanethiols are patterned on gold substrates. The hydrophobic alkanethiolate SAMs promote the adhesion of extracellular matrix (ECM) and attachment of cells while the background area, covered by the oligo-ethylene glycol terminated SAMs, resists protein adsorption. While effective, widespread use of this technique for forming micropatterned materials for tissue engineering has been limited by the difficult synthesis of oligo-ethyleneglycol terminal thiols and nonbiodegradibility of the metal substrates. To address this deficiency, we have extended soft lithography-microcontact printing methodology to form chemical and topographical micropatterns directly onto two biocompatible, and biodegradable materials, chitosan and gelatin, using only inexpensive commercially available chemicals and polymers for surface modification.

Figure 1 A illustrates the procedure used to form these topographically patterned biomaterials. Features of interest are initially patterned onto a silicon master using traditional photolithography technique. PDMS precursor solution is poured over the mold and cured to form a flexible and reusable PDMS mold. Micropatterned chitosan films are then formed by introducing a small volume of 2% chitosan solution with acetic acid as solvent onto the PDMS mold. After drying, the chitosan films with complementary topological features to the PDMS mold can be peeled off easily from the mold. Patterned gelatin films can be prepared in a similar way, but are crosslinked with 0.5% glutaraldehyde solution.

View larger version (21K): [in this window] [in a new window]   Figure 1. A) Schematic of the two step soft lithography procedure for creating topographical patterns on biomaterials. B) Schematic of the microcontact printing procedure to coat the plateau regions with trichlorovinylsilane modified Pluronic F127. Contacting the Pluronic F127 coated glass slides onto the topographical patterned chitosan film coats only plateau regions with Pluronic F127.

To spatially control attachment and proliferation of endothelial cells exclusively within the grooves, plateaus of the chitosan film were chemically modified using trichlorovinylsilane coupling agent and a protein resistant triblock copolymer of ethylene and polypropylene glycols, Pluronic F127, commercially available from BASF with a nominal formula of [(PEO)₁₀₀-(PPO)₆₅-(PEO)₁₀₀] and overall molecular mass of 12,600 daltons.

Figure 1B shows a schematic of the microcontact printing procedure we have developed to selectively modify the plateaus of the patterned chitosan and gelatin substrates with TCVS modified PEO/PPO/PEO block copolymers while leaving the surface of the grooves unmodified. Selective chemical modification was accomplished by contacting topographical plateaus of the chitosan surface with glass slides coated with mixtures of Pluronic F127 and TCVS. This chemical modification procedure has previously been used to graft pluronic F127 onto glass and pyrolytic carbon surfaces.

The efficacy of this approach for patterning proteins on biomaterials was monitored by fluorescence microscopy. Pluronic-coated chitosan films with 10, 20, 30, and 50 µm grooves were incubated with fluorescently labeled BSA protein for 30 min then rinsed with water to wash off non-adsorbed BSA. The resulting patterns of protein on the chitosan surface confirm that BSA adsorbs exclusively within the grooves not coated with Pluronic F127. Sharp edges demarcating the borders of regions where BSA protein adsorbs reveal exceptional spatial control of protein adsorption possible using this technique. Moreover, the trichlorovinylsilane coupled Pluronic F127 remains robustly attached to chitosan substrate and resists protein adsorption or cell attachment after more than two weeks in culture medium.

2. Spatially control the attachment and spreading of endothelial cells on biomaterials
To demonstrate the efficacy of this method in controlling the spatial distribution of cells, human microvascular endothelial cells were plated onto Pluronic-coated biomaterials. Figure 2 shows that endothelial cells selectively attach and spread along the 10, 20, 30, and 50 µm grooves. However, cell culture studies performed on identical topographically patterned chitosan films without selective Pluronic coating on the plateaus show completely random attachment of cells on both groove and plateau regions. Micron scale topographical features, by themselves, inadequately control the spatial organization of cells.

View larger version (39K): [in this window] [in a new window]   Figure 2. Cytoskeletal alignment in microvascular endothelial cells cultured in A) 50, B) 30, C) 20, D) 10 µm grooves, and E) unpatterned chitosan. Microfilaments aligned parallel to grooves within 72 h. Actin microfilaments (green) were visualized by Alexra-488-labeled phalloidin. Cell nuclei were visualized by DAPI (blue).

Cell culture studies on these micropatterned chitosan substrates also revealed that confinement within the grooves significantly alters spreading of endothelial cells. Human microvascular endothelial cells were found to naturally spread on unpatterned chitosan substrates to a mean cell area of 2558 ± 295 µm². When confined within grooves, the spreading of endothelial cells decreases as grooves are made narrower. Mean spreading areas of 2550 ± 220, 2240 ± 190, 1830 ± 200, and 1280 ± 220 µm² were observed on 50, 30, 20, and 10 µm wide grooves respectively. These studies also revealed that the width of the 10 and 20 µm grooves is never spanned by more than a single cell.

The cytoskeleton of the patterned cells visualized using fluorescence microscopy is shown in Fig. 2 . F-actin components of the cytoskeleton were labeled using Alexra488 linked phalloidin, while the cell nuclei were visualized by DAPI staining. Compared with cells on unpatterned chitosan, cells patterned within the 10, 20, 30, and 50 µm grooves become oriented along the grooves after 72 h.

The efficacy of this new method in controlling the spatial organization, spreading, and orientation of cells supports the use of this technique for developing a new generation of micropatterned and biodegradable scaffolds for tissue engineering applications.

CONCLUSIONS

We have demonstrated here a new approach for controlling spatial organization, spreading, and orientation of cells on two micropatterned biomaterials: chitosan and gelatin. Unlike traditional cell patterning techniques that make use of gold, silver, palladium, or silicone substrates, cells patterned on these biomaterials have much broader tissue engineering applications. Advantages of this method include 1) ease in forming micrometer-scale topographical features on biomaterials; 2) effective control on the spatial organization, spreading, and orientation of cells; 3) direct attachment of cells without the need for pre-coating substrates with extracellular matrix; 4) the substrates are transparent and attached cells can be directly visualized using conventional light microscopy. The approach can be used to create many other micron-size patterns on biomaterials to suit specific tissue engineering applications.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0490fje;

This Article Full Text (PDF) All Versions of this Article: 18/3/525 03-0490fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by WANG, Y. C. Articles by HO, C.-C. Search for Related Content PubMed PubMed Citation Articles by WANG, Y. C. Articles by HO, C.-C.