Associate Professor University of New Hampshire, United States
Introduction:: The field of tissue engineering is rapidly adopting the principles of additive manufacturing (i.e. 3D bioprinting), which provides a means to generate complex functional tissue constructs in a programmable manner within micron-scale precision. However, the low mechanical stiffness and strength of 3D printed hydrogels significantly limits the application of 3D bioprinting in tissue engineering. Here, we propose the use of a novel bioink consisting of Gelatin, Methacrylated methacrylated gelatin (GelMA) and a\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. Enhanced mechanical properties of the IPN hydrogels are demonstrated by rheology and mechanical testing. Biocompatibility of the bioink and the subsequent crosslinking process was demonstrated by cytotoxicity and proliferation assays. Cell morphologies in the 3D structures were monitored by live/dead assay in conjunction with confocal microscopy. Cell viability was measured by calculating the live cell count/total cell count.
Materials and Methods:: Characterization of bioink and crosslinked hydrogels: The bioink was made from 5% (w/v) gelatin, 5% GelMA and 2% alginate dissolved in DMEM. The shear thinning property of the bioink was measured by rheometer, and the stiffness and strength of the crosslinked hydrogels were measured by rheometer and mechanical tester, respectively.
3D bioprinting: For 3D bioprinting, human dermal fibroblasts (hDFs) were dispersed in the bioink at a final concentration of 5×10^5 cells/ml. A photoinitiator (Irgacure 2959) was added to the bioink for photopolymerization.
The BioAssemblyBotTM was used to extrude the bioink from a syringe needle of a diameter 120 µm and used the in-built software package, TSIM, to construct and subsequently print a grid-structure with a specified dimensions of 2.4cm (L)×3.0cm(W)×0.1cm(H) with each square having dimensions of 2mm×2mm. The grid was printed via a layer-by-layer extrusion method with each layer having a specified width of 0.25 mm. The constructs were crosslinked via UV (365 nm) irradiation and submerging them in the cell culture media with 10% microbial transglutaminase (mTG) or mTG supplemented with calcium chloride (24 mM).
Biocompatibility: LDH and Cell Viability count were performed to assess cytotoxicity of the crosslinking methods and hDF viability on day 1 and day 7 post encapsulation. Live/dead assay in conjunction with confocal microscopy was performed on day 1 and day 7 to visualize cytotoxicity and cellular morphologies.
One way ANOVA was performed on the cell viability data. All pairs, Tukey-Kramer HSD was chosen to test for testing statistical significance amongst all sample pairs.
Results, Conclusions, and Discussions:: Results:
The bioink clearly showed shear-thinning property, which makes it proper for extrusion-based 3D bioprinting. Stiffness of the IPN hydrogels 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’ (19000 Pa) than the hydrogel crosslinked by UV only (UV-only, G’ = 2450 Pa) or the hydrogel crosslinked by UV followed by mTG (UV+mTG, G’ = 4100 Pa) (Fig. 1a). The maximum strength of the hydrogel also followed a similar trend (Data-not-shown). The bioprinted structures remained stable throughout the study in all groups. Cell counting indicates an increase in viability for all groups by day 7 except UV+mTG+Ca which showed a decrease in viability from day 1 (Fig. 1b). Cytotoxicity measured by LDH from the encapsulated hDFs gradually decreased over 7 days for all groups (Fig. 1c). 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 (Fig. 1d). However, a delayed calcium crosslinking of UV+mTG on day 3 (UV+mTG+Ca3d) resulted in more increased cell spreading while achieving a stiff and strong structures (Fig. 1d). Tukey-Kramer HSD for the day 7 indicate pairs with UV+mTG+Ca have p-values < 0.05: UV-only (p-value = 0.0497), UV+mTG (p-value = 0.0410), UV+mTG+Ca3d (p-value = 0.0402).
Discussion:
The results indicates a wide range of morphologies corresponding to the stiffness of the constructs. As expected the UV-only group had very high cell spreading. It is well known that lower number of crosslinks/stiffness results in higher spreading. However, a delayed crosslinking by calcium on day 3 allowed us to maintain high stiffness while also allowing for ample cell spreading. This is significant because it will eventually pave way for the construction of functional bone tissue constructs which requires highly stiff gels and high cell viability for osteogenic differentiation.
Conclusions:
We have demonstrated the use of multiple crosslinking mechanisms to bioprint mechanically stiff and strong cell-hydrogel constructs with minimal cytotoxicity. Stiff hydrogels resulted in less spreading. However, a delayed application of calcium crosslinking induced more spreading.
Acknowledgements (Optional): : Dr. Jeong\ Patrick Earley UNH TA assistanship
