Alginate-based hydrogels with improved adhesive properties for cell encapsulation

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Abstract

Hydrogel-based biomaterials are ideal scaffolding matrices for microencapsulation, but they need to be modified to resemble the mechanical, structural and chemical properties of the native extracellular matrix. Here, we compare the mechanical properties and the degradation behavior of unmodified and modified alginate hydrogels in which cell adhesive functionality is conferred either by blending or covalently cross-linking with gelatin. Furthermore, we measure the spreading and proliferation of encapsulated osteoblast-like MG-63 cells. Alginate hydrogels covalently crosslinked with gelatin show the highest degree of cell adhesion, spreading, migration, and proliferation, as well as a faster degradation rate, and are therefore a particularly suitable material for microencapsulation.

Introduction

Cell encapsulation – the immobilization of cells in polymeric hydrogels – is a promising technique in tissue engineering [1]. Hydrogels can act as a semipermeable membrane, which protect the encapsulated cells from the host's immune system while allowing for the bidirectional diffusion of oxygen, nutrients and waste. Moreover, hydrogels attenuate the mechanical stress and friction not only on encapsulated cells but also on adjacent tissue upon transplantation [1], [2], [3], [4]. The material of choice for many cell encapsulation applications is alginate because of its biocompatibility and rapid ionic gelation property with divalent cation [5], [6]. Alginate represents a family of anionic polysaccharides extracted from brown algae or bacteria. They are linear unbranched copolymers composed of (1-4)-linked β-d-mannuronic acid (M) and α-l-guluronic acid (G) monomers [7], [8], [9]. The fraction and sequence of M- and G-monomers vary with the origin of alginate and contribute to different chemical and physical properties [10]. When the G-blocks of neighboring polymer chains are linked with each other through divalent cation bridges, for example Ca2+, alginate forms a hydrogel with viscoelastic properties [11]. However, alginate does not promote cell adhesion and proliferation due to the absence of cell adhesion motifs. It has been shown in previous studies that cell adhesion to alginate hydrogels can be achieved by modification of alginate through functionalization with gelatin [12], [13]. Gelatin is a biodegradable protein, produced by denaturation of collagen, which transforms the triple helix structure of collagen into a random coil structure [14] and thereby exposes the cell adhesion motif RGD (Arg-Gly-Asp) of collagen [15], [16].

In a previous study, we established that alginate hydrogels that were functionalized by crosslinking with gelatin promoted better adhesion, proliferation, and migration of encapsulated osteoblast-like MG-63 cells compared to pure alginate hydrogels or to hydrogels functionalized with the specific integrin binding sequence RGD [12]. However, it has remained an open question whether the presence of gelatin on its own, e.g. by blending with alginate, would be sufficient for cell adhesion, migration, and proliferation, or whether gelatin must be crosslinked with alginate. Furthermore, it is unknown if the presence of gelatin speeds up the degradation behavior of the hydrogel over time and thereby promotes cell migration and proliferation. Finally, it is unknown how stably the gelatin is bound or contained in the hydrogel, or how quickly it is released over time into the surrounding medium.

In this study we compared the behavior of osteoblast-like MG-63 cells in two alginate hydrogel systems that were functionalized with gelatin either by covalent crosslinking of alginate di-aldehyde and gelatin (ADA-GEL-x), or by simple blending of alginate and gelatin prior to polymerization (ALG-GEL-b). We embedded the cells in hydrogel capsules of 800 μm diameter, and characterized cell viability, mitochondrial activity, spreading morphology and hydrogel degradation over a time course of up to 28 days. ADA-GEL-x hydrogels facilitated superior adhesion, proliferation, migration, and morphology of encapsulated cells compared to ALG-GEL-b. Moreover, the gelatin release kinetics was slower in ADA-GEL-x compared to ALG-GEL-b, yet the overall hydrogel degradation rate as evaluated from the decrease in stiffness of microcapsules over time was higher. Thus, we conclude that the better adhesion, proliferation, and migration of encapsulated osteoblast-like MG-63 cells in gelatin-crosslinked alginate hydrogel microcapsules arises as a combination of a stronger binding of gelatin to the hydrogel matrix, and a faster mechanical degradation behavior that better accommodates the proliferation and migration of embedded cells.

Section snippets

Preparation of functionalized alginate gels

Sodium alginate (sodium salt of alginic acid from brown algae, suitable for immobilization of micro-organisms, guluronic acid content 65–70%) and gelatin (from porcine skin, suitable for cell culture, Type A, Bloom 300) were obtained from Sigma–Aldrich, Germany. Ethanol, ethylene glycol, sodium metaperiodate and calcium chloride dehydrate (CaCl2·2H2O) were purchased from VWR International, Germany. Silver nitrate was obtained from Alfa Aesar, USA.

Sodium alginate was dissolved in PBS, and

Mechanical analysis

Fig. 1 shows the storage modulus and loss tangent of all materials measured by DMTA immediately after their preparation and over time of up to 28 days of incubation in DMEM at 37 °C.

ALG exhibited the highest storage modulus (570 kPa at 1 Hz) immediately after preparation, as there is no additional material hindering the cross-linking of alginate G-blocks. This value is found to be higher compared to the storage modulus (∼100 kPa) of ALG as reported by Hunt et al. [20]. However, it should be

Conclusions

The analysis of three different alginate-based hydrogels revealed similar mechanical properties initially but different degradation rates and cell responses. Pure alginate and gelatin blended alginate did not facilitate cell adhesion and migration, and showed suppressed metabolic activity after longer incubation times. Cells in the alginate–gelatin crosslinked hydrogels showed good cell adhesion and spreading, and increasing mitochondrial activity after longer incubation times. Alginate–gelatin

Acknowledgement

This work was supported by the Emerging Fields Initiative (EFI) of the University of Erlangen-Nuremberg, Germany (project TOPbiomat). Bapi Sarker acknowledges the German Academic Exchange Service (DAAD) for financial support.

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