The NLRP3 inflammasome is a multimeric protein complex that initiates an inflammatory form of cell death and triggers the release of proinflammatory cytokines IL-1β and IL-18. The NLRP3 inflammasome has been implicated in a wide range of diseases, including Alzheimer’s disease, Prion diseases, type 2 diabetes, and some infectious diseases. It has been found that a variety of stimuli including danger-associated molecular patterns (DAMPs, such as silica and uric acid crystals) and pathogen-associated molecular patterns (PAMPs) can activate NLRP3 inflammasome, but the specific regulatory mechanisms of NLRP3 inflammasome activation remain unclear. Understanding the mechanisms of NLRP3 activation will enable the development of its specific inhibitors to treat NLRP3-related diseases. In this review, we summarize current understanding of the regulatory mechanisms of NLRP3 inflammasome activation as well as inhibitors that specifically and directly target NLRP3.

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Existing techniques to encapsulate cells into microscale hydrogels generally yield high polymer-to-cell ratios and lack control over the hydrogel’s mechanical properties1. Here, we report a microfluidic-based method for encapsulating single cells in a ~6 micron layer of alginate that increases the proportion of cell-containing microgels by 10-fold, with encapsulation efficiencies over 90%. We show that in vitro cell viability was maintained over a three-day period, that the microgels are mechanically tractable, and that for microscale cell assemblages of encapsulated marrow stromal cells cultured in microwells, osteogenic differentiation of encapsulated cells depends on gel stiffness and cell density. We also show that intravenous injection of singly-encapsulated marrow stromal cells into mice delays clearance kinetics and sustains donor-derived soluble factors in vivo. The encapsulation of single cells in tunable hydrogels should find use in a variety of tissue engineering and regenerative medicine applications.

Direct injection is a minimally invasive method of stem cell transplantation for numerous injuries and diseases. However, despite its promising potential, its clinical translation is diffi cult due to the low cell retention and engraftment after injection. With high versatility, high-resolution control and injectability, microfabrication of stem-cell laden biomedical hydrogels holds great potential as minimally invasive technology. Herein, a strategy of microfl uidicsassisted technology entrapping bone marrow-derived mesenchymal stem cells (BMSCs) and growth factors in photocrosslinkable gelatin (GelMA) microspheres to ultimately generate injectable osteogenic tissue constructs is presented. Additionally, it is demonstrated that the GelMA microspheres can sustain stem cell viability, support cell spreading inside the microspheres and migration from the interior to the surface as well as enhance cell proliferation. This fi nding shows that encapsulated cells have the potential to directly and actively participate in the regeneration process. Furthermore, it is found that BMSCs encapsulated in GelMA microspheres show enhanced osteogenesis in vitro and in vivo, associated with a signifi cant increase in mineralization. In short, the proposed strategy can be utilized to facilitate bone regeneration with minimum invasiveness, and can potentially be applied along with other matrices for extended applications. One potential attractive strategy for stem cell delivery that overcomes these limitations is to suspend the stem cells in hydrogels which can be injected and solidifi ed in situ . Hydrogels have a high water content, similar to tissues, which not only enables homogeneous encapsulation of cells and growth factors, but also allows for facile delivery via injection. Their readily tunable degradation properties provide further control over the release behavior of incorporated cargo material. [ 5 ] Hydrogels of synthetic origin, poly (ethylene glycol) diacrylate (PEGDA), [ 6 ] and of natural origin, such as hyaluronic acid (HA), [ 7 ] alginate, [ 8 ] collagen, [ 9 ] and gelatin [ 10 ] have been tested. However, their clinical success has frequently been impeded by insuffi cient oxygen and nutrient supply due to the large size of the bone defects, which compromises cell survival and performance, resulting in poor bone regeneration. [ 11 ] Furthermore, the bulk environment and the limited interfacial interactions between the cells and the hydrogel material restrict tissue regeneration. Therefore, development of alternate hydrogel geometries for stem cell delivery is required to further drive clinical translation of BMSC-based bone repair strategies. DOIOne such geometry is hydrogel microspheres which can encapsulate both stem cells and their growth factors; they facilitate nutrient and waste transfer and thereby maintain the viability of preseeded cells, while also preserving the scaffold's injectability. [ 12,13 ] In addition, such 3D scaffolds have large surface area which improves cell-matrix interactions. Thus the us...

biodegradable gels, since physical crosslinking based on electrostatic interactions is generally favored over the use of chemical crosslinking to achieve sustained drug release, [ 17 ] cell attachment, [ 18 ] or hydrogel formation. [ 19 ] Recently, oppositely charged dextran microspheres [ 20 ] or poly(lactic-co -glycolic acid) (PLGA) nanospheres [ 21 ] have been used to form moldable scaffolds, but only indirect proof for electrostatic self-assembly was provided based on rheological characterization, while the underlying gel formation mechanism was not elucidated. Moreover, disadvantages of these gels include: i) the necessity to derivatize dextran or PLGA by grafting charged groups onto the polymer backbone, which moreover induced cytotoxicity; [20b] ii) the release of harmful degradation byproducts, such as lactic and glycolic acid (in case of PLGA nanospheres), which can denature entrapped signaling proteins [ 22 ] and cause infl ammatory responses [ 23 ] of the host tissue; and iii) the absence of cell-adhesive peptide sequences required for the attachment of anchorage-dependent mesenchymal stem cells such as fi bro-and osteoblasts. [ 17 ]

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The design of bioactive three-dimensional (3D) scaffolds is a major focus in bone tissue engineering. Incorporation of growth factors into bioprinted scaffolds offers many new possibilities regarding both biological and architectural properties of the scaffolds. This study investigates whether the sustained release of bone morphogenetic protein 2 (BMP-2) influences osteogenicity of tissue engineered bioprinted constructs. BMP-2 loaded on gelatin microparticles (GMPs) was used as a sustained release system, which was dispersed in hydrogel-based constructs and compared to direct inclusion of BMP-2 in alginate or control GMPs. The constructs were supplemented with goat multipotent stromal cells (gMSCs) and biphasic calcium phosphate to study osteogenic differentiation and bone formation respectively. BMP-2 release kinetics and bioactivity showed continuous release for three weeks coinciding with osteogenicity. Osteogenic differentiation and bone formation of bioprinted GMP containing constructs were investigated after subcutaneous implantation in mice or rats. BMP-2 significantly increased bone formation, which was not influenced by the release timing. We showed that 3D printing of controlled release particles is feasible and that the released BMP-2 directs osteogenic differentiation in vitro and in vivo.

Cell-laden microgels with highly uniform sizes have significant potential in tissue engineering and cell therapy due to the capability to provide physiologically relevant three-dimensional (3D) microenvironment for living cells. In this work, we present a simple and efficient microfluidic approach to produce monodisperse cell-laden microgels through the use of double emulsion drops with an ultra-thin oil shell as the sacrificial template. Specifically, the thin oil shell in double emulsion spontaneously dewets upon polymerization of the innermost precursor drop and subsequent transfer into an aqueous solution, resulting in direct dispersion of microgels in an aqueous phase. Compared to conventional single emulsion-based techniques for cell encapsulation, this one-step approach prevents prolonged exposure of cells to the oil phase, leading to high-throughput cell encapsulation in microgels without compromising the cell viability. Moreover, this approach allows us to culture cells within a 3D microgel which mimics the extracellular matrix, thus enabling long-term cell functionality. This microfluidic technique represents a significant step forward in high-throughput cell microencapsulation technology and offers a potentially viable option to produce cell-laden microgels for widespread applications in tissue engineering and cell therapies.