Table 2.

Preclinical evaluation of in situ forming injectable HA-based hydrogels.

Material	HA a MW b (KDa)/ Functional Groups	DM (%) c/ Functionalization	Cross-Linking Reaction/Cross-Linker/Gelation Onset (s)	Bioactive Agent/Stimulation/Extra	Cell Type/Cell Number per mL	Outcome
Redox/enzymatic reaction
HTG d [57]	-/COOH	13.38/-	Enzymatic/tyrosinase/108–132	EGCG e/-	Porcine chondrocytes/2 × 107	The HTG d hydrogel was found to promote accumulation of ECM f EGCG e-loaded hydrogel protected cartilage from inflammation and diminished cartilage loss in an OA g mouse model
HA-GEL h [59]	350/COOH	-/-	Redox/HRP i and H2O2/-	-/electrical	Porcine MSCs j/1 × 106	The electrical stimulation was revealed to enhance the chondrogenic potential of the HA-GEL h hydrogel
HA-TA k [52]	70/COOH	24	Oxidative coupling reaction (redox)/HRP i and H2O2/10–500	Platelet lysate	MSCs j/107	This study showed that hMSC-laden HA-TA k hydrogels that were enriched with platelet lysate favored a cartilage-like ECM f deposition in vitro. Hydrogel degradation happened at the same time with ECM f deposition leading to the formation of a dense matrix. The results of this study confirmed the possibility of using HA-TA-PL l hydrogel as a cell delivery system for cartilage tissue engineering applications.
HA a [60]	1010–1800/COOH	/transglutaminase substrate peptides	Enzymatic/thrombin, factor XIII, transglutaminase-modified heparin/60–120	TGF-β m/-	Human infant chondrocytes/5, 10 or 15 × 106,	Cartilaginous matrix was produced by polydactyly chondrocytes in the developed biomimetic hydrogels
HA a [50]	1010–1800/COOH	/transglutaminase substrate peptides, heparin	Enzymatic/thrombin, transglutaminase factor XIII/900	TGF-β m/-	Human chondroprogenitor cells (fetal origin)/15 × 106	Matrix deposition was shown to be stimulated by a slow release of TGF-β m.
HA-MA-PNIPAAm-CL n [61]	2000/OH	30/-	Redox/-/-	-/-	Rabbit adipose-derived stem cells/1 × 106	Enhancement of chondrogenesis of adipose-derived stem cells in HA-PNIPAAm-CL n hydrogel for cartilage regeneration in rabbits
PVCL-g-HA o (methacrylate HA) [62]	58 and 1100/OH	-/-	Redox/VA-057 p initiator/-	-/-	Bovine chondrocytes/3.65 × 106	In this study, thermosensitive injectable hydrogels were developed to be used for cartilage tissue engineering applications. These hydrogels appeared to be promising materials favoring the viability of chondrocytes as well as the biochemical synthesis of ECM f proteins under hypoxia.
HA-Tyr k [63]	90/COOH	6/-	Oxidative coupling reaction (Redox)/HRP i and H2O2/60		Caprine MSCs j/107	The 3D microenvironment of the HA-Tyr k hydrogels controlled cellular condensation throughout chondrogenesis and influenced the spatial organization of cells, ECM f biosynthesis and histogenesis of cartilage tissue.
Michael-type addition reaction
MeHA q [2]	66–99/OH	46.5 ± 5.5/- 46.5 ± 5.5/CS r-binding peptide 46.5 ± 5.5/-	Michael-type addition/MMP7 s-degradable peptide/457 ± 68.1	-/-	MSCs j/1 × 106 chondrocytes/1.25 × 106	Differentiation of MSCs j towards a chondrogenic phenotype Enhancement of cell differentiation towards a chondrogenic phenotype Arrangement of cell clusters in isogenous groups, distinctive of hyaline cartilage morphology and deposition of glycosaminoglycans
Hyper-branched PEGDA t-thiolated HA [56]	-/COOH	-/-	Michael-type addition/-/120	-/-	Human AFF-MSCs u/5 × 106	AFF-MSCs u were differentiated towards a chondrogenic phenotype Full-thickness cartilage defects were successfully repaired in 8 weeks.
MA-HA v [64]	59/COOH	30/-	Michael-type addition/MMP w-cleavable peptides/-	-/-	Human MSCs j/20 × 106	Enhanced chondrogenesis and suppressed hypertrophy of human MSCs j encapsulated in MA-HA v hydrogels were the result of cell-mediated hydrogel degradation via MMPs w.
Schiff base reaction
Glycol chitosan-oxidized HA a [65]	100/OH	33.4/-	Schiff base reaction/-/-	Cartilage ECM f particles/-	BMSCs x/2 × 107	The presence of cartilage ECM f particles resulted in the formation of more mature cartilage tissue containing higher levels of GAGs y and collagen II
Collagen-HAD z [66]	1500–1800/	-/-	Schiff base reaction/-/-	-/-	Rabbit chondrocytes/5 × 104	Both healthy and osteoarthritic cartilage in vitro models were developed by varying HAD z concentration in the hydrogels.
CH-HAD aa [53]	-/OH	50/-	Schiff base raction//25–60	-/-	Rabbit chondrocytes/5 × 106	The results of this study demonstrated that hydrogel stiffness had a huge impact on maintaining the phenotype of chondrocytes as well as the production of ECM f.
OHA/GC ab [67]	1000/OH	~6.8–33.8/-	Schiff base raction//97–120	-/-	ATDC5 chondrogenic cell line/106	OHA/GC ab hydrogels exhibited efficient biocompatibility and resistance under natural conditions, and they could be used as an injectable cell delivery system for cartilage tissue engineering applications.
Photocross-linking
AHAMA ac [55]	100–200/OH	24/-	Photopolymerization/Irgacure 2959/-	-/-	BMSCs x/5 × 106	AHAMA ac hydrogel was shown to significantly promote neocartilage integration with host tissue and cartilage regeneration in osteochondral defects in rats.
mGL/mHA ad [58]	66–99/OH		Photocross-linking/LAP ae		Human BMSCs x/ 20 × 106	Chondrogenesis and cartilage formation were favored for MSCs j encapsulated in mGL/mHA ad hydrogels at a ratio of 9:1. The implantation of mGL/mHA ad hydrogel inside the defect exhibited cartilage and bone formation after 12 weeks, indicating its potential use for the repair of osteochondral defects.
GelMA af/HAMA ag [68]	860/OH		Photocross-linking/LAP ae and visible light (405 nm), Irgacure 2959 and UV ah light (365 nm)	/MEW-mPCL ai reinforcement	Human articular chondrocytes/107	In this study, photocross-linking based on UV ah light exhibited enhanced chondrocyte cell behavior compared to visible light cross-linking. Bovine-derived GelMA af photocross-linked with Irgacure 2959 showed properties that resembled native articular cartilage tissue.
MeHA ag [69]	75/OH	37/± HAV, ADAM-cleavable domain	Photocross-linking/Irgacure 2959		MSCs j/20 × 106	This study showed the possibility of a hydrogel material mimicking the complicated microenvironment throughout embryogenesis towards the formation of stem-cell-based cartilage.
MeHA ag/ELP aj [70]	1600/OH		Photocross-linking/	ZnO ak (antimicrobial)	Human MSCs j, NIH-3T3/2 × 106, 5 × 106	This study confirmed that MeHA ag/ELP aj-ZnO ak hydrogels can be used for tissue engineering applications due to their tunable natural characteristics and their adhesive and antimicrobial properties.
MeHA ag [71]	1000/OH	1.2/	Photocross-linking/Irgacure 2959	TGFβ3 m/DCC al or DVC am microparticles	Rat BMSC x/20 × 106	This study demonstrated that DVC am microparticles showed enhanced chondroinductivity and rheological performance of hydrogel precursors in comparison to DCC al.
MeHA ag [72]	74/OH		Photocross-linking/Irgacure 2959	TGFβ3 m	Allogeneic MSCs j/60 × 106	The data from this study indicated that combining MSCs j with growth factors and hydrogel materials followed by a preculture period and utilizing standard tissue engineering techniques could give a more promising outcome in comparison with directly implanting cells and growth factors.
MeHA ag, MeHA ag+ColI an, MeHA ag+MeCS ao [73]	74/OH	30	Photocross-linking/Irgacure 2959		Human MSCs j/20 × 106	The results of this study showed that by controlling the formula of cartilage specific biopolymers embedded into cell-laden hydrogels, it was possible to tune the level of maturation and calcification of the newly formed cartilage.
MeHA ag [74]	74/OH	29	Photocross-linking/Irgacure 2959		Human MSCs j/20 × 106	The study showed that HA a concentration, and not cross-linking density, can affect the hypoxia-mediated positive or negative control of the hypertrophic differentiation of cells encapsulated in HA a hydrogels after chondrogenic induction. The outcome of this study could be useful for the design and optimization of hydrogels and tissue culture protocols.
Fibrinogen/HA-MA ag [75]	1500–1800/OH	95 ± 13/-	Ionic and chemical interactions, Photocross-linking/Irgacure 2959/	TGFβ m/-	BMSCs x/104/well	Fibrin/HA-MA ag hydrogel could be used for the delivery of BMSCs x. Fibrin/HA-MA ag hydrogel favors the differentiation of BMSC x into chondrocytes and it could be helpful for the repair of articular cartilage tissue in OA g patients.
GelMA af and HA-MA ag [76]	860/OH		Photocross-linking/Irgacure 2959/900		Human chondrocytes/107	The mixtures of GelMA af and HA-MA ag are considered promising materials for cartilage tissue engineering applications.
MeHA ag [77]	74/OH	27	Photocross-linking/Irgacure 2959		MSCs j and/or chondrocytes/20 × 106	The study demonstrated that the coculture of hMSCs j and chondrocytes encapsulated in HA a hydrogels increased the mechanical properties and cartilage-specific ECM f content of tissue-engineered cartilage.
Self-cross-linking and other reactions
ColI an/HA-sNHS ap [78]	61/COOH	32, 50, 83/	Self-cross-linking/no initiators and no cross-linkers/93–130		Rabbit chondrocytes/5 × 106	These self-cross-linkable and injectable hydrogels with adjustable physical properties could be used for cartilage tissue engineering applications.
HA-SH aq/GelSH ar, HA-SH aq/GelMA af, HA-SH aq/Gel as [79]	340/COOH	35.3/	Strong disulfide bonding between HA-SH aq and GelSH ar/7.19, Michael addition between HA-SH aq and GelMA af/7.31, Physical interaction/7.27	-/-	Rabbit chondrocytes/3 × 106	The strong disulfide bonding was shown to enhance the performance/biological function of the encapsulated chondrocytes
Thiolated HA a—collagen [32]	100, 300, 1000/COOH	-/-	Formation of disulfide bonds/thiolated icariin/1800	-/-	Chondrocytes/5 × 106	The developed hydrogels were found to facilitate cell proliferation, maintain the chondrocyte phenotype and promote the secretion of cartilage ECM j.
Thiolated HA—collagen I [54]	300/COOH	-/-	Self-cross-linking/10	-/-	Rabbit chondrocytes/5 × 106	The hydrogels facilitated cell adhesion and proliferation The encapsulated chondrocytes were shown to maintain their phenotype and to secrete a great amount of ECM j.
HA a-ADH at/PAD au, HA a-ADH at/PAD-RGD av [80]	740/COOH	41.5/-	Hydrazone reaction/PAD-RGD av/112–399	-/-	Chondrocytes/6 × 106	HA a-ADH at/PAD-RGD av hydrogel with a 5/5 weight ratio was characterized as the most promising microenvironment that could mimic host tissue and maintain chondrocyte phenotype as well as favoring chondrogenesis

^a Hyaluronic acid, ^b molecular weight, ^c degree of modification, ^d tyramine-modified hyaluronic acid-gelatin, ^e epigallocatechin-3-gallate, ^f extracellular matrix, ^g osteoarthritis, ^h tyramine-modified hyaluronic acid—tyramine-modified gelatin, ⁱ horseradish peroxidase, ^j mesenchymal stem cells, ^k hyaluronic acid tyramine hydrogel, ^l hyaluronic acid tyramine hydrogel with platelet lysate, ^m transforming growth factor beta, ⁿ methacrylated hyaluronic acid cross-linked poly(N-isopropylacrylamide), ^o poly(N-vinylcaprolactam) and methacrylated hyaluronic acid, ^p 2,20-azobis[N-(2-carboxyethyl)22-methylpropionamidine]hydrate, ^q methacrylated hyaluronic acid, ^r chondroitin sulfate, ^s matrix metalloproteinase 7, ^t poly(ethylene glycol) diacrylate, ^u arthroscopic flushing-fluid-derived mesenchymal stem cells, ^v maleimide-modified HA, ^w matrix metalloproteinase, ^x bone marrow mesenchymal stem cells, ^y glycosaminoglycans, ^z hyaluronic acid dialdehyde, ^aa chitosan–hyaluronic acid dialdehyde, ^ab oxidized hyaluronate/glycol chitosan, ^ac methacrylated aldehyde-modified hyaluronic acid, ^ad methacrylated gelatin-methacrylated hyaluronic acid, ^ae lithium phenyl-2,4,6-trimethylbenzoylphosphinate, ^af gelatin methacryloyl, ^ag hyaluronic acid methacrylate (or methacrylated hyaluronic acid), ^ah ultraviolet, ^ai melt-electrowritten medical-grade polycaprolactone, ^aj elastin-like polypeptide, ^ak zinc oxide, ^al decellularized cartilage, ^am devitalized cartilage, ^an type I collagen, ^ao methacrylated chondroitin sulfate, ^ap N-hydroxysulfosuccinimide-activated hyaluronic acid, ^aq thiolated hyaluronic acid, ^ar thiolated gelatin, ^as gelatin, ^at adipic dihydrazide, ^au pectin dialdehyde, ^av aldehyde groups of G4RGDS-grafted aldehyde pectin.