(W0930-07-46) Harnessing Vanillin for Development of 3D Printable Chitosan/β-Glycerophosphate Hydrogels

Purpose: Chitosan/β-glycerophosphate (CS/GP) hydrogels are biocompatible and thermally sensitive, which make them a promising composite for tissue engineering and drug delivery. However, their slow gelation and poor mechanical strength restrict clinical applications, necessitating a crosslinker to enhance gelation kinetics and structural integrity while maintaining biocompatibility. This study explored vanillin, a naturally derived compound with antioxidant, anti-apoptotic, and antimicrobial properties, to improve the gelation and mechanical properties of CS/GP hydrogels, aiming to design gelling-property controllable biomaterials for 3D printable biomimetic scaffolds.

Methods: Hydrogels were formulated using chitosan (1.1%–1.3% w/v) and β-glycerophosphate (10%–15% w/v), and optimized via harnessing vanillin (0.05%–0.15% w/v) as a crosslinker. Formulation variables including chitosan, β-glycerophosphate, and vanillin concentrations, were combined in  various ratio and optimized to assess their effects on hydrogel properties. Gelation properties, such as pH, gelation onset time, and complete gelation time, were evaluated at 37°C using the test tube inversion method, while gel strength was qualitatively evaluated and categorized into three types (i.e., soft, partial, solid). Statistical analysis was performed using one-way ANOVA to compare gelation times and pH across formulations to finalize the gel formation suitable for biomedical applications. The subsequent studies on the final product to examine and validate the rheological properties, microstructural characterization via scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and mechanical stress testing were followed.

Results: The produced CS/GP-hydrogels showed a varying range of properties according to the CS/GP ratio. Formulations with higher β-glycerophosphate concentrations reduced gelation onset time from 5 to 1 minute and complete gelation times from 23 to 9 minutes, as determined by the test tube inversion method. Incorporation of vanillin (0.05%–0.15% w/v) into 1.3% chitosan and 15% β-glycerophosphate formulations further reduced complete gelation time to 4–7 minutes, as assessed by the test tube inversion method. Vanillin-containing hydrogels maintained solid gel strength, as compared to the controls, and exhibited a biocompatible with the pH range of 7.29–7.3. Optimized formulations demonstrated thermosensitivity, consistent gelation kinetics and solid structural integrity (i.e., suitable porosity, osmolality, swelling behavior and biodegradability), assuring their gelling properties suitable for tissue engineering and drug delivery applications.

Conclusion: This study optimized CS/GP hydrogels using vanillin as a crosslinker, and has achieved  enhanced gelation kinetics while maintaining solid gel strength and biocompatibility. The incorporation of vanillin offers a promising approach for development of gelling-property controllable, thermosensitive and biocompatible hydrogels for 3D printable biomimetic scaffolds.

References: 1. Qin, H., Wang, J., Wang, T., Gao, X., Wan, Q., & Pei, X. (2018). Preparation and characterization of chitosan/β-glycerophosphate thermal-sensitive hydrogel reinforced by graphene oxide. Frontiers in Chemistry, 6, 565.
2. Sirangelo, I., Sapio, L., Ragone, A., Naviglio, S., Iannuzzi, C., Barone, D., Giordano, A., & Borriello, M. (2020). Vanillin prevents doxorubicin-induced apoptosis and oxidative stress in rat H9c2 cardiomyocytes. Nutrients, 12(8), 2317.
3. Deng, A., Kang, X., Zhang, J., Yang, Y., & Yang, S. (2017). Enhanced gelation of chitosan/β-sodium glycerophosphate thermosensitive hydrogel with sodium bicarbonate and biocompatibility evaluated. Materials Science and Engineering: C, 78, 1147–1154.

Acknowledgements: This work was supported by funds provided by the School of Graduate Studies, University of Missouri-Kansas City.


Figure 1. Schematic illustration of chitosan, β-glycerophosphate, and vanillin self-assembly into a hydrogel for biomedical applications. At 25°C (left), chitosan, β-glycerophosphate, and vanillin are dispersed, whereas at 37°C (right), they form a hydrogel network. The arrow, labeled "Sol-to-Gel Transition," indicates the temperature-driven process.