Table 2.

Dual crosslinking strategies for 3D bioprinting applications.

Bioink composition	Crosslinking mechanism & parameters	Significant observations	Ref
Gelatin methacrylol (GelMA), microbial transglutaminase (mTGase), Irgacure 2959	Step 1: Enzymatic crosslinking via Ca2+ independent mTGase Step 2: UV irradiation (365 nm, 1.5 mW/cm2) for 5 min	• Reduced gelation time and enhanced viscoelastic property observed for increased mTGase concentration	[69]
Tyramine modified alginate (Alg/Tyr), Riboflavin (RF), sodium persulfate (SPS)	Homogenous ionic crosslinking by CaCO3 and photo-crosslinking using visible light (440 nm, 2500 mW/cm2) for 3 min	• Dual crosslinked hydrogels formed in a shorter gelation period had interconnected porous network, increased compressive strength of 74 kPa, and 564% swelling ratio with 36% physiological stability when compared to mono-crosslinked (photo-crosslinked) hydrogels	[70]
Tyramine modified methylcellulose (MC-Tyr), Riboflavin (RF), Riboflavin 5′-monophosphate (RFp), NIH3T3 fibroblasts	Step 1: Chemical crosslinking via conjugated carboxyl group of MC and amine groups of Tyramine; thermal crosslinking via methoxy groups of methyl cellulose Step 2: Visible light (440 nm, 2500 mW/cm2) for 120 s	• Hydrophobic and covalent interactions observed in dual crosslinked hydrogels • Gelation temperature (~40 °C) of MC-Tyr remains close to physiological temperature • Compressive strength and elastic modulus (4.01 kPa) of RFp crosslinked hydrogels were higher when compared to RF crosslinked hydrogels due to the salting-out effect of the RFp photo-initiator • Bioink exhibited good printability and increased structural stability of the 3D printed scaffolds (20 × 20 × 0.2 mm) for up to 60 days in PBS • Increased NIH-3T3 cell viability (~90%) was observed within the printed constructs	[71]
Hydroxy butyl methacrylated chitosan (HBC-MA), Eosin Y, triethanolamine, 1-vinyl-2-pyrrolidinone, NIH3T3 cells	Step 1: Chemical conjugation of acrylate and hydroxy butyl groups to chitosan; Thermal crosslinking at ~37 °C Step 2: Visible light (450–550 nm, 10 mW/cm2) for 30 s	• Hydrogels showed compact and regular porous morphology, thermo-responsiveness (20 °C), good injectability, swellability, increased storage modulus (G′), increased compressive strength, low-temperature degradability • In vitro studies showed increased fibroblasts viability and proliferation for up to 72 h and dependent on pre-polymer concentrations • Subcutaneous injection of dual crosslinked hydrogels in mice showed degradation at physiological temperature	[72]
Gelatin methacrylol (GelMA), microbial transglutaminase (mTGase), Irgacure 2959, human breast cancer cell line HCC1806	Step 1: Photo-crosslinking via acrylate groups of GelMA Step 2: Enzymatic crosslinking via lysine and glutamine groups of GelMA	• Dual crosslinked hydrogels exhibited the tunable rheological and mechanical property • Suitable for disease modeling applications • Good printing fidelity with the visco-elastic nature of the dual crosslinked hydrogels for 3D printing applications • Cytocompatible	[73]
Tyramine modified hyaluronic acid (HA-Tyr), Eosin Y, Horse Radish Peroxidase (HRP), hydrogen peroxide (H2O2), hTERT fibroblasts/human mesenchymal stem cells (hMSCs)/bovine chondrocytes	Step 1: Enzymatic crosslinking by HRP and H2O2 Step 2: Visible light (505 nm) crosslinking mediated by Eosin Y	• Cell-laden bioinks exhibited shear thinning behavior with good printability • Cylindrical construct with criss-cross inner pattern (diameter—20 mm, height—1.4 mm) was printed with cells • After 24 h, cell viability of extruded hMSCs, chondrocytes, and hTERT—laden bioinks was 78%, 70%, and 75% respectively • Chondrocyte laden printed constructs showed higher swelling when compared to other cell-laden printed constructs	[74]
Methacrylated chitosan, β-glycerophosphate disodium salt hydrate (β-GP), LAP, fibroblasts (NIH/3T3)/osteoblast-like cells (Saos-2)/neuronal-like cells (SH-SY5Y)	Step 1: Thermal crosslinking via β-GP Step 2: UV crosslinking (365 nm, 30 mW/cm2) for 2 min	• Developed ptCS hydrogels showed increased storage modulus and anti-oxidative property • ptCS cell encapsulated hydrogels showed good compatibility for up to 7 days • Four layered grid-shaped constructs were printed successfully	[75]