Gel formulation and long term mechanical stability analysis of cellularized gelatin-based hydrogel using ElastoSens™ Bio
MATERIALS AND METHODS
RESULTS AND DISCUSSION
The amount of enzyme was optimized by real-time measurements of the shear storage modulus over crosslinking time (12 h in total). Fig. 1A shows that the shear storage modulus of 15 % gelatin hydrogels increased with the amount of enzymes (2.5, 5, and 10 U/mL). The condition of 10 U/mL led to the highest storage moduli values; however, it was also responsible for the highest variation in the measurements associated with a fast and inhomogeneous gelation and difficulties in sample manipulation. Therefore, the condition of 5 U/mL was selected for the following experiments. The concentration of gelatin (10, 15 and 20 %) was then varied with the optimized amount of enzyme. Fig. 1B shows that the storage modulus increased with the increasing concentration of gelatin due to the higher densities of carbonyl and amino groups available for bonding to each other. Following the completion of gelation, the complex (G*) and loss (G”) moduli (Fig. 1C) of the hydrogels showed dominant solid-like behavior at all concentrations (tan(δ) < 1).
Keratinocytes were then seeded on top of the different hydrogels (10, 15 and 20 % of gelatin with 5 U/mL of TG) (Fig. 2A) and the storage modulus was measured over 14 days of culture (Fig. 2B). Fig. 2B shows that the 10 % gelatin gel lost its integrity after 7 days of incubation suggesting that degradation was higher than ECM production. For 15 % gelatin, the hydrogel maintained its integrity for 14 days, although its storage modulus decreased by 50 %. On the other hand, the storage modulus of 20 % gelatin remained unchanged over 14 days of culture with respect to the storage modulus of unseeded gelatin. Interestingly, this condition (20 % gelatin) was the one that most matched the human skin storage modulus (40-60 kPa ). Due to the more physiological storage modulus and the mechanical stability over time, the concentration of 20 % gelatin was selected for the preparation of the epidermis model. In the study, the model was infected with Escherichia coli to investigate the inflammatory response of keratinocytes by measuring the expression level of pro-inflammatory cytokines. The authors mentioned that the model has great potential for modeling wound infections and drug testing.
Fig. 2 shows the shear storage modulus (G’) of PhotoHA® and PhotoGel® after exposure to UV light for 5 or 10 minutes at room temperature. Their increasing G’ shows that they were not fully crosslinked after 5 minutes of exposure to UV light. At these concentrations, the HA-based hydrogels were softer than gelatin-based UV-crosslinked hydrogels.
 Liaw, C. Y., Ji, S., & Guvendiren, M. (2018). Engineering 3D hydrogels for personalized in vitro human tissue models. Advanced healthcare materials, 7(4), 1701165.
 Jahanshahi, M., Hamdi, D., Godau, B., Samiei, E., Sanchez-Lafuente, C. L., Neale, K. J., … & Akbari, M. (2020). An Engineered Infected Epidermis Model for In Vitro Study of the Skin’s Pro-Inflammatory Response. Micromachines, 11(2), 227.
 Kearney, S. P., Khan, A., Dai, Z., & Royston, T. J. (2015). Dynamic viscoelastic models of human skin using optical elastography. Physics in Medicine & Biology, 60(17), 6975.
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