Biomacromolecules 2008, 9, 2315–2321
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Biomimetic Carbohydrate Substrates of Tunable Properties Using Immobilized Dextran Hydrogels Mark H. Lee,*,†,‡,‡ David Boettiger,†,‡ and Russell J. Composto†,‡ Institute for Medicine and Engineering, Department of Materials Science and Engineering, and Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Received February 24, 2008; Revised Manuscript Received May 17, 2008
Glycidylmethacrylate-modified dextran macromers (Dex-GMA) of different degrees of substitution (DS) were synthesized. The elastic modulus of the hydrogels produced using one-component and two-component macromer systems was measured using rheometry. When one macromer of DS 1/10 was used, a hydrogel modulus in the range of 0.2 Pa to 42 kPa was obtained by varying the Dex-GMA concentration from 80 to 200 mg/mL. When a mixture of two macromers of different DS (1/10 and 1/23) was used, a more uniform variation of modulus in the range of 0.4 Pa to 42 kPa was achieved by controlling the ratio of the two macromers. When dextran hydrogels were functionalized with fibronectin and immobilized onto glass substrates, the attachment, spreading, and growth of human aortic smooth muscle cells were modulated by the elastic properties of the dextran matrix. The dextran hydrogel system with tunable mechanical and biochemical properties appears promising for applications in cell culture and tissue engineering.
Introduction Historically, interactions of the extracellular matrix (ECM) with cells have largely focused on the complex set of biochemical and topographical interactions occurring at the interface. Recent studies have generated much interest in what role the mechanical properties of the matrix play in determining cell function. The findings from these studies link many of the fundamental behaviors of cells such as adhesion, spreading, growth, and differentiation with matrix elasticity or stiffness.1–4 This has important implications for the production of substrates for biotechnological and tissue engineering products. It is clear that the next generation of matrices and substrates developed to interact with cells need to be designed with mechanical properties, such as the elastic modulus, being a key parameter. Hydrogels are used extensively for biomaterials applications and a wide variety of synthetic and biopolymers have been and continue to be developed into this class of materials.5–8 The usage of synthetic polymers is advantageous in that they can easily be manipulated to yield the desired physicochemical characteristics however many are hindered by low biocompatibility. On the other hand, biopolymers often benefit from their inherent similarity to the matrix material found in vivo but often suffer from practical limitations in material availability, lot-tolot variation, and excessive cost. Polysaccharides, in particular, are attractive for biotechnological applications due to their inherently biomimetic physicochemical properties that allow for more optimal interactions with proteins and cells. The biopolymer used in this work, Dextran, is readily available and possesses a long history of both commercial and clinical use as plasma volume expanders,9 wound cleansing and healing agents,10,11 and drug delivery carriers.12,13 Unlike most other polysaccharides, the physical structure and properties of the B-512(F) dextran used in the work has been well-character* To whom correspondence should be addressed. Phone: (215) 898-6501. Fax: (215) 898-9557. E-mail:
[email protected]. † Institute for Medicine and Engineering. ‡ Department of Materials Science and Engineering. ‡ Department of Microbiology.
ized.14 Therefore, dextran effectively combines the advantages offered by synthetic and natural polymers and is an ideal material to be used as matrices in cell culture and tissue engineering applications. In this work, we developed immobilized dextran hydrogels of tunable mechanical properties from macromer precursors with the objective to develop platforms for cell culture and other tissue engineering applications. Rheometric measurements were used to characterize the elastic modulus of dextran hydrogels in situ and to optimize a novel two-component macromer system capable of producing more uniform, physiologically relevant properties at constant gel density. A complimentary method for activating glass substrates to polymerize with and, hence, more effectively immobilize the hydrogel is also reported herein. Lastly, we immobilize fibronectin to these matrices and determine its effect on the attachment and function of human aortic smooth muscle cells.
Experimental Methods Materials. Commercially available dextran from Leuconostoc mesenteroides NRRL B-512(F) was used for this work. Notably, dextran from this source is known to possess low degree of (1f3) branching (5%) and, therefore, possess more uniform properties.13,14 Dextran (MW ∼ 15-20 kDa), glycidyl methacrylate (GMA), ammonium persulfate (APS), N,N,N′,N′-tetramethylethylenediamine (TEMED), and all other reagents were purchased from Sigma-Aldrich unless otherwise noted. 4-(Dimethylamino)pyridine (DMAP), dimethylsulfoxide (DMSO), and ethanol were purchased from Acros Organics. Macromer Preparation and Characterization. Dextran macromers of different degrees of substitution (DS) were prepared using the procedure by De Smedt et al.15 Briefly, 50 g of dextran and 10 g of DMAP were fully dissolved in 450 mL of anhydrous DMSO under argon. GMA (7.3 g for DS 1/10; 2.9 g for DS
