Table 1.

Representative click chemistry reaction methods, reactants, cell culture details, and their advantages and disadvantages. (In table format)

S.no	Hydrogel preparation methods	Reactants	Cells/in vivo/in vitro	Gelation time and degradation	Advantages	Disadvantages	References
1	Copper-catalyzed click chemistry reactions	HA-hydrazine, collagen, and HA-aldehyde or HA-benzaldehyde, Copper (II) sulfate pentahydrate	hMSCs	Gelation in 5 min	Enhanced focal adhesion, cell spreading, fiber remodeling	Copper ions toxicity, reactive oxygen species generation, regional variation in viscosity due to rapid gelation, non-homogeneous gels	[51]
2	Diels–Alder click reactions	Furan-linked gelatin against maleimide-linked PEG, chitosan-Pluronic F127)	Cardiomyocyte cells, in vitro and in vivo	Gelation in 2 h, slower degradation rate in vitro	Injectable gels, fully-interpenetrating network, thermosensitive, cell adhesive	Slow gelation, reduced solubility of the functional groups, inability to be injected in vivo in few cases	[59]
		N-maleoyl alanine terminated F127, furan grafted chondroitin sulfate, oxidized chondroitin sulfate, bone morphogenetic protein-4	Rat mesenchymal stem cells, bone cells (repair), in vitro and in vivo	Gelation in 3 days, 14 days degradation in vitro	Easily modifiable, improved viscoelastic properties and rheological properties. Swellable, injectable, self-healing ability		[61]
		Sodium alginate, bio glass and modified chondroitin sulphate	Cranial bone defect repair, in vitro and in vivo	Gelation in 3 days, 4 weeks at neutral pH, faster degradation at basic pH in vitro	Triple cross-linked injectable hydrogel, better physio-chemical properties		[60]
		Furfurylamine linked chondroitin sulfate, F127 linked maleimido and PEG-AMI, bone morphogenetic protein-4	Bone cells (repair)	Gelation in Less than one min	Non-covalent and covalent crosslinking, good biocompatibility		[17]
		Furan linked HA, dexamethasone and maleimide linked HA	Human adipose-derived stem cells, in vitro	Gelation in 60 min, more than 21 days for degradation in vitro	Thermo-responsible hydrogels, dexamethasone-controlled release in local environment, non-cytotoxic, can deliver adipogenic factors		[57]
		HA with furan adipic dihydrazide, HA with furan CHO and followed by addition of dimaleimide PEG	Chondrocyte cells (cartilage), in vitro	Gelation in 5 min	Mechanical properties, tissue adhesive, self-healing, pH responsiveness		[53]
3	Strain-promoted azide-alkyne cycloaddition (SP-AAC) reactions	Azadibenzocyclooctyne-modified dextran and azide-modified dextran	Chondrocyte cells(cartilage), in vitro	Gelation in 1.1 to 10.2 min, slow degradation rate up to 21 days in vitro	Gelation time modifiable using concentration variation and substitution degree of dextran, encapsulation of cells and cell spheroids	Reaction rate is lower than copper catalyzed reactions, alkynes are larger, trigger high perturbation, less ligation rate, high side reactions	[72, 114]
		Dibenzocyclooctyl (DBCO)-modified HA, 4-arm PEG azide	Chondrocyte cells (cartilage), in vitro and in vivo	Gelation in 10–14 min, slow degradation up to 35 days in vivo	Nontoxic cross linker, good biocompatibility, in situ physical gelation, elastic modulus can be modified by varying concentration		[73]
		Hyperbranched poly(ε-caprolactone) (hyPCL)32-(1R,8S,9 s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol (hyPCL32-BCN) and hyPCL32-azide (hyPCL32-N3)	Bone (MC3T3 preosteoblast) cells	Gelation in 30 min	Injectable hyperbranched PCL, biocompatible, excellent support for cell adhesion and proliferation		[71]
		4-dibenzocyclooctynol functionalized PEG, 4 arm PEG tetraazide, protein igands (laminin), neurogenic differentiation factor (interferon-γ)	Neural stem cell, In vitro	Gelation in less than 5 min	Additional differentiation factors not required in medium, high cell viability, no UV light required		[16]
4	Thiol-ene-based click chemistry reactions	PEGDA, dithiothreitol, borox	Endothelial cells and neural stem cells, in vitro	Gelation within few min of stirring	Injectable, 3D printable, sacrificial removal enables tubular structure formation	Polymer substrates lack alkenes with electron deficiency, acid condition required for the reaction	[77]
		4-arm PEG norbornene, PEG dithiol, Protein array printing of collagen I, collagen III, collagen IV, fibronectin, laminin, elastin and hyaluronan	Smooth muscle cells, in vitro		Cellular stiffness and increased functional contractility, cell attachment and 3D infiltration	UV source needed in radical mediated reactions which may damage cells, oxygen sensitivity may result in disulfide formation; the presence of cysteine and amine group residues may compete with thiol group while hydrogel formation occurs.	[82]
		Alkene-linked (allyl and norbornene residues), poly(oligoethylene glycol methacrylate)), cell adhesive peptides RGD and REDV	Human umbilical vein endothelial cells (HUVECs), in vitro		Photo-patterned peptides at the surface, development of cell array is achieved		[80]
		Norbornene-linked pectin macromer, monocysteine RGD peptide, biscysteine peptide with matrix metalloproteinase cleavage site	Human neonatal dermal fibroblasts, in vitro	Degradation9 h in enzymatic treatment in vitro	Cell surface receptors in pectin may increase cell attachment and integration, simple, fast, robust, independent modification of the biochemical or biophysical cues in the system is possible		[79]
		8-arm-PEG thiol macromer, thioester di(vinyl ether), caged thioester catalyst, norbornene RGD	Primary hMSCs, in vitro		Dynamic, modifiable visco-elastic properties, using pH, stoichiometry and crosslinker structure the hydrogel network can be modified, thiol-ester resembles biological reactions		[78]
5	Oxime based click chemistry reactions	Aminooxy-terminated PEGs, aldehyde modified HA and collagen I	Schwann cells, HMSCs, in vitro		Less toxic to the cells, tunable properties, peptide, protein bonding is easier	Not bio-orthogonal	[22]