Table 1.

Natural hydrogels.

	Natural Hydrogels
Author	Hydrogel Used	Type of Study	Hydrogel Modification	Hydrogel Properties	Cells Used	Upregulated Biological Molecules	Outcomes
	Collagen Hydrogel
Souron et al., 2014 [104]	Collagen	In vivo		3D collagen matrices, mimic in vivo cell–cell and cell–matrix interactions, and regulate cell growth	Rat pulp cells labeled with indium-111-oxine		1 month following implantation, active fibroblasts, new blood vessels and nervous fibers were present in the cellularized 3D collagen hydrogel.
Kwon et al., 2017 [60]	Collagen	In vitro	Crosslinked with cinnamaldehyde (CA).	Crosslinked collagen with shorter gelation time enhances cellular adhesion. Higher stiffness enhances odontogenic differentiation.	Human dental pulp stem cells (DPSCs)	Dentin sialophosphoprotein (DSPP), Dentin matrix protein 1 (DMP-1), Matrix extracellular phosphoglycoprotein (MEPE), Osteonectin (ON)	CA shortened the setting time, increased compressive strength and surface roughness of collagen hydrogels. CA-crosslinked hydrogels promoted the proliferation and odontogenic differentiation of human DPSCs
Pankajakshan et al., 2020 [61]	Collagen	In vitro	Varying hydrogel stiffnesses through varying oligomer concentrations. Incorporation of Vascular endothelial growth factor (VEGF) into 235 Pa collagen or Bone morphogenetic protein 2 (BMP-2) into the 800 Pa ones.	Stiffness affect cytoskeletal organization and cell shape and specify stem cell lineage.	DPSCs	von Willebrand Factor (vWF), platelet endothelial cell adhesion molecule 1 (PECAM-1), vascular endothelial-cadherin	Collagen hydrogels with tunable stiffness supported cell survival, and favored differentiation of cells to a specific lineage. DPSCs cultured in 235 Pa matrices showed an increased expression of endothelial markers, cells cultured in 800 Pa showed increased alkaline phosphatase (ALP) activity and Alizarin S staining.
	Gelatin Hydrogel
Kikuchi et al., 2007 [84]	Gelatin hydrogel	In vivo	Crosslinked gelatin hydrogel microspheres were impregnated with fibroblast growth factor 2 (FGF-2) and mixed with collagen sponge pieces.	Gelatin hydrogel microspheres (the water content is 95 vol%; diameters ranged from 5–15 µm; the average of diameter was 10 µm)		DSPP	Controlled release of FGF2 from gelatin hydrogels induced the formation of dentin-like particles with dentin defects above exposed pulp.
Ishimatsu et al., 2009 [117]	Gelatin hydrogel	In vivo	Gelatin hydrogel microspheres incorporating FGF-2.	Gelatin hydrogel microspheres (the water content is 95 vol%, the average diameter was 10 µm)		DMP-1	Dentin regeneration on amputated pulp can be regulated by adjusting the dosage of FGF-2 incorporated in biodegradable gelatin hydrogels.
Nageh et al., 2013 [85]	Gelatin hydrogel	Clinical trial	FGF incorporated in gelatin hydrogel.	Acidic gelatin hydrogel microspheres with a mean diameter of 59 µm and 95.2% water content.			Follow-up X-ray revealed an increase in root length and width with a reduction in apical diameter confirming the root’s development.
Bhatnagar et al., 2015 [62]	Gelatin hydrogel	In vitro	enzymatically crosslinked with microbial transglutaminase (mTG).	Hard and soft gelatin–mTG gels consist of 1.125 mL and 1.488 mL of a 10% gelatin solution with 0.375 mL (3:1 (v/v) gelatin: mTG), and 0.012 mL (125:1 (v/v) gelatin: mTG) of mTG respectively	DPSCs	Osteocalcin (OCN), ALP, DSPP	Enzymatically crosslinked gelatin hydrogels are a potential effective scaffold for dentin regeneration regardless of matrix stiffness or chemical stimulation using dexamethasone.
Miyazawa et al., 2015 [63]	Gelatin hydrogel	In vitro In vivo	Simvastatin-lactic acid grafted gelatin micelles, mixed with gelatin, followed by chemical crosslinking to form gelatin hydrogels.	Carboxymethylcellulose (CMC) value of micelles was 79 μg/mL. Water solubilization of Simvastatin 43 wt.%. Simvastatin in the gelatin 3.23 wt.%. The sizes of granules 500 μm with rough surfaces and uniformly sized pores	DPSCs	ALP, Dentin sialoprotein (DSP), BMP-2	It is possible to achieve odontoblastic differentiation of DPSCs through the controlled release of Simvastatin from gelatin hydrogel.
	Gelatin Methacrylate Hydrogel
Athirasala et al., 2017 [68]	Gelatin Methacrylate hydrogel (GelMA)	In vitro	Gelatin with methacrylic anhydride.	GelMA hydrogels of 5, 10 and 15% (w/v) concentrations showed a honeycomb-like structure. Both 10% and 15% hydrogel groups appeared to have smaller pore sizes than 5% GelMA.	Odontoblasts like cells (OD21) and endothelial colony- forming cells		Pre-vascularized hydrogel scaffolds with microchannels fabricated using GelMA is a simple and effective strategy for dentin–pulp complex regeneration.
Khayat et al., 2017 [64]	GelMA	In vivo	Gelatin with methacrylic anhydride.		DPSCs and human umbilical vein endothelial cells (HUVECs)		GelMA hydrogel combined with human DPSC/human umbilical vein endothelial cells as promising pulpal revascularization treatment to regenerate human dental pulp tissues.
Ha et al., 2020 [72]	GelMA	In vitro	Gelatin with methacrylic anhydride hydrogels of increasing concentrations.	Increasing polymer concentrations from 5% to 10% and 15% (w/v), resulting in increasing extents of crosslinking. The elastic moduli of hydrogels, increased with increase in polymer concentration from 1.7 kPa for 5% GelMA to 7 kPa and 16.4 kPa for 10% and 15% GelMA hydrogels, respectively.	stem cells of the apical papilla (SCAP)		Substrate mechanics and geometry have a statistically significant influence on SCAP response.
Park et al., 2020 [65]	GelMA	In vitro	GelMA conjugated with synthetic BMP-2 mimetic peptide prepared into bioink.	GelMA exhibited a ~2 kPa storage modulus (G’) before crosslinking and ~4 kPa after crosslinking.	DPSCs	DSPP, OCN	BMP peptide-tethering bioink could accelerate the differentiation of human DPSCs in 3D bioprinted dental constructs.
Jang et al., 2020 [67]	GelMA	In vitro In vivo	Thrombin solution added to GelMA hydrogel.		DPSCs		Gelatin hemostatic hydrogels may serve as a viable regenerative scaffold for pulp regeneration.
	Fibrin Hydrogel
Meza 2019 [249]	Platelet Rich Fibrin (PRF)	Case report			DPSCs		Autologous DPSCs isolated from extirpated autologous inflamed dental pulp were loaded on autologous PRF in lower premolar tooth with irreversible pulpitis for successful regeneration.
Ducret et al., 2019 [123]	Fibrin–chitosan	In vitro	Enriching the fibrin-hydrogel with chitosan.	10 mg/mL fibrinogen and 0.5% (w/w), 40% DA chitosan, formed a hydrogel at physiological pH (≈7.2), which was sufficiently fluid to preserve its injectability without affecting fibrin biocompatibility	DPSCs		Chitosan imparted antibacterial activity to fibrin hydrogel, reducing E. faecalis growth. The blending of chitosan in fibrin hydrogels did not affect the viability, proliferation and collagen-forming capacity of encapsulated DPSCs as compared to unmodified fibrin.
Mittal et al., 2019 [250]	PRF	Clinical trial					PRF and collagen are better scaffolds than placentrex and chitosan for apexogenesis of immature necrotic permanent teeth.
Bekhouche et al., 2020 [82]	Fibrin	In vitro	Incorporation of clindamycin loaded Poly (D, L) Lactic Acid nanoparticles (CLIN-loaded PLA NPs).	Fibrin hydrogel constituted a reservoir of CLIN-loaded PLA NPs inhibiting E. faecalis growth without affecting cell viability and function.	DPSCs		Fibrin hydrogels containing CLIN-loaded PLA NPs showed an antibacterial effect against E. faecalis and inhibited biofilm formation. DPSCs viability and type I collagen synthesis in cellularized hydrogels were similar to the unmodified groups.
Renard et al., 2020 [131]	Fibrin-chitosan	In vivo	Same formulation of fibrin–chitosan hydrogel as used by Ducret et al., 2019 [123]	40% DA chitosan incorporation in the fibrin hydrogel did not modify modify dental pulpi inflammatory/immune response and triggered polarization of pro-regenerative M2 macrophages.			In in vivo model of rat incisor pulpotomy, fibrin–chitosan hydrogels imparted a similar inflammatory response in the amputated pulp as unmodified fibrin. Both groups enhanced the polarization of pro-regenerative M2 macrophages.
Zhang et al., 2020 [71]	Fibrin	In vitro	Fibrin hydrogel loaded with DPSCs-derived extracellular vesicles (EVs).	2 mg/mL fibrinogen formed a hydrogel which was able to retain and preserve the activity of EVs. Forming the most extensive tubular network forming at an EVs concentration of 200 µg/mL	Co-culture of DPSCs and HUVECs	VEGF	Investigated hydrogels enhanced rapid neovascularization under starvation culture, increased deposition of collagen I, III, and IV, and promoted the release of VEGF.
	Matrigel 3D
Mathieu et al., 2013 [143]	Matrigel	In vitro			DPSCs		Encapsulating transforming growth factor beta1 (TGF-b1) and FGF-2 in a biodegradable Poly glycolide-co-lactide (PGLA) microsphere
Ito et al., 2017 [77]	Matrigel	In vivo			Bone marrow mesenchymal stem cells (BMMSCs)	Nestin, DSPP	Pulp tissue regeneration was successfully achieved.
Sueyama et al., 2017 [78]	Matrigel	In vivo			BMMSCs and endothelial cells (ECs).	DSPP, Nestin Bcl-2, Cxcl1, Cxcr2, VEGF	The implantation of ECs with mesenchymal stem cells accelerated pulp tissue regeneration/healing and dentin bridge formation.
Gu et al., 2018 [79]	Matrigel	In vivo			BMMSCs		M1-to-M2 transition of macrophages plays an important role in creating a favorable microenvironment necessary for pulp tissue regeneration.
Kaneko et al., 2019 [80]	Matrigel	In vivo			BMMSCs nucleofected with pVectOZ-LacZ plasmid encoding β-galactosidase	DSPP	BMMSCs could differentiate into cells involved in mineralized tissue formation in the functionally relevant region.
	Keratin Hydrogel
Sharma et al., 2016 [69]	Keratin hydrogel	In vitro		Highly branched interconnected porous micro-architecture with a maximum average pore size of 160 µm and minimum pore size of 25 µm. G′ > G″ indicates the elastic solid-like nature of the gel. After 3 months the degradation rate was 68%.	odontoblast-like cells (MDPC-23)	ALP, DMP-1	Keratin enhanced proliferation and odontoblastic differentiation of odontoblast-like cells. Keratin hydrogels may be a potential scaffold for pulp–dentin regeneration.
Sharma et al., 2016 [70]	Keratin hydrogel	In vitro In vivo		Highly branched interconnected porous micro-architecture with a maximum average pore size of 160 µm and minimum pore size of 25 µm. G′ > G′′ indicates the elastic solid-like nature of the gel. After 3 months the degradation rate was 68%.	odontoblast-like cells (MDPC-23) and DPSCs		Keratin hydrogel enhanced odontogenic differentiation of odontoblast-like cells and enhanced reparative dentin formation.
Sharma et al., 2017 [150]	Keratin hydrogel	In vivo		Branched interconnected porous micro-architecture with average pore size 163.5 and porosity 82.8%. There was a gradual increase in G’ from 7% to 20% (w/v) gel concentration. The average contact angle was 35.5°.			Keratins hydrogel can be a source for biological treatment options for dentin–pulp complex.
	Alginate Hydrogel
Dobie et al., 2002 [86]	Alginate	In vitro	TGF- β1 or HCL acid-treatment of the hydrogels.	Alginate hydrogels are valuable for delivery of growth factors (GFs) (or agents to release endogenous GFs) to enhance reparative processes of dentin–pulp complex.			Alginate hydrogel acted as an efficient carrier for TGF- β1. Furthermore, acid treatment of the hydrogel aided in the release of TGF- β1 from dentin matrix. Alginate–TGF–β1 blends stimulated reactionary dentinogenic responses with increased predentin width.
Bhoj et al., 2015 [83]	Alginate	In vitro	Arginine-glycine-aspartic acid (RGD)-modified alginate hydrogels, loaded with VEGF and FGF-2.	RGD–alginate matrix acted as pulp replacement, compatible with the DPSCs and HUVECs, and can deliver VEGF and FGF-2.	Co-culture of DPSCs and HUVECs		Combined addition of FGF and VEGF led to an increased proliferation of both DPSCs and HUVECs in the hydrogels. RGD-modified alginate can efficiently retain VEGF and FGF-2.
Smith et al., 2015 [81]	Alginate	In vitro	Alginate hydrogel doped with bovine dental pulp extracellular matrix (pECM).	3D Alginate hydrogel doped with pECM formed 3D matrices. pECM provides additional signals for differentiation.	Primary dental pulp cells		Induced differentiation in the mineralizing medium resulted in time-dependent mineral deposition at the periphery of the hydrogel.
Verma et al., 2017 [127]	Alginate–fibrin	In vivo	Oxidized alginate–fibrin hydrogel microbeads.	7.5% oxidized alginate coupled with fibrinogen concentration of 0.1% enhanced microbead degradation, cell release, and proliferation.	DPSCs		Oxidized alginate–fibrin hydrogel microbeads encapsulating DPSCs showed similar regenerative potential to traditional revascularization protocol in ferret teeth. In both groups, the presence of residual bacteria affected root development.
Athirasala et al., 2018 [168]	Alginate	In vitro	Blending alginate hydrogels with soluble and insoluble fractions of the dentin matrix as a bioink for 3D printing.	Dentin matrix proteins preserve the natural cell-adhesive (RGD) and MMP-binding sites, which are lacking in unmodified alginate, that are important for viability, proliferation, and differentiation.	SCAP	ALP, Runt-related transcription factor -2 (RUNX2)	Alginate and insoluble dentin matrix (in 1:1 ratio) hydrogels bioink significantly enhanced odontogenic differentiation of SCAP under the effect of the soluble dentin molecules in the hydrogel.
Yu et al., 2019 [169]	Alginate and gelatin hydrogels	In vitro	3D bioprinted crosslinked composite alginate and gelatin hydrogels (4% and 20% by weight, respectively).	3D printing accurately controls the interconnected porosity and pore diameter of the scaffold, and imitate natural cell tissue in vivo.	DPSCs	ALP, OCN, DSPP	3D-printed alginate and gelatin hydrogels aqueous extracts are more suitable for the growth of DPSCs, and can better promote cell proliferation and differentiation.
	Chitosan Hydrogel
Park et al., 2013 [179]	Chitosan hydrogel	In vitro	N-acetylation of glycol chitosan	Glycol chitosan (0.2 g) and acetic anhydride (0.87 g) were dissolved in 50 mL of a mixture of distilled water and methanol (50/50, v/v) degree of acetylation 90% pore size ranged from 5 to 40 mm.	Human DPSCs	DSPP, DMP-1, ON, osteopontin	Glycol chitin-based thermo-responsive hydrogel scaffold promoted the proliferation and odontogenic differentiation of human DPSCs.
El Ashiry et al., 2018 [182]	Chitosan hydrogel	In vivo		Chitosan 1 g; 77% deacylation, high molecular weight, was dissolved in 2% acetic acid.	DPSCs		DPSCs and GFs incorporated in chitosan hydrogel can regenerate pulp–dentin-like tissue in non-vital immature permanent teeth with apical periodontitis in dogs.
Wu et al., 2019 [180]	Chitosan hydrogel	In vitro	beta-sodium glycerophosphate added to chitosan (CS/β-GP).	viscosity: 200–400 m Pa·s 2% (w/v) chitosan solution 56% (w/v) beta-sodium glycerophosphate (β-GP) solution CS: β-GP is 5/L	DPSCs	VEGF, ALP, OCN, Osterix, DSPP	CS/β-GP hydrogel could release VEGF continually and promote odontogenic differentiation of DPSCs.
Zhu et al., 2019 [181]	Chitosan hydrogel	In vitro	Ag-doped bioactive glass micro-size powder particles added to chitosan (Ag-BG/CS).	Ag-BG/CS pore diameter reaching around 60–120 μm.	DPSCs	OCN, ALP, RUNX-2	Ag-BG/CS enhanced the odontogenic differentiation potential of lipopolysaccharide-induced inflammatory-reacted dental pulp cells and expressed antibacterial and anti-inflammatory activity.
	Hyaluronic Acid Hydrogel
Chrepa et al., 2016 [198]	Hyaluronic acid (HA) hydrogel	In vitro		SCAP/Restylane 1:10 concentration SCAP/Matrigel mixture at 1:1 concentration 1,4-butanediol diglycidyl ether; DVS, divinyl sulphone crosslinking agent	SCAP	ALP, DSPP, DMP-1, MEPE gene	HA injectable hydrogel promoted SCAP survival, mineralization and differentiation into an odontoblastic phenotype.
Yang et al., 2016 [193]	Hyaluronic acid hydrogel	In vivo	HA crosslinked with 1,4-butanediol diglycidyl ether	HA (1.5 × 106 Da) 1,4-Butanediol diglycidyl ether crosslinking agent HA concentration of 20 mg mL−1 gel particles of 0– 400 mm.	Dental mesenchymal cells		HA is an injectable scaffold that can regenerate cartilage and dentin–pulp complex.
Almeida et al., 2018 [195]	Hyaluronic acid hydrogel	In vitro	Photo crosslinking of methacrylated HA incorporated with PL.	High molecular weight (1.5–1.8 MDa) HA 1% (w/v) HA solution (in distilled water) reacted with methacrylic anhydride (10 times molar excess) methacrylated disaccharides% was 10.9 ± 1.07% HA hydrogels incorporating 100% (v/v) PL Met-HA was dissolved at a concentration of 1.5% (w/v) in both of the PBS and PL photoinitiator solutions.	Human DPSCs	ALP, collagen type I A 1 strand (COLIA1)	HA hydrogels incorporating PL increased the cellular metabolism and stimulate the mineralized matrix deposition by hDPSCs.
Silva et al., 2018 [194]	Hyaluronic acid hydrogel	In vitro Ex vivo	HA hydrogels incorporating cellulose nanocrystals and enriched with Platelet lysate (PL).	1 wt. % ADH-HA, 1 wt. % a-HA, 0.125 to 0.5 wt. % a-CNCs in 50 v/v% PL solution.	Human DPSCs		HA hydrogel enabled human DPSCs survival and migration.
Zhu et al., 2018 [197]	Hyaluronic acid hydrogel	In vivo	Crosslinked HA hydrogel	Crosslinked HA gel (24 mg/mL) mixed with cells at 1:1–1:1.4 (v/v, i.e., gel/cells) ratio with final cell concentration of *2 · 107/mL 1,4-butanediol diglycidyl ether; DVS, divinyl sulphone crosslinking agent 9%	DPSCs	Nestin, DSPP, DMP-1, Bone sialoprotein	HA hydrogel regenerated pulp-like tissue with a layer of dentin-like tissue or osteodentin along the canal walls.
Niloy et al., 2020 [196]	Hyaluronic acid hydrogel	In vitro	Converting sodium salt of HA into Tetrabutylammonium salt and subsequent conjugation of Aminoethyl methacrylate (AEMA) to HA backbone.	AEMA-HA macromers of two different molecular weights (18 kD and 270 kD) 1 g of H/100 mL deionized water, mixed with 12.5 g of ion exchange resin was converted from its hydrogen form to its TBA form AEMA hydrochloride (0.25 equivalent to HA repeat units)	DPSCs	NANOG, SOX2	HA hydrogels have great potential to mimic the in vivo 3D environment to maintain the native morphological property and stemness of DPSCs.
	Agarose Hydrogel
Cao et al., 2016 [214]	Agarose hydrogel	In vitro	Calcium chloride (CaCl 2) Agarose hydrogel	1.0 g of Agarose powder, 1.9 g of CaCl 2 H20			Agarose hydrogel promoted occlusion of dentinal tubules and formation of enamel prisms-like tissue on human dentin surface.
	Cellulose Hydrogel
Teti et al., 2015 [227]	Cellulose hydrogel	In vitro	Hydroxyapatite was loaded inside CMC hydrogel	Degree of carboxymethylation of 95% (CMC) (average MW 700 KDa)	DPSCs	ALP, RUNX2, COL-IA1, SPARC, DMP-1, DSPP	CMC–hydroxyapatite hydrogel up regulated the osteogenic and odontogenic markers expression and promoted DPSCs adhesion and viability.
Aubeux et al., 2016 [226]	Cellulose hydrogel	In vitro	Silanes grafted along the hydroxy-propyl-methyl-cellulose chains.	nanoporous macromolecular structure. Pores have an average diameter of 10 nm		TGF-b1	Cellulose hydrogel enhanced non-collagenous matrix proteins release from smashed dentin powder.
Iftikhar et al., 2020 [228]	Cellulose hydrogel	In vitro		The surface area, average pore size and particle size of BAG (45S5 Bioglass®) were 65m2/g 5.7 nm, and 92 nm, respectively.	MC3T3-E1 cells differentiated into osteoblasts and osteocytes.		The prepared injectable bioactive glass, hydroxypropylmethyl cellulose (HPMC) and Pluronic F127 was biocompatible in an in vitro system and has the ability to regenerate dentin.
	Extracellular Matrix Hydrogel
Chatzistavrou et al., 2014 [246]	Extracellular matrix (ECM) hydrogel	In vitro	silver-doped bioactive glass (Ag-BG) incorporated into ECM	Ag-BG powder form with particle size < 35μm. ECM concentration of 10 mg/mL ECM60/Ag-BG40, ECM50/Ag-BG50, ECM30/Ag-BG70 weight ratio.	DPSCs		Ag-BG/ECM presented enhanced regenerative properties and anti-bacterial action.
Wang et al., 2015 [245]	ECM hydrogel	In vivo In vitro	silver-doped bioactive glass (Ag-BG) incorporated into ECM	Ag-BG powder form with particle size < 35μm. ECM pepsin digest stock solutions of 10 mg ECM/mL (dry weight) Ag-BG: ECM = 1:1 in wt. %.	DPSCs		Ag-BG/ECM showed antibacterial property, induced dental pulp cells proliferation and differentiation. The in vivo results supported the potential use of Ag-BG/ECM as an injectable material for the restoration of lesions involving pulp injury.
Li et al., 2020 [247]	ECM hydrogel	In vitro		Pre-gel solution was diluted into 0.75% w/v and 0.25% w/v.	Human DPSCs	DSPP, DMP-1	decellularized matrix hydrogel derived from human dental pulp effectively contributed to promoting human DPSCs proliferation, migration, and induced multi-directional differentiation.
Holiel et al., 2020 [248]	ECM hydrogel	Clinical trial	Human treated dentin matrix hydrogel was dispersed in sodium alginate solution	Particle sized powder (range 350–500 μm) 5% (w/v) of sodium alginate 0.125 g of sterile human treated dentin matrix was dispersed in the sodium alginate solution with a mass ratio of 1:1			Treated dentin matrix hydrogel attained dentin regeneration and conservation of pulp vitality.