The applicability of furfuryl-gelatin as a novel bioink for tissue engineering applications

Abstract

Three-dimensional bioprinting is an innovative technique in tissue engineering, to create layer-by-layer structures, required for mimicking body tissues. However, synthetic bioinks do not generally possess high printability and biocompatibility at the same time. So, there is an urgent need for naturally derived bioinks that can exhibit such optimized properties. We used furfuryl-gelatin as a novel, visible-light crosslinkable bioink for fabricating cell-laden structures with high viability. Hyaluronic acid was added as a viscosity enhancer and either Rose Bengal or Riboflavin was used as a visible-light crosslinker. Crosslinking was done by exposing the printed structure for 2.5 min to visible light and confirmed using Fourier transform infrared spectroscopy and rheometry. Scanning electron microscopy revealed a highly porous networked structure. Three different cell types were successfully bioprinted within these constructs. Mouse mesenchymal stem cells printed within monolayer and bilayer sheets showed viability, network formation and proliferation (∼5.33 times) within 72 h of culture. C2C12 and STO cells were used to print a double layered structure, which showed evidence of the viability of both cells and heterocellular clusters within the construct. This furfuryl-gelatin based bioink can be used for tissue engineering of complex tissues and help in understanding how cellular crosstalk happens in vivo during normal or diseased pathology. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 314-323, 2019.

Keywords: bilayer sheets; biocompatibility; furfuryl-gelatin; hyaluronic acid; visible-light crosslinkable bioink.

Figure 1

(A) Bioink (no cells) showing a viscous mixture prior to crosslinking. (B) Pre- (left) and post-crosslinked (right) hydrogel (sheet) (C) Versatility of patterns being printed (ring: left and sheet: right) (D) Double layered sheet printing feasibility (en-face: top and cross-section: bottom). In B, C and D, yellow corresponds to RF and pink to RB being used. Scale bar in all images corresponds to 2 cm.

Figure 2

Rheology analysis of f-gelatin based hydrogels. Shown is a characteristic dataset obtained from a disc shaped (8 mm) sample of printed crosslinked f-gelatin hydrogel sample.

Figure 3

(A) FTIR spectrum of crosslinked f-gelatin in the presence of RB and HA. (B) FTIR spectra of f-gelatin and (C) of HA respectively.

Figure 4

Degree of swelling of a printed sheet sample of f-gelatin after crosslinking.

Figure 5

Representative image acquired using SEM of a printed sheet sample of f-gelatin after crosslinking.

Figure 6

(A) Viability of the mouse MSC stained with Hoechst (blue), post printing after 24 hr. (B) Retention of mouse MSC, pre-stained with PKH67 (green), within the bioprinted construct after 5 days of culture.

Figure 7

FACS analysis to show cell proliferation and biocompatibility of the printed sheet structures of f-gelatin after crosslinking. Cells pre-stained with cell trace violet were cultured upto (A) 24 hrs and (B) 72 hrs within printed constructs.

Figure 8

In A and B, shown are bright field z-scans of STO fibroblasts (elongated spindle shaped) co-cultured with C2C12 myoblasts cells (rounded enlarged, confirmed in C and in E). Scale bar is 150 μm in A, B and 200 μm in C and E. In C, a single plane (cross section) was imaged whereas in E, a Z-scan was run spanning several planes as indicated with the arrow (right hand side). In D, shown is a single slice of z-stack section showing top layer (fluorescent: green for C2C12) and bottom layer (non-fluorescent: STO).