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The dual role of reactive oxygen and nitrogen species (RONS) in physiological and pathological processes in biological systems has been widely reported. It has been recently suggested that the regulation of RONS levels under physiological and pathological conditions is a potential therapy to promote health and treat diseases, respectively. Injectable hydrogels have been emerging as promising biomaterials for RONS-related biomedical applications owing to their excellent biocompatibility, three-dimensional and extracellular matrix-mimicking structures, tunable properties and easy functionalization. These hydrogels have been developed as advanced injectable platforms for locally generating or scavenging RONS, depending on the specific conditions of the target disease. In this review article, the design principles and mechanism by which RONS are generated/scavenged from hydrogels are outlined alongside a discussion of their in vitro and in vivo evaluations. Additionally, we highlight the advantages and recent developments of these injectable RONS-controlling hydrogels for regenerative medicines and tissue engineering applications.

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.

Despite their high efficacy and safety, long-acting contraceptive methods are underutilized among women in some settings because they usually require injection or implantation by healthcare personnel. Here, we report a self-administrable microneedle (MN) patch for the rapid administration of a sustained-release contraceptive hormone delivery system into the skin that increases the simplicity and reliability of the MN delivery. We developed an immediate microneedle detachment system using a porous patch backing that has sufficient strength during MN insertion into skin under compression, but enables immediate detachment (1 s) of the MNs due to fracture at the MN - backing interface upon patch removal under tension from the skin surface. After patch application, the removed patch produced no biohazardous sharps waste, and was designed to achieve long-acting contraception by formulating poly(lactic-co-glycolic acid) MNs to slowly release the contraceptive hormone levonorgestrel for up to 1 month. Our combined strategy using immediate MN detachment in the skin and sustained drug delivery from the MNs could facilitate greater access to long-acting contraception by providing a simple and convenient option for self-administered, long-acting contraception.

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.

Retinoic acid (RA) is a well-known morphogen in human development. However, how an RA gradient distribution influences cardiac development remains obscure due to the lack of appropriate experimental apparatus. To address this issue, a polymeric micelle system with covalently attached RA was engineered to deliver gradient quantities of RA upon photo-irradiation. A photo-degradable polymeric nanoparticle (NP) composed of an amphiphilic methoxy(polyethylene glycol)-b-poly(e-caprolactone)-co-poly(azido-e-caprolactone-g-ortho nitrobenzyl retinoic ester) copolymer was synthesized, and hanging RA was covalently attached through a photo-sensitive o-nitrobenzyl (ONB) linker. The ONB linker was efficiently cleaved when exposed to a light (365 nm)-gradient, and the consequent gradient release of RA from the micelles was demonstrated. The efficacy of the photo-gradient-mediated RA release was validated across different concentrations of polymer micelles over varied irradiation periods. It was confirmed that polymer micelles demonstrated minimal cytotoxicity when exposed to mouse embryoid bodies (EBs). Finally, when the photo-gradient release of polymer micelles was applied, GFP-cardiac troponin T reporter mouse EBs demonstrated a concurrent gradient-like pattern of cardiac differentiation, verifying the utility of our novel photo-gradient approach to study morphogen gradients not only for cardiac development but also for other potential biological microenvironments subject to morphogen presentation with highly defined spatial and temporal geometries.

Exposure of aged mice to a young systemic milieu revealed remarkable rejuvenation effects on aged tissues, including skeletal muscle. Although some candidate factors have been identified, the exact identity and the underlying mechanisms of putative rejuvenating factors remain elusive, mainly due to the complexity ofin vivoparabiosis. Here, we present anin vitromuscle parabiosis system that integrates young- and old-muscle stem cell vascular niche on a three-dimensional microfluidic platform designed to recapitulate key features of native muscle stem cell microenvironment. This innovative system enables mechanistic studies of cellular dynamics and molecular interactions within the muscle stem cell niche, especially in response to conditional extrinsic stimuli of local and systemic factors. We demonstrate that vascular endothelial growth factor (VEGF) signaling from endothelial cells and myotubes synergistically contribute to the rejuvenation of the aged muscle stem cell function. Moreover, with the adjustable on-chip system, we can mimic both blood transfusion and parabiosis and detect the time-varying effects of anti-geronic and pro-geronic factors in a single organ or multi-organ systems. Our unique approach presents a complementaryin vitromodel to supplementin vivoparabiosis for identifying potential anti-geronic factors responsible for revitalizing aging organs.

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.

Introducing antifouling property to biomaterial surfaces has been considered an effective method for preventing the failure of implanted devices. In order to achieve this, the immobilization of zwitterions on biomaterial surfaces has been proven to be an excellent way of improving anti-adhesive potency. In this study, poly(sulfobetaine-co-tyramine), a tyramine-conjugated sulfobetaine polymer, was synthesized and simply grafted onto the surface of polyurethane via a tyrosinase-mediated reaction. Surface characterization by water contact angle measurements, X-ray photoelectron spectroscopy and atomic force microscopy demonstrated that the zwitterionic polymer was successfully introduced onto the surface of polyurethane and remained stable for 7 days. In vitro studies revealed that poly(sulfobetaine-co-tyramine)-coated surfaces dramatically reduced the adhesion of fibrinogen, platelets, fibroblasts, and S. aureus by over 90% in comparison with bare surfaces. These results proved that polyurethane surfaces grafted with poly(sulfobetaine-co-tyramine) via a tyrosinase-catalyzed reaction could be promising candidates for an implantable medical device with excellent bioinert abilities. Statement of Significance Antifouling surface modification is one of the key strategy to prevent the thrombus formation or infection which occurs on the surface of biomaterial after transplantation. Although there are many methods to modify the surface have been reported, necessity of simple modification technique still exists to apply for practical applications. The purpose of this study is to modify the biomaterial’s surface by simply immobilizing antifouling zwitterion polymer via enzyme tyrosinase-mediated reaction which could modify versatile substrates in mild aqueous condition within fast time period. After modification, pSBTA grafted surface becomes resistant to various biological factors including proteins, cells, and bacterias. This approach appears to be a promising method to impart antifouling property on biomaterial surfaces.

Administration of dendritic cells (DCs) combined with oncolytic adenovirus (Ad) expressing antitumor cytokines induces a potent antitumor effect and antitumor immunity by ameliorating the immunosuppressive tumor microenvironment. However, this combination therapy has significant limitations due to rapid dissemination and inactivation of the therapeutics at the tumor site, necessitating multiple injections of both therapeutics. To overcome these limitations, we have utilized gelatin-based hydrogel to co-deliver oncolytic Ad co-expressing interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (oAd) and DCs for sustained release of both therapeutics. The injectable and biodegradable hydrogels were prepared by mixing the polymer solutions containing horseradish peroxidase and hydrogen peroxide. Gel matrix enabled sustained release of both oAd and DCs while preserving their biological activity over a considerable time period, leading to efficient retention of both therapeutics in tumor tissue. Further, tumors treated with oAd- and DC-loaded gel (oAd+DC/gel) showed a significantly greater expression level of IL-12, GM-CSF, and interferon-γ (IFN-γ) than either single treatment (oAd or DC) or oAd in combination with DC (oAd+DC), resulting in efficient activation of both endogenous and exogenous DCs, migration of DCs to draining lymph nodes, and tumor infiltration of CD4+ and CD8+ T cells. Moreover, oAd+DC/gel resulted in a significantly higher number of tumor-specific IFN-γ-secreting immune cells compared with oAd+DC. Lastly, oAd+DC/gel significantly attenuated tumor-mediated thymic atrophy, which is associated with immunosuppression in the tumor microenvironment, compared with oAd+DC. Taken together, these results demonstrate that gelatin gel-mediated co-delivery of oncolytic Ad and DCs might be a promising strategy to efficiently retain both therapeutics in tumor tissue and induce a potent antitumor immune response for an extended time period via a single administration.

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...

Vascular disease is a leading cause of death and disability worldwide. Current surgical intervention and treatment options for vascular diseases have exhibited limited long-term success, emphasizing the need to develop advanced treatment paradigms for early detection and more effective treatment of dysfunctional cells in a specific blood vessel lesion. Advances in targeted nanoparticles mediating cargo delivery enables more robust prevention, screening, diagnosis, and treatment of vascular disorders. In particular, nanotheranostics integrates diagnostic imaging and therapeutic function into a single agent, and is an emerging platform towards more effective and localized vascular treatment. This review article highlights recent advances and current challenges associated with the utilization of targeted nanoparticles for real-time diagnosis and treatment of vascular diseases. Given recent developments, nanotheranostics offers great potential to serve as an effective platform for targeted, localized, and personalized vascular treatment.

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

Supplemental material, JBA899787 Supplemental Material2 for Horseradish peroxidase-catalyzed hydrogelation of fish gelatin with tunable mechanical properties and biocompatibility by Phuong Le Thi, Yunki Lee, Dieu Linh Tran, Thai Thanh Hoang Thi and Ki Dong Park in Journal of Biomaterials Applications

A practical strategy to strengthen the inherent tissue adhesiveness of bioglues was investigated without compromising their chemical structure. A simple blending of α-cyclodextrins (α-CDs) in gelatin glues substantially improved the adhesiveness of the glues upon contact with porcine skin. Interestingly, the adhesiveness was even further enhanced when the glues were supplemented with γ-CDs through the formation of multivalent supramolecular networks inside of the glue. In contrast, the effect of β-CDs was rather limited because their water solubility is relatively low. A model study using modified gold substrates demonstrated that CDs also interacted with biomolecules naturally present in skin, thereby improving the adhesiveness. Altogether, we suggest that the supramolecular networking of CDs, both inside the glue and at the glue-skin interface, could reinforce tissue adhesiveness of bioglues, which allows them to be of great relevance to a primary treatment modality for larger skin incisions.

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.

Nanoparticle-based imaging and therapy are of interest for theranostic nanomedicine. In particular, superparamagnetic iron oxide (SPIO) nanoparticles (NPs) have attracted much attention in cancer imaging, diagnostics, and treatment because of their superior imagability and biocompatibility (approved by the Food and Drug Administration). Here, we developed SPIO nanoparticles (NPs) that self-assembled into magnetic nanoclusters (SAMNs) in aqueous environments as a theranostic nano-system. To generate multi-functional SPIO NPs, we covalently conjugated β-cyclodextrin (β-CD) to SPIO NPs using metal-adhesive dopamine groups. Polyethylene glycol (PEG) and paclitaxel (PTX) were hosted in the β-CD cavity through high affinity complexation. The core-shell structure of the magnetic nanoclusters was elucidated based on the condensed SPIO core and a PEG shell using electron microscopy and the composition was analyzed by thermogravimetric analysis (TGA). Our results indicate that nanocluster size could be readily controlled by changing the SPIO/PEG ratio in the assemblies. Interestingly, we observed a significant enhancement in magnetic resonance contrast due to the large cluster size and dense iron oxide core. In addition, tethering a tumor-targeting peptide to the SAMNs enhanced their uptake into tumor cells. PTX was efficiently loaded into β-CDs and released in a controlled manner when exposed to competitive guest molecules. These results strongly indicate that the SAMNs developed in this study possess great potential for application in image-guided cancer chemotherapy. Statement of Significance In this study, we developed multi-functional SPIO NPs that self-assembled into magnetic nanoclusters (SAMNs) in aqueous conditions as a theranostic nano-system. The beta-cyclodextrin (β-CD) was immobilized on the surfaces of SPIO NPs and RGD-conjugated polyethylene glycol (PEG) and paclitaxel (PTX) were hosted in the β-CD cavity through high affinity complexation. We found that nanocluster size could be readily controlled by varying the SPIO/PEG ratio in the assemblies, and also demonstrated significant improvement of the functional nanoparticles for theranostic systems; enhanced magnetic resonance, improved cellular uptake, and efficient PTX loading and sustained release at the desired time point. These results strongly indicate that the SAMNs developed in this study possess great potential for application in image-guided cancer chemotherapy.

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...

Exposure of aged mice to a young systemic milieu revealed remarkable rejuvenation effects on aged tissues, including skeletal muscle. Although some candidate factors have been identified, the exact identity and the underlying mechanisms of putative rejuvenating factors remain elusive, mainly due to the complexity of in vivo parabiosis. Here, we present an in vitro muscle parabiosis system that integrates young- and old-muscle stem cell vascular niche on a three-dimensional microfluidic platform designed to recapitulate key features of native muscle stem cell microenvironment. This innovative system enables mechanistic studies of cellular dynamics and molecular interactions within the muscle stem cell niche, especially in response to conditional extrinsic stimuli of local and systemic factors. We demonstrate that vascular endothelial growth factor (VEGF) signaling from endothelial cells and myotubes synergistically contribute to the rejuvenation of the aged muscle stem cell function. Moreover, with the adjustable on-chip system, we can mimic both blood transfusion and parabiosis and detect the time-varying effects of anti-geronic and pro-geronic factors in a single organ or multi-organ systems. Our unique approach presents a complementary in vitro model to supplement in vivo parabiosis for identifying potential anti-geronic factors responsible for revitalizing aging organs.

Parathyroid glands are compact glandular bodies attached to the thyroid gland and have a major function of producing parathyroid hormone (PTH), which regulates calcium and phosphate homeostasis. Over- or underproduction of PTH leads to “hyperparathyroidism” or “hypoparathyroidism,” respectively; of the two, hypoparathyroidism occurs more frequently due to surgical/radiational operations for thyroid/laryngeal disorder. Hypoparathyroidism results in severe chronic hypocalcemia that is fatal to humans. A few therapeutic approaches, including lifetime intake of calcium and vitamin D, recombinant PTH injection, and autograft of parathyroid tissues, have been developed. Recently, the use of stem cell engineering using human embryonic stem cells and mesenchymal stem cells derived from adipocytes and particularly tonsils, in combination with scaffold systems, has become an appealing option, offering improved biocompatibility. This chapter overviews the new paradigm of parathyroid regeneration using stem cell engineering with scaffolds and its potential therapeutic applications in the treatment of hypoparathyroidism. Recent publication employing a scaffold-free system is also reviewed.

We investigated therapeutic potential of human tonsil-derived mesenchymal stem cells (TMSC) subcutaneously delivered to ovariectomized (OVX) mice for developing more safe and effective therapy for osteoporosis. TMSC were isolated from tonsil tissues of children undergoing tonsillectomy, and TMSC-embedded in situ crosslinkable gelatin-hydroxyphenyl propionic acid hydrogel (TMSC-GHH) or TMSC alone were delivered subcutaneously to the dorsa of OVX mice. After 3 months, three-dimensionally reconstructed micro-computed tomographic images revealed better recovery of the femoral heads in OVX mice treated with TMSC-GHH. Serum osteocalcin and alkaline phosphatase were also recovered, indicating bone formation only in TMSC-GHH-treated mice, and absence in hypercalcemia or other severe macroscopic deformities showed biocompatibility of TMSC-GHH. Additionally, visceral fat reduction effects by TMSC-GHH further supported their therapeutic potential. TMSC provided therapeutic benefits toward osteoporosis only when embedded in GHH, and showed potential as a supplement or alternative to current therapies.

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.

Liposomes containing nanogels (liponanogels) were fabricated by sonicating heparin-Pluronic (HP) nanogels with pegylated lipids for ribonuclease (RNase) delivery. Liponanogels with an average diameter of 316 nm were obtained and their spherical morphology was elucidated by atomic force microscopy (AFM). Confocal laser scanning microscopy (CLSM) revealed the core-shell structure of the liponanogels using two different fluorescent dyes, showing that HP nanogels were localized in the core of the liposomes. Interestingly, the hybridization of these two systems remedies the drawbacks of each system while they hold their strengths called “WIN-WIN effect”. When HP nanogels were used to encapsulate RNase in liponanogels, the loading of RNase was almost doubled as compared with the loading in liposomes without nanogels. Due to the presence of a lipid bilayer on the nanogels, the release of RNase was prolonged over 4 days whereas it was much faster (82% after 21 h) for bare HP nanogels. The cytotoxicity of the RNase-loaded liponanogels was much higher than that of free RNase because of the endocytic cellular uptake of the particles. We believe that these hybrid liponanogel systems can potentially be utilized for the hereditary diseases and targeted cancer therapy since they can efficiently load RNases and sustainly release in target cells. Open image in new window

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.】

The leading cause of synthetic graft failure includes thrombotic occlusion and intimal hyperplasia at the site of vascular anastomosis. Herein, we report a co- immobilization strategy of heparin and potent anti-neointimal drug (Mitogen Activated Protein Kinase II inhibitory peptide; MK2i) by using a tyrosinase-catalyzed oxidative reaction for preventing thrombotic occlusion and neointimal formation of synthetic vascular grafts. The binding of heparin–tyramine polymer (HT) onto the polycarprolactone (PCL) surface enhanced blood compatibility with significantly reduced protein absorption (64.7% decrease) and platelet adhesion (82.2% decrease) compared to bare PCL surface. When loading MK2i, 1) the HT depot surface gained high MK2i- loading efficiency through charge-charge interaction, and 2) this depot platform enabled long-term, controlled release over 4 weeks (92–272 μg/mL of MK2i). The released MK2i showed significant inhibitory effects on VSMC migration through down-regulated phosphorylation of target proteins (HSP27 and CREB) associated with intimal hyperplasia. In addition, it was found that the released MK2i infiltrated into the tissue with a cumulative manner in ex vivo human saphenous vein (HSV) model. This present study demonstrates that enzymatically HT-coated surface modification is an effective strategy to induce long-term MK2i release as well as hemocompatibility, thereby improving anti- neointimal activity of synthetic vascular grafts.