On-Demand Local Immunomodulation via Epigenetic Control of Macrophages Using an Inflammation-Responsive Hydrogel for Accelerated Wound Healing.

Effective resolution of inflammation contributes to favorable tissue regenerative therapeutic outcomes. However, fine coordination of local immunomodulation in a timely manner is limited because of the lack of strategies for controlling disease dynamics. We developed an inflammation-responsive hydrogel (IFRep gel) as an effective therapeutic strategy for on-demand epigenetic modulation against disease dynamics in wound healing. The IFRep gel is designed to control drug release by cathepsins according to the state of inflammation for active disease treatment. The gel loaded with an inhibitor of the epigenetic reader bromodomain (BRD)4 regulates the translocation of nuclear factor erythroid 2 to the nucleus, where it promotes antioxidant gene expression to reverse the inflammatory macrophage state in vitro. In addition, on-demand BRD inhibition using the responsive hydrogel accelerates wound healing by controlling the early inflammatory phase and keratinocyte activation in vivo. Our data demonstrate the clinical utility of using the IFRep gel as a promising strategy for improving therapeutic outcomes in inflammation-associated diseases.

Induction of ferroptosis using functionalized iron-based nanoparticles for anti-cancer therapy.

Ferroptosis, a cell death pathway that is induced in response to iron, has recently attracted remarkable attention given its emerging therapeutic potential in cancer cells. The need for a promising modality to improve chemotherapy's efficacy through this pathway has been urgent in recent years, and this non-apoptotic cell death pathway accumulates reactive oxygen species (ROS) and is subsequently involved in lipid peroxidation. Here, we report cancer-targeting nanoparticles that possess highly efficient cancer-targeting ability and minimal systemic toxicity, thereby leading to ferroptosis. To overcome the limit of actual clinical application, which is the ultimate goal due to safety issues, we designed safe nanoparticles that can be applied clinically. Nanoparticles containing ferroptosis-dependent iron and FDA-approved hyaluronic acid (FHA NPs) are fabricated by controlling physicochemical properties, and the FHA NPs specifically induce ROS production and lipid peroxidation in cancer cells without affecting normal cells. The excellent in vivo anti-tumor therapeutic effect of FHA NPs was confirmed in the A549 tumor-bearing mice model, indicating that the induction of FHA NP-mediated cell death via the ferroptosis pathway could serve as a powerful platform in anticancer therapy. We believe that this newly proposed FHA NP-induced ferroptosis strategy is a promising system that offers the potential for efficient cancer treatment and provides insight into the safe design of nanomedicines for clinical applications.

Cardiovascular tissue regeneration system based on multiscale scaffolds comprising double-layered hydrogels and fibers.

We report a technique to reconstruct cardiovascular tissue using multiscale scaffolds incorporating polycaprolactone fibers with double-layered hydrogels comprising fibrin hydrogel surrounded by secondary alginate hydrogel. The scaffolds compartmentalized cells into the core region of cardiac tissue and the peripheral region of blood vessels to construct cardiovascular tissue, which was accomplished by a triple culture system of adipose-derived mesenchymal stem cells (ADSCs) with C2C12 myoblasts on polycaprolactone (PCL) fibers along with human umbilical vein endothelial cells (HUVECs) in fibrin hydrogel. The secondary alginate hydrogel prevented encapsulated cells from migrating outside scaffold and maintained the scaffold structure without distortion after subcutaneous implantation. According to in vitro studies, resultant scaffolds promoted new blood vessel formation as well as cardiomyogenic phenotype expression of ADSCs. Cardiac muscle-specific genes were expressed from stem cells and peripheral blood vessels from HUVECs were also successfully developed in subcutaneously implanted cell-laden multiscale scaffolds. Furthermore, the encapsulated stem cells modulated the immune response of scaffolds by secreting anti-inflammatory cytokines for successful tissue construction. Our study reveals that multiscale scaffolds can be promising for the remodeling and transplantation of cardiovascular tissue.

A novel 3D indirect co-culture system based on a collagen hydrogel scaffold for enhancing the osteogenesis of stem cells.

In this study, the paracrine effect between adipose-derived mesenchymal stem cells (ADSCs) and osteoblasts was investigated in collagen-based three-dimensional (3D) scaffolds. 3D encapsulation of mesenchymal stem cells in hydrogel scaffolds was conducted for bone tissue regeneration. Osteoblasts were encapsulated in alginate microbeads with uniform size, which could be controlled by varying the supply voltage using electrostatic droplet extrusion. Osteoblast-encapsulated microbeads were embedded with ADSCs in collagen bulk hydrogel scaffolds with a high survival rate. The separated space between the two types of cells made it possible to confirm ADSC differentiation into osteogenic lineages in the 3D collagen hydrogel scaffold by the paracrine effect in vitro. Furthermore, co-cultured ADSC and osteoblasts showed enhanced bone formation compared with the ADSC monoculture group in the rat calvarial defect model. The system developed in this study provides a novel in vitro tissue model for bone regeneration without exogenous factors, and it has the potential to be used to study the paracrine effect in various co-culture systems in the near future.

Mesenchymal stem cell 3D encapsulation technologies for biomimetic microenvironment in tissue regeneration.

Mesenchymal stem cell (MSC) encapsulation technique has long been emerged in tissue engineering as it plays an important role in implantation of stem cells to regenerate a damaged tissue. MSC encapsulation provides a mimic of a three-dimensional (3D) in vivo environment to maintain cell viability and to induce the stem cell differentiation which regulates MSC fate into multi-lineages. Moreover, the 3D matrix surrounding MSCs protects them from the human innate immune system and allows the diffusion of biomolecules such as oxygen, cytokines, and growth factors. Therefore, many technologies are being developed to create MSC encapsulation platforms with diverse materials, shapes, and sizes. The conditions of the platform are determined by the targeted tissue and translation method. This review introduces several details of MSC encapsulation technologies such as micromolding, electrostatic droplet extrusion, microfluidics, and bioprinting and their application for tissue regeneration. Lastly, some of the challenges and future direction of MSC encapsulation technologies as a cell therapy-based tissue regeneration method will be discussed.

Metal enhanced fluorescence (MEF) for biosensors: General approaches and a review of recent developments.

Fluorescence-based biosensor platforms have been intensively investigated not only to increase the sensitivity but also to improve the performance of biosensors. By exploiting metal from the macroscopic down to the nanoscopic surface, various architectures have been devised to manipulate fluorescence signals (enhancement, quenching) within near-optical fields. The interaction of a metallic surface with proximal fluorophores (in the range of 5-90 nm) has beneficial effects on optical properties such as an increased quantum yield, improved photostability and a reduced lifetime of fluorophores. This phenomenon called metal-enhanced fluorescence (MEF) has been extensively used in biosensory applications. However, their applications for biological analysis practically remain challenging in biological microenvironments. Therefore, this review primarily provides a general overview of MEF biosensor systems from the basic mechanism to state-of-the-art biological applications. The review also covers the pros and cons of MEF biosensor as well as discussions about further directions in biological perspectives.

Promotion of Vascular Morphogenesis of Endothelial Cells Co-Cultured with Human Adipose-Derived Mesenchymal Stem Cells Using Polycaprolactone/Gelatin Nanofibrous Scaffolds.

New blood vessel formation is essential for tissue regeneration to deliver oxygen and nutrients and to maintain tissue metabolism. In the field of tissue engineering, in vitro fabrication of new artificial vessels has been a longstanding challenge. Here we developed a technique to reconstruct a microvascular system using a polycaprolactone (PCL)/gelatin nanofibrous structure and a co-culture system. Using a simple electrospinning process, we fabricated three-dimensional mesh scaffolds to support the sprouting of human umbilical vein endothelial cells (HUVECs) along the electrospun nanofiber. The co-culture with adipose-derived mesenchymal stem cells (ADSCs) supported greater sprouting of endothelial cells (ECs). In a two-dimensional culture system, angiogenic cell assembly produced more effective direct intercellular interactions and paracrine signaling from ADSCs to assist in the vascular formation of ECs, compared to the influence of growth factor. Although vascular endothelial growth factor and sphingosine-1-phosphate were present during the culture period, the presence of ADSCs was the most important factor for the construction of a cell-assembled structure in the two-dimensional culture system. On the contrary, HUVECs co-cultured on PCL/gelatin nanofiber scaffolds produced mature and functional microvessel and luminal structures with a greater expression of vascular markers, including platelet endothelial cell adhesion molecule-1 and podocalyxin. Furthermore, both angiogenic factors and cellular interactions with ADSCs through direct contact and paracrine molecules contributed to the formation of enhanced engineered blood vessel structures. It is expected that the co-culture system of HUVECs and ADSCs on bioengineered PCL/gelatin nanofibrous scaffolds will promote robust and functional microvessel structures and will be valuable for the regeneration of tissue with restored blood vessels.

Design of biomimetic cellular scaffolds for co-culture system and their application.

The extracellular matrix of most natural tissues comprises various types of cells, including fibroblasts, stem cells, and endothelial cells, which communicate with each other directly or indirectly to regulate matrix production and cell functionality. To engineer multicellular interactions in vitro, co-culture systems have achieved tremendous success achieving a more realistic microenvironment of in vivo metabolism than monoculture system in the past several decades. Recently, the fields of tissue engineering and regenerative medicine have primarily focused on three-dimensional co-culture systems using cellular scaffolds, because of their physical and biological relevance to the extracellular matrix of actual tissues. This review discusses several materials and methods to create co-culture systems, including hydrogels, electrospun fibers, microfluidic devices, and patterning for biomimetic co-culture system and their applications for specific tissue regeneration. Consequently, we believe that culture systems with appropriate physical and biochemical properties should be developed, and direct or indirect cell-cell interactions in the remodeled tissue must be considered to obtain an optimal tissue-specific microenvironment.

Functional spheroid organization of human salivary gland cells cultured on hydrogel-micropatterned nanofibrous microwells.

Development of a tissue-engineered, salivary bio-gland will benefit patients suffering from xerostomia due to loss of fluid-secreting acinar cells. This study was conducted to develop a bioengineering system to induce self-assembly of human parotid epithelial cells (hPECs) cultured on poly ethylene glycol (PEG) hydrogel-micropatterned polycaprolactone (PCL) nanofibrous microwells. Microwells were fabricated by photopatterning of PEG hydrogel in the presence of an electrospun PCL nanofibrous scaffold. hPECs were plated on plastic dishes, Matrigel, PCL nanofibers, or PCL nanofibrous microwells. When the cells were plated onto plastic, they did not form spheres, but aggregated to form 3D acinar-like spheroids when cultured on Matrigel, PCL, and PCL microwells, with the greatest aggregating potency being observed on the PCL microwells. The 3D-assembled spheroids in the PCL microwells expressed higher levels of salivary epithelial markers (α-amylase and AQP5), tight junction proteins (ZO-1 and occludin), adherence protein (E-cadherin), and cytoskeletal protein (F-actin) than those on the Matrigel and PCL. Furthermore, the 3D-assembled spheroids in the PCL microwells showed higher levels of α-amylase secretion and intracellular calcium concentration ([Ca2+]i) than those on the Matrigel and PCL nanofibers, suggesting more functional organization of hPECs. We established a bioengineering 3D culture system to promote robust and functional acinar-like organoids from hPECs. PCL nanofibrous microwells can be applied in the future for bioengineering of an artificial bio-salivary gland for restoration of salivary function. STATEMENT OF SIGNIFICANCE: Three dimensional (3D) cultures of salivary glandular epithelial cells using nanofibrous bottom facilitate the formation of acinar-like organoids. In this study, we adapted a PEG hydrogel-micropatterned PCL nanofibrous microwell for the efficient bioengineering of human salivary gland organoids, in which we could easily produce uniform size of 3D organoids. This 3D culture system supports spherical organization, gene and protein expression of acinar markers, TJ proteins, adherence, and cytoskeletal proteins, as well as to promote epithelial structural integrity and acinar secretory functions, and results showed superior efficiency relative to Matrigel and nanofibrous scaffold culture. This 3D culture system has benefits in terms of inert, non-animal and serum-free culture conditions, as well as controllable spheroid size and scalable production of functional SG organoids and is applicable to bioengineering approaches for an artificial bio-gland, as well as to investigations of salivary gland physiology and regeneration.