Photoactivated growth factor release from bio-orthogonally crosslinked hydrogels for the regeneration of corneal defects.

In situ-forming hydrogels are an attractive option for corneal regeneration, and the delivery of growth factors from such constructs have the potential to improve re-epithelialization and stromal remodeling. However, challenges persist in controlling the release of therapeutic molecules from hydrogels. Here, an in situ-forming bio-orthogonally crosslinked hydrogel containing growth factors tethered via photocleavable linkages (PC-HACol hydrogel) was developed to accelerate corneal regeneration. Epidermal growth factor (EGF) was conjugated to the hydrogel backbone through photo-cleavable (PC) spacer arms and was released when exposed to mild intensity ultraviolet (UV) light (2-5 mW/cm2, 365 nm). The PC-HACol hydrogel rapidly gelled within a few minutes when applied to corneal defects, with excellent transparency and biocompatibility. After subsequent exposure to UV irradiation, the hydrogel promoted the proliferation and migration of corneal epithelial cells in vitro. The rate of re-epithelialization was positively correlated to the frequency of irradiation, verified through ex vivo rabbit cornea organ culture studies. In an in vivo rat corneal wound healing study, the PC-HACol hydrogel exposed to UV light significantly promoted re-epithelialization, the remodeling of stromal layers, and exhibited significant anti-scarring effects, with minimal α-SMA and robust ALDH3A1 expression. Normal differentiation of the regenerated epithelia after healing was evaluated by expression of the corneal epithelial biomarker, CK12. The remodeled cornea exhibited full recovery of corneal thickness and layer number without hyperplasia of the epithelium.

Secured delivery of basic fibroblast growth factor using human serum albumin-based protein nanoparticles for enhanced wound healing and regeneration.

BACKGROUND: Basic fibroblast growth factor (bFGF) is one of the critical components accelerating angiogenesis and tissue regeneration by promoting the migration of dermal fibroblasts and endothelial cells associated with matrix formation and remodeling in wound healing process. However, clinical applications of bFGF are substantially limited by its unstable nature due to rapid decomposition under physiological microenvironment. RESULTS: In this study, we present the bFGF-loaded human serum albumin nanoparticles (HSA-bFGF NPs) as a means of enhanced stability and sustained release platform during tissue regeneration. Spherical shape of the HSA-bFGF NPs with uniform size distribution (polydispersity index < 0.2) is obtained via a simple desolvation and crosslinking process. The HSA-bFGF NPs securely load and release the intact soluble bFGF proteins, thereby significantly enhancing the proliferation and migration activity of human dermal fibroblasts. Myofibroblast-related genes and proteins were also significantly down-regulated, indicating decrease in risk of scar formation. Furthermore, wound healing is accelerated while achieving a highly organized extracellular matrix and enhanced angiogenesis in vivo. CONCLUSION: Consequently, the HSA-bFGF NPs are suggested not only as a delivery vehicle but also as a protein stabilizer for effective wound healing and tissue regeneration.

A small-data-driven model for predicting adsorption properties in polymeric thin films.

Artificial intelligence allowing data-driven prediction of physicochemical properties of polymers is rapidly emerging as a powerful tool for advancing material science. Here, we developed a methodology to use polymer adsorption data as predictable data by analyzing causal relationships between polymer properties and experimental results instead of using big polymer data.

Nano-structure of vitronectin/heparin on cell membrane for stimulating single cell in iPSC-derived embryoid body.

Individual cell environment stimulating single cell is a suitable strategy for the generation of sophisticated multicellular aggregates with localized biochemical signaling. However, such strategy for induced pluripotent stem cell (iPSC)-derived embryoid bodies (EBs) is limited because the presence of external stimulation can inhibit spontaneous cellular communication, resulting in misdirection in the maturation and differentiation of EBs. In this study, a facile method of engineering the iPSC membrane to stimulate the inner cell of EBs while maintaining cellular activities is reported. We coated the iPSC surface with nanoscale extracellular matrix fabricated by self-assembly between vitronectin and heparin. This nano-coating allowed iPSC to retain its in vitro properties including adhesion capability, proliferation, and pluripotency during its aggregation. More importantly, the nano-coating did not induce lineage-specific differentiation but increased E-cadherin expression, resulting in promotion of development of EB. This study provides a foundation for future production of sophisticated patient-specific multicellular aggregates by modification of living cell membranes.

2D graphene oxide particles induce unwanted loss in pluripotency and trigger early differentiation in human pluripotent stem cells.

The potential health hazards of particulates, such as micro/nano-sized plastics and carbon materials have recently received extensive attention. However, their toxicological properties in association with stem cell differentiation is still relatively unexplored. In this study, we elucidated the cytotoxic effects of 2D graphene oxide (GO), in relation to differentiation of human induced pluripotent stem cells (hiPSCs). Supplementation of GO to hiPSCs demonstrated uptake of GO through the plasma membrane and intracellular accumulation was observed. Increasing the concentration of GO led to reduced viability and increased likelihood of hiPSC colony detachment. Moreover, treatment of GO resulted in significant loss in pluripotency markers, OCT-4 and NANOG. In particular, when hiPSCs were cultured with GO in cardiomyocyte induction medium, upregulation of cardiomyocyte marker, NKX2.5, along with observation of early triggering of differentiation were observed. Taken together, our results highlight the risk in the uptake and accumulation of GO on the stem cell development by unwanted loss in pluripotency and accelerated initiation of differentiation.

Transmission and regulation of biochemical stimulus via a nanoshell directly adsorbed on the cell membrane to enhance chondrogenic differentiation of mesenchymal stem cell.

A nanoscale artificial extracellular matrix (nanoshell) formed by layer-by-layer adsorption can enhance and modulate the function of stem cells by transferring biochemical stimulus to the cell directly. Here, the nanoshell composed of fibronectin (FN) and chondroitin sulfate (CS) is demonstrated to promote chondrogenic differentiation of mesenchymal stem cells (MSCs). The multilayer structure of nanoshell is formed by repeating self-assembly of FN and CS, and its thickness can be controlled through the number of layers. The expression of chondrogenic markers in MSCs coated with the FN/CS nanoshell was increased as the number of bilayers in the nanoshell increased until four, but when it exceeds five bilayers, the effect began to decrease. Finally, the MSCs coated with optimized four bilayers of FN/CS nanoshell have high chondrogenic differentiation efficiency and showed the potential to increase formation of cartilage tissue when it is transplanted into mouse kidney. So, the precise regulation of stem cell fate at single cell level can be possible through the cellular surface modification by self-assembled polymeric film.

Construction of nano-scale cellular environments by coating a multilayer nanofilm on the surface of human induced pluripotent stem cells.

Interactions with peripheral environments, such as extracellular matrix (ECM) and other cells, and their balance play a crucial role in the maintenance of pluripotency and self-renewal of human pluripotent stem cells. In this study, we focused on a nano-sized artificial cellular environment that is directly attached to the cytoplasmic membrane as a facile method that can effect intercellular interactions at the single-cell level. We designed multilayered nanofilms that are self-assembled on the surface of human induced pluripotent stem cells (iPSCs), by repetitive adsorption of fibronectin and heparin or chondroitin sulfate. However, the surface modification process could also lead to the loss of cell-cell adhesion, which may result in apoptotic cell death. We investigated the proliferation and pluripotency of the iPSCs coated with the nanofilm in order to establish the suitable nanofilm structure and coating conditions. As a result, the cell viability reduced with the increase in the duration of the coating process, but the undifferentiated state and proliferation of the cells were maintained until 2 bilayers were coated. To suppress the dissociation-induced apoptosis, Y-27632, the Rho-associated kinase inhibitor (ROCKi), was added to the coating solution; this allowed the coating of up to 4 bilayers of the nanofilm onto the iPSCs. These results are expected to accelerate the pace of iPSC studies on 3-dimensional cultures and naïve pluripotency, in which the regulation of cellular interactions plays a critical role.

Artificial cellular nano-environment composed of collagen-based nanofilm promotes osteogenic differentiation of mesenchymal stem cells.

In regenerative medicine, the generation of therapeutic stem cells and tissue engineering are important for replacing damaged tissues. Numerous studies have attempted to produce cellular components that mimic the native tissue for gaining optimal function. Particularly, the extracellular matrix (ECM) composition plays an important role in cellular functions including determining the fates of mesenchymal stem cells (MSCs). Here, we evaluated the osteogenic effects of a nanofilm in which oppositely charged polyelectrolytes were alternately adsorbed onto the cell surface to create an artificial ECM environment for single MSCs. Interestingly, nanofilm composed of collagen (Col) and alginate (AA) showed relatively high stiffness and MSCs coated with the Col/AA nanofilm showed increased osteogenic differentiation efficiency compared to other nanofilm-coated MSCs. Further analysis revealed that the Col/AA nanofilm coating stimulated osteogenesis by activating transcriptional coactivators with the PDZ binding motif through extracellular signal-related kinase and p38 MAPK signaling. This nano-sized cellular coating will facilitate the development of nanotechnology for controlling cellular functions and advance stem cell-based clinical applications for regenerative medicine. STATE OF SIGNIFICANCE: In this study, we developed an artificial cellular nano-environment formed by multilayer nanofilms. We demonstrated that the nanofilms introduced to mesenchymal stem cells (MSCs) stimulate osteogenic differentiation by regulating intracellular signaling. Among the various nanofilm combinations, the induction of osteogenic gene transcription in collagen (Col) and alginate (AA) film-coated MSCs was the most pronounced compared to that on other nanofilms. A minimum number of Col/AA nanofilm bilayers (n = 2) was required for effective induction of MSC osteogenic differentiation. In addition, we observed the correlation between the promoting effect of osteogenic differentiation and stiffness of the nanofilm. Our results may be useful for developing a cell coating model system widely applicable in bioengineering and regenerative medicine.

Layer-by-layer assembled polymeric thin films as prospective drug delivery carriers: design and applications.

BACKGROUND: The main purpose of drug delivery systems is to deliver the drugs at the appropriate concentration to the precise target site. Recently, the application of a thin film in the field of drug delivery has gained increasing interest because of its ability to safely load drugs and to release the drug in a controlled manner, which improves drug efficacy. Drug loading by the thin film can be done in various ways, depending on type of the drug, the area of exposure, and the purpose of drug delivery. MAIN TEXT: This review summarizes the various methods used for preparing thin films with drugs via Layer-by-layer (LbL) assembly. Furthermore, additional functionalities of thin films using surface modification in drug delivery are briefly discussed. There are three types of methods for preparing a drug-carrying multilayered film using LbL assembly. First methods include approaches for direct loading of the drug into the pre-fabricated multilayer film. Second methods are preparing thin films using drugs as building blocks. Thirdly, the drugs are incorporated in the cargo so that the cargo itself can be used as the materials of the film. CONCLUSION: The appropriate designs of the drug-loaded film were produced in consideration of the release amounts and site of the desired drug. Furthermore, additional surface modification using the LbL technique enabled the preparation of effective drug delivery carriers with improved targeting effect. Therefore, the multilayer thin films fabricated by the LbL technique are a promising candidate for an ideal drug delivery system and the development possibilities of this technology are infinite.

Structure of a Multilayer Nanofilm To Increase the Encapsulation Efficiency of Basic Fibroblast Growth Factor.

In this study, we established the structure of a multilayer nanofilm that more efficiently encapsulates basic fibroblast growth factor (bFGF). First, a positively charged layer material was selected from biocompatible polymers such as collagen (Col), poly(beta-amino ester) (Poly2), and chitosan (Chi), while considering the film thickness. We then investigated the change in bFGF encapsulation efficiency when the multilayer structure was changed from a tetralayer to a trilayer. As a result, we obtained a highly improved bFGF encapsulation efficiency in the nanofilm using a positively charged layer formed by a blend of Col and Poly2 and a negatively charged poly(acrylic acid) (PAA) layer within a trilayered structure. In particular, we found that a significant amount of adsorbed bFGF was desorbed again during the film fabrication process of a tetralayered nanofilm. In the conventional nanofilm, bFGF was regarded as a polycation and formed a multilayer nanofilm that was composed of a tetralayered structure and was represented as (polycation/polyanion/bFGF/polyanion) n where n = number of repeated tetralayers. Here, we suggested that bFGF should not be considered a polycation, rather it should be considered as a small quantity of molecule that exists between the polyanion and polycation layers. In this case, the nanofilm is composed of repeating units of (polycation/polyanion/bFGF/polycation/polyanion), because the amount of adsorbed bFGF is considerably lower than that of other building blocks.

Efficient Encapsulation and Sustained Release of Basic Fibroblast Growth Factor in Nanofilm: Extension of the Feeding Cycle of Human Induced Pluripotent Stem Cell Culture.

Basic fibroblast growth factor (bFGF) has an established pivotal function in biomedical engineering, especially for the human pluripotent stem cells (iPSCs). However, the limitation of bFGF is the ease of denaturation under normal physiological conditions, inducing loss of its activity. In this study, we designed multi-trilayered nanofilm composed of a repeating polycation/polyanion/bFGF structure, which has high loading efficiency and short buildup time. We also investigated that the loading and release of bFGF from the nanofilm with two parameters (counter-polyanion and film architectures). Then, we prepared the optimized nanofilm which maintains a sustained bFGF level in physiological condition to apply the nanofilm to human iPSCs culture. The amount of bFGF release from 12 trilayer nanofilm was 36.4 ng/cm2, and activity of bFGF encapsulated into the nanofilm was maintained (60%) until 72 h during incubation at 37 °C. As a result, the iPSCs grown in the presence of the nanofilm with tridaily replacement of growth medium maintained undifferentiated morphology and expression levels of pluripotency marker proteins.

Multilayer Nanofilms via Inkjet Printing for Stabilizing Growth Factor and Designing Desired Cell Developments.

Biologically versatile basic fibroblast growth factor (bFGF), well known for roles of signaling molecules between cells and regulating various cellular processes, has been proven to utilize specific functionalities. However, the remarkable functions are inclinable to dwindle with decrease of bFGFs' activity. In nanoscale, developing thin films with intrinsic characteristics of building molecules can facilitate handling various materials for desired purposes. Fabricating nanofilm and handling sensitive materials without detriment to activity via highly productive manufacturing are significant for practical uses in the field of biomedical applications. Herein, a multilayered nanofilm fabricating system is developed by inkjet printing to incorporate bFGF successfully. It is demonstrated that water mixed with glycerol as biological ink maintains stability of bFGFs through simulation and experimental study. With highly stable bFGFs, the proliferation of human dermal fibroblast is enhanced and the undifferentiated state of induced pluripotent stem cell is maintained by the controlled release of bFGF.

Effect of pH on the structure and drug release profiles of layer-by-layer assembled films containing polyelectrolyte, micelles, and graphene oxide.

Layer by layer (lbl) assembled multilayer thin films are used in drug delivery systems with attractive advantages such as unlimited selection of building blocks and free modification of the film structure. In this paper, we report the fundamental properties of lbl films constructed from different substances such as PS-b-PAA amphiphilic block copolymer micelles (BCM) as nano-sized drug vehicles, 2D-shaped graphene oxide (GO), and branched polyethylenimine (bPEI). These films were fabricated by successive lbl assembly as a result of electrostatic interactions between the carboxyl group of BCM and amine group of functionalized GO or bPEI under various pH conditions. We also compared the thickness, roughness, morphology and degree of adsorption of the (bPEI/BCM) films to those in the (GO/BCM) films. The results showed significant difference because of the distinct pH dependence of each material. In addition, drug release rates of the GO/BCM film were more rapid those of the (bPEI/BCM) film in pH 7.4 and pH 2 PBS buffer solutions. In (bPEI/BCM/GO/BCM) film, the inserted GO layers into bPEI/BCM multilayer induced rapid drug release. We believe that these materials &pH dependent film properties allow developments in the control of coating techniques for biological and biomedical applications.