Transplantation of Stem Cell Spheroid-Laden 3-Dimensional Patches with Bioadhesives for the Treatment of Myocardial Infarction.

Myocardial infarction (MI) is treated with stem cell transplantation using various biomaterials and methods, such as stem cell/spheroid injections, cell sheets, and cardiac patches. However, current treatment methods have some limitations, including low stem cell engraftment and poor therapeutic effects. Furthermore, these methods cause secondary damage to heart due to injection and suturing to immobilize them in the heart, inducing side effects. In this study, we developed stem cell spheroid-laden 3-dimensional (3D) patches (S_3DP) with biosealant to treat MI. This 3D patch has dual modules, such as open pockets to directly deliver the spheroids with their paracrine effects and closed pockets to improve the engraft rate by protecting the spheroid from harsh microenvironments. The spheroids formed within S_3DP showed increased viability and expression of angiogenic factors compared to 2-dimensional cultured cells. We also fabricated gelatin-based tissue adhesive biosealants via a thiol-ene reaction and disulfide bond formation. This biosealant showed stronger tissue adhesiveness than commercial fibrin glue. Furthermore, we successfully applied S_3DP using a biosealant in a rat MI model without suturing in vivo, thereby improving cardiac function and reducing heart fibrosis. In summary, S_3DP and biosealant have excellent potential as advanced stem cell therapies with a sutureless approach to MI treatment.

Composite Spheroid-Laden Bilayer Hydrogel for Engineering Three-Dimensional Osteochondral Tissue.

A combination of hydrogels and stem cell spheroids has been used to engineer three-dimensional (3D) osteochondral tissue, but precise zonal control directing cell fate within the hydrogel remains a challenge. In this study, we developed a composite spheroid-laden bilayer hydrogel to imitate osteochondral tissue by spatially controlled differentiation of human adipose-derived stem cells. Meticulous optimization of the spheroid-size and mechanical strength of gelatin methacryloyl (GelMA) hydrogel enables the cells to homogeneously sprout within the hydrogel. Moreover, fibers immobilizing transforming growth factor beta-1 (TGF-ß1) or bone morphogenetic protein-2 (BMP-2) were incorporated within the spheroids, which induced chondrogenic or osteogenic differentiation of cells in general media, respectively. The spheroids-filled GelMA solution was crosslinked to create the bilayer hydrogel, which demonstrated a strong interfacial adhesion between the two layers. The cell sprouting enhanced the adhesion of each hydrogel, demonstrated by increase in tensile strength from 4.8 ± 0.4 to 6.9 ± 1.2 MPa after 14 days of culture. Importantly, the spatially confined delivery of BMP-2 within the spheroids increased mineral deposition and more than threefold enhanced osteogenic genes of cells in the bone layer while the cells induced by TGF-ß1 signals were apparently differentiated into chondrocytes within the cartilage layer. The results suggest that our composite spheroid-laden hydrogel could be used for the biofabrication of osteochondral tissue, which can be applied to engineer other complex tissues by delivery of appropriate biomolecules.

O2 variant chip to simulate site-specific skeletogenesis from hypoxic bone marrow.

The stemness of bone marrow mesenchymal stem cells (BMSCs) is maintained by hypoxia. The oxygen level increases from vessel-free cartilage to hypoxic bone marrow and, furthermore, to vascularized bone, which might direct the chondrogenesis to osteogenesis and regenerate the skeletal system. Hence, oxygen was diffused from relatively low to high levels throughout a three-dimensional chip. When we cultured BMSCs in the chip and implanted them into the rabbit defect models of low-oxygen cartilage and high-oxygen calvaria bone, (i) the low oxygen level (base) promoted stemness and chondrogenesis of BMSCs with robust antioxidative potential; (ii) the middle level (two times ≥ low) pushed BMSCs to quiescence; and (iii) the high level (four times ≥ low) promoted osteogenesis by disturbing the redox balance and stemness. Last, endochondral or intramembranous osteogenesis upon transition from low to high oxygen in vivo suggests a developmental mechanism-driven solution to promote chondrogenesis to osteogenesis in the skeletal system by regulating the oxygen environment.

Oxygen-supplying syringe to create hyperoxia-inducible hydrogels for in situ tissue regeneration.

Recent trends in the design of regenerative materials include the development of bioactive matrices to harness the innate healing ability of the body using various biophysicochemical stimuli (defined as in situ tissue regeneration). Among these, hyperoxia (>21% pO2) is a well-known therapeutic factor for promoting tissue regeneration, such as immune cell recruitment, cell proliferation, angiogenesis, and fibroblast differentiation into myofibroblast. Although various strategies to induce hyperoxia are reported, developing advanced hyperoxia-inducing biomaterials for tissue regeneration is still challenging. In this study, a catalase-immobilized syringe (defined as an Oxyringe) via calcium peroxide-mediated surface modification is developed as a new type of oxygen-supplying system. Hyperoxia-inducible hydrogels are fabricated utilizing Oxyringe. This hydrogel plays a role as a physical barrier for hemostasis. In addition, hyperoxic matrices induce transient hyperoxia in vivo (up to 46.0% pO2). Interestingly, the hydrogel-induced hyperoxia boost the initial macrophage recruitment and rapid inflammation resolution. Furthermore, hyperoxic oxygen release of hydrogels facilitates neovascularization and cell proliferation involved in the proliferation phase, expediting tissue maturation related to the remodeling phase in wound healing. In summary, Oxyringe has excellent potential as an advanced oxygen-supplying platform to create hyperoxia-inducing hydrogels for in situ tissue regeneration.

Advances in gelatin-based hydrogels for wound management.

Skin wounds can be classified into two categories, namely acute and chronic wounds. While acute wounds are healed by the normal wound healing process, chronic wounds are not normally healed, causing inflammation, pain, discomfort, serious complications, and economic burden owing to treatment costs. To alleviate and treat chronic wounds, various biomaterials have been developed. Among them, in situ forming polymeric hydrogels have been widely used as a promising wound care material due to their beneficial properties, including sol-gel phase transition, moisturizing effect on the surrounding environment, biocompatibility, and structural similarity to the native extracellular matrix. The development of bioactive hydrogels that provide artificial cellular microenvironments or stimulate surrounding tissues through physicochemical and biological stimuli is an emerging trend in the fabrication of hydrogels. Notably, gelatin-based hydrogels have attracted much attention as bioactive matrices owing to their biocompatibility, biodegradability, and various functional moieties for chemical modification. In this review, we discuss the development and use of advanced gelatin-based hydrogels for wound management and tissue regeneration.

Three-Dimensional Printable Gelatin Hydrogels Incorporating Graphene Oxide to Enable Spontaneous Myogenic Differentiation.

Three-dimensional (3D) bioprinting has attracted considerable attention for producing 3D engineered cellular microenvironments that replicate complex and sophisticated native extracellular matrices (ECM) as well as the spatiotemporal gradients of numerous physicochemical and biological cues. Although various hydrogel-based bioinks have been reported, the development of advanced bioink materials that can reproduce the complexity of ECM accurately and mimic the intrinsic property of laden cells is still a challenge. This paper reports 3D printable bioinks composed of phenol-rich gelatin (GHPA) and graphene oxide (GO) as a component for a myogenesis-inducing material, which can form a hydrogel network in situ by a dual enzyme-mediated cross-linking reaction. The in situ curable GO/GHPA hydrogel can be utilized successfully as 3D-printable bioinks to provide suitable cellular microenvironments with facilitated myogenic differentiation of C2C12 skeletal myoblasts. Overall, we suggest that functional bioinks may be useful in muscle tissue engineering and regenerative medicine.

Design of Advanced Polymeric Hydrogels for Tissue Regenerative Medicine: Oxygen-Controllable Hydrogel Materials.

Engineered polymeric hydrogels have been extensively utilized in tissue engineering and regenerative medicine because of their biocompatibility, tunable properties, and structural similarity in their native extracellular microenvironment. The native extracellular matrix (ECM) has been implicated as a crucial factor in the regulation of cellular behaviors and their fate. The emerging trend in the design of hydrogels involves the development of advanced materials to precisely recapitulate the native ECM or to stimulate the surrounding tissues via physical, chemical, or biological stimuli. The ECM presents various parameters such as ECM components, soluble factors, cell-to-cell and cell-to-matrix interactions, physical forces, and physicochemical environments. Among these environmental factors, oxygen is considered as an essential signaling molecule. In particular, abnormal oxygen tension such as a lack of oxygen (defined as hypoxia) and an excess supply of oxygen (defined as hyperoxia) plays a pivotal role during early vascular development, tissue regeneration and repair, and tumor progression and metastasis. In this chapter, we discuss how engineered polymeric hydrogels serve as either an artificial extracellular microenvironment to create engineered tissues or as an acellular matrix to stimulate the native tissues for a wide range of biomedical applications including tissue engineering and regenerative medicine, wound healing, and engineered disease models. Specifically, we focus on emerging technologies to create advanced polymeric hydrogel materials that accurately mimic or stimulate the native ECM.

In situ forming and reactive oxygen species-scavenging gelatin hydrogels for enhancing wound healing efficacy.

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.

Hydrogen Peroxide-Releasing Hydrogels for Enhanced Endothelial Cell Activities and Neovascularization.

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.