A tunable silk-alginate hydrogel scaffold for stem cell culture and transplantation

Abstract

One of the major challenges in regenerative medicine is the ability to recreate the stem cell niche, which is defined by its signaling molecules, the creation of cytokine gradients, and the modulation of matrix stiffness. A wide range of scaffolds has been developed in order to recapitulate the stem cell niche, among them hydrogels. This paper reports the development of a new silk-alginate based hydrogel with a focus on stem cell culture. This biocomposite allows to fine tune its elasticity during cell culture, addressing the importance of mechanotransduction during stem cell differentiation. The silk-alginate scaffold promotes adherence of mouse embryonic stem cells and cell survival upon transplantation. In addition, it has tunable stiffness as function of the silk-alginate ratio and the concentration of crosslinker--a characteristic that is very hard to accomplish in current hydrogels. The hydrogel and the presented results represents key steps on the way of creating artificial stem cell niche, opening up new paths in regenerative medicine.

Keywords: Alginate; Elasticity; Laminin; Scaffold; Silk; Stem cells.

Figure 1. Addition of ECM materials promotes cell adherence to the scaffold

D3 (mESC) stably expressing Fluc were plated on different scaffold composites and imaged 3 days after plating. A. Representative BLI images of the different scaffolds with D3 cells B. Quantification of BLI signal (* p<0.05 unpaired 2 tails Ttest, n=3)

Figure 2. Scaffold structural changes following incubation in media and transplantation in a mouse

Scaffold samples pore size was evaluated using E-SEM (scale bar 20μm) B. Scaffolds labeled with FITC were transplanted in BalbC mice using the window chamber model. Scaffold fluorescence was evaluated using IVM. C. Quantification of fluorescent signal, scaffold to background ratio.

Figure 3. The silk:alginate:laminin scaffold induces a very mild immune response

Mice were treated with PBS injection (negative control n=5), LPS intra-nasal exposure (positive control n=4), and transplanted with the scaffold (n=4). Mice were sacrificed, blood and tissue samples were taken for histology evaluation and TNFα analysis. A. representative images of skin wound healing and regional lymph node following treatment. There was no significant difference in wound healing response or lymph node size due to scaffold transplantation. B. Histology section of the scaffold area, mild immune reaction was observed. Inset in B shows higher magnification: small numbers of reactive macrophages and multinucleated giant cells (arrows), small amounts of fibroplasia. C. Serum samples were analyzed for TNFα. The scaffold did not induce an elevation in TNFα levels.

Figure 4. In vivo survival of rMSC

rMSC stably expressing Fluc were transplanted in nude mice hind limb with PBS, matrigel and scaffold. A. BLI images of representative mice at days 1, 4 7, 14, 21 and 28 post transplantation. B & C. Quantification of BLI signal (* p<0.05 unpaired 2 tails Ttest, n=5 per group)

Figure 5. Tuning the elasticity of the scaffold

The scaffold’s elasticity was measured using a rheometer. The elasticity values are varying under different conditions. A. Variation of the silk:alginate ratio allows tuning of the the scaffold’s elasticity. B. Incubation of scaffolds (S3:A2) in 95% FBS and 95% FBS supplemented with 25mM CaCl₂ for 20–360 min. C. The scaffold’s elasticity is tunable when it was incubated in alternating conditions (S3:A2).