Table 2.

Hydrogels for wound healing applications.

Type of Hydrogel	Analysis	Animal Model/Cell Line/Microorganism	Biological Activity	References
poly(vinyl alcohol) (PVA)/β-glucan (β-1,6-branched-β-1,3-glucan)	-determination of cellular morphology - assessment of cell viability - wound healing evaluation in vivo - assessment of skin regeneration (in vitro scratch wound healing assay) - evaluation of granulation tissue formation (hematoxylin and eosin staining) - epidermal thickness measurements - assessment of localization of cytokeratin proteins in the skin tissue - examination of protein expression (Western blotting	in vitro: - human dermal fibroblasts - human keratinocytes (HaCaT) in vivo: - mice	- wound healing acceleration - development of skin appendages in regenerated skin tissue - formation of capillary vessel - better granulation and reepithelialization - hierarchical arrangement of dermal layers - increase in expression level of transforming growth factor (TGF)-β3,cytokeratin 10 (K10), and cytokeratin 14 (K14) in the skin tissue - no detectable changes in cell morphology - migration and proliferation of keratinocytes and fibroblasts enhancement - skin regeneration - significantly faster wound closure - skin regeneration around the wound site - increase intranslational levels of K10, K14, and TGF-β3 proteins	[126]
dextran hydrogel	- evaluation of progress in wound healing - assessment of cell infiltration - assessment of macrophages and neutrophils accumulation - examination of aniogenic response - assessment of neovascularization - immunohistochemical analysis (Masson’s trichrome and alpha-smooth muscle actin staining) - assessment of blood flow surrounding the wound (laser Doppler) - analysis of the regenerated skin structure	in vivo: - mice - murine burn wound model	- promotion of remarkable neovascularization - promotion of dermal regeneration with complete skin appendages - facilitating early infiltration and degradation of inflammatory cells - promotion of the infiltration of angiogenic cells into healing wounds - mature epithelial structure with hair follicles and sebaceous glands development - acceleration of the recruitment of endothelial and cell progenitors into the wound area	[127]
self-crosslink able dextran-isocyanatoethyl methacrylate-ethylamine hydrogel (DexIEME	- evaluation of new skin regeneration in murine and porcine models - assessment of newly regenerated hair follicles - polarization of pro-inflammatory (M1)/anti-inflammatory (M2) macrophages - assessment of regenerative capacity in burn scars on mice - histological examination (hematoxylin and eosin staining) - assessment of regenerative capacity in deep full skin injury in pigs	in vitro: - human monocytic leukemia (THP-1)cell line in vivo: - murine and porcine models	- low pro-inflammatory response in murine and porcine models - regeneration of full skin structures with appendages on both pre-existing scars and acute wounds - promotion of complete skin regeneration with hair regrowth on preexisting scars - promotion of M2 macrophage phenotype - more mature wound - retention of the reticulated epithelial layer by new skin - more adipose tissue within the newly regenerated skin in both animal models - attenuation of scar formation	[128]
hybrid dextran hydrogel with incorporated curcumin encapsulated PEG-PLA(poly(lactide)-block-poly(ethylene glycol))	- evaluation of capacity to control curcumin release - histological study (hematoxylin and eosin staining) - collagen staining using Masson’s trichrome method - evaluation of neovascularization - assessment of expression of CD31 and vimentin in wound tissue	in vivo: - male BALB/C mice - murine full thickness wound model	- sustained release of curcumin from dextran hydrogel - significantly augment the reepithelialization of epidermis; increase in collagen deposition in the wound area - increase in fibroblast/mesenchymal cell density - acceleration of angiogenesis and fibroblast accumulation - increase tissue granulation - more mature epithelial structure with hair follicles and sebaceous glands - acceleration of the wound healing process	[129]
PVA/dextran/chitosan	- assessment of cell proliferation - determination of cytotoxicity (WST-1 assay) - evaluation of antimicrobial effect	in vitro: - mouse NIH-3T3 fibroblasts - Escherichia coli - Staphylococcus aureus	- antimicrobial ability to both Gram (+) and Gram (−) bacteria - no adverse effect on cell growth - dextran concentration-dependent stimulation of NIH-3T3 cell proliferation - improvement of cell adhesion to hydrogel with higher dextran concentration	[130]
cyclodextrin/cellulose hydrogel with gallic acid	- evaluation of antibacterial activity - assessment of cytotoxicity	in vitro: - 3T3 fibroblasts - Staphylococcus epidermidis - Staphylococcusaureus - Klebsiellapneumoniae	- sustained gallic acid release - significant reduction in the growth of all three bacteria - no cytotoxic effect on fibroblasts - no release of substances potentially toxic to fibroblasts	[125]
hydroxyethyl cellulose (HEC) supplemented hydrogel loaded with tungsten oxide (WO3)	- assessment of cytotoxicity of hydroxyethyl cellulose (HEC) with/without WO3 - determination of healing capacity - evaluation of anti-inflammatory activity(lipopolysaccharide-stimulated inflammation) - evaluation of antibacterial activity	in vitro: - human dermal fibroblast cells - white blood cells - Shigella sp. - Salmonella sp. - Pseudomonas aeruginosa - Bacillus cereus -Staphylococcus aureus	- antibacterial activities against pathogenic Gram-negative and Gram-positive strains - no morphological changes in the examined cells - improving tungsten oxide safety toward normal human cells (white blood cells and dermal fibroblast) - suppressed an abnormal immune response via normalization proinflammatory cytokine	[131]
bacterial cellulose/acrylic acid hydrogel containing keratinocytes and fibroblasts	- assessment of wound closure in vivo - histological analysis (hematoxylin-eosin and Masson’s trichrome staining) - determination of COL-1, CK-14, involucrin, and α-SMA expression in the tissue - assessment of regenerated skin ultrastructure (transmission electron microscopy)	in vitro: - human epidermal keratinocytes - human dermal fibroblasts in vivo: - male athymic mice(CrTac:NCr-Foxn1nu) -skin samples from six consenting patients	- acceleration on burn wound healing in vivo - confirmed by the presence of more mature keratinocytes - increase in involucrin expression - complete reepitheliation of the wound area by day 13 - decrease in cytokeratin 14 antibody and α-SMA expression - increased expression of collagen type I - more organized skin and collagen structures in the skin layer - a greater deposition of collagen in the mice - promotion of skin regeneration - the appearance of regenerated skin similar to normal skin	[132]
chitosan–cellulose nanofiber (CS–CNF) composite self-healing hydrogels	- investigation of cell proliferation rate by confocal microscopy and CCK-8 assay - evaluation of the oxygen metabolism and mitochondrial function of the cells - determination of nestin, glialfibrillary acidic protein (GFAP), CNPase, β-tubulin and microtubule-associated protein 2 (MAP2) gene expression for cells in chitosan–cellulose nanofiber (CS–CNF) hydrogels - assessment of nestin, GFAP, CNPase, β-tubulin and MAP2 protein expression of NSCs in CS–CNF hydrogels by immunostaining - evaluation of therapeutic function in vivo	in vitro: - neural stem cells (NSCs) from adult mouse brains in vivo: - adult wild-type zebrafish	- promotion of neural regeneration - the increase in the efficiency of mitochondrial electron transfer - strong relationship between oxygen metabolism and hydrogel self-healing properties - the increase in nestin, GFAP, CNPase, and MAP2 gene expression - lower level of β-tubulin (the early neural marker) - increase in the expression level of nestin, GFAP, CNPase, and MAP2 proteins - better regeneration in cerebellar injury of adult zebrafish	[133]
chitosan hydrogel containing fibroblast growth factor-2	- assessment of contraction and speed of wound closure in mice - evaluation of granulation tissue formation - assessment of capillary formation and epithelialization - histological examination (hematoxylin-eosin staining)	in vitro: - human umbilical vein endothelial cells (HUVECs) in vivo: - healing-impaired diabetic (db/db) C57BL/6female mice and their normal (db/+) littermates	- significant induction of wound contraction and accelerated wound closure in mice - accelerating wound closure by adding FGF-2 to the chitosan hydrogel in db/db mice, but not in db/+ mice - advanced granulation tissue and capillary formation - replacement of almost all necrotic tissues with new granulation tissue after injury on day 16 - significant epithelialization - stimulation of HUVEC growth, but loss of this ability after washing with phosphate-buffered saline (PBS) hydrogels for more than 3 days	[134]
rutin-conjugated chitosan-poly(ethylene glycol)-tyramine (RCPT) hydrogel	- evaluation of cell cytotoxicity - assessment of wound healing - evaluation of neoepithelium and granulation tissue formation - assessment of proteins formation	in vitro: - L929 mouse fibroblasts in vivo: - male Sprague-Dawley rats	- increase in fibroblasts proliferation at low concentration - very low cytotoxicity at high concentrations - increase in fibroblasts proliferation by releasing rutin - enhancement of wound healing - induction of better defined formation of neoepithelium and thicker granulation - more new blood vessels in wound - increase in the formation of extracellular proteins, primarily collagen	[135]
gelatin/CM-chitosan hydrogel	- assessment of hydrogel cytotoxicity - evaluation of the ability of NIH 3T3 cells to proliferate on or into the three-dimensional structure of hydrogel	in vitro: - L929 mouse fibroblasts - NIH/3T3 fibroblasts	- promotion of cell attachment on the hydrogel surface - acceleration of NIH 3T fibroblasts growth on the hydrogel - excellent cytocompatibility of the hydrogels	[136]
alginate/chitin/chitosan/fucoidan hydrogel	- assessment of hydrogel cytotoxicity and stimulatory effects on dermal fibroblast cells (DFCs) and dermal microvascular endothelial cells (DMVECs) - histological examination of removed skin and wound tissue samples (hematoxylin-eosin staining) - histological observations on repair of healing-impaired wounds	in vitro: - human dermal fibroblast cells (DFCs) - dermal microvascular endothelial cells (DMVECs) in vivo: - male Sprague-Dawley rats	- no cytotoxicity to DFCs and DMVECs - minor effect on healing of wounds not treated with mitomycin C - significant stimulation of repair of mitomycin C-treated healing-impaired wounds in rats - promotion of tissue granulation and capillary formation - positive effects on wound closure - progress in wound contraction and reepithelialization	[137]
chitosan hydrogel/honey	- assessment of antimicrobial activity - evaluation of granulation and fibrotic tissue formation - evaluation of inflammatory response - determination of reepithelialization - evaluation of wound shrinkage effects	in vitro: - Staphylococcus aureus - Bacillus cereus - Escherichia coli - Pseudomonas aeruginosa - Candida albicans in vivo: - male Wistar rats - rat full-thickness wound model	- significant decrease in the minimum inhibitory concentration against bacteria after the combination of a chitinase hydrogel with honey - significant improvement in the inflammatory index on days 3 and 7 - increase in granulation tissue formation - significantly higher fibrotic tissue formation index - increase in the level of reepithelialization	[138]
water-soluble chitin hydrogel	- histological examination of the wounded skins (hematoxylin and eosin staining, Masson’s trichrome staining) - determination of collagen-hydroxyproline in wounded skin - assessment of reepithelialization	in vivo: - animal model: rats	- acceleration of wound healing - significant reepithelialization - granulation tissue replacement in the wound by fibrosis - healing of hair follicles in rats - high skin tensile strength and the formation of the correct collagen fiber system	[139]
alginate hydrogels coated with chitosan	- assessment of cell morphology - determination of cytotoxicity - evaluation of antibacterial activity	in vitro: - human mesenchymal stromal cells (MSCs) - Escherichia coli	- unmodified cell morphology - no inhibition of MSC growth and proliferation - bacterial inactivation higher than 99% after 3 h - complete killing of bacteria after 24 h	[140]
gelatin-alginate hydrogel	- assessment of cytotoxicity (MTT assay) - determination of cell adhesion - live/dead staining (calcein acetoxymethyl–propidium iodide)	in vitro: - HFF-1 foreskin fibroblast cells	- time-dependent partial inhibition of cell viability - reduction of cell adhesion properties - presence of only living cells, no dead cells	[141]
collagen-hyaluronic acid hydrogel	- assessment of cytotoxicity (MTT assay) - evaluation of cells proliferation encapsulated into hydrogels at different time using confocal laser scanning microscopy - measurement of the cells’ attachment with scanning electron microscope - measurement of vascular endothelial growth factor secretion by human microvascular endothelial cells (HMECs) with enzyme-linked immunosorbent assay (vascular endothelial growth factor (VEGF)-ELISA) - assessment of antibacterial activity - measurement of biocompatibility and inflammatory in vivo - assessment of wound healing	in vitro: - human microvascular endothelial cells (HMECs) - fibroblasts (COS-7) - Escherichia coli - Staphylococcus aureus in vivo: - mice - mice full-thickness skin wound model	- good viabilities of HMECs and COS-7 cells - biocompatibility - excellent differential behaviors of cells in hydrogel - increase in the amount of vascular endothelial growth factor (VEGF) up to day 7 of culture - no foreign body reaction like immigration of giant cells into hydrogel - only few inflammatory infiltrations in the interface of hydrogel and tissue - gradual reduction in the number and infiltration of inflammatory cells - normal morphology with no signs of pathology for all organs, including heart, liver, spleen, lungs, and kidney - good adhesion of the hydrogel to the skin - killing about 55% of E. coli and 47% of S. aureus after incubating for 3 h - less colony-forming units of E. coli or S. aureus - disappearance of redness of wounds	[142]
hyaluronic acid-based hydrogels	- measurements of the white blood cell (WBC) level - histological staining with hematoxylin - immunochemistry analyses - determination of α-SMA, VEGF, and TGF-β1 gene expression (fluorescence-based quantitative PCR)	in vivo: - New Zealand white rabbits	- wound reduction - reduction of fluid secretion and thinner scab formation - faster recovery of the animals - increase in the level of white blood cells after surgery, reaching a maximum on day 7, followed by a decrease to a constant value - hair follicle renewal - increase in α-SMA expressions, which alleviated wound inflammation in the first few days after surgery and alleviated scar formation by reducing TGF-β1 levels. - faster self-healing compared to control, manifested by a faster increase in TGF-β1 level and higher VEGF levels after 7 days and lower after 14 days compared to controls - increase in α-SMA, TGF-β1, and VEGFmRNA levels from day 3, reaching the highest level on day 7 or 14,followed by a continuous decrease - increase in VEGF expression, which promotes skin regeneration through neovascularization	[143]
heparin-based hydrogel with loaded human epidermal growth factor (hEGF)	- evaluation of human epidermal growth factor (hEGF) release in vitro - histological examination (hematoxylin and eosin staining) - determination of the total amount of collagen in the regenerated skin(hydroxyproline assay) - immunochemistry staining with an anti-wide spectrum cytokine antibody by using LSAB+system-HRP kit	in vivo: - male BALB/c mice	- sustained release profile of hEGF in vitro - significantly accelerated closure of wounds - closing the epidermal layer and regenerating the extracellular matrix - denser epithelialization, blood vessel, hair follicles, and sebaceous gland formation - high similarity in the structure of the skin tissue of mice after injury after applying the hydrogel to normal skin tissue - accelerating wound closing and facilitating wound remodeling into normal skin tissue - increased proliferation and differentiation of keratinocytes - improvement of keratinocyte migration to the site of injury - hair follicles formation at the recovered tissue	[144]
collagen hydrogel	- assessment of wound morphology - determination of scarring shape and physical properties - histological examination (hematoxylin and eosin staining) - assessment of angiogenesis changes	in vivo: - Sprague-Dawley diabetic rat	- faster wound healing - increasing the speed and quality of full thickness wound healing in diabetic rats - significantly higher quantity of fibroblasts - increase in the number of new capillaries	[157]
collagen gel	- assessment of Mrc-1 (a reparative M2 macrophage marker) gene expression - assessment of anti-inflammatory cytokine interleukin (IL)-10 and fibroblast growth factor-basic (β-FGF)expression - immunohistochemical staining (with anti-vimentin and) DAPI)(4′,6-diamidino-2-phenylindole) - evaluation of mature collagen deposition - analyses of wound tissues	in vitro: - human THP-1 monocytes in vivo: - Yorkshire pigs - swine model of chronic ischemic wounds	- upregulation of Mrc-1 expression in vitro - induction of (IL)-10 and (β-FGF) expression in vitro - increased expression of CCR2 (M2 macrophage marker) - upregulation of transforming growth factor-β, vascular endothelial growth factor, von Willebrand’s factor, and collagen type I expression in ischemic wounds - increase in endothelial cells proliferation - significant increase in fibroblasts in ischemic wound-edge tissue - higher abundance of mature collagen	[158]
injectable tannic acid-loaded gelatin-based hydrogels blended with gellan gum	- evaluation of antibacterial activity using agar disc diffusion test - assessment of wound healing efficacy - histological examination (hematoxylin and eosin (H&E) and Masson’s trichrome staining) - measurements of epidermal thickness - assessment of skin tissue formation - evaluation of collagen deposition - examination of cell cytotoxicity and migration	in vitro: -murine L929 fibroblasts - Escherichia coli - Staphylococcus aureus - methicillin-resistant Staphylococcus aureus in vivo: - BALB/c mice	- long-term antibacterial efficacy - sustained release of tannic acid in vitro - significant wound reduction - no scars - skin fully covered with hair - 100% healing rate - increase in the thickness of the epidermis - effective granulation tissue formation - greater accumulation and collagen fiber content - effective skin regeneration and function restoration - increase in L929 cell viability	[159]
gelatin/oxidized alginate-tyramine hydrogels	- examination of tissue adhesive strength - assessment of residue H2O2 level - evaluation of antibacterial activity - measurements of cytotoxicity in vitro (WST-1 assay)	in vitro: - human dermal fibroblasts (hDFBs) - Escherichia coli - Staphylococcus aureus in vivo: - porcine skin	- high antibacterial ability due to the constant release of H2O2 - low adhesive strength of hydrogel allowing for the easy dressing removal - no cytotoxic effect on test cells	[160]
mesenchymal stem cells (MSCs) spheroid-containing fibrin hydrogels	- evaluation of cytokine bioactivity to stimulate endothelial cells and macrophages - assessment of anti-inflammatory potential - capacity of entrappedMSC spheroids to promote angiogenesis in a three-dimensional skin-like environment assessment	in vitro: - human bone marrow-derived MSCs - diabetic human microvascular cells (HMVECs) - raw 264.7 mouse macrophages in vivo: - human skin equivalent (HSE) model	- cytokine secretion with potential proangiogenic and anti-inflammatory effects - enhancing angiogenesis in the equivalent of human skin - increased endothelial cell penetration - stimulation of sprouts and greater invasion distance into the wound - dependence of VEGF and prostaglandin E2 (PGE2) secretion on the mechanical properties of hydrogels	[161]
human hair keratin-based hydrogel	- assessment of cell proliferation - immunocytochemical staining - evaluation of the cellular interaction of keratin with HaCaT cells - assessment of cell migration - measurements of gene expression	in vitro: - human keratinocyte cell line (HaCaT) in vivo: -male C57BL/6J mice	- stronger expression of vimentin and fibroblastic spindle shape in HaCaT cells - increasing the number of cells migrating into the space between confluent cell colonies - upregulation of the expressions of migration-related genes such as integrin αV, integrin α5, integrin β1, and integrin β6 - increase in mRNA expressions of integrin αV, integrin β5, integrin β6, fibronectin, Snail and vimentin - induction of molecular expressions of integrin β1 and vimentin - fully recovering the skin aspect without any signs of scab and skin contraction in animal test - reepithelialization of skin and the proliferation of dermal fibroblast with adnexa in dermis in vivo - effective wound remodeling with skin adnexa in vivo - high rate of the proliferation of dermal fibroblasts with skin adnexa and dense dermal fiber in vivo - full wound repair with skin appendages - regeneration of muscle tissue - increasing the number of hair follicles	[162]
feather keratin hydrogel	- evaluation of wound healing in vivo - assessment of cellular responses and vascularization (hematoxylin and eosin staining) - determination of collagen deposition in the wound skin (trichrome staining) - determination of the inflammatory cytokines IL-1β, IL-6, and TNF-α levels in the serum of rats (ELISA)	in vivo: - male Sprague-Dawley rats	- significant acceleration of wound healing - approximately 90% wound closure within 10 days - complete reepithelialization after 21 days - more hair follicles - more new capillaries around the inflammatory cells - maturation of the epidermis over the wound and restoration of new skin to normal after 28 days - acceleration of collagen deposition - thicker collagen fibers - no adverse systemic toxicity in rats after hydrogel implantation - no obvious organ damage and significant histopathological differences in the tissue organs in rats - no or minimal inflammatory response - no significant elevations in cytokines levels	[163]
injectable silk fibroin hydrogel	- assessment of cell migration - evaluation of wound closure - histological analysis - determination of collagen type I and III, TNF-α, CD68, CD163, and glyceraldehyde-3- phosphate- dehydrogenase (GAPDH) expression (qRT-PCR analysis) - determination of suprabasal keratin marker cytokeratin 10 (CK10), basal keratin marker cytokeratin 14 (CK14), and marker of terminally differentiated keratinocytes involucrin (INV) expression	in vitro: - HaCaT cells in vivo: - female Wistar albino rats - full thickness third-degree burn wounds	- complete wound closure after 21 days - two-fold accelerated healing rate in a regenerative manner - rapid development of granulation tissue - enhanced, early reepithelialization - mature epidermo-dermal regeneration - increase in HaCaT cells proliferation - formation of small blood vessels on day 7 - ten-fold increase in vessel density - significant upregulation and downregulation of collagen type I and III during different stages of wound healing - increase in collagen type I secretion from day 7 to 21 - higher secretion of collagen type III - visible expression of cytokines CK10 and CK14	[164]
heparinized silk fibroin hydrogels loading FGF1 (fibroblast growth factor 1	- assessment of platelet-derived growth factor (PDGF) and growth factor TGF-β expression (ELISA) - histological evaluation using hematoxylin and eosin and Masson’s trichrome staining - assessment of fibroblast L929 cells proliferation and migration	in vitro: - fibroblast L929 cells in vivo: - Sprague-Dawley rats - full-thickness excisional wounds	- increase in expression level of PDGF on day 7 - no statistically significant difference for expression of TGF-β1 - acceleration of dermis formation - acceleration of epidermal differentiation into hair follicles and sebaceous glands - improvement in small scars healing	[165]