Microgels for Cell Delivery in Tissue Engineering and Regenerative Medicine.

Microgels prepared from natural or synthetic hydrogel materials have aroused extensive attention as multifunctional cells or drug carriers, that are promising for tissue engineering and regenerative medicine. Microgels can also be aggregated into microporous scaffolds, promoting cell infiltration and proliferation for tissue repair. This review gives an overview of recent developments in the fabrication techniques and applications of microgels. A series of conventional and novel strategies including emulsification, microfluidic, lithography, electrospray, centrifugation, gas-shearing, three-dimensional bioprinting, etc. are discussed in depth. The characteristics and applications of microgels and microgel-based scaffolds for cell culture and delivery are elaborated with an emphasis on the advantages of these carriers in cell therapy. Additionally, we expound on the ongoing and foreseeable applications and current limitations of microgels and their aggregate in the field of biomedical engineering. Through stimulating innovative ideas, the present review paves new avenues for expanding the application of microgels in cell delivery techniques.

High-Resolution Additive Manufacturing of a Biodegradable Elastomer with A Low-Cost LCD 3D Printer.

Artificial organs and organs-on-a-chip (OoC) are of great clinical and scientific interest and have recently been made by additive manufacturing, but depend on, and benefit from, biocompatible, biodegradable, and soft materials. Poly(octamethylene maleate (anhydride) citrate (POMaC) meets these criteria and has gained popularity, and as in principle, it can be photocured and is amenable to vat-photopolymerization (VP) 3D printing, but only low-resolution structures have been produced so far. Here, a VP-POMaC ink is introduced and 3D printing of 80 µm positive features and complex 3D structures is demonstrated using low-cost (≈US$300) liquid-crystal display (LCD) printers. The ink includes POMaC, a diluent and porogen additive to reduce viscosity within the range of VP, and a crosslinker to speed up reaction kinetics. The mechanical properties of the cured ink are tuned to match the elastic moduli of different tissues simply by varying the porogen concentration. The biocompatibility is assessed by cell culture which yielded 80% viability and the potential for tissue engineering illustrated with a 3D-printed gyroid seeded with cells. VP-POMaC and low-cost LCD printers make the additive manufacturing of high resolution, elastomeric, and biodegradable constructs widely accessible, paving the way for a myriad of applications in tissue engineering and 3D cell culture as demonstrated here, and possibly in OoC, implants, wearables, and soft robotics.

Liquid-embedded (bio)printing of alginate-free, standalone, ultrafine, and ultrathin-walled cannular structures.

While there has been considerable success in the three-dimensional bioprinting of relatively large standalone filamentous tissues, the fabrication of solid fibers with ultrafine diameters or those cannular featuring ultrathin walls remains a particular challenge. Here, an enabling strategy for (bio)printing of solid and hollow fibers whose size ranges could be facilely adjusted across a broad spectrum, is reported, using an aqueous two-phase embedded (bio)printing approach combined with specially designed cross-linking and extrusion methods. The generation of standalone, alginate-free aqueous architectures using this aqueous two-phase strategy allowed freeform patterning of aqueous bioinks, such as those composed of gelatin methacryloyl, within the immiscible aqueous support bath of poly(ethylene oxide). Our (bio)printing strategy revealed the fabrication of standalone solid or cannular structures with diameters as small as approximately 3 or 40 µm, respectively, and wall thicknesses of hollow conduits down to as thin as <5 µm. With cellular functions also demonstrated, we anticipate the methodology to serve as a platform that may satisfy the needs for the different types of potential biomedical and other applications in the future, especially those pertaining to cannular tissues of ultrasmall diameters and ultrathin walls used toward regenerative medicine and tissue model engineering.

Freeform Cell-Laden Cryobioprinting for Shelf-Ready Tissue Fabrication and Storage.

One significant drawback of existing bioprinted tissues is their lack of shelf-availability caused by complications in both fabrication and storage. Here, we report a cryobioprinting strategy for simultaneously fabricating and storing cell-laden volumetric tissue constructs through seamlessly combining extrusion bioprinting and cryopreservation. The cryobioprinting performance was investigated by designing, fabricating, and storing cell-laden constructs made of our optimized cryoprotective gelatin-based bioinks using a freezing plate with precisely controllable temperature. The in situ freezing process further promoted the printability of cell-laden hydrogel bioinks to achieve freeform structures otherwise inconvenient with direct extrusion bioprinting. The effects of bioink composition on printability and cell viability were evaluated. The functionality of the method was finally investigated using cell differentiation and chick ex ovo assays. The results confirmed the feasibility and efficacy of cryobioprinting as a single-step method for concurrent tissue biofabrication and storage.

Immunomodulatory Microgels Support Proregenerative Macrophage Activation and Attenuate Fibroblast Collagen Synthesis.

Scars composed of fibrous connective tissues are natural consequences of injury upon incisional wound healing in soft tissues.  Hydrogels that feature a sustained presentation of immunomodulatory cytokines are known to modulate wound healing. However, existing immunomodulatory hydrogels lack interconnected micropores to promote cell ingrowth. Other limitations include invasive delivery procedures and harsh synthesis conditions that are incompatible with drug molecules. Here, hybrid nanocomposite microgels containing interleukin-10 (IL-10) are reported to modulate tissue macrophage phenotype during wound healing. The intercalation of laponite nanoparticles in the polymer network yields microgels with tissue-mimetic elasticity (Young's modulus in the range of 2-6 kPa) and allows the sustained release of IL-10 to promote the differentiation of macrophages toward proregenerative phenotypes. The porous interstitial spaces between microgels promote fibroblast proliferation and fast trafficking (an average speed of ≈14.4 µm h-1 ). The incorporation of hyaluronic acid further enhances macrophage infiltration. The coculture of macrophages and fibroblasts treated with transforming growth factor-beta 1 resulted in a twofold reduction in collagen-I production for microgels releasing IL-10 compared to the IL-10 free group. The new microgels show potential toward regenerative healing by harnessing the antifibrotic behavior of host macrophages.

Vertical Extrusion Cryo(bio)printing for Anisotropic Tissue Manufacturing.

Due to the poor mechanical properties of many hydrogel bioinks, conventional 3D extrusion bioprinting is usually conducted based on the X-Y plane, where the deposited layers are stacked in the Z-direction with or without the support of prior layers. Herein, a technique is reported, taking advantage of a cryoprotective bioink to enable direct extrusion bioprinting in the vertical direction in the presence of cells, using a freezing plate with precise temperature control. Of interest, vertical 3D cryo-bioprinting concurrently allows the user to create freestanding filamentous constructs containing interconnected, anisotropic microchannels featuring gradient sizes aligned in the vertical direction, also associated with enhanced mechanical performances. Skeletal myoblasts within the 3D-cryo-bioprinted hydrogel constructs show enhanced cell viability, spreading, and alignment, compared to the same cells in the standard hydrogel constructs. This method is further extended to a multimaterial format, finding potential applications in interface tissue engineering, such as creation of the muscle-tendon unit and the muscle-microvascular unit. The unique vertical 3D cryo-bioprinting technique presented here suggests improvements in robustness and versatility to engineer certain tissue types especially those anisotropic in nature, and may extend broad utilities in tissue engineering, regenerative medicine, drug discovery, and personalized therapeutics.

Injectable, Pore-Forming, Perfusable Double-Network Hydrogels Resilient to Extreme Biomechanical Stimulations.

Biological tissues hinge on blood perfusion and mechanical toughness to function. Injectable hydrogels that possess both high permeability and toughness have profound impacts on regenerative medicine but remain a long-standing challenge. To address this issue, injectable, pore-forming double-network hydrogels are fabricated by orchestrating stepwise gelation and phase separation processes. The interconnected pores of the resulting hydrogels enable direct medium perfusion through organ-sized matrices. The hydrogels are amenable to cell encapsulation and delivery while promoting cell proliferation and spreading. They are also pore insensitive, tough, and fatigue resistant. When tested in biomimetic perfusion bioreactors, the hydrogels maintain physical integrity under prolonged, high-frequency biomechanical stimulations (>6000 000 cycles at 120 Hz). The excellent biomechanical performance suggests the great potential of the new injectable hydrogel technology for repairing mechanically dynamic tissues, such as vocal folds, and other applications, such as tissue engineering, biofabrication, organs-on-chips, drug delivery, and disease modeling.

Emerging Technologies in Multi-Material Bioprinting.

Bioprinting, within the emerging field of biofabrication, aims at the fabrication of functional biomimetic constructs. Different 3D bioprinting techniques have been adapted to bioprint cell-laden bioinks. However, single-material bioprinting techniques oftentimes fail to reproduce the complex compositions and diversity of native tissues. Multi-material bioprinting as an emerging approach enables the fabrication of heterogeneous multi-cellular constructs that replicate their host microenvironments better than single-material approaches. Here, bioprinting modalities are reviewed, their being adapted to multi-material bioprinting is discussed, and their advantages and challenges, encompassing both custom-designed and commercially available technologies are analyzed. A perspective of how multi-material bioprinting opens up new opportunities for tissue engineering, tissue model engineering, therapeutics development, and personalized medicine is offered.

Composite Inks for Extrusion Printing of Biological and Biomedical Constructs.

Extrusion-based three-dimensional (3D) printing is an emerging technology for the fabrication of complex structures with various biological and biomedical applications. The method is based on the layer-by-layer construction of the product using a printable ink. The material used as the ink should possess proper rheological properties and desirable performances. Composite materials, which are extensively used in 3D printing applications, can improve the printability and offer superior performances for the printed constructs. Herein, we review composite inks with a focus on composite hydrogels. The properties of different additives including fibers and nanoparticles are discussed. The performances of various composite inks in biological and biomedical systems are delineated through analyzing the synergistic effects between the composite ink components. Different applications, including tissue engineering, tissue model engineering, soft robotics, and four-dimensional printing, are selected to demonstrate how 3D-printable composite inks are exploited to achieve various desired functionality. This review finally presents an outlook of future perspectives on the design of composite inks.

Author Correction: Carbon nanotubes promote cell migration in hydrogels.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Carbon nanotubes promote cell migration in hydrogels.

Injectable hydrogels are increasingly used for in situ tissue regeneration and wound healing. Ideally, an injectable implant should promote the recruitment of cells from the surrounding native tissue and allow cells to migrate freely as they generate a new extracellular matrix network. Nanocomposite hydrogels such as carbon nanotube (CNT)-loaded hydrogels have been hypothesized to promote cell recruitment and cell migration relative to unloaded ones. To investigate this, CNT-glycol chitosan hydrogels were synthesized and studied. Chemoattractant-induced cell migration was studied using a modified Boyden Chamber experiment. Migrated cells were counted using flow cytometry. Cell adhesion was inferred from the morphology of the cells via an image segmentation method. Cell migration and recruitment results confirmed that small concentrations of CNT significantly increase cell migration in hydrogels, thereby accelerating tissue regeneration and wound healing in situations where there is insufficient migration in the unloaded matrix.

Triggered micropore-forming bioprinting of porous viscoelastic hydrogels.

Cell-laden scaffolds of architecture and mechanics that mimic those of the host tissues are important for a wide range of biomedical applications but remain challenging to bioprint. To address these challenges, we report a new method called triggered micropore-forming bioprinting. The approach can yield cell-laden scaffolds of defined architecture and interconnected pores over a range of sizes, encompassing that of many cell types. The viscoelasticity of the bioprinted scaffold can match that of biological tissues and be tuned independently of porosity and stiffness. The bioprinted scaffold also exhibits superior mechanical robustness despite high porosity. The bioprinting method and the resulting scaffolds support cell spreading, migration, and proliferation. The potential of the 3D bioprinting system is demonstrated for vocal fold tissue engineering and as an in vitro cancer model. Other possible applications are foreseen for tissue repair, regenerative medicine, organ-on-chip, drug screening, organ transplantation, and disease modeling.

Carbon nanotube composite hydrogels for vocal fold tissue engineering: Biocompatibility, rheology, and porosity.

Porous composite hydrogels were prepared using glycol chitosan as the matrix, glyoxal as the chemical crosslinker, and carbon nanotubes (CNTs) as the fibers. Both carboxylic and hydroxylic functionalized CNTs were used. The homogeneity of CNTs dispersion was evaluated using scanning electron microscopy. Human vocal fold fibroblasts were cultured and encapsulated in the composite hydrogels with different CNT concentrations to quantify cell viability. Rheological tests were performed to determine the gelation time and the storage modulus as a function of CNT concentration. The gelation time tended to decrease for low concentrations and increase at higher concentrations, reaching a local minimum value. The storage modulus obeyed different trends depending on the functional group. The porosity of the hydrogels was found to increase by 120% when higher concentrations of carboxylic CNTs were used. A high porosity may promote cell adhesion, migration, and recruitment from the surrounding native tissue, which will be investigated in a future work aiming at applying this injectable biomaterial for vocal fold tissue regeneration.