Page 369 - IJB-10-3

International Journal of Bioprinting Multi-physical field control inkjet bioprinting

viable option for tissue regeneration and replacement in In this study, we proposed a more straightforward
in vivo experiments. 24,25 Numerous studies have utilized method to use 5% GelMA for inkjet bioprinting. The inkjet
GelMA to conduct printing on human tissues such as printing process can be classified as drop-on-demand
skin, blood vessels, lungs, and bones, verifying its safety in (DOD) or continuous inkjet (CIJ), and the DOD printing
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vivo and highlighting its significant potential for practical can be divided into three types: hot bubble, mechanical,
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application. Table 1 illustrates that Yoon et al. can improve and piezoelectric printing. We chose a piezoelectric
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the biocompatibility of biological ink by combining inkjet printhead, which allows the control of microdroplet
sodium alginate with GelMA, but GelMA still necessitates diameter and velocity by tweaking the pressure field, and the
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saponification treatment for printing. Obviously, using control of proposed bioink’s temperature. A new printing
GelMA as ink for inkjet printing is excellent. The 5% approach, multi-physical field control piezoelectric inkjet
concentration of GelMA is beneficial for cell growth and bioprinting (MFCPIB), was developed for making 3D
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is commonly used in extrusion bioprinting. However, tissue-like structures with 5% GelMA. This new printing
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GelMA is in a liquid state at high temperatures and a method provided a high level of bioactivity and was used
gelatinous state at low temperatures, implying that it is a to make cell-laden hydrogel 3D tissue-like structures
thermosensitive material. Besides, a higher concentration from GelMA. We developed an inkjet bioprinting system
results in a higher temperature sensitivity. Because inkjet with MFCPIB and optimized the inkjet printing method.
printing differs from extrusion printing, it is not feasible Finally, we used 5% GelMA to fabricate 3D structures with
for GelMA to have a pre-gel structure at low temperatures a high aspect ratio that can potentially mimic the functions
inside the printhead because it would clog the nozzle of native tissues such as blood vessels and skin.
and prevent the formation of a jet. Maintaining the
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right temperature can be challenging when using inkjet 2. Materials and methods
bioprinting to print with 5% GelMA. GelMA must be 2.1. Biomaterial inks
liquefied in the high-temperature inkjet printhead and The preparation process of GelMA was as follows. Gelatin
then cooled in microdroplets in cold air after injection. (type A from porcine, Sigma-Aldrich, St. Louis, MO, USA)
Temperature control is crucial throughout printing, was dissolved in phosphate-buffered saline (PBS; Adamas,
including the printing head, feeding system, printing Shanghai, China) and stirred at 60°C to dissolve fully and
substrate, and ambient air. While extrusion printing is obtain a 10% w/v gelatin solution. Methacrylic anhydride
often used to regulate the printing head temperature, inkjet (Sigma-Aldrich, St. Louis, MO, USA) was added at a 0.5
printing lacks sufficient research on temperature control mL/min rate at 50°C and reacted for 1 h. The reaction was
for both the printing head and feeding system due to the terminated by adding five times the volume of PBS, and then
complex structure of the printhead. As a result, some two- the mixture was dialyzed for 1 week at 40°C to remove salts
dimensional images were only printed after modification and methacrylic acid. The reaction solution was aliquoted,
of lower-concentration GelMA. Unfortunately, a lower lyophilized, and stored at -80°C. Lyophilized GelMA was
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concentration is unsuitable for cell growth and does dissolved in PBS at 50°C for 4 h. After adding 0.15% w/v
not offer enough strength to maintain the geometry or lithium phenyl-(2, 4, 6-trimethyl benzoyl) phosphinate
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function of 3D tissue-like structures. On the other hand, (LAP; Sigma-Aldrich, St. Louis, MO, USA), the solution
the process of curing GelMA heavily relies on precise was filtered using a 0.22 μm filter (Millipore, BOS, USA).
temperature control. However, current bioprinting devices The mixture was exposed to 5 mW/cm ultraviolet light
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can only control the temperature of the substrate, but it (365 nm) for 60 s to form stable GelMA hydrogels.
cannot control the ambient air above it. Because the cells To prepare cell-laden bioink, we added 1 mL of the cell
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will perish if the substrate temperature drops below 0°C, suspension to 4 mL of GelMA solution to form cell-laden
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the minimum temperature of the bottom plate cannot be hydrogels. The final concentration of cells was 5 × 10 /mL,
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lower than 4°C. Given that the air temperature above the and the final concentration of GelMA was 5% w/v.
bottom plate exceeds 4°C, the temperature of microdroplet
passing through the air cannot be decreased to a printable 2.2. Cell culture
temperature to prevent model collapse. Achieving high Smooth muscle cells (SMCs; ScienCell, San Diego, USA)
aspect ratios in printed structures can prove to be a were used to prepare the bioink for cell printing studies.
challenging task in the absence of appropriate ambient SMCs were maintained in DMEM/F12-Dulbecco’s
air temperature regulation. Therefore, the temperature Modified Eagle Medium (ThermoFisher Scientific,
field must be strictly controlled during the GelMA inkjet Waltham, USA) with 10% fetal bovine serum (FBS) in a 5%
printing process, rendering the implementation of inkjet CO atmosphere at 37°C. Culture medium was exchanged
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printing with 5% GelMA impossible. every 2 days.

Volume 10 Issue 3 (2024) 361 doi: 10.36922/ijb.2120