Associate Professor University Of New Hampshire, United States
Introduction: The field of tissue engineering is rapidly adopting the principles of additive manufacturing, which provides a means to generate complex functional tissue constructs in a programmable manner with micron-scale precision. However, the low mechanical stiffness and strength of 3D printed constructs significantly limits the application of 3D bioprinting in tissue engineering. Here, we propose a novel bioprinting process using a bioink consisting of gelatin, methacrylated gelatin (GelMA) and alginate. Once printed, this bioink is crosslinked by (i) photopolymerization of GelMA by UV radiation, (ii) enzymatic crosslinking of gelatin by microbial transglutaminase (mTG) and (iii) ionic crosslinking of alginate by calcium ions, to form an interconnected polymer network (IPN), which exhibits much improved mechanical properties with excellent biocompatibility in vitro.
Materials and
Methods: Characterization of bioink and crosslinked constructs: The bioink was made from 5% (w/v) gelatin, 5% GelMA and 2% alginate dissolved in DMEM. Viscosity of the bioink and stiffness of crosslinked hydrogels were measured by rheometer. Mechanical strength of the crosslinked constructs was measured by mechanical tester under the compressive mode. 3D bioprinting: For 3D bioprinting, human dermal fibroblasts (hDFs) were dispersed in the bioink at a final concentration of 1×10^6 cells/ml. BioAssemblyBot 400 was used to print a grid-structure with a specified dimensions of 2.4 cm (L) x 3.0 cm (W) x 0.1 cm (H) with each square having dimensions of 2 mm x 2 mm. The grid was printed via a layer-by-layer extrusion method with each layer having a specified width of 0.25 mm. The printed constructs were crosslinked via UV irradiation and submerging them in the cell culture media with 5% microbial transglutaminase (mTG) or mTG supplemented with calcium chloride (24 mM). Biocompatibility: LDH assay was performed to assess cytotoxicity of the printing and crosslinking processes on day 1, 3 and 7 post-printing. Live/dead assay in conjunction with confocal microscopy was performed on day 1 and day 7 to visualize cell viability and cellular morphologies.
Results, Conclusions, and Discussions:
Results: The bioink showed a shear-thinning property, which makes it proper for extrusion-based 3D bioprinting. Stiffness of the IPN constructs significantly increased as more crosslinking mechanisms were incorporated: the hydrogel that was crosslinked by UV, mTG and Ca2+ (UV+mTG+Ca) had much higher G’ (18.9 kPa) than the hydrogel crosslinked by UV only (UV only, G’ = 2.34 kPa) or the hydrogel crosslinked by UV followed by mTG (UV+mTG, G’ = 4.00 kPa) (Figure 1A). The ultimate strength of the constructs also followed a similar trend (Figure 1B). The bioprinted structures remained stable throughout the study in all groups. Cytotoxicity measured by LDH from the encapsulated hDFs gradually decreased over 7 days for all groups (Figure 1C). Confocal imaging showed high cell viability in all groups. However, different cell morphologies were observed depending on the stiffness of the hydrogel. UV-only displayed the highest spreading by day 7, followed by UV+mTG, while UV+mTG+Ca showed minimal cell spreading (Figure 1D). However, when mTG and calcium crosslinking were implemented at later time points (mTG and calcium on day 2, UV+mTG2+Ca2 or mTG on day 2 and calcium on day 4, UV+mTG2+Ca4), cell spreading was on par with UV-only construct while achieving stiff and strong structures.
Conclusions: We have demonstrated the use of multiple crosslinking mechanisms to bioprint mechanically stiff and strong cell-hydrogel constructs with minimal cytotoxicity. As expected, stiff constructs resulted in less cell spreading. However, delayed applications of mTG and calcium crosslinking successfully addressed this issue. We believe that this project will eventually pave way for the construction of even more complex structures armed with improved mechanical properties.
