Microfluidics-assisted fabrication of natural killer cell-laden microgel enhances the therapeutic efficacy for tumor immunotherapy.

Recently, interest in cancer immunotherapy has increased over traditional anti-cancer therapies such as chemotherapy or targeted therapy. Natural killer (NK) cells are part of the immune cell family and essential to tumor immunotherapy as they detect and kill cancer cells. However, the disadvantage of NK cells is that cell culture is difficult. In this study, porous microgels have been fabricated using microfluidic channels to effectively culture NK cells. Microgel fabrication using microfluidics can be mass-produced in a short time and can be made in a uniform size. Microgels consist of photo cross-linkable polymers such as methacrylic gelatin (GelMa) and can be regulated via controlled GelMa concentrations. NK92 cell-laden three-dimensional (3D) microgels increase mRNA expression levels, NK92 cell proliferation, cytokine release, and anti-tumor efficacy, compared with two-dimensional (2D) cultures. In addition, the study confirms that 3D-cultured NK92 cells enhance anti-tumor effects compared with enhancement by 2D-cultured NK92 cells in the K562 leukemia mouse model. Microgels containing healthy NK cells are designed to completely degrade after 5 days allowing NK cells to be released to achieve cell-to-cell interaction with cancer cells. Overall, this microgel system provides a new cell culture platform for the effective culturing of NK cells and a new strategy for developing immune cell therapy.

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.

Structural evaluation of Poly(lactic acid) degradation at standardized composting temperature of 58 degrees.

The accumulation of petroleum-based plastics on our planet is causing serious environmental pollution. Biodegradable plastics, promoted as eco-friendly solutions, hold the potential to address this issue. However, their impact on the environment and the mechanisms of their natural degradation remain inadequately understood. Furthermore, the specific conditions set forth in international standards for evaluating the biodegradability of biodegradable plastics have led to misconceptions about their real-world behavior. To properly elucidate the relationship between their degradability and structure, this study mimics the thermal effect on poly(lactic acid) (PLA) under standardized composting temperature. The higher the crystallinity of PLA, the lower the degradation rate, which suggests that crystallinity is a key factor in determining degradation. The composting temperature of 58 °C induces crystallization by having a structural effect on the polymer, which in turn reduces the degradation rate of PLA. Therefore, control over temperature and crystallization during the processing and degradation of PLA is crucial, as it not only determines the biodegradability but also enhances the utility.

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.

Network of cyano-p-aramid nanofibres creates ultrastiff and water-rich hydrospongels.

The structure-property paradox of biological tissues, in which water-rich porous structures efficiently transfer mass while remaining highly mechanically stiff, remains unsolved. Although hydrogel/sponge hybridization is the key to understanding this phenomenon, material incompatibility makes this a challenging task. Here we describe hydrogel/sponge hybrids (hydrospongels) that behave as both ultrastiff water-rich gels and reversibly squeezable sponges. The self-organizing network of cyano-p-aramid nanofibres holds approximately 5,000 times more water than its solid content. Hydrospongels, even at a water concentration exceeding 90 wt%, are hard as cartilage with an elastic modulus of 50-80 MPa, and are 10-1,000 times stiffer than typical hydrogels. They endure a compressive strain above 85% through poroelastic relaxation and hydrothermal pressure at 120 °C. This performance is produced by amphiphilic surfaces, high rigidity and an interfibrillar, interaction-driven percolating network of nanofibres. These features can inspire the development of future biofunctional materials.

Exploring stimuli-responsive elastin-like polypeptide for biomedicine and beyond: potential application as programmable soft actuators.

With the emergence of soft robotics, there is a growing need to develop actuator systems that are lightweight, mechanically compliant, stimuli-responsive, and readily programmable for precise and intelligent operation. Therefore, "smart" polymeric materials that can precisely change their physicomechanical properties in response to various external stimuli (e.g., pH, temperature, electromagnetic force) are increasingly investigated. Many different types of polymers demonstrating stimuli-responsiveness and shape memory effect have been developed over the years, but their focus has been mostly placed on controlling their mechanical properties. In order to impart complexity in actuation systems, there is a concerted effort to implement additional desired functionalities. For this purpose, elastin-like polypeptide (ELP), a class of genetically-engineered thermoresponsive polypeptides that have been mostly utilized for biomedical applications, is being increasingly investigated for stimuli-responsive actuation. Herein, unique characteristics and biomedical applications of ELP, and recent progress on utilizing ELP for programmable actuation are introduced.

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.

Manipulating Physicochemical Properties of Biosensor Platform with Polysuccinimide-Silica Nanocomposite for Enhanced Protein Detection.

As point-of-care testing (POCT) is becoming the new paradigm of medical diagnostics, there is a growing need to develop reliable POCT devices that can be conveniently operated in a minimally invasive manner. However, the clinical potential of POCT diagnostics is yet to be realized, mainly due to the limited and inconsistent amount of collected samples on these devices, undermining their accuracy. This study proposes a new biosensing platform modified with a functional polysuccinimide (PSI)-silica nanoparticle (SNP) composite system that can substantially increase the protein conjugation efficiency by modulating physicochemical interaction with proteins by several hundred percent from an unmodified device. The efficacy of this PSI-SNP system is further validated by applying it on the surface of a microneedle array (MN), which has emerged as a promising POCT device capable of accessing interstitial fluid through minimal penetration of the skin. This PSI-SNP MN is demonstrated to detect a wide array of proteins with high sensitivity on par with conventional whole serum analysis, validated by in vivo animal testing, effectively displaying broad applicability in biomedical engineering.

Tunable Physicomechanical and Drug Release Properties of In Situ Forming Thermoresponsive Elastin-like Polypeptide Hydrogels.

With the continued advancement in the design and engineering of hydrogels for biomedical applications, there is a growing interest in imparting stimuli-responsiveness to the hydrogels in order to control their physicomechanical properties in a more programmable manner. In this study, an in situ forming hydrogel is developed by cross-linking alginate with an elastin-like polypeptide (ELP). Lysine-rich ELP synthesized by recombinant DNA technology is reacted with alginate presenting an aldehyde via Schiff base formation, resulting in facile hydrogel formation under physiological conditions. The physicomechanical properties of alginate-ELP hydrogels can be controlled in a wide range by the concentrations of alginate and ELP. Owing to the thermoresponsive properties of the ELP, the alginate-ELP hydrogels undergo swelling/deswelling near the physiological temperature. Taking advantage of these highly attractive properties of alginate-ELP, drug release kinetics were measured to evaluate their potential as a thermoresponsive drug delivery system. Furthermore, an ex vivo model was used to demonstrate the minimally invasive tissue injectability.

Precision Control of Programmable Actuation of Thermoresponsive Nanocomposite Hydrogels with Multilateral Engineering.

Hydrogels capable of stimuli-responsive deformation are widely explored as intelligent actuators for diverse applications. It is still a significant challenge, however, to "program" these hydrogels to undergo highly specific and extensive shape changes with precision, because the mechanical properties and deformation mechanism of the hydrogels are inherently coupled. Herein, two engineering strategies are simultaneously employed to develop thermoresponsive poly(N-isopropyl acrylamide) (PNIPAm)-based hydrogels capable of programmable actuation. First, PNIPAm is copolymerized with poly(ethylene glycol) diacrylate (PEGDA) with varying molecular weights and concentrations. In addition, graphene oxide (GO) or reduced graphene oxide (rGO) is incorporated to generate nanocomposite hydrogels. These strategies combine to allow the refined control of mechanical and diffusional properties of hydrogels over a broad range, which also directly influences variable thermoresponsive actuation. It is expected that this comprehensive design principle can be applied to a wide range of hydrogels for programmable actuation.

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.

Programmable multilayer printing of a mechanically-tunable 3D hydrogel co-culture system for high-throughput investigation of complex cellular behavior.

Hydrogels are widely used as a 3D cell culture platform, as they can be tailored to provide suitable microenvironments to induce cellular phenotypes with physiological significance. Hydrogels are especially deemed attractive as a co-culture platform, in which two or more different types of cells are cultured together in close proximity, since the spatial distribution of different cell types can be rendered possible by advanced microfabrication schemes. Herein, programmable multilayer photolithography is employed to develop a 3D hydrogel-based co-culture system in an efficient and scalable manner, which consists of an inner microgel array containing one cell type covered by an outer hydrogel overlay containing another cell type. In particular, the mechanical properties of microgel array and hydrogel overlay are independently controlled in a wide range, with elastic moduli ranging from 1.7 to 31.6 kPa, allowing the high-throughput investigation of both individual hydrogel mechanics and mechanical gradients generated at their interface. Utilizing this system, phenotypical changes (i.e. proliferation, spheroid formation and Mφ polarization) of macrophages encapsulated in microgel array, in response to complex mechanical microenvironment and co-cultured fibroblasts, are comprehensively explored.

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.

Graft Architecture Guided Simultaneous Control of Degradation and Mechanical Properties of In Situ Forming and Fast Dissolving Polyaspartamide Hydrogels.

Polyaspartamide, derived from polysuccinimide (PSI), has the advantage of conveniently presenting desired functional groups by ring-opening addition of amine-based nucleophiles to the succinimidyl ring moieties of PSI. Using diamines with varying lengths of poly(ethylene glycol) linker, polyaspartamide presenting amine groups with controllable grafting density and length, namely, poly(2-hydroxyethyl aspartamide)-g-amino-poly(ethylene glycol) (PHEA-PEGAm) could be synthesized. This PHEA-PEGAm was then used to develop in situ forming hydrogels by Schiff base formation with aldehyde-containing alginate (Alg-ALD). By modulating the graft architecture (i.e., grafting length and density), the mechanical properties of the resulting Alg-PHEA hydrogels could be controlled in a broad range. Remarkably, the hydrogels were shown to undergo facile degradation and complete dissolution in physiological conditions, regardless of hydrogel mechanics, by the expedited hydrolysis through the action of remaining amine groups, which was also heavily influenced by the graft architecture. Moreover, the rate of degradation could be further controlled by additional ionic cross-linking of alginate. The potential application as an injectable drug delivery system was demonstrated by measuring drug release kinetics and monitoring degradation ex vivo.

Cell subtype-dependent formation of breast tumor spheroids and their variable responses to chemotherapeutics within microfluidics-generated 3D microgels with tunable mechanics.

Tumor spheroids have been considered valuable miniaturized three dimensional (3D) tissue models for fundamental biological investigation as well as drug screening applications. Most tumor spheroids are generated utilizing the inherent aggregate behavior of tumor cells, and the effect of microenvironmental factors such as extracellular matrix (ECM) on tumor spheroid formation has not been extensively elucidated to date. Herein, uniform-sized spherical microgels encapsulated with different subtypes of breast tumor cells, based on tumor aggressiveness, are developed by flow-focusing microfluidics technology. Mechanical properties of microgels are controlled in a wide range via polymer concentration, and their influence on tumor physiology and spheroid formation is shown to be highly dependent on cell subtype. Specifically, the formation of polyploid/multinucleated giant cancer cells is a key early step in determining initial proliferation and eventual tumor spheroid generation within microgels with varying mechanics. In addition, chemotherapeutic screening performed on these tumor spheroids in microgels also display significantly variable cytotoxic effects based on microgel mechanics for each cell subtype, further highlighting the importance of microenvironmental factors on tumor spheroid physiology.

In situ facile-forming chitosan hydrogels with tunable physicomechanical and tissue adhesive properties by polymer graft architecture.

In situ forming hydrogels generated upon spontaneous and biocompatible reaction under physiological conditions are widely investigated as injectable and implantable biomaterials. However, it is still a significant challenge to control their mechanics while maintaining their gelation behavior, due to the interdependency between gelation kinetics and mechanics. Herein, physicomechanical properties of in situ forming chitosan hydrogels via Schiff base formation are tuned in a wide range, while maintaining gelation kinetics, via polymer graft architecture. By introducing poly(ethylene glycol) (PEG) grafts with varying lengths and densities, the resulting PEG-grafted chitosan ('PEG-g-Cs') not only dissolve readily in neutral aqueous media, but also effectively control the mechanical properties of hydrogels, while maintaining facile gelation kinetics. These properties are further controlled by the chain length of polymeric crosslinker, PEG-dialdehyde. In addition, tissue adhesive properties of the hydrogels are further examined using ex vivo and in vivo models for their potential applications as tissue sealants.