I dont think it would work well as GA concentration is too low, from the other side increase o the concentration enhance toxicity effect of GA. We used that concentration and system for cryogel preparation

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Dear Jessica Samir

Fabricating a stable injectable hydrogel with 2% chitosan and 1% PVA can be challenging due to factors influencing gelation, such as cross-linking efficiency, pH, temperature, and the compatibility of components.

Possible Reasons for Failure

* Inadequate Cross-Linking Reaction: (1) Glutaraldehyde concentration: At 0.1%, the concentration might be insufficient to initiate or complete cross-linking for gelation. (2) Reaction environment: The pH and temperature of the solution may not be optimal for glutaraldehyde to effectively cross-link chitosan and PVA.

* Chitosan's Solubility and Ionization: (1) Chitosan requires an acidic environment (typically pH 4–6) to dissolve and maintain protonated amine groups (-NH₃⁺), essential for cross-linking. (2) If the pH is too high, chitosan may precipitate, preventing interaction with the cross-linker.

* Polymer Compatibility: Chitosan and PVA may not interact synergistically at the given concentrations, leading to phase separation or insufficient physical entanglement.

* Insufficient Gelation Time: Gelation may require more time, particularly with mild cross-linkers like glutaraldehyde at low concentrations.

Recommendations to Achieve Hydrogel Gelation

* Optimize Cross-Linking: (1) Increase the concentration of glutaraldehyde incrementally (e.g., try 0.5% or 1%) while monitoring for signs of gelation. (2) Ensure thorough mixing to allow uniform distribution of the cross-linker.

* Adjust Reaction Conditions: (1) pH: Keep the solution pH in the range of 4.5–6.0 to ensure chitosan remains soluble and reactive. (2) Temperature: Carry out the cross-linking reaction at room temperature (~25°C) or slightly elevated (~37°C) to facilitate gelation without degrading the polymers.

* Improve Polymer Interactions: (1) Pre-blend chitosan and PVA thoroughly by dissolving each in their respective solvents (e.g., acetic acid for chitosan, water for PVA) before mixing. (2) Consider adjusting the ratios of chitosan to PVA (e.g., 1:1, 3:2) to identify the best composition for gelation.

* Alternative Cross-Linking Strategies: (1) Physical Cross-Linking: Use freeze-thaw cycles with PVA, which can induce physical entanglement and hydrogen bonding without chemical cross-linkers. (2) Dual Cross-Linking: Combine glutaraldehyde with ionic cross-linkers (e.g., tripolyphosphate for chitosan) to enhance gelation.

* Characterize the Solution Before Gelation: (1) Measure viscosity and monitor changes during cross-linking to understand the gelation kinetics. (2) Conduct Fourier-transform infrared (FTIR) spectroscopy to confirm cross-linking reactions.