Table 1.

Key silk fibroin hydrogel fabrication techniques.

Methods	Fabrication Techniques	Comments
Chemically induced gelation	Salts	Salts can promote protein-protein association for example the addition of Ca2+ ions reduce the gelation time of RSF solution [122,123].
	Polymer agents	Polymer agents, such as polyethylene glycols and PEO, have been shown to promote protein-protein associations, and protein aggregation through volume exclusion and movement of water by osmosis [122,123].
	Organic solvents	Alcohols are the most common used among organic solvents, which can induce structural conformation changes of RSF from α-helix to β-sheet structures [124].
	Surface active agents	Surface active agents readily bind with proteins leading to protein unfolding and aggregation [125]. For example, adding the anionic surfactant sodium dodecyl sulfate (SDS) into RSF solutions and incubating at 60 °C can induce stable hydrogels with good mechanical properties [100].
	Small neutral additives	Small neutral additives through their ionic strength and/or specific interactions with proteins can influence protein aggregation.125 For example, the addition of glycerol (30%; v/v) can reduce the gelation time of RSF solution and has been applied in biomedical applications [126,127].
	pH	As the pH of RSF solution is adjusted near the isoelectric point (PI = 3.8–3.9), stable hydrogels can be formed as well as reduced gelation time of RSF scaffolds [128]. This is because the pH of protein solution near its isoelectric point can induce protein precipitation [32].
	High pressure CO2	High-pressure CO2 as a volatile acid can be used as a fine tuning adjustment of the solutions pH, therefore, RSF solutions subjected to high-pressure CO2 at 60 bar, has been shown to form stable hydrogels within 2 h [129].
	Chemical crosslinking	Chemical crosslinking agents (e.g., hydrogen peroxide and horseradish peroxidase) can be used to covalently crosslink phenol groups of tyrosine residues on silk fibroin proteins to form highly elastic RSF hydrogels [130].
	Chemical coupling	Diazonium coupling chemistry can functionalize tyrosine residues of SF protein, resulting in an adjustment of the hydrophobic and hydrophilic properties, giving rise to the ability to rapidly produce controlled RSF hydrogels from as little as 5 min to two hours [131].
Physically induced gelation	Temperature	The gelation time of RSF solutions decreases with increasing temperature, this is because molecular collisions increase with respect to temperature [117,123].
	Shear force	A strong enough shear force applied to an RSF solution can promote molecule-molecule interactions and improve concentration fluctuation, resulting in gelation and aggregation phenomena [132,133]. Vortex mixing is the way to initiate RSF gelation due to the high shear forces applied to the solution [134].
	Ultrasound	Sonication can lead to local areas of extreme pressure and temperature, resulting in gelation and aggregation [135].
	Electric fields	Applying electric fields across RSF solutions leads to local pH decreases and thus silk protein aggregates [134].