Microfluidics-assisted fabrication of gelatin-silica core-shell microgels for injectable tissue constructs

Abstract

Microfabrication technology provides a highly versatile platform for engineering hydrogels used in biomedical applications with high-resolution control and injectability. Herein, we present a strategy of microfluidics-assisted fabrication photo-cross-linkable gelatin microgels, coupled with providing protective silica hydrogel layer on the microgel surface to ultimately generate gelatin-silica core-shell microgels for applications as in vitro cell culture platform and injectable tissue constructs. A microfluidic device having flow-focusing channel geometry was utilized to generate droplets containing methacrylated gelatin (GelMA), followed by a photo-cross-linking step to synthesize GelMA microgels. The size of the microgels could easily be controlled by varying the ratio of flow rates of aqueous and oil phases. Then, the GelMA microgels were used as in vitro cell culture platform to grow cardiac side population cells on the microgel surface. The cells readily adhered on the microgel surface and proliferated over time while maintaining high viability (∼90%). The cells on the microgels were also able to migrate to their surrounding area. In addition, the microgels eventually degraded over time. These results demonstrate that cell-seeded GelMA microgels have a great potential as injectable tissue constructs. Furthermore, we demonstrated that coating the cells on GelMA microgels with biocompatible and biodegradable silica hydrogels via sol-gel method provided significant protection against oxidative stress which is often encountered during and after injection into host tissues, and detrimental to the cells. Overall, the microfluidic approach to generate cell-adhesive microgel core, coupled with silica hydrogels as a protective shell, will be highly useful as a cell culture platform to generate a wide range of injectable tissue constructs.

Figure 1

(a) Microfluidic fabrication of GelMA microgels. Aqueous droplets made of GelMA pregel solution, generated from a microfluidic flow-focusing device, were photopolymerized to form GelMA microgels. (b, c) Microscopic images of a microfluidic flow-focusing device generating GelMA droplets (b) and GelMA microgels fabricated by UV-initiated photopolymerization of GelMA droplets (c).

Figure 2

(a) Diameter of GelMA microgels (8 wt %), before and after swelling in PBS, was controlled by the ratio of flow rates of aqueous phase (GelMA pregel solution) to oil phase (Q_Aq/Q_O). (b) Force-displacement curve of a GelMA microgel measured by an AFM-assisted nanoindentation. Elastic modulus (E) was calculated using Hertz contact mechanics theory (eq 1).

Figure 3

(a) GelMA microgels were used as in vitro platform to culture cells on the surface. (b) Microscopic images of CSP cells cultured on GelMA microgels. The cells adhered on the surface of GelMA microgels and proliferated over time (scale bar: 100 μm). (c) A confocal fluorescent microscopic image of CSP cells on GelMA microgel. The cells were stained with Alexa488-phalloidin and DAPI to visualize actin and nuclei, respectively (scale bar: 20 μm). A cross-sectional image is shown on the right panel. (d) The number of cells (N_t) at time, t, normalized with the initial number of cells (N₀) were plotted and fitted with eq 2 (k_P: proliferation rate).

Figure 4

CSP cells on GelMA microgels were placed on a cell adhesive surface, and their adhesion and proliferation over time were monitored (scale bar: 50 μm).

Figure 5

(a) Schematic description of fabrication of protective silica hydrogel shell on the GelMA microgels. (b) Optical microscopic images of GelMA microgels and silica-coated GelMA microgels. (c) The silica hydrogel was further characterized with scanning electron microscopy (SEM). Inset shows the magnified view of the silica hydrogel shell on top of GelMA microgel core, identified with an arrow. (d) The thickness of the silica hydrogel measured over the reaction time. (e) (Left) A microscopic image of CSP cells on GelMA microgels coated with silica gel. A fluorescent image of the cell nuclei stained with DAPI (blue) was overlaid to identify the cells. (Right) SEM image shows the surface of cells covered with silica hydrogel.

Figure 6

(a) Fluorescent images of CSP cells on GelMA microgels (left) and GelMA microgels coated with silica hydrogel (right), subjected to oxidative stress. The cells were stained with calcein-AM (green) and ethidium homodimer-1 (red) to visualize live and dead cells, respectively (scale bar: 50 μm). (b) Viability of the CSP cells was quantified as the percentage of the live cells (*p < 0.05).

Figure 7

(a) Degradation product, silicic acid, from silica hydrogel was monitored by measuring the absorbance of silicomolybdate complex at 400 nm (A₄₀₀). The plot was fitted with a first-order degradation kinetics model. (b) Silica-coated CSP cell-seeded microgels were placed on a cell adhesive surface, and monitored their adhesion and proliferation over time. (Scale bar: 50 μm).