Expression Microdissection for the Analysis of miRNA in a Single-Cell Type.

Cell-specific microRNA (miRNA) expression estimates are important in characterizing the localization of miRNA signaling within tissues. Much of these data are obtained from cultured cells, a process known to significantly alter miRNA expression levels. Thus, our knowledge of in vivo cell miRNA expression estimates is poor. We previously demonstrated expression microdissection-miRNA-sequencing (xMD-miRNA-seq) to acquire in vivo estimates, directly from formalin-fixed tissues, albeit with a limited yield. In this study, we optimized each step of the xMD process, including tissue retrieval, tissue transfer, film preparation, and RNA isolation, to increase RNA yields and ultimately show strong enrichment for in vivo miRNA expression by qPCR array. These method improvements, such as the development of a noncrosslinked ethylene vinyl acetate membrane, resulted in a 23- to 45-fold increase in miRNA yield, depending on the cell type. By qPCR, miR-200a increased by 14-fold in xMD-derived small intestine epithelial cells, with a concurrent 336-fold reduction in miR-143 relative to the matched nondissected duodenal tissue. xMD is now an optimized method to obtain robust in vivo miRNA expression estimates from cells. xMD will allow formalin-fixed tissues from surgical pathology archives to make theragnostic biomarker discoveries.

Recent advances of two-dimensional material additives in hybrid perovskite solar cells.

Perovskite solar cells (PSCs) have become one of the state-of-the-art photovoltaic technologies due to their facile solution-based fabrication processes combined with extremely high photovoltaic performance originating from excellent optoelectronic properties such as strong light absorption, high charge mobility, long free charge carrier diffusion length, and tunable direct bandgap. However, the poor intrinsic stability of hybrid perovskites under environmental stresses including light, heat, and moisture, which is often associated with high defect density in the perovskite, has limited the large-scale commercialization and deployment of PSCs. The use of process additives, which can be included in various subcomponent layers in the PSC, has been identified as one of the effective approaches that can address these issues and improve the photovoltaic performance. Among various additives that have been explored, two-dimensional (2D) materials have emerged recently due to their unique structures and properties that can enhance the photovoltaic performance and device stability by improving perovskite crystallization, defect passivation, and charge transport. Here, we provide a review of the recent progresses in 2D material additives for improving the PSC performance based on key representative 2D material systems, including graphene and its derivatives, transitional metal dichalcogenides, and black phosphorous, providing a useful guideline for further exploiting unique nanomaterial additives for more efficient and stable PSCs in the near future.

Enhancing Crystallization in Hybrid Perovskite Solar Cells Using Thermally Conductive 2D Boron Nitride Nanosheet Additive.

Controlling crystallization and grain growth is crucial for realizing highly efficient hybrid perovskite solar cells (PSCs). In this work, enhanced PSC photovoltaic performance and stability by accelerating perovskite crystallization and grain growth via 2D hexagonal boron nitride (hBN) nanosheet additives incorporated into the active perovskite layer are demonstrated. In situ X-ray scattering and infrared thermal imaging during the perovskite annealing process revealed the highly thermally conductive hBN nanosheets promoted the phase conversion and grain growth in the perovskite layer by facilitating a more rapid and spatially uniform temperature rise within the perovskite film. Complementary structural, physicochemical, and electrical characterizations further showed that the hBN nanosheets formed a physical barrier at the perovskite grain boundaries and the interfaces with charge transport layers, passivating defects, and retarding ion migration. As a result, the power conversion efficiency of the PSC is improved from 17.4% to 19.8%, along with enhanced device stability, retaining ≈90% of the initial efficiency even after 500 h ambient air storage. The results not only highlight 2D hBN as an effective additive for PSCs but also suggest enhanced thermal transport as one of the pathways for improved PSC performance by 2D material additives in general.

Potentiometric Biosensors Based on Molecular-Imprinted Self-Assembled Monolayer Films for Rapid Detection of Influenza A Virus and SARS-CoV-2 Spike Protein.

Rapid, yet accurate and sensitive testing has been shown to be critical in the control of spreading pandemic diseases such as COVID-19. Current methods which are highly sensitive and can differentiate different strains are slow and cannot be conveniently applied at the point of care. Rapid tests, meanwhile, require a high titer and are not sufficiently sensitive to discriminate between strains. Here, we report a rapid and facile potentiometric detection method based on nanoscale, three-dimensional molecular imprints of analytes on a self-assembled monolayer (SAM), which can deliver analyte-specific detection of both whole virions and isolated proteins in microliter amounts of bodily fluids within minutes. The detection substrate with nanoscale inverse surface patterns of analytes formed by a SAM identifies a target analyte by recognizing its surface nano- and molecular structures, which can be monitored by temporal measurement of the change in substrate open-circuit potential. The sensor unambiguously detected and differentiated H1N1 and H3N2 influenza A virions as well as the spike proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle-East respiratory syndrome (MERS) coronavirus in human saliva with limits of detection reaching 200 PFU/mL and 100 pg/mL for the viral particles and spike proteins, respectively. The demonstrated speed and specificity of detection, combined with a low required sample volume, high sensitivity, ease of potentiometric measurement, and simple sample collection and preparation, suggest that the technique can be used as a highly effective point-of-care diagnostic platform for a fast, accurate, and specific detection of various viral pathogens and their variants.

Fabrication of ordered bi-metallic array with superstructure of gold micro-rings via templated-self-assembly procedure and its SERS application.

Bi-metallic patterns with array of Pt discs decorated by Au rings were fabricated onto the substrate by a templated-self-assembly procedure in which multi-step self-assembly processes were involved. The original pattern was established by using the breath figure method. The bi-metallic sample with Au rings exhibited rather high sensitivity as well as great reproducibility within the array in surface-enhanced Raman scattering test.

In Situ Time-Resolved X-ray Scattering Study of Isotactic Polypropylene in Additive Manufacturing.

Using a specially designed apparatus, which collects simultaneous temperature and X-ray scattering data, we performed in situ measurements of the filament during MatEx 3D printing. The data show that the MatEx 3D printing extrusion process provides sufficient shear to form shish-kebab structures, which initially nucleate at the filament surface and spread into the filament core. Time-resolved measurements show that the kebab component near the surface relaxes after deposition of the second filament and enhances chain diffusion across the interface. SEM images indicate near complete interfacial merging of the filaments, which results in excellent mechanical properties.

A new strategy to engineer polymer bulk heterojunction solar cells with thick active layers via self-assembly of the tertiary columnar phase.

We report that the addition of a non-photoactive tertiary polymer phase in the binary bulk heterojunction (BHJ) polymer solar cell leads to a self-assembled columnar nanostructure, enhancing the charge mobilities and photovoltaic efficiency with surprisingly increased optimal active blend thicknesses over 300 nm, 3-4 times larger than that of the binary counterpart. Using the prototypical poly(3-hexylthiophene) (P3HT):fullerene blend as a model BHJ system, we discover that the inert poly(methyl methacrylate) (PMMA) added in the binary BHJ blend self-assembles into vertical columns, which not only template the phase segregation of electron acceptor fullerenes but also induce the out-of-plane rotation of the edge-on-orientated crystalline P3HT phase. Using complementary interrogation methods including neutron reflectivity, X-ray scattering, atomic force microscopy, transmission electron microscopy, and molecular dynamics simulations, we show that the enhanced charge transport originates from the more randomized molecular stacking of the P3HT phase and the spontaneous segregation of fullerenes at the P3HT/PMMA interface, driven by the high surface tension between the two polymeric components. The results demonstrate a potential method for increasing the thicknesses of high-performance polymer BHJ solar cells with improved photovoltaic efficiency, alleviating the burden of stringently controlling the ultrathin blend thickness during the roll-to-roll-type large-area manufacturing environment.

The influence of surface chemistry on adsorbed fibrinogen conformation, orientation, fiber formation and platelet adhesion.

Thrombosis is a clear risk when any foreign material is in contact with the bloodstream. Here we propose an immunohistological stain-based model for non-enzymatic clot formation that enables a facile screen for the thrombogenicity of blood-contacting materials. We exposed polymers with different surface chemistries to protease-free human fibrinogen. We observed that on hydrophilic surfaces, fibrinogen is adsorbed via αC regions, while the γ400-411 platelet-binding dodecapeptide on the D region becomes exposed, and fibrinogen fibers do not form. In contrast, fibrinogen is adsorbed on hydrophobic surfaces via the relatively hydrophobic D and E regions, exposing the αC regions while rendering the γ400-411 inaccessible. Fibrinogen adsorbed on hydrophobic surfaces is thus able to recruit other fibrinogen molecules through αC regions and polymerize into large fibrinogen fibers, similar to those formed in vivo in the presence of thrombin. Moreover, the γ400-411 is available only on the large fibers not elsewhere throughout the hydrophobic surface after fibrinogen fiber formation. When these surfaces were exposed to gel-sieved platelets or platelet rich plasma, a uniform monolayer of platelets, which appeared to be activated, was observed on the hydrophilic surfaces. In contrast, large agglomerates of platelets were clustered on fibers on the hydrophobic surfaces, resembling small nucleating thrombi. Endothelial cells were also able to adhere to the monomeric coating of fibrinogen on hydrophobic surfaces. These observations reveal that the extent and type of fibrinogen adsorption, as well as the propensity of adsorbed fibrinogen to bind platelets, may be modulated by careful selection of surface chemistry. STATEMENTS OF SIGNIFICANCE: Thrombosis is a well-known side effect of the introduction of foreign materials into the bloodstream, as might exist in medical devices including but not limited to stents, valves, and intravascular catheters. Despite many reported studies, the body's response to foreign materials in contact with the blood remains poorly understood. Current preventive methods consist of drug eluting coatings on the devices or the systemic administration of standard anticoagulants. Here we present a potential mechanism by which surface chemistry can affects fibrinogen conformation and thus affects platelet adhesion and consequently thrombus formation. Our findings suggest a possible coating which enables endothelial cell adhesion while preventing platelet adhesion.

Enhancing the Mechanical Properties of Biodegradable Polymer Blends Using Tubular Nanoparticle Stitching of the Interfaces.

"Green" polymer nanocomposites were made by melt blending biodegradable poly(lactic acid) (PLA) and poly(butylene adipate-co-butylene terephthalate) (PBAT) with either montmorillonite clays (Cloisite Na(+)), halloysite nanotubes (HNTs), the resorcinol diphenyl phosphate (RDP)-coated Cloisite Na(+), and coated HNTs. A technique for measuring the work of adhesion (Wa) between nanoparticles and their matrixes was used to determine the dispersion preference of the nanoparticles in the PLA/PBAT blend system. Transmission electron microscopy (TEM) images of thin sections indicated that even though both RDP-coated nanotubes and clay platelets segregated to the interfacial regions between the two immiscible polymers, only the platelets, having the larger specific surface area, were able to reduce the PBAT domain sizes. The ability of clay platelets to partially compatibilize the blend was further confirmed by the dynamic mechanical analysis (DMA) which showed that the glass transition temperatures of two polymers tended to shift closer. No shift was observed with either coated or uncoated HNTs samples. Izod impact testing demonstrated that the rubbery PBAT phase greatly increased the impact strength of the unfilled blend, but addition of only 5% of treated clay decreased the impact strength by nearly 50%. On the other hand, an increase of 9% relative to the unfilled blend sample was observed with the addition of 5% treated nanotubes. TEM cross-section analysis confirmed that the RDP-coated clay platelets covered most of the interfacial area. On one hand, this enabled them to reduce the interfacial tension effectively; on the other hand, it prevented chain entanglements across the phase boundary and increased the overall brittleness, which was confirmed by rheology measurements. In contrast, the RDP-coated HNTs were observed to lie perpendicular to the interface, which made them less effective in reducing interfacial tension but encouraged interfacial entanglements across the interface, resulting in "stitching" of the interface and an increase in the Izod impact of the blend.

Establishing correlations in the en-mass migration of dermal fibroblasts on oriented fibrillar scaffolds.

Wound healing proceeds via fibroblast migration along three dimensional fibrillar substrates with multiple angles between fibers. We have developed a technique for preparation of three dimensional fibrillar scaffolds with where the fiber diameters and the angles between adjacent fiber layers could be precisely controlled. Using the agarose droplet method we were able to make accurate determinations of the dependence of the migration speed, focal adhesion distribution, and nuclear deformation on the fiber diameter, fiber spacing, and angle between adjacent fiber layers. We found that on oriented single fiber layers, whose diameters exceeded 1 µm, large focal adhesion complexes formed in a linear arrangement along the fiber axis and cell motion was highly correlated. On multi layered scaffolds most of the focal adhesion sites reformed at the junction points and the migration speed was determined by the angle between adjacent fiber layers, which followed a parabolic function with a minimum at 30°. On these surfaces we observed a 25% increase in the number of focal adhesion points and a similar decrease in the degree of nuclear deformation, both phenomena associated with decreased mobility. These results underscore the importance of substrate morphology on the en-mass migration dynamics. STATEMENT OF SIGNIFICANCE: En-mass fibroblast migration is an essential component of the wound healing process which can determine rate and scar formation. Yet, most publications on this topic have focused on single cell functions. Here we describe a new apparatus where we designed three dimensional fibrillar scaffolds with well controlled angles between junction points and highly oriented fiber geometries. We show that the motion of fibroblasts undergoing en-mass migration on these scaffolds can be controlled by the substrate topography. Significant differences in cell morphology and focal adhesions was found to exist between cells migrating on flat versus fibrillar scaffolds where the migration speed was found to be a function of the angle between fibers, the fiber diameter, and the distance between fibers.

Continual cell deformation induced via attachment to oriented fibers enhances fibroblast cell migration.

Fibroblast migration is critical to the wound healing process. In vivo, migration occurs on fibrillar substrates, and previous observations have shown that a significant time lag exists before the onset of granulation tissue. We therefore conducted a series of experiments to understand the impact of both fibrillar morphology and migration time. Substrate topography was first shown to have a profound influence. Fibroblasts preferentially attach to fibrillar surfaces, and orient their cytoplasm for maximal contact with the fiber edge. In the case of en-mass cell migration out of an agarose droplet, fibroblasts on flat surfaces emerged with an enhanced velocity, v = 52µm/h, that decreases to the single cell value, v = 28µm/h within 24 hours and remained constant for at least four days. Fibroblasts emerging on fibrillar surfaces emerged with the single cell velocity, which remained constant for the first 24 hours and then increased reaching a plateau with more than twice the initial velocity within the next three days. The focal adhesions were distributed uniformly in cells on flat surfaces, while on the fibrillar surface they were clustered along the cell periphery. Furthermore, the number of focal adhesions for the cells on the flat surfaces remained constant, while it decreased on the fibrillar surface during the next three days. The deformation of the cell nuclei was found to be 50% larger on the fiber surfaces for the first 24 hours. While the mean deformation remained constant on the flat surface, it increased for the next three days by 24% in cells on fibers. On the fourth day, large actin/myosin fibers formed in cells on fibrillar surfaces only and coincided with a change from the standard migration mechanism involving extension of lamellipodia, and retraction of the rear, to one involving strong contractions oriented along the fibers and centered about the nucleus.

Biomineralization on enzymatically cross-linked gelatin hydrogels in the absence of dexamethasone.

A mechanical stimulus and chemical induction by dexamethasone have been important factors in dental pulp stem cell (DPSC) differentiation and biomineralization. We have demonstrated that the enzymatically crosslinked gelatin hydrogels are extremely effective substrates for DPSC differentiation towards odontoblasts. DPSCs were seeded on the crosslinked hard (∼8 kPa) and soft (∼0.15 kPa) gelatin hydrogels for 35 days with and without dexamethasone. Odontogenic differentiation markers such as OCN, ALP and DSPP were upregulated after 35 days of culture on crosslinked hydrogels with and without dexamethasone. SEM and Alizarin red staining of the crosslinked hydrogels showed a biomineralized sheet of hydroxyapatite deposits laid by the DPSCs on the top surface and inside the hydrogel. We found that the DPSC differentiation and biomineralization were independent of the hydrogel stiffness and dexamethasone. We hypothesize that this biomineralization was indeed triggered by the surface chemistry of the crosslinked gelatin hydrogels since we did not observe any biomineralization on the uncrosslinked gelatin or mTG. We also showed that the DPSCs, when removed from hard hydrogel surfaces and re-seeded on a TCPS, retained their odontogenic lineage and showed a permanent mineralization effect. Our results show the potential of enzymatically crosslinked gelatin hydrogels as scaffolds for dentin regeneration.

Gold nanoparticles cellular toxicity and recovery: adipose Derived Stromal cells.

Gold nanoparticles (AuNPs) are currently used in numerous medical applications. Herein, we describe their in vitro impact on human adipose-derived stromal cells (ADSCs) using 13 nm and 45 nm citrate-coated AuNPs. In their non-differentiated state, ADSCs were penetrated by the AuNPs and stored in vacuoles. The presence of the AuNPs in ADSCs resulted in increased population doubling times, decreased cell motility and cell-mediated collagen contraction. The degree to which the cells were impacted was a function of particle concentration, where the smaller particles required a sevenfold higher concentration to have the same effect as the larger ones. Furthermore, AuNPs reduced adipogenesis as measured by lipid droplet accumulation and adiponectin secretion. These effects correlated with transient increases in DLK1 and with relative reductions in fibronectin. Upon removal of exogenous AuNPs, cellular NP levels decreased and normal ADSC functions were restored. As adiponectin helps regulate energy metabolism, local fluctuations triggered by AuNPs can lead to systemic changes. Hence, careful choice of size, concentration and clinical application duration of AuNPs is warranted.

Deformation gradients imprint the direction and speed of en masse fibroblast migration for fast healing.

En masse cell migration is more relevant compared with single-cell migration in physiological processes of tissue formation, such as embryogenesis, morphogenesis, and wound healing. In these situations, cells are influenced by the proximity of other cells including interactions facilitated by substrate mechanics. Here, we found that when fibroblasts migrated en masse over a hydrogel, they established a well-defined deformation field by traction forces and migrated along a trajectory defined by field gradients. The mechanics of the hydrogel determined the magnitude of the gradient. For materials stiff enough to withstand deformation related to cellular traction forces, such patterns did not form. Furthermore, migration patterns functioned poorly on very soft matrices where only a minimal traction gradient could be established. The largest degree of alignment and migration velocity occurred on the gels with the largest gradients. Granulation tissue formation in punch wounds of juvenile pigs was correlated strongly with the modulus of the implanted gel, in agreement with in vitro en masse cell migration studies. These findings provide basic insight into the biomechanical influences on fibroblast movement in early wounds and relevant design criteria for the development of tissue-engineered constructs that aim to stimulate en masse cell recruitment for rapid wound healing.

Platinum folate nanoparticles toxicity: cancer vs. normal cells.

Almost for two decades metallic nanoparticles are successfully used for cancer detection, imaging and treatment. Due to their high electron density they can be easily observed by electron microscopy and used in laser and radiofrequency therapy as energy releasing agents. However, the limitation for this practice is an inability to generate tumor-specific heating in a minimally invasive manner to the healthy tissue. To overcome this restraint we proposed to use folic acid coated metallic nanoparticles and determine whether they preferentially penetrate cancer cells. We developed technique for synthesizing platinum nanoparticles using folic acid as stabilizing agent which produced particles of relatively narrow size distribution, having d=2.3 ± 0.5 nm. High resolution TEM and zeta potential analysis indicated that the particles produced by this method had a high degree of crystalline order with no amorphous outer shell and a high degree of colloidal stability. The keratinocytes and mammary breast cells (cancer and normal) were incubated with platinum folate nanoparticles, and the results showed that the IC50 was significantly higher for the normal cells than the cancer cells in both cases, indicating that these nanoparticles preferentially target the cancer cells. TEM images of thin sections taken from the two types of cells indicated that the number of vacuoles and morphology changes after incubation with nanoparticles was also larger for the cancer cells in both types of tissue studied. No preferential toxicity was observed when folic acid receptors were saturated with free folic acid prior to exposure to nanoparticles. These results confirm our hypothesis regarding the preferential penetration of folic acid coated nanoparticles to cancer cells due to receptor mediated endocytosis.

The effects of UV emission from compact fluorescent light exposure on human dermal fibroblasts and keratinocytes in vitro.

Compact fluorescent light (CFL) bulbs can provide the same amount of lumens as incandescent light bulbs, using one quarter of the energy. Recently, CFL exposure was found to exacerbate existing skin conditions; however, the effects of CFL exposure on healthy skin tissue have not been thoroughly investigated. In this study, we studied the effects of exposure to CFL illumination on healthy human skin tissue cells (fibroblasts and keratinocytes). Cells exposed to CFLs exhibited a decrease in the proliferation rate, a significant increase in the production of reactive oxygen species, and a decrease in their ability to contract collagen. Measurements of UV emissions from these bulbs found significant levels of UVC and UVA (mercury [Hg] emission lines), which appeared to originate from cracks in the phosphor coatings, present in all bulbs studied. The response of the cells to the CFLs was consistent with damage from UV radiation, which was further enhanced when low dosages of TiO(2) nanoparticles (NPs), normally used for UV absorption, were added prior to exposure. No effect on cells, with or without TiO(2) NPs, was observed when they were exposed to incandescent light of the same intensity.

Control of anti-thrombogenic properties: surface-induced self-assembly of fibrinogen fibers.

Wound healing is a complex process initiated by the formation of fibrin fibers and endothelialization. Normally, this process is triggered in a wound by thrombin cleavage of fibrinopeptides on fibrinogen molecules, which allows them to self spontaneously-assemble into large fibers that provide the support structure of the clot and promote healing. We have found that the fibrous structures can also form without thrombin on most polymer or metal surfaces, including those commonly used for stents. We show that the relatively hydrophobic E and D regions of the fibrinogen molecule are adsorbed on these surfaces, exposing the αC domains, which in turn results in the formation of large fiber structures that promote endothelial cell adhesion. We show that the entire process can be suppressed when stents or other substrates are coated with polymers that are functionalized to bind the αC domains, leading to the development of potentially nonthrombogenic implant materials.

Polymer dynamics in semidilute solution during electrospinning: a simple model and experimental observations.

Electrospun polymer nanofibers demonstrate outstanding mechanical and thermodynamic properties as compared to macroscopic-scale structures. Our previous work has demonstrated that these features are attributed to nanofiber microstructure [Nat. Nanotechnol. 2, 59 (2007)]. It is clear that this microstructure is formed during the electrospinning process, characterized by a high stretching rate and rapid evaporation. Thus, when studying microstructure formation, its fast evolution must be taken into account. This study focuses on the dynamics of a highly entangled semidilute polymer solution under extreme longitudinal acceleration. The theoretical modeling predicts substantial longitudinal stretching and transversal contraction of the polymer network caused by the jet hydrodynamic forces, transforming the network to an almost fully stretched state. This prediction was verified by x-ray phase-contrast imaging of electrospinning jets of poly(ethylene oxide) and poly(methyl methacrylate) semidilute solutions, which revealed a noticeable increase in polymer concentration at the jet center, within less than 1 mm from the jet start. Thus, the proposed mechanism is applicable to the initial stage of the microstructure formation.

The role of moderate static magnetic fields on biomineralization of osteoblasts on sulfonated polystyrene films.

We have investigated the effects of moderate static magnetic fields (SMFs) on murine MC3T3-E1 osteoblasts, and found that they enhance proliferations and promote differentiation. The increase in proliferation rates in response to SMFs was greater in cultures grown on partially sulfonated polytstyrene (SPS, degree of sulfonation: 33%) than in cultures grown on tissue culture plastic. We have previously shown that when the degree of sulfonation exceeded a critical value (12%) [1], spontaneous fibrillogenesis occured which allowed for direct observation of the ECM fibrillar organization under the influence of external fields. We found that the ECM produced in cultures grown on the SPS in the presence of the SMFs assembled into a lattice with larger dimensions than the ECM of the cultures grown in the absence of SMFs. During the early stages of the biomineralization process (day 7), the SMF exposed cultures also templated mineral deposition more rapidly than the control cultures. The rapid response is attributed to orientation of diamagnetic ECM proteins already present in the serum, which could then initiate further cellular signaling. SMFs also influenced late stage osteoblast differentiation as measured by the increased rate of osteocalcin secretion and gene expression beginning 15 days after SFM exposure. This correlated with a large increase in mineral deposition, and in cell modulus. GIXD and EDXS analysis confirmed early deposition of crystalline hydroxyapatite. Previous studies on the effects of moderate SMF had focused on cellular gene and protein expression, but did not consider the organization of the ECM fibers. Our ability to form these fibers has allowed us explore this additional effect and highlight its significance in the initiation of the biomineralization process.