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The overexpression of reactive oxygen species (ROS) contributes to the pathogenesis of numerous diseases such as atherosclerosis, myocardial infarction, cancer, and chronic inflammation. Therefore, the development of materials that can locally control the adverse effects resulting from excessive ROS generation is of great significance. In this study, the antioxidant gallic acid-conjugated gelatin (GGA) was introduced into gelatin-hydroxyphenyl propionic (GH) hydrogels to create an injectable hydrogel with enhanced free radical scavenging properties compared to pure GH hydrogels. The modified hydrogels were rapidly formed by an HRP-catalyzed cross-linking reaction with high mechanical strength and biodegradability. The resulting GH/GGA hydrogels effectively scavenged the hydroxyl radicals and DPPH radicals, and the scavenging capacity could be modulated by varying GGA concentrations. Moreover, in an in vitro H2O2-induced ROS microenvironment, GH/GGA hydrogels significantly suppressed the oxidative damage of human dermal fibroblast (hDFBs) and preserved their viability by reducing intracellular ROS production. More importantly, the ROS scavenging hydrogel efficiently accelerated the wound healing process with unexpected regenerative healing characteristics, shown by hair follicle formation; promoted neovascularization; and highly ordered the alignment of collagen fiber in a full-thickness skin defect model. Therefore, we expect that injectable GH/GGA hydrogels can serve as promising biomaterials for tissue regeneration applications, including wound treatment and other tissue repair related to ROS overexpression. STATEMENT OF SIGNIFICANCE: Recently, many researchers have endeavored to develop injectable hydrogel matrices that can modulate the ROS level to normal physiological processes for the treatment of various diseases. Here, we designed an injectable gelatin hydrogel in which gallic acid, an antioxidant compound, was conjugated onto a gelatin polymer backbone. The hydrogels showed tunable properties and could scavenge the free radicals in a controllable manner. Because of the ROS scavenging properties, the hydrogels protected the cells from the oxidative damage of ROS microenvironment and effectively accelerated the wound healing process with high quality of healed skin. We believe that this injectable ROS scavenging hydrogel has great potential for wound treatment and tissue regeneration, where oxidative damage by ROS contributes to the pathogenesis.

Current stem cell-based therapy for cardiac repair and regeneration after myocardial infarction (MI) is not readily translatable into clinical scenarios due to the low retention and survival of the transplanted cells. Here, we evaluated a simple and feasible design of gelatin-hydroxyphenyl propionic acid (GH) hydrogel as an in situ cross-linkable and injectable cell delivery platform for cardiac tissue regeneration. The GH hydrogel exhibited improved cell retention and survival in vitro and in vivo when encapsulating mouse bone marrow-derived mesenchymal stem cells (MSCs) that were used as promising therapeutic candidates for stem cell therapy. Moreover, we demonstrated that MSC-encapsulating GH hydrogels led to a significant improvement in cardiac functional metrics, such as the fractional shortening (FS), ejection fraction (EF), and end-systolic volume (ESV). Similarly, MSC-encapsulating GH hydrogels induced favorable effects in the cardiac structures of the infarcted heart, producing less fibrosis and thicker infarcted walls. These results suggest that GH hydrogels can be used as an instructive cell delivery platform to provide a suitable microenvironment for transplanted cells; therefore, their in vivo applications combined with MSCs may provide great potential for repair and regeneration of injured cardiac tissues after MI.

Hydrogels that mimic the composition and properties of the extracellular matrix have attracted great attention as potential polymeric scaffolds for tissue engineering applications. In this study, an injectable hydrogel composed of hyaluronic acid and gelatin was developed via the oxidative coupling reaction using horseradish peroxidase (HRP). The hydrogel was prepared by mixing two phenol-conjugated polymer solutions, hyaluronic acid-tyramine (HA-TA) and gelatin-hydroxyphenyl propionic acid (GH), in the presence of HRP and hydrogen peroxide (H202). The gelation rate and mechanical properties of composite hydrogel were controlled by adjusting the HRP and H202 concentrations, respectively Compared to the pure HA-TA hydrogels, the stability and cellular behaviors of composite hydrogel improved significantly In addition, the injectable hydrogel system showed good performance in 3D printing with high cell viability after one day of printing. The results suggest that enzymatically crosslinked HA-TA and GH hybrid (HA-TA/GH) composite of HA-TA and GH hydrogel has the potential as a material for tissue engineering and 3D printing biofabrication.

Injectable hydrogels can serve as therapeutic vehicles and implants for the treatment of various diseases as well as for tissue repair/regeneration. In particular, the horseradish peroxidase (HRP) and hydrogen peroxide (H2O2)-catalyzed hydrogelation system has attracted much attention, due to its ease of handling and controllable gel properties. In this study, we introduce calcium peroxide (CaO2) as a H2O2-generating reagent to gradually supply a radical source for the HRP-catalyzed crosslinking reaction. This novel therapy can create stiff hydrogels without compromising the cytocompatibility of the hydrogels due to the use of initially high concentrations of H2O2. The physico-chemical properties of the hydrogels can be controlled by varying the concentrations of HRP and CaO2. In addition, the controlled and sustained release of bioactive molecules, including H2O2, O2, and Ca2+ ions, from the hydrogels could stimulate the cellular behaviors (attachment, migration, and differentiation) of human mesenchymal stem cells. Moreover, the hydrogels exhibited killing efficacy against both Gram-negative and Gram-positive bacteria, dependent on the H2O2 and Ca2+ release amounts. These positive results suggest that hydrogels formed by HRP/CaO2 can be used as potential matrices for a wide range of biomedical applications, such as bone regeneration and infection treatment.

Adhesive hydrogels offer considerable potential in biomedical applications because of the excellent integration between materials and tissues. Horseradish peroxidase (HRP)-catalyzed hydrogelation, with advantages that include ease of handling, simplicity in material design, high biocompatibility, and processability, is becoming a promising candidate to be engineered as an adhesive hydrogel. This review summarizes recent development of HRP-mediated hydrogelation towards the bioadhesive field, and other applications requiring the tissue adhesion of hydrogels. From the viewpoint of adhesive hydrogel designation, the basic chemistry of the adhesive mechanism, combinations of interactions, and currently commercial bioglues are summarized. The review recapitulated the adhesive hydrogel applications, the clinical translation ability, the controlling hydrogelation kinetics, the related physicochemical characteristics, cytotoxicity, and animal studies. Looking to the future, to make more substantial contributions to the bioadhesive field, or to various biomedical applications, higher adhesion should be continuously improved, and greater emphasis should be placed on clinical trials.

The development of antibacterial wound dressing with easily removable characteristic and highly exudate absorption is becoming a major requirement in the treatment of chronic wounds. In this study, the in situ forming gelatin/oxidized alginate-tyramine hydrogels (GH/OATA) were designed to overcome the current limitations. These hybrid hydrogels were quickly and controllably formed from several seconds to a few minutes by horseradish peroxidase/H2O2. The hybrid hydrogels at the optimal composition of GH/OATA 50/50 v/v showed the unique feature including stiffness being similar to skin, high swelling ratio due to the hydrophilic property of oxidized alginate, low adhesive strength allowing for the easy dressing removal without pain. In addition, the hybrid hydrogels could release the H2O2 amount sustainably to kill bacteria. Furthermore, 3D culture of human dermal fibroblasts inside hybrid hydrogels achieved more than 80% cell proliferation which implies the cell-compatibility.

Recently, human hair-derived keratin protein has been recognized as biomaterial with high potential due to its excellent bioactivity and biocompatibility. Here, we designed human hair-derived keratin-based in situ cross-linkable hydrogels that can serve as a dynamic matrix for the enhanced wound healing. We demonstrated that our developed the keratin-based hydrogels accelerated re-epithelization and wound healing process in a full-thickness animal. Also, we investigated the molecular mechanism underlying the enhanced wound healing. In conclusion, our study proposes that human hair-derived keratin-based hydrogels with excellent bioactivity have great potential for use as a wound healing material, along with its other biomedical applications.

Nitric oxide (NO) is an endogenous molecule with many critical biological functions that depend on its concentration. At high levels, NO provides broad-spectrum antibacterial effects through both its pathogen inhibition and killing abilities. However, its short half-life has been a great challenge to its clinical application in pharmaceutical forms. In this study, we incorporated the NO donor S-nitrosothiolated gelatin (GelSNO) into injectable gelatin-based hydrogels (GHs) to controllably release NO. Under catalysis by horseradish peroxidase, H2O2 oxidizes phenol moieties functionalized on gelatin to quickly form phenol-phenol crosslinks that encapsulate GelSNO. Through thermal, visible light, and oxidizing agent-driven mechanisms, NO is released from the GH/GelSNO hydrogels. By varying the GelSNO concentration, the release of NO was controllable in a wide range, 0.054–2.050 μmol/mL, for up to 14 days. In addition, NO release was fine-tunable as a function of H2O2 concentration. Notably, the in situ formation of peroxynitrite (ONOO−) that produces potent antibacterial effects originated from H2O2 residues and nitrous acid formed by NO and oxygen in aqueous solution. The Kirby-Bauer method indicated that there was an inhibition zone against both Escherichia coli and Staphylococcus aureus incubated with GH/GelSNO hydrogels. The AlarmaBlue assay showed that E. coli and S. aureus were completely killed at NO concentrations of 0.39 and 0.58 μmol/mL. Cytotoxicity tests of GH/GelSNO hydrogels on human dermal fibroblasts at the indicated bactericidal NO concentrations induced no cell toxicity. In summary, GH/GelSNO hydrogels may provide a new platform for topical delivery of NO in treating wound infections and for various biomedical applications. Statement of Significance NO is an effective antibacterial agent even in cases of antibiotic-resistant bacteria. Moreover, its intermediate, peroxynitrite, has been reported to have a much higher ability to kill bacteria. In this study, we utilized injectable GH/GelSNO hydrogels formed by HRP/H2O2 reaction not only to control NO release but also to generate peroxynitrite in situ from released NO and H2O2 residues. The GH/GelSNO hydrogels showed significant antibacterial ability on both gram-positive and negative bacteria, while no cytotoxicity was induced on human dermal fibroblasts. In addition, their tunable chemico-physical properties and controllable NO release within a wide range but narrow scale will make the hydrogels useful in various biomedical applications.

Various types of commercialized wound dressings (e.g., films, foams, gels, and nanofiber meshes) have been clinically used as a physical barrier against bacterial invasion and as wound-healing materials. Although these dressings can protect the wounded tissue from the external environment, they cannot treat the wounds that are already infected with bacteria. Herein, we report in situ H2O2-releasing hydrogels as an active wound dressing with antibacterial properties for treatment of drug-resistant bacterial infection. In this study, H2O2 was used for two major purposes: (1) in situ gel formation via a horseradish peroxidase (HRP)/H2O2-triggered cross-linking reaction, and (2) antibacterial activity of the hydrogel via its oxidative effects. We found that there were residual H2O2 in the matrix after in situ HRP-catalyzed gelling, and varying the feed amount of H2O2 (1–10 mM; used to make hydrogels) enabled control of H2O2 release kinetics within a range of 2–509 μM. In addition, although the gelatin-hydroxy...

Horseradish peroxidase-catalyzed injectable gelatin hydrogels have attracted much attention in various biomedical fields because of their processability, biodegradability, and excellent biocompatib...

A practical approach toward developing dual-functional hydrogels with high adhesiveness and hydrophobic drug delivery is described. An additional Schiff base reaction was introduced into a phenol-phenol crosslinked gelatin hydrogel to markedly increase adhesiveness. Oxidized beta-cyclodextrin (o beta-CD) functionalized with aldehyde groups and possessing a hydrophobic cavity was exploited as a crosslinker in the Schiff base reaction to solubilize the hydrophobic drug. Simply blending gelatin-tyramine (GTA) and ob-CD in the presence of horseradish peroxidase (HRP)/H2O2 rapidly and controllably formed GTAo beta-CD hydrogels in situ. The optimal composition of GTA-o beta-CD hydrogels was found to be 5 wt% GTA (GTA5) with 1 wt% ob-CD. Their adhesiveness was 2.3-and 6.2-fold higher than those of GTA-only hydrogels and fibrin glue, respectively. Their elastic modulus and degradation rate were 1.8-and 1.5-fold higher than those of GTA hydrogels owing to additional imine bonds. Hydrophobic drugs (dexamethasone and curcumin) could be dissolved homogeneously in GTA-o beta-CD matrices with greater loading efficiencies than in GTA matrices. An in vitro test of cell viability using human dermal fibroblasts demonstrated that GTA-o beta-CD hydrogels were cytocompatible. In summary, dual-functional injectable GTA-o beta-CD hydrogels can be used as a promising platform to improve tissue adhesion and hydrophobic drug delivery.

In situ forming hydrogels show promise as therapeutic implants and carriers in a wide range of biomedical applications. They can easily seal or fill damaged tissue, thereby functioning as cell/drug delivery vehicles or hemostats. In this regard, strong and stable adhesion to the surrounding tissue is considered an important parameter to improve the in vivo performance of in situ forming hydrogels. In this study, tissue-adhesive gelatin-based hydrogels were prepared by dual-enzymatic cross-linking, where horseradish peroxidase (HRP) and tyrosinase (Tyr) were used. Tyr was employed to convert phenol groups of gelatin derivatives into o-quinone, which can readily react with nucleophiles (e.g., amines or thiols) on tissue surfaces, thereby resulting in strong tissue adhesion. Incorporating Tyr (0.25 kU mL−1) did not affect the gelation rate or mechanical strength of HRP-cross-linked hydrogels. Importantly, the dual-enzymatically cross-linked hydrogels (GH/HRP/Tyr) exhibited significantly improved adhesive strength (34 kPa), which was superior to single HRP-cross-linked hydrogels (GH/HRP; 19 kPa) and commercially available fibrin glues (7 kPa). These dual-enzymatically cross-linked gelatin-based hydrogels with strong adhesiveness could act as promising bio-adhesives for tissue-regeneration applications.

Hydrogels are widely used as implantable scaffolds and drug delivery carriers for biomedical applications. In particular, in situ cross-linkable hydrogels synthesized via enzyme-mediated reaction have received great attention in the field of injectable biomedical research as they have applications in minimally invasive procedures and have easily controllable physicochemical properties (e.g., gelation time, mechanical properties, etc.) under mild conditions. In this study, we synthesized poly(ethylene glycol) (PEG)-co-polymerized poly(glycerol sebacate) (PGS) polymers (PEG-co-PGS) capable of dissolving in aqueous environments and developed injectable hydrogel platforms via a horseradish peroxidase (HRP)-catalyzed cross-linking reaction. To induce in situ gelling, HRP-reactive phenol moieties (tyramine) were covalently conjugated to the PEG-co-PGS polymers, and hydrogel networks were formed in the presence of HRP and hydrogen peroxide (H2O2). The chemical structures of synthesized polymers were confirmed by 1H NMR spectroscopy, and the physicochemical properties of the hydrogels were assessed under varying concentrations of HRP and H2O2 solutions. The gelation time of PEG-co-PGS hydrogels ranged from 12 s to 237 s based on the HRP concentration (0.02-0.25 mg/mL), and the elastic modulus (16-41 Pa) depended on H2O2 concentration. In vitro cytocompatibility studies in human dermal fibroblasts revealed that PEG-co-PGS hydrogels were highly cytocompatible, with no negative effects on cell morphology and viability. In conclusion, our results suggest that PGS-based injectable hydrogels with multi-tunable properties and good cytocompatibility have tremendous potential as injectable scaffolds for tissue engineering applications.

Recently, numerous studies have focused on the development of scaffolds for skeletal tissue engineering and regeneration with various structures. Among various structures, in situ forming hydrogels have attracted considerable attention because they can provide 3D microenvironments for cells, and their stiffness and elasticity can be easily controlled by physical or chemical means. Over the last decade, graphene oxide (GO) has been widely explored as a potential candidate for biomaterials because of its excellent physicochemical properties and outstanding biocompatibility. In this study, horseradish peroxide-reactive gelatin polymer (GH) hydrogels incorporated with GO were prepared and their physicochemical and biomechanical properties were characterized by scanning electron microscopy, Raman and Fourier transform-infrared spectroscopy, ther-mogravimetric analysis, and rheological study. The cellular behaviors of the C2C12 myoblasts within the GO-incorporated GH (GH/GO) hydrogels were examined by a...

Reactive oxygen species (ROS) have been implicated as a critical modulator for various therapeutic applications such as treatment of vascular disorders, wound healing, and cancer treatment. Specifically, growing evidence has recently demonstrated that transient or low levels of hydrogen peroxide (H2O2) facilitates tissue regeneration and wound repair through acute oxidative stress that can evaluate intracellular ROS levels in cells or tissues. Herein, we report a gelatin-based H2O2-releasing hydrogel formed by dual enzyme-mediated reaction using horseradish peroxidase and glucose oxidase (GO x). The release behavior of H2O2 from the hydrogel matrices can be precisely controlled by varying the GO x concentrations. We demonstrate that H2O2-releasing hydrogels with the optimal condition increase transient upregulation of intracellular ROS levels in the endothelial cells (ECs), enhance proliferative activities of ECs in vitro, and facilitate neovascularization in ovo. We suggest that our H2O2-releasing hydrogels hold great potential as an injectable and dynamic matrix for the treatment of vascular disorders as well as in tissue regenerative medicine.

Oxidized polysaccharide-based hydrogels have recently attracted much attention for tissue regeneration because of their biocompatibility and tissue-adhesive property. In this study, we introduce a new type of chitosan-based hydrogel as a tissue sealant, which was prepared by enzymatic mediation from periodate-oxidized chitosan–polyethylene glycol–tyramine. The oxidized chitosan backbone was expected to enhance the interconnection between the hydrogel layer and collagen in the tissues via the Schiff-base reaction. Proton nuclear magnetic resonance spectra indicated that tyramine-functionalized polyethylene glycol-nitrophenyl carbonate ester was conjugated to the oxidized chitosan. The degree of oxidation of the chitosan backbone was around 14% of the glucosamine units by proton nuclear magnetic resonance. The hydrogel was rapidly formed in situ (within a few seconds) in the presence of horseradish peroxidase and hydrogen peroxide. In vitro experiments with live/dead cell assays showed that the oxidized chitosan-based hydrogel was cytobiocompatible. The hydrogel exhibited high tissue adhesion strength on porcine skin models as well as good tissue-adhesive ability and wound healing properties on rabbit skin. These positive results could be promising for the application of oxidized chitosan-based hydrogels as a wound sealant.

【Facile immobilization of growth factors in hyaluronic acid (HA) hydrogels using dual enzymes is reported in the paper. The hydrogels were formed by using horseradish peroxidase (HRP) and hydrogen peroxide ( $H_2O_2$ ) and transforming growth factor- ${\beta}3$ (TGF- ${\beta}3$ ) was covalently conjugated on the hydrogels in situ using tyrosinase (Ty) without any modifications. For the preparation of hydrogels, HA was grafted with poly(ethylene glycol) (PEG), which was modified with a tyrosine. The gelation times of the HA hydrogels were ranging from 415 to 17 s and the storage moduli was dependent on the concentration of $H_2O_2$ and Ty (470-1600 Pa). A native TGF- ${\beta}3$ (200 ng/mL) was readily encapsulated in the HA hydrogels and 17% of the TGF- ${\beta}3$ was released over 1 month at the Ty concentration of 0.5 KU/mL, while the release was faster when 0.3 KU/mL of Ty was used for the encapsulation (27%). It can be suggested that the growth factors resident in the hydrogels for a long period of time may lead cells proliferating and differentiating, whereas the growth factors that are initially released from the hydrogels can induce the ingrowth of cells into the matrices. Therefore, the dual enzymatic methods as facile gel forming and loading of various native growth factors or therapeutic proteins could be highly promising for tissue regenerative medicines.】

Recently, the interest in antimicrobial hydrogels with impregnated antibacterial agents has significantly increased because of their ability to combat infection in biomedical applications, including wound management, tissue engineering, and biomaterial surface coating. Among these antibacterial reagents, silver nanoparticles (AgNP) show good antibacterial activity against both gram-negative and gram-positive bacteria, including highly multi-resistant strains. However, the entrapment of AgNP within a hydrogel matrix is often associated with toxicity issues because of the use of chemical reductants (e.g., commonly sodium borohydride), burst leaching, or unwanted agglomeration of AgNP in the absence of surfactants or stabilizers. In this study, we present catechol-rich gelatin hydrogels with in situ hybridization of AgNP for enhanced antimicrobial activities. AgNP were formed through a redox reaction between silver ions and the catechol moieties of a gelatin derivative polymer, without the addition of any chemical reductants. The AgNP with an average size of 20 nm were entrapped within hydrogel matrices and showed sustained release from the hydrogel matrix (8.7% for 14 days). The resulting hydrogels could kill both gram-negative and gram-positive bacteria, depending on the amount of AgNP released from the hydrogels and did not have a significant influence on mammalian cell viability. We believe that our catechol-rich hydrogels in situ hybridizations with AgNP have great potential for biomedical applications, such as wound management and surface coating, because of their excellent antibacterial activities and biocompatibility.

Development of bioadhesives with tunable mechanical strength, high adhesiveness, biocompatibility, and injectability is greatly desirable in all surgeries to replace or complement the sutures and staples. Herein, the dual catalytic activity of horseradish peroxidase is exploited to in situ form the hydroxyphenyl propionic acid-gelatin/thiolated gelatin (GH/GS) adhesive hydrogels including two alternative crosslinks (phenol-phenol and disulfide bonds) with fast gelation (few seconds - several minutes) and improved physicochemical properties. Their elastic moduli increase from 6.7 to 10.3 kPa by adding GS polymer that leads to the better stability of GH/GS hydrogels than GH ones. GH/GS adhesive strength is respectively 6.5-fold and 15.8-fold higher than GH-only and fibrin glue that is due to additional disulfide linkages between hydrogels and tissues. Moreover, in vitro cell study with human dermal fibroblast showed the cell-compatibility of GH/GS hydrogels. Taken together, GH/GS hydrogels can be considered as promising potential adhesive materials for various biomedical applications.

Biomimetic parathyroid regeneration with sustained release of parathyroid hormone (PTH) into the blood stream is a considerable challenge in hypoparathyroidism treatment. We recently reported that tonsil-derived mesenchymal stem cells (TMSCs), if these cells were both differentiated in vitro before implantation and incorporated into a scaffold Matrigel, are a good cell source for parathyroid regeneration in a parathyroidectomized (PTX) animal model. Here, we present a new strategy for improved clinical application that enhances the sustained release of PTH by controlling mechanical stiffness using in situ-forming gelatin-hydroxyphenyl propionic acid (GH) hydrogels (GHH). Differentiated TMSCs (dTMSCs) embedded in a GHH with a strength of 4.4 kPa exhibited the best sustained release of PTH and were the most effective in hypoparathyroidism treatment, showing improved blood calcium homeostasis compared with Matrigel-embedded dTMSCs. Interestingly, undifferentiated control TMSCs (cTMSCs) also released PTH in a sustained manner if incorporated into GHH. Collectively, these findings may establish a new paradigm for parathyroid regeneration that could ultimately evolve into an improved clinical application. Copyright © 2017 John Wiley & Sons, Ltd.

In situ-forming hydrogels are an attractive class of implantable biomaterials that are used for biomedical applications. These injectable hydrogels are versatile and provide a convenient platform for delivering cells and drugs via minimally invasive surgery. Although several crosslinking methods for preparing in situ forming hydrogels have been developed over the past two decades, most hydrogels are not sufficiently versatile for use in a wide variety of tissue-engineering applications. In recent years, enzyme-catalysed crosslinking approaches have been emerged as a new approach for developing in situ-forming hydrogels. In particular, the horseradish peroxidase (HRP)-catalysed crosslinking approach has received increasing interest, due to its highly improved and tunable capacity to obtain hydrogels with desirable properties. The HRP-catalysed crosslinking reaction immediately occurs upon mixing phenol-rich polymers with HRP and hydrogen peroxide (H2O2) in aqueous media. Based on this unique gel-forming feature, recent studies have shown that various properties of formed hydrogels, such as gelation time, stiffness and degradation rate, can be easily manipulated by varying the concentrations of HRP and H2O2. In this review, we outline the versatile properties of HRP-catalysed in situ-forming hydrogels, with a brief introduction to the crosslinking mechanisms involved. In addition, the recent biomedical applications of HRP-catalysed in situ-forming hydrogels for tissue regeneration are described.

Many women around the world are suffering from urinary incontinence, defined as the unintentional leakage of urine by external abnormal pressure. Although various kinds of materials have been utilized to treat this disease, therapies that are more effective are still needed for the treatment of urinary incontinence. Here, we present a macro/nanogel composed of in situ forming gelatin-based macrogels and self-assembled heparin-based nanogels, which can serve as an injectable and bioactive bulking material for the treatment of urinary incontinence. The hybrid hydrogels were prepared via enzymatic reaction in the presence of horseradish peroxidase and hydrogen peroxide. Incorporating a growth factor (GF)-loaded heparin nanogel into a gelatin gel matrix enabled the hybrid gel matrix to release GF continuously up to 28 days. Moreover, we demonstrated that the hydrogel composites stimulated the regeneration of the urethral muscle tissue surrounding the urethral wall and promoted the recovery of their biological function when injected in vivo. Thus, the macro/nanohydrogels may provide an advanced therapeutic technique for the treatment of urinary incontinence as well as an application for regenerative medicine.

In this study, in situ forming gelatin hydrogels via horseradish peroxidase (HRP)-catalyzed cross-linking were developed to serve as bioactive wound dressings with suitable tissue adhesive properties to deliver dermal fibroblasts (DFBs). The DFB-encapsulated gelatin hydrogels with different stiffnesses, GH-soft (1.1 kPa) and GH-hard (6.2 kPa), were prepared by controlling the hydrogen peroxide (H2O2) concentrations. The GH-soft hydrogel was capable of facilitating the proliferation of DFBs and the synthesis of extracellular components, as compared to GH-hard hydrogels. In addition, the subcutaneously injected GH-soft hydrogel with bioluminescent reporter cells provided enhanced cell survival and local retention over 14 days. In vivo transplantation of DFB-encapsulated GH-soft hydrogels accelerated wound contraction, and promoted collagen deposition and neovascularization within the incisions performed on mice skin. Therefore, we expect that HRP-catalyzed in situ forming gelatin hydrogels can be useful for local delivery of cells with high viability in wounds, which holds great promise for advancing wound healing technologies and other tissue engineering applications.

In situ formation of horseradish peroxidase (HRP)-free gelatin hydrogels was achieved via ferromagnetic microbead-assisted enzymatic cross-linking. Gelation time and mechanical stiffness of the hydrogels can be tuned in situ, which makes HRP-free gelatin hydrogels suitable for injectable cell delivery.

Horseradish peroxidase (HRP) and hydrogen peroxide (H2 O2 )-mediated crosslinking reaction has become an attractive method to create in situ forming hydrogels. While the crosslinking system has been widely utilized, there are certain issues require improvement to extend their biomedical applications, including creation of stiff hydrogels without compromising cytocompatibility due to initially high concentrations of H2 O2 . A gelatin-based hydrogels formed through a dual enzyme-mediated crosslinking reaction using HRP and glucose oxidase (GOx) as an H2 O2 -generating enzyme to gradually supply a radical source in HRP-mediated crosslinking reaction is reported. The physicochemical properties can be controlled by varying enzyme concentrations. Furthermore the hydrogel matrices provide 3D microenvironments for supporting the growth and spreading of human dermal fibroblasts with minimized cytotoxicity, despite the cells being encapsulated within stiff hydrogels. These hydrogels formed with HRP/GOx have great potential as artificial microenvironments for a wide range of biomedical applications.

Tissue engineering therapies require biocompatible and bioactive biomaterials that are capable of encouraging an angiogenic response for effective tissue regeneration. In this study, a SVVYGLR peptide, which functions as a potent angiogenic factor, was conjugated into injectable gelatin-poly(ethylene glycol)-tyramine (GPT) hydrogels in situ to enhance endothelial cell activities and neo-vascularization. SVVYGLRGGY (SV-Y) conjugated GPT (SV-GPT) hydrogels were formed in situ via enzyme-mediated reaction using horseradish peroxidase (HRP) and hydrogen peroxide (H(2)O(2)). The physico-chemical properties were characterized and could be controlled depending on the feed peptide and H(2)O(2) concentration. The concentration of conjugated peptide ranged from 0.37 to 0.81 μmol/mL, and the elastic moduli (G') of the hydrogels were 600-4900 Pa. In vitro cell studies using human umbilical vein endothelial cells (HUVECs) and in vivo subcutaneous injection studies were performed to confirm the effect of the SVVYGLR peptide on HUVEC activity and neo-vascularization. Obtained results demonstrated that the in situ conjugation of SVVYGLR sequences into phenol residues of GPT hydrogels enhanced the activity of HUVECs in vitro and stimulated the formation of new blood vessels in the hydrogel matrices in vivo. From the results, we suggest that in situ conjugation of SV-Y to GPT hydrogels via the enzymatic reaction may be an efficient tool to prepare injectable bioactive hydrogels that can enhance endothelial cell activities and promoting angiogenesis for tissue regeneration.

In situ cross-linkable hybrid hydrogels composed of gelatin and 4-arm-polypropylene oxide-polyethylene oxide (Tetronic) was developed as an injectable scaffold for tissue regeneration. The gelatin was modified by hydroxyphenyl propionic acid (HPA) and the Tetronic was conjugated with tyramines (Tet-TA). The hydrogels were rapidly formed by mixing the polymer solutions containing horseradish peroxidase (HRP) and hydrogen peroxide (H(2)O(2)). The gelation time and mechanical properties of the hydrogels could be controlled by varying the HRP and H(2)O(2) concentrations. In vitro degradation study of the hybrid hydrogels was carried out using collagenase and the prolonged proteolytic degradation was obtained due to the presence of the Tetronic. Human dermal fibroblast (hDFB) was cultured in the hydrogel matrices to evaluate the cyto-compatibility. The encapsulated cells were shown to be highly viable and spread over the gel matrices, suggesting that the hybrid hydrogels have an excellent cyto-compatibility. The hydrogels were also subcutaneously injected in the back of mice and the results demonstrated that the hydrogels were rapidly formed at the injected site. From these results, we demonstrate that the in situ cross-linkable hydrogels formed by hybridization of gelatin and Tetronic via enzyme-mediated reactions hold great promise for use as injectable matrices for tissue regenerative medicine due to their tunable physico-chemical properties and excellent bioactivity.

Background Hydrogels can serve as three-dimensional (3D) scaffolds for cell culture and be readily injected into the body. Recent advances in the image technology for 3D scaffolds like hydrogels have attracted considerable attention to overcome the drawbacks of ordinary imaging technologies such as optical and fluorescence microscopy. Multiphoton microscopy (MPM) is an effective method based on the excitation of two-photons. In the present study, C2C12 myoblasts differentiated in 3D gelatin hydroxyphenylpropionic acid (GHPA) hydrogels were imaged by using a custom-built multiphoton excitation fluorescence microscopy to compare the difference in the imaging capacity between conventional microscopy and MPM. Results The physicochemical properties of GHPA hydrogels were characterized by using scanning electron microscopy and Fourier-transform infrared spectroscopy. In addition, the cell viability and proliferation of C2C12 myoblasts cultured in the GHPA hydrogels were analyzed by using Live/Dead Cell and CCK-8 assays, respectively. It was found that C2C12 cells were well grown and normally proliferated in the hydrogels. Furthermore, the hydrogels were shown to be suitable to facilitate the myogenic differentiation of C2C12 cells incubated in differentiation media, which had been corroborated by MPM. It was very hard to get clear images from a fluorescence microscope. Conclusions Our findings suggest that the gelatin-based hydrogels can be beneficially utilized as 3D scaffolds for skeletal muscle engineering and that MPM can be effectively applied to imaging technology for tissue regeneration.

In this study, we developed horseradish peroxidase (HRP)-catalyzed sprayable gelatin hydrogels (GH) as a bioactive wound dressing that can deliver cell-attracting chemotactic cytokines to the injured tissues for diabetic wound healing. We hypothesized that topical administration of chemokines using GH hydrogels might improve wound healing by inducing recruitment of the endogenous cells. Two types of chemokines (interleukin-8; IL-8, macrophage inflammatory protein-3α; MIP-3α) were simply loaded into GH hydrogels during in situ cross-linking, and then their wound-healing effects were evaluated in streptozotocin-induced diabetic mice. The incorporation of chemokines did not affect hydrogels properties including swelling ratio and mechanical stiffness, and the bioactivities of IL-8 and MIP-3α released from hydrogel matrices were stably maintained. In vivo transplantation of chemokine-loaded GH hydrogels facilitated cell infiltration into the wound area, and promoted wound healing with enhanced re-epithelialization/neovascularization and increased collagen deposition, compared with no treatment or the GH hydrogel alone. Based on our results, we suggest that cell-recruiting chemokine-loaded GH hydrogel dressing can serve as a delivery platform of various therapeutic proteins for wound healing applications. Statement of Significance Despite development of materials combined with therapeutic agents for diabetic wound treatment, impaired wound healing by insufficient chemotactic responses still remain as a significant problem. In this study, we have developed enzyme-catalyzed gelatin (GH) hydrogels as a sprayable dressing material that can deliver cell-attracting chemokines for diabetic wound healing. The chemotactic cytokines (IL-8 and MIP-3α) were simply loaded within hydrogel during in situ gelling, and wound healing efficacy of chemokine-loaded GH hydrogels was investigated in STZ-induced diabetic mouse model. These hydrogels significantly promoted wound-healing efficacy with faster wound closure, neovascularization, and thicker granulation. Therefore, we expect that HRP-catalyzed in situ forming GH hydrogels can serve as an injectable/sprayable carrier of various therapeutic agents for wound healing applications.

Graphene-based nanomaterials with different oxidation degrees were incorporated into Tetronic–tyramine (Tet–TA) hydrogels via enzymatic cross-linking. The molecular oxidation of graphene in combination with amphiphilic Tet–TA significantly improved the water dispersibility of graphene oxide (GO), resulting in a significant reinforcement of Tet–TA/GO composite hydrogels that can be used as an injectable biomaterial platform.

Directing angiogenic differentiation of mesenchymal stem cells (MSCs) still remains challenging for successful tissue engineering. Without blood vessel formation, stem cell-based approaches are unable to fully regenerate damaged tissues due to limited support for cell viability and desired tissue/organ functionality. Herein, we report in situ cross-linkable gelatin−hydroxyphenyl propionic acid (GH) hydrogels that can induce pro-angiogenic profiles of MSCs via purely material-driven effects. This hydrogel directed endothelial differentiation of mouse and human patient-derived MSCs through integrin-mediated interactions at the cell-material interface, thereby promoting perfusable blood vessel formation in vitro and in vivo. The causative roles of specific integrin types (α1 and αvβ3) in directing endothelial differentiation were verified by blocking the integrin functions with chemical inhibitors. In addition, to verify the material-driven effect is not species-specific, we confirmed in vitro endothelial differentiation and in vivo blood vessel formation of patient-derived human MSCs by this hydrogel. These findings provide new insight into how purely material-driven effects can direct endothelial differentiation of MSCs, thereby promoting vascularization of scaffolds towards tissue engineering and regenerative medicine applications in humans.

Clinical trials utilizing mesenchymal stem cells (MSCs) for severe vascular diseases have highlighted the need to effectively engraft cells and promote pro-angiogenic activity. A functional material accomplishing these two goals is an ideal solution as spatiotemporal and batch-to-batch variability in classical therapeutic delivery can be minimized, and tissue regeneration would begin rapidly at the implantation site. Gelatin may serve as a promising biomaterial due to its excellent biocompatibility, biodegradability, and non-immuno/antigenicity. However, the dissolution of gelatin at body temperature and quick enzymatic degradation in vivo have limited its use thus far. To overcome these challenges, an injectable, in situ crosslinkable gelatin was developed by conjugating enzymatically-crosslinkable hydroxyphenyl propionic acid (GHPA). When MSCs are cultured in 3D in vitro or injected in vivo in GHPA, spontaneous endothelial differentiation occurs, as evidenced by marked increases in endothlelial cell marker expressions (Flk1, Tie2, ANGPT1, vWF) in addition to forming an extensive perfusable vascular network after 2-week subcutaneous implantation. Additionally, favorable host macrophage response is achieved with GHPA as shown by decreased iNOS and increased MRC1 expression. These results indicate GHPA as a promising soluble factor-free cell delivery template which induces endothelial differentiation of MSCs with robust neovasculature formation and favorable host response.

The present study describes enzymatically cross-linked gelatin-based hydrogels as in situ forming tissue adhesives. A series of gelatin derivatives with different phenolic contents were synthesized by conjugating hydroxyphenyl propionic acid and tyramine to gelatin backbones. Two gelatin derivatives, gelatin–hydroxyphenyl propionic acid (GH) and gelatin–hydroxyphenyl propionic acid–tyramine (GHT) with maximum obtainable phenolic contents (146.6 μmol g−1 GH and 395.7 μmol g−1 GHT), were used to prepare gelatin-based hydrogels via horseradish peroxidase (HRP)-mediated reactions in the presence of hydrogen peroxide (H2O2). By changing the HRP and H2O2 concentrations, the gelation time, mechanical strength, and degradation rate of the hydrogels were fairly well controlled, indicating a tunable rate and degree of cross-linking. In addition, we found that an increase in phenolic content led to increased mechanical strength of the hydrogels. Lap-shear test results clearly showed that the GH and GHT hydrogels exhibited 2–3 times greater tissue adhesiveness compared to fibrin glues. On the basis of these results, we conclude that in situ forming gelatin-based hydrogels, which are both injectable and sprayable, can be used as an alternative to conventional tissue adhesives.