Hydrogel based on interpenetrating polymer networks of dextran and gelatin for vascular tissue engineering

Abstract

Hydrogel networks are highly desirable as three-dimensional (3-D) tissue engineering scaffolds for cell encapsulation due to the high water content and ability to mimick the native extracellular matrix. However, their application is limited by their nanometer-scale mesh size, which restricts the spreading and proliferation of encapsulated cells, and their poor mechanical properties. This study seeks to address both limitations through application of a novel cell-encapsulating hydrogel family based on the interpenetrating polymer network (IPN) of gelatin and dextran bifunctionalized with methacrylate (MA) and aldehyde (AD) (Dex–MA–AD). The chemical structure of the synthesized Dex–MA–AD was verified by ¹H-NMR and the degrees of substitution of MA and AD were found to be 14 and 13.9 ± 1.3 respectively. The water contents in all these hydrogels were approximately 80%. Addition of 40 mg/ml to 60 mg/ml gelatin to neat Dex–MA–AD increased the compressive modulus from 15.4 ± 3.0 kPa to around 51.9 ± 0.1 kPa (about 3.4-fold). Further, our IPN hydrogels have higher dynamic storage moduli (i.e. on the order of 10⁴ Pa) than polyethylene glycol-based hydrogels (around10²–10³ Pa) commonly used for smooth muscle cells (SMCs) encapsulation. Our dextran-based IPN hydrogels not only supported endothelial cells (ECs) adhesion and spreading on the surface, but also allowed encapsulated SMCs to proliferate and spread in the bulk interior of the hydrogel. These IPN hydrogels appear promising as 3-D scaffolds for vascular tissue engineering.

Introduction

Hydrogel networks are widely used as three-dimensional (3-D) soft tissue engineering scaffolds to encapsulate cells due to their high water content and physical properties emulating the native extracellular matrix (ECM) [1], [2], [3]. When encapsulated inside hydrogels, cells requiring extensive cell spreading, such as fibroblasts and smooth muscle cells (SMCs), unlike chondrocytes, show a round morphology and delayed proliferation, migration and matrix production due to the hydrogel initial nanometer-scale mesh size [4], [5], [6], [7].

Degradable hydrogels have been investigated for generating space in order to allow cells to spread and migrate as the gel network degrades. For hydrolytically degradable hydrogels, synchronization of the rate of degradation with cellular growth and matrix accumulation to match tissue evolution is not trivial [2], [5], [8], [9], [10]. Enzymatically labile hydrogels that degrade by cell-secreted or exogenously applied enzymes is an alternative family of degradable hydrogels which has been shown to support 3-D cell spreading [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26]. Many of these hydrogels are based on peptide/protein modified polyethylene glycol (PEG), which have been shown to support spreading, migration and cell–cell contacts of fibroblasts, SMCs and mesenchymal stem cells (MSCs) within them and promote dorsal root ganglion cell outgrowth and bone formation [12], [19], [20], [26]. The pericellularly localized degradation due to the protein components enables tuning of the degradation rate to closely match the tissue formation rate [23]. However, many of these PEG-based hydrogels are quite soft with elastic moduli on the order of 10²–10³ Pa [12], [22], [24], [25], making them difficult to handle. Besides, soft gels are unable to support high level of cell-generated tension required to sustain stress fibers and focal adhesion, which would eventually influence cell function and tissue formation [24].

Dextran, a natural hydrophilic degradable polysaccharide, is an alternative to PEG for hydrogel formation [27], [28], [29], [30], [31], [32], [33], [34], [35]. Although it is resistant to protein adsorption and cell adhesion [36], dextran possesses abundant pendant hydroxyl functional groups making it amenable to chemical modification [34], [37]. It has been used as two-dimensional (2-D) or porous soft tissue engineering scaffold and in drug-delivery [33], [35], [38]. RGD modified dextran has been used for encapsulation of aggregates (rather than individual) of human embryonic stem cells (hESCs) by Langer's group [31]. Although enhanced vascular differentiation was reported, cellular proliferation was not observed. Rather than using adhesive peptide, gelatin which has good biodegradability and low level of immunogenicity and cytotoxicity [39], can also be incorporated into dextran hydrogel [28], [29], [40].

In this paper, we have synthesized a methacrylate- and aldehyde-bifunctionalized dextran (Dex–MA–AD) (Scheme 1) and demonstrated a new cell-encapsulating hydrogel family based on the interpenetrating network of Dex–MA–AD and gelatin (Scheme 2). The hydrogel is formed by ultraviolet (UV)-crosslinking between pendant methacrylate groups on Dex–MA–AD and Schiff base reaction between Dex–MA–AD and gelatin. The synthesized Dex–MA–AD was characterized by ¹H-NMR. The physical properties (i.e. sol content, compressive and dynamic mechanical properties and swelling ratio) were also measured. Adhesion and spreading of ECs in 2-D culture and spreading and proliferation of SMCs in 3-D culture were observed with these hydrogels. Also, this hydrogel possesses relatively high elastic modulus (on the order of 10⁴ Pa) making it desirable for vascular tissue engineering.