Multiscale Control of Nanofiber-Composite Hydrogel for Complex 3D Cell Culture by Extracellular Matrix Composition and Nanofiber Alignment.

In order to manipulate the complex behavior of cells in a 3-dimensional (3D) environment, it is important to provide the microenvironment that can accurately portray the complexity of highly anisotropic tissue structures. However, it is technically challenging to generate a complex microenvironment using conventional biomaterials that are mostly isotropic with limited bioactivity. In this study, the gelatin-hyaluronic acid hydrogel incorporated with aqueous-dispersible, short nanofibers capable of in situ alignment is developed to emulate the native heterogeneous extracellular matrix consisting of fibrous and non-fibrous components. The gelatin nanofibers containing magnetic nanoparticles, which could be aligned by external magnetic field, are dispersed and embedded in gelatin-hyaluronic acid hydrogel encapsulated with dermal fibroblasts. The aligned nanofibers via magnetic field could be safely integrated into the hydrogel, and the process could be repeated to generate larger 3D hydrogels with variable nanofiber alignments. The aligned nanofibers in the hydrogel can more effectively guide the anisotropic morphology (e.g., elongation) of dermal fibroblasts than random nanofibers, whereas myofibroblastic differentiation is more prominent in random nanofibers. At a given nanofiber configuration, the hydrogel composition having intermediate hyaluronic acid content induces myofibroblastic differentiation. These results indicate that modulating the degree of nanofiber alignment and the hyaluronic acid content of the hydrogel are crucial factors that critically influence the fibroblast phenotypes. The nanofiber-composite hydrogel capable of directional nanofiber alignment and tunable material composition can effectively induce a wide array of phenotypic plasticity in 3D cell culture.

Injectable Self-Oxygenating Cardio-Protective and Tissue Adhesive Silk-Based Hydrogel for Alleviating Ischemia After Mi Injury.

Myocardial infarction (MI) is a significant cardiovascular disease that restricts blood flow, resulting in massive cell death and leading to stiff and noncontractile fibrotic scar tissue formation. Recently, sustained oxygen release in the MI area has shown regeneration ability; however, improving its therapeutic efficiency for regenerative medicine remains challenging. Here, a combinatorial strategy for cardiac repair by developing cardioprotective and oxygenating hybrid hydrogels that locally sustain the release of stromal cell-derived factor-1 alpha (SDF) and oxygen for simultaneous activation of neovascularization at the infarct area is presented. A sustained release of oxygen and SDF from injectable, mechanically robust, and tissue-adhesive silk-based hybrid hydrogels is achieved. Enhanced endothelialization under normoxia and anoxia is observed. Furthermore, there is a marked improvement in vascularization that leads to an increment in cardiomyocyte survival by ≈30% and a reduction of the fibrotic scar formation in an MI animal rodent model. Improved left ventricular systolic and diastolic functions by ≈10% and 20%, respectively, with a ≈25% higher ejection fraction on day 7 are also observed. Therefore, local delivery of therapeutic oxygenating and cardioprotective hydrogels demonstrates beneficial effects on cardiac functional recovery for reparative therapy.

Flexible, Biodegradable, and Wireless Magnetoelectric Paper for Simple In Situ Personalization of Bioelectric Implants.

Bioelectronic implants delivering electrical stimulation offer an attractive alternative to traditional pharmaceuticals in electrotherapy. However, achieving simple, rapid, and cost-effective personalization of these implants for customized treatment in unique clinical and physical scenarios presents a substantial challenge. This challenge is further compounded by the need to ensure safety and minimal invasiveness, requiring essential attributes such as flexibility, biocompatibility, lightness, biodegradability, and wireless stimulation capability. Here, a flexible, biodegradable bioelectronic paper with homogeneously distributed wireless stimulation functionality for simple personalization of bioelectronic implants is introduced. The bioelectronic paper synergistically combines i) lead-free magnetoelectric nanoparticles (MENs) that facilitate electrical stimulation in response to external magnetic field and ii) flexible and biodegradable nanofibers (NFs) that enable localization of MENs for high-selectivity stimulation, oxygen/nutrient permeation, cell orientation modulation, and biodegradation rate control. The effectiveness of wireless electrical stimulation in vitro through enhanced neuronal differentiation of neuron-like PC12 cells and the controllability of their microstructural orientation are shown. Also, scalability, design flexibility, and rapid customizability of the bioelectronic paper are shown by creating various 3D macrostructures using simple paper crafting techniques such as cutting and folding. This platform holds promise for simple and rapid personalization of temporary bioelectronic implants for minimally invasive wireless stimulation therapies.

Ultrasound standing wave spatial patterning of human umbilical vein endothelial cells for 3D micro-vascular networks formation.

Generating functional and perfusable micro-vascular networks is an important goal for the fabrication of large and three-dimensional tissues. Up to now, the fabrication of micro-vascular networks is a complicated multitask involving several different factors such as time consuming, cells survival, micro-diameter vasculature and strict alignment. Here, we propose a technique combining multi-material extrusion and ultrasound standing wave forces to create a network structure of human umbilical vein endothelial cells within a mixture of calcium alginate and decellularized extracellular matrix. The functionality of the matured microvasculature networks was demonstrated through the enhancement of cell-cell adhesion, angiogenesis process, and perfusion tests with microparticles, FITC-dextran, and whole mouse blood. Moreover, animal experiments exhibited the implantability including that the pre-existing blood vessels of the host sprout towards the preformed vessels of the scaffold over time and the microvessels inside the implanted scaffold matured from empty tubular structures to functional blood-carrying microvessels in two weeks.

Double-layered blood vessels over 3 mm in diameter extruded by the inverse-gravity technique.

One of the most promising techniques for treating severe peripheral artery disease is the use of cellular tissue-engineered vascular grafts (TEVGs). This study proposes an inverse-gravity (IG) extrusion technique for creating long double-layered cellular TEVGs with diameters over 3 mm. A three-layered coaxial laminar hydrogel flow in an 8 mm-diameter pipe was realised simply by changing the extrusion direction of the hydrogel from being aligned with the direction of gravity to against it. This technique produced an extruded mixture of human aortic smooth muscle cells (HASMCs) and type-I collagen as a tubular structure with an inner diameter of 3.5 mm. After a 21 day maturation period, the maximal burst pressure, longitudinal breaking force, and circumferential breaking force of the HASMC TEVG were 416 mmHg, 0.69 N, and 0.89 N, respectively. The HASMC TEVG was endothelialised with human umbilical vein endothelial cells to form a tunica intima that simulated human vessels. Besides subcutaneous implantability on mice, the double-layered blood vessels showed a considerably lower adherence of platelets and red blood cells once exposed to heparinised mouse blood and were considered nonhaemolytic. The proposed IG extrusion technique can be applied in various fields requiring multilayered materials with large diameters.

Emerging Technology of Nanofiber-Composite Hydrogels for Biomedical Applications.

Hydrogels and nanofibers have been firmly established as go-to materials for various biomedical applications. They have been mostly utilized separately, rarely together, because of their distinctive attributes and shortcomings. However, the potential benefits of integrating nanofibers with hydrogels to synergistically combine their functionalities while attenuating their drawbacks are increasingly recognized. Compared to other nanocomposite materials, incorporating nanofibers into hydrogel has the distinct advantage of emulating the hierarchical structure of natural extracellular environment needed for cell and tissue culture. The most important technological aspect of developing "nanofiber-composite hydrogel" is generating nanofibers made of various polymers that are cross-linked and short enough to maintain stable dispersion in hydrated environment. In this review, recent research efforts to develop nanofiber-composite hydrogels are presented, with added emphasis on nanofiber processing techniques. Several notable examples of implementing nanofiber-composite hydrogels for biomedical applications are also introduced.

Multiscale Engineering of Nanofiber-Aerogel Composite Nanogenerator with Tunable Triboelectric Performance Based on Multifunctional Polysuccinimide.

Multiscale polymer engineering, involving chemical modification to control their triboelectric polarities as well as physicomechanical modification to maximize charge transfer and structural durability, is paramount to developing a high-performance triboelectric nanogenerator (TENG). This report introduces a highly efficient and comprehensive strategy to engineer high-performance TENG based on multifunctional polysuccinimide (PSI). With the ability of PSI to undergo facile nucleophilic addition with amines, sodium sulfate and quaternary ammonium chlorides having opposite charged groups are conjugated to PSI in varying densities. The resulting Sulfo-PSI and TMAC-PSI, respectively, processed into nanofibrous films, demonstrate highly enhanced and variable triboelectric properties based on the charge type and density. To further enhance the mechanical toughness and biocompatibility necessary for wearable applications, these PSI nanofibers are processed into alginate aerogel (AG). The sustained triboelectric performance of this nanofiber-AG TENG as a wearable energy harvester and biosensor is examined and validated in detail.

Mechanotopography-Driven Design of Dispersible Nanofiber-Laden Hydrogel as a 3D Cell Culture Platform for Investigating Tissue Fibrosis.

Fibrosis is one of the most frequent occurrences during one's lifetime, identified by various physiological changes including, most notably, excessive deposition of extracellular matrix (ECM). Despite its physiological importance, it is still a significant challenge to conduct a systematic investigation of tissue fibrosis, mainly due to the lack of in vitro 3D tissue model that can accurately portray the characteristic features of fibrotic events. Herein, a hybrid hydrogel system incorporating dispersible nanofibers is developed to emulate highly collagenous deposits formed within a fibrotic tissue leading to altered mechanotopographical properties. Micrometer-length, aqueous-stable nanofibers consisting of crosslinked gelatin network embedded with graphene oxide (GO) or reduced graphene (rGO) are infused into hydrogel, resulting in controllable mechanotopographical properties while maintaining permeability sufficiently enough for various cellular activities. Ultimately, the ability to induce fibrotic behavior of fibroblasts cultured in these mechanotopography-controlled, nanofiber-laden hydrogels is investigated in detail.

Synergistic control of mechanics and microarchitecture of 3D bioactive hydrogel platform to promote the regenerative potential of engineered hepatic tissue.

Culturing autologous cells with therapeutic potential derived from a patient within a bioactive scaffold to induce functioning tissue formation is considered the ideal methodology towards realizing patient-specific regenerative medicine. Hydrogels are often employed as the scaffold material for this purpose mainly for their tunable mechanical and diffusional properties as well as presenting cell-responsive moieties. Herein, a two-fold strategy was employed to control the physicomechanical properties and microarchitecture of hydrogels to maximize the efficacy of engineered hepatic tissues. First, a hydrophilic polymeric crosslinker with a tunable degree of reactive functional groups was employed to control the mechanical properties in a wide range while minimizing the change in diffusional properties. Second, photolithography technique was utilized to introduce microchannels into hydrogels to overcome the critical diffusional limit of bulk hydrogels. Encapsulating hepatic progenitor cells derived via direct reprogramming of tissue-harvested fibroblasts, the application of this strategy to control the mechanics, diffusion, and architecture of hydrogels in a combinatorial manner could allow the optimization of their hepatic functions. The regenerative capacity of this engineered hepatic tissue was further demonstrated using an in vivo acute liver injury model.

Dual-functional alginate crosslinker: Independent control of crosslinking density and cell adhesive properties of hydrogels via separate conjugation pathways.

Alginate is an abundant natural polysaccharide widely utilized in various biomedical applications. Alginate also possesses numerous hydroxyl and carboxylate functional groups that allow chemical modifications to introduce different functionalities. However, it is difficult to apply various chemical reactions to alginate due to limited solubility in organic solvents. Herein, functional moieties for radical polymerization and cell adhesion were separately conjugated to hydroxyl and carboxylate groups of alginate, respectively, in order to independently control the crosslinking density and cell adhesive properties of hydrogels. Sodium counterions of alginate are first substituted with tetrabutylammonium ions to facilitate the dissolution in an organic solvent, followed by in situ conjugations of (1) cell adhesion molecules (CAM) via carbodiimide-mediated amide formation and (2) methacrylate via ring-opening nucleophilic reaction. The resulting CAM-linked methacrylic alginate was able to not only crosslink different monomers to form hydrogels with varying mechanical properties, but also induce stable cell adhesion to the hydrogels.