On-demand expansion fluorescence and photoacoustic microscopy (ExFLPAM).

Expansion microscopy (ExM) is a promising technology that enables nanoscale imaging on conventional optical microscopes by physically magnifying the specimens. Here, we report the development of a strategy that enables i) on-demand labeling of subcellular organelles in live cells for ExM through transfection of fluorescent proteins that are well-retained during the expansion procedure; and ii) non-fluorescent chromogenic color-development towards efficient bright-field and photoacoustic imaging in both planar and volumetric formats, which is applicable to both cultured cells and biological tissues. Compared to the conventional ExM methods, our strategy provides an expanded toolkit, which we term as expansion fluorescence and photoacoustic microscopy (ExFLPAM), by allowing on-demand fluorescent protein labeling of cultured cells, as well as non-fluorescent absorption contrast-imaging of biological samples.

On-Demand Expansion Fluorescence and Photoacoustic Microscopy (ExFLPAM).

Expansion microscopy (ExM) is a promising technology that enables nanoscale imaging on conventional optical microscopes by physically magnifying the specimens. Here, we report the development of a strategy that enables i) on-demand labeling of subcellular organelles in live cells for ExM through transfection of fluorescent proteins that are well-retained during the expansion procedure; and ii) non-fluorescent chromogenic color-development towards efficient bright-field and photoacoustic imaging in both planar and volumetric formats, which is applicable to both cultured cells and biological tissues. Compared to the conventional ExM methods, our strategy provides an expanded toolkit, which we term as expansion fluorescence and photoacoustic microscopy (ExFLPAM), by allowing on-demand fluorescent protein labeling of cultured cells, as well as non-fluorescent absorption contrast-imaging of biological samples.

Begonia-Inspired Slow Photon Effect of a Photonic Crystal for Augmenting Algae Photosynthesis.

Plant photosynthesis is considered to be an environmentally friendly and effective measure for reducing carbon dioxide levels to meet the global objective of carbon neutrality. However, the light energy utilization of photosynthetic pigments is insufficient. Begonia pavonine (B. pavonina) with blue leaves exhibits a photosynthetic quantum yield 10% higher than those of other plants by virtue of their photonic crystal (PC) thylakoids. Inspired by this property, we prepared non-angle-dependent PC hydrogels and assembled them with algae Chlorella pyrenoidosa (C. pyre). The band edge of PC hydrogels matched the absorption peaks of C. pyre, and the resulting slow photon effect increased the interaction time between incident light and photosynthetic pigments, which in turn induced the expression of light-harvesting proteins and the synthesis of pigments, thereby improving the light energy utilization. Further, we introduced an artificial antenna into the assembly, which assisted the slow photon effect in increasing the oxygen evolution and carbon sequestration rate by more than 200%. This method avoids the photobleaching problems faced by methods of synthesizing artificial antenna pigments and the biosafety problems faced by genetically engineered methods of editing pigments or proteins.

Kinetically Controlled Synthesis of Nonspherical Polystyrene Nanoparticles with Manipulatable Morphologies.

The morphology of nanoparticles plays a critical role in determining their properties and applications. Herein, we report a versatile approach to the fabrication of nonspherical polystyrene (PS) nanoparticles with controlled morphologies on the basis of kinetically controlled seed-mediated polymerization. By manipulating parameters related to the reaction kinetics including the concentration of monomers, injection rate of reactants, and reaction temperature, the monomers could be directed to polymerize on the selective sites of PS seeds, and after the removal of the second polymer, nonspherical nanoparticles with a variety of thermodynamically unfavored morphologies could be synthesized. We systematically investigated the formation mechanism of these nonspherical nanoparticles by monitoring the evolution of seeds during the reaction. Moreover, we have also successfully extended this strategy to reaction systems containing monomers with different combinations and seeds with different sizes. We believe this work will provide a promising route to the fabrication of nonspherical polymer nanoparticles with controlled morphologies for various applications.

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.

Reversed-engineered human alveolar lung-on-a-chip model.

Here, we present a physiologically relevant model of the human pulmonary alveoli. This alveolar lung-on-a-chip platform is composed of a three-dimensional porous hydrogel made of gelatin methacryloyl with an inverse opal structure, bonded to a compartmentalized polydimethylsiloxane chip. The inverse opal hydrogel structure features well-defined, interconnected pores with high similarity to human alveolar sacs. By populating the sacs with primary human alveolar epithelial cells, functional epithelial monolayers are readily formed. Cyclic strain is integrated into the device to allow biomimetic breathing events of the alveolar lung, which, in addition, makes it possible to investigate pathological effects such as those incurred by cigarette smoking and severe acute respiratory syndrome coronavirus 2 pseudoviral infection. Our study demonstrates a unique method for reconstitution of the functional human pulmonary alveoli in vitro, which is anticipated to pave the way for investigating relevant physiological and pathological events in the human distal lung.

Expanding sacrificially printed microfluidic channel-embedded paper devices for construction of volumetric tissue models in vitro.

We report a method for expanding microchannel-embedded paper devices using a precisely controlled gas-foaming technique for the generation of volumetric tissue models in vitro. We successfully fabricated hollow, perfusable microchannel patterns contained in a densely entangled network of bacterial cellulose nanofibrils using matrix-assisted sacrificial three-dimensional printing, and demonstrated the maintenance of their structural integrity after gas-foaming-enabled expansion in an aqueous solution of NaBH4. The resulting expanded microchannel-embedded paper devices showed multilayered laminar structures with controllable thicknesses as a function of both NaBH4 concentration and expansion time. With expansion, the thickness and porosity of the bacterial cellulose network were significantly increased. As such, cellular infiltration was promoted comparing to as-prepared, non-expanded devices. This simple technique enables the generation of truly volumetric, cost-effective human-based tissue models, such as vascularized tumor models, for potential applications in preclinical drug screening and personalized therapeutic selection.

Bioinspired multistructured paper microfluidics for POCT.

The rapid development of and the large market for medical diagnostics necessitate point-of-care testing (POCT) with superior sensitivity, miniaturization, multiple functionalities and high integration. Thus, flexible substrates with complex structures that provide multiple functions are in demand. Herein, we present multistructured pseudo-papers (MSPs) as a platform for building flexible microfluidics. Flexible and freestanding MSPs are generated by the self-assembly of colloidal silica crystals or core-shell copolymer elastic colloidal crystals on microcavity PDMS molds to form photonic crystals (PCs). Nitrocellulose (NC) multistructured pseudo-papers (NC MSPs) were obtained by etching SiO2 PCs after NC precursor infiltration, while elastic copolymer (EC) multistructured pseudo-papers (EC MSPs) were directly peeled off the mold; both types of freestanding MSPs have ordered micropillars and nanocrystal structures and presented unique properties such as pumpless liquid transport and fluorescence and chemiluminescence (CL) enhancement. MSPs with designed patterns were fabricated by patterned PDMS molds, and complicated microfluidic chips were used to generate MSPs by utilizing these patterns as liquid channels. The MSPs were used for fabricating microfluidic sensors for human cardiac marker and cancer marker sensing; the features of these bioinspired MSPs indicate their potential for sensitive sensing, which will enable them to find broader applications in many fields.

Programmable Liquid Adhesion on Bio-Inspired Re-Entrant Structures.

Surfaces combining antispreading and high adhesion can find wide applications in the manipulation of liquid droplets, generation of micropatterns and liquid enrichment. To fabricate such surfaces, almost all the traditional methods demand multi-step processes and chemical modification. And even so, most of them cannot be applied for some liquids with extremely low surface energy. In the past decade, multiply re-entrant structures have aroused much attention because of their universal and modification-independent antiadhesion or antipenetration ability. Unfortunately, theories and applications about their liquid adhesion behavior are still rare. In this work, inspired by the springtail skin and gecko feet in the adhered state, it is demonstrated that programmable liquid adhesion is realized on the 3D-printed micro doubly re-entrant arrays. By arranging the arrays reasonably, three different Cassie adhesion behaviors can be obtained: I) no residue adhesion, II) tunable adhesion, and III) absolute adhesion. Furthermore, various arrays are designed to tune macro/micro liquid droplet manipulation, which can find applications in the transportation of liquid droplets, liquid enrichment, generation of tiny droplets, and micropatterns.

Decreased microRNA-182-5p helps alendronate promote osteoblast proliferation and differentiation in osteoporosis via the Rap1/MAPK pathway.

Osteoporosis (OP) is a serious health problem that contributes to osteoporotic structural damage and bone fragility. MicroRNAs (miRNAs) can exert important functions over bone endocrinology. Therefore, it is of substantial significance to clarify the expression and function of miRNAs in bone endocrine physiology and pathology to improve the potential therapeutic value for metabolism-related bone diseases. We explored the effect of microRNA-182-5p (miR-182-5p) on osteoblast proliferation and differentiation in OP rats after alendronate (ALN) treatment by targeting adenylyl cyclase isoform 6 (ADCY6) through the Rap1/mitogen-activated protein kinase (MAPK) signaling pathway. Rat models of OP were established to observe the effect of ALN on OP, and the expression of miR-182-5p, ADCY6 and the Rap1/MAPK signaling pathway-related genes was determined. To determine the roles of miR-182-5p and ADCY6 in OP after ALN treatment, the relationship between miR-182 and ADCY6 was initially verified. Osteoblasts were subsequently extracted and transfected with a miR-182-5p inhibitor, miR-182-5p mimic, si-ADCY6 and the MAPK signaling pathway inhibitor U0126. Cell proliferation, apoptosis and differentiation were also determined. ALN treatment was able to ease the symptoms of OP. miR-182-5p negatively targeted ADCY6 to inhibit the Rap1/MAPK signaling pathway. Cells transfected with miR-182 inhibitor decreased the expression of ALP, BGP and COL I, which indicated that the down-regulation of miR-182-5p promoted cell differentiation and cell proliferation and inhibited cell apoptosis. In conclusion, the present study shows that down-regulated miR-182-5p promotes the proliferation and differentiation of osteoblasts in OP rats through Rap1/MAPK signaling pathway activation by up-regulating ADCY6, which may represent a novel target for OP treatment.

Up-regulation of microRNA-340 promotes osteosarcoma cell apoptosis while suppressing proliferation, migration, and invasion by inactivating the CTNNB1-mediated Notch signaling pathway.

Osteosarcoma (OS) is the most common histological form of primary bone cancer. It is most prevalent in teenagers and young adults. The present study aims at exploring the regulatory effect of microRNA-340 (miR-340) on OS cell proliferation, invasion, migration, and apoptosis via regulating the Notch signaling pathway by targeting ß-catenin (cadherin-associated protein) 1 (CTNNB1). OS tissues belonging to 45 patients and normal femoral head tissues of 45 amputees were selected. Cells were allocated to different groups. In situ hybridization was performed to determine the positive rate of miR-340 expression while immunohistochemistry was used to determine that of CTNNB1 and B-cell lymphoma 2 (Bcl-2). We used a series of experiments to measure the expressions of related factors and assess rates of cell proliferation, migration, invasion, cycle, and apoptosis respectively. Our results show that miR-340 was expressed a higher level in normal tissue than OS tissue. Expression of Notch, CTNNB1, hairy and enhancer of split 1 (Hes1), Bcl-2, Runt-related transcription factor 2 (Runx2), and osteocalcin increased and that of miR-340, Bcl-2 interacting mediator of cell death (BIM), and Bcl-2 associated protein X (Bax) decreased in OS tissues. U-2OS cell line had the highest miR-340 expression. We also found that the up-regulation of miR-340 had increased expression of miR-340, BIM, and Bax but decreased expression of Notch, CTNNB1, Hes1, Bcl-2, Runx2, and osteocalcin. Up-regulation of miR-340p lead to increased cell apoptosis, suppressed cell proliferation, migration, and invasion. Our study demonstrates that overexpression of miR-340 could suppress OS cell proliferation, migration, and invasion as well as promoting OS cell apoptosis by inactivating the Notch signaling pathway via down-regulating CTNNB1. Functional miR-340 overexpression might be a future therapeutic strategy for OS.