Stable gelatin hydrogels without photo-crosslinking

Published : 01/12/2021 16:42:10

Gelatin is produced from partial hydrolysis of collagen. The cytocompatibility of gelatin promotes a favorable environment for cell proliferation (Davidenko et al. 2016). On cooling, gelatin partially recovers the collagen helical structure (so-called collagen-fold) when the concentration is sufficiently high (Ward and Courts 1977). Nevertheless, 3D printed gelatin hydrogels can easily collapse under physiological conditions (T = 37oC and pH = 7.4). For applications where photo-crosslinking is not allowed, the use of chemical agents such as genipin is recommended.

Genipin is a molecule extracted from the fruit of the gardenia plant. The reaction mechanism with gelatin is a two-step process. In the first step, the genipin molecule undergoes a nucleophilic attack by the primary amines of gelatin, resulting in the heterocyclic linking of genipin to the gelatin amine. The second reaction occurs when the ester group on genipin undergoes a nucleophilic substitution. The resultant covalent crosslinks between the primary amine residues leave minimal residual toxicity (Solorio et al. 2010).

We evaluated the effect of genipin as a crosslinker for preparing 3D printed gelatin hydrogels. Fig. 1 illustrates the printing behavior of ClaroBGI600 in combination with genipin (dry-basis mass ratio gelatin:genipin, 1:1). Extrudable consistency was achieved up to 2hrs of printing (left nozzle).


Fig. 1: ClaroBGI600 + genipin (1:1) printing the 6th lattice after 2hrs (left nozzle). Regular gelatin + genipin (1:1) in the right nozzle. T=25°C. Nozzle 25G was used.

The enhanced printing shown by ClaroBGI600 enabled not only printing longer but with lower pressure. Fig. 2 shows that the maximum printing pressure while printing ClaroBGI600/genipin formulation was about half of the pressure required to print with regular gelatin. This means that the physical gel formed by ClaroBGI600 required fewer shear-stresses to flow through the nozzle. Although the filament formed could retain its shape after deposition with the ideal recovery time to allow the formation of a 3D construct.

Fig. 2: ClaroBGI600 and regular gelatin + genipin (1:1) maximum printing pressures using an Inkredible+ printer  T=25°C. Nozzle 25G was used.

References

Davidenko, N., C. F. Schuster, D. V. Bax, R. W. Farndale, S. Hamaia, S. M. Best, and R. E. Cameron. 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).

Solorio, L., C. Zwolinski, A. W. Lund, M. J. Farrell, and J. P. Stegemann. 2010. “Gelatin Microspheres Crosslinked with Genipin for Local Delivery of Growth Factors.” Journal of Tissue Engineering and Regenerative Medicine 4(7):514–23.

Ward, A. G. and A. Courts. 1977. The Science and Technology of Gelatin. Food scien. Academic Press.

Yang, Gang, Zhenghua Xiao, Haiyan Long, Kunlong Ma, Junpeng Zhang, Xiaomei Ren, and Jiang Zhang. 2018. “Assessment of the Characteristics and Biocompatibility of Gelatin Sponge Scaffolds Prepared by Various Crosslinking Methods.” Scientific Reports 8(1):1616.

Zhu, D., Q. Wu, and N. Wang. 2011. “3.02 - Industrial Enzymes.” Pp. 3–13 in, edited by M. B. T.-C. B. (Second E. Moo-Young. Burlington: Academic Press.

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