Under Pressure ?

Published : 11/03/2020 16:30:33

Cell viability is negatively affected with increasing printing pressure

Bioprinting structures using cell-laden bioinks remains a challenge. Shape fidelity with minimal effects on cell viability can only be achieved with a delicate balance between material properties and printing parameters. Gelatin methacryloyl (GelMA) is a well-known photocurable biomaterial with proven cell-viability due to the presence of the peptide motif arginine-glycine-asparagine (RGD) in the gelatin’s backbone (Davidenko et al., 2016). The methacrylamide group that is present in GelMA allows the matrix to be covalently cross-linked under the presence of a photoinitiator and UV light. This feature ensures the stability of the 3D printed construction under physiological conditions of temperatures (T~37 °C) which are ideal for cell growth and proliferation. When GelMA is used to formulate cell-laden bioinks for extrusion-based bioprinting protocols, the temperature-dependent gelation mechanism of GelMA needs to be controlled in order to prevent undesired shear stresses on cells while printing. On cooling, and depending on the concentration, the gelatin part of GelMA partially recovers into a collagen helical structure (this is the so-called collagen-fold). The gelation process is thermo-reversible, i.e. above 35 °C, gelatin exists as random coiled structure that is unable to form interchain hydrogen bonds.

Below the gelation temperature and at high concentrations, the gelation behavior of GelMA results in viscosity increments which, in turn, will require higher extrusion pressure to make the bioink flow through the nozzle. Koti et al (2019) studied the effects of GelMA concentration, extruder pressure and the duration of UV exposure on the survival of cardiac myocytes and fibroblasts. The authors demonstrated an upward and linear dependency of the printing pressure within an increasing concentration range of GelMA (from 10 % to 25 % w/v with printing pressures ranging from 138 kPa to 276 kPa, respectively), using a 23-gauge needle at 20 °C for a pneumatic plunger-based 3D printer. The increased printing pressures led to a decay in cell viability that in some cases was not noticed immediately after printing, but which became prominent after keeping the cell-laden 3D-construct in culture for 6 days. Therefore, Koti’s study calls attention to the long-term effect of the printing pressure on cell viability (Koti et al., 2019).

The interesting work by Liu et al (2019) on the regeneration of osteochondral defect shows a tri-layered scaffold printed with an extrusion-based multi-nozzle bioprinter. The top layer was printed with a 15 % GelMA hydrogel for cartilage, the interfacial layer consisted of a combination of 20 % GelMA and 3 % nanohydroxyapatite (nHA) (20/3 % GelMA/nHA) hydrogel, and the subchondral bone at the bottom layer was composed of a 30/3 % GelMA/nHA hydrogel. The printing pressure that was necessary to extrude the GelMA-based bioink on the top layer was approximately 120-140 kPa, which corroborates with the values obtained in the work conducted by Koti et al (2019), under similar printing parameters of temperature and nozzle diameter (Liu et al., 2019).

The patented technology developed by Claro allows for printing with GelMA at pressures 30-50 % lower than the values reported by the literature (Koti et al., 2019; Liu et al., 2019). Our ClaroBGI800 was tested in a Cellink® Inkredible+ bioprinter with a narrow nozzle (27G) and without temperature control (Tambient = 21 ± 1 °C). The printing pressure was 60 kPa and 100 kPa for a concentration of 10 % and 15 %, respectively. The enhanced flow is a result of materials science applied to modify the gelatin structure in such a way as to slow down the gelation mechanism upon cooling. In other words, the intrinsic cytocompatibility of our GelMA-based biomaterial (ClaroBGI800) is preserved. The lower printing pressures depicted by ClaroBGI800 suggests a great potential for the long-term preservation of cell viability in cell-laden 3D-printed tissues.

REFERENCES

Davidenko, N., Schuster, C. F., Bax, D. V., Farndale, R. W., Hamaia, S., Best, S. M., & Cameron, R. E. (2016). Evaluation of cell binding to collagen and gelatin: a study of the effect of 2D and 3D architecture and surface chemistry. Journal of Materials Science: Materials in Medicine, 27(10). https://doi.org/10.1007/s10856-016-5763-9
Koti, P., Muselimyan, N., Mirdamadi, E., Asfour, H., & Sarvazyan, N. A. (2019). Use of GelMA for 3D printing of cardiac myocytes and fibroblasts. Journal of 3D Printing in Medicine, 3(1), 11–22. https://doi.org/10.2217/3dp-2018-0017
Liu, J., Li, L., Suo, H., Yan, M., Yin, J., & Fu, J. (2019). 3D printing of biomimetic multi-layered GelMA/nHA scaffold for osteochondral defect repair. Materials & Design, 171, 107708. https://doi.org/10.1016/J.MATDES.2019.107708

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