Preparation and characterization of nanoclay-hydrogel composite support-bath for bioprinting of complex structures

Abstract

Three-dimensional bioprinting of cell-laden hydrogels in a sacrificial support-bath has recently emerged as a potential solution for fabricating complex biological structures. Physical properties of the support-bath strongly influence the bioprinting process and the outcome of the fabricated constructs. In this study, we reported the application of a composite Pluronic-nanoclay support-bath including calcium ions as the crosslinking agent for bioprinting of cell-laden alginate-based hydrogels. By tuning the rheological properties, a shear-thinning composite support-bath with fast self-recovery behavior was yielded, which allowed continuous printing of complex and large-scale structures. The printed structures were easily and efficiently harvested from the support-bath without disturbing their shape fidelity. Moreover, the results showed that support-bath assisted bioprinting process did not influence the viability of cells encapsulated within hydrogel. This study demonstrates that Pluronic-nanoclay support-bath can be utilized for bioprinting of complex, cell-laden constructs for vascular and other tissue engineering applications.

Figure 1

Temperature and time sweep measurements of the support-bath showing storage and loss moduli over time for different concentrations of PF. Laponite and CaCl₂ concentrations were set to 3% and 1%, respectively. Storage (G′) modulus (filled symbols) and loss (G″) modulus (open symbols). Vertical dashed line seperates two measurements. Region (1) represents temperature sweep test from 4 to 37 °C and region (2) shows time sweep measurement at 37 °C for 2 h.

Figure 2

Dynamic rheological characterization of the support-bath representing the effect of PF concentration on flow behavior and recoverability of the structure at 37 °C (Laponite-RDS and CaCl₂ concentrations were constant at 3 and 1%, respectively except for control samples). Control 1 and control 2 included 10% PF, and 3% RDS, respectively at constant 1% CaCl₂ (a) Strain amplitude sweep profiles of supporting mediums, (b) frequency sweep profiles within the linear viscoelastic range, (c) viscosity vs. shear rate plots revealing the shear thinning behavior of the support material, (d) cyclic strain measurements at high (50%) and low (0.6%) strains showing storage (G′) moduli of the samples in 4 cycles. Storage (G′) modulus (filled symbols) and loss (G″) modulus (open symbols).

Figure 3

Dynamic rheological characterization of the support-bath representing the effect of CaCl₂ concentration on network characteristics, flow behavior and recoverability at 37 °C (a-d were included samples with constant PF and Laponite concentrations of 10 and 3%, respectively.) (e–f were included only 3% Laponite-RDS.) (a,e) Strain amplitude sweep profiles of supporting medium, (b,f) frequency sweep profiles within the linear viscoelastic range. (c,g) viscosity vs. shear rate plots revealing the shear thinning behavior of the support material, (d,h) cyclic strain measurements at high (50%) and low (0.6%) strains showing storage (G′) moduli of the samples in four cycles. Storage (G′) modulus (filled symbols) and loss (G″) modulus (open symbols).

Figure 4

Characterization of PF-RDS support-bath for printability of tubular structures in various angular configurations. Digital images of the printed tubular alginate structures using 25 gauge nozzle in the support-bath angled at (a) 90°, (b) 60° and (c) 45°, and (d) a conical structure with 60° angle with respect to the surface. Digital images of front and top views of (a1, a2) 90°, (b1, b2) 60° and (c1, c2) 45° bended tubular structures and (d1, d2) conical structure after removal from support-bath. Scale bars indicate 5 mm.

Figure 5

Fabrication of 3D complex constructs. CAD models of (a) star shape, (b) 0–90° grid pattern, (c) branched vascular structure, and (d) nose shape. Digital images of the fabricated structures (a1, b1, c1, d1) before and (a2, b2, c2, d2) after recovery from PF-RDS support-bath. Scale bars indicate 5 mm.

Figure 6

Fabrication of cell-laden alginate constructs using PF-RDS support-bath. (a) Image of harvested bioprinted tubular structure from support bath. (b) Confocal microscopy image of live/dead cells encapsulated in the alginate hydrogel in a complete 3D bioprinted hollow structure at Day 3 and the zoomed images of cells obtained on Day 1, Day 3 and Day 7. (c) Quantitative viability analysis of cells for Day 1, 3 and 7 after bioprinting. Two tail Students t-test was used to analyze the significant change in the cell-viability after bioprinting process. P-values *<0.05 were considered as significant. Scale bars indicate 1 mm for (a,b) and 0.5 mm for the zoomed images.