Engineering biomaterials by inkjet printing of hydrogels with functional particulates.

Hydrogels with particulates, including proteins, drugs, nanoparticles, and cells, enable the development of new and innovative biomaterials. Precise control of the spatial distribution of these particulates is crucial to produce advanced biomaterials. Thus, there is a high demand for manufacturing methods for particle-laden hydrogels. In this context, 3D printing of hydrogels is emerging as a promising method to create numerous innovative biomaterials. Among the 3D printing methods, inkjet printing, so-called drop-on-demand (DOD) printing, stands out for its ability to construct biomaterials with superior spatial resolutions. However, its printing processes are still designed by trial and error due to a limited understanding of the ink behavior during the printing processes. This review discusses the current understanding of transport processes and hydrogel behaviors during inkjet printing for particulate-laden hydrogels. Specifically, we review the transport processes of water and particulates within hydrogel during ink formulation, jetting, and curing. Additionally, we examine current inkjet printing applications in fabricating engineered tissues, drug delivery devices, and advanced bioelectronics components. Finally, the challenges and opportunities for next-generation inkjet printing are also discussed.

Inkjet-printed morphogenesis of tumor-stroma interface using bi-cellular bioinks of collagen-poly(N-isopropyl acrylamide-co-methyl methacrylate) mixture.

Recent advances in biomaterials and 3D printing/culture methods enable various tissue-engineered tumor models. However, it is still challenging to achieve native tumor-like characteristics due to lower cell density than native tissues and prolonged culture duration for maturation. Here, we report a new method to create tumoroids with a mechanically active tumor-stroma interface at extremely high cell density. This method, named "inkjet-printed morphogenesis" (iPM) of the tumor-stroma interface, is based on a hypothesis that cellular contractile force can significantly remodel the cell-laden polymer matrix to form densely-packed tissue-like constructs. Thus, differential cell-derived compaction of tumor cells and cancer-associated fibroblasts (CAFs) can be used to build a mechanically active tumor-stroma interface. In this methods, two kinds of bioinks are prepared, in which tumor cells and CAFs are suspended respectively in the mixture of collagen and poly (N-isopropyl acrylamide-co-methyl methacrylate) solution. These two cellular inks are inkjet-printed in multi-line or multi-layer patterns. As a result of cell-derived compaction, the resulting structure forms tumoroids with mechanically active tumor-stroma interface at extremely high cell density. We further test our working hypothesis that the morphogenesis can be controlled by manipulating the force balance between cellular contractile force and matrix stiffness. Furthermore, this new concept of "morphogenetic printing" is demonstrated to create more complex structures beyond current 3D bioprinting techniques.

A scaling law of particle transport in inkjet-printed particle-laden polymeric drops.

Hydrogels with embedded functional particulates are widely used to create soft materials with innovative functionalities. In order to advance these soft materials to functional devices and machines, critical technical challenges are the precise positioning of particulates within the hydrogels and the construction of the hydrogels into a complex geometry. Inkjet printing is a promising method for addressing these challenges and ultimately achieving hydrogels with voxelized functionalities, so-called digital hydrogels. However, the development of the inkjet printing process primarily relies on empirical optimization of its printing and curing protocol. In this study, a general scaling law is proposed to predict the transport of particulates within the hydrogel during inkjet printing. This scaling law is based on a hypothesis that water-matrix interaction during the curing of inkjet-printed particle-laden polymeric drops determines the intra-drop particle distribution. Based on the hypothesis, a dimensionless similarity parameter of the water-matrix interaction is proposed, determined by the hydrogel's water evaporation coefficient, particle size, and mechanical properties. The hypothesis was tested by correlating the intra-drop particle distribution to the similarity parameter. The results confirmed the scaling law capable of guiding ink formulation and printing and curing protocol.

Manipulating polymer composition to create low-cost, high-fidelity sensors for indoor CO2 monitoring.

Carbon dioxide (CO2) has been linked to many deleterious health effects, and it has also been used as a proxy for building occupancy measurements. These applications have created a need for low-cost and low-power CO2 sensors that can be seamlessly incorporated into existing buildings. We report a resonant mass sensor coated with a solution-processable polymer blend of poly(ethylene oxide) (PEO) and poly(ethyleneimine) (PEI) for the detection of CO2 across multiple use conditions. Controlling the polymer blend composition and nanostructure enabled better transport of the analyte gas into the sensing layer, which allowed for significantly enhanced CO2 sensing relative to the state of the art. Moreover, the hydrophilic nature of PEO resulted in water uptake, which provided for higher sensing sensitivity at elevated humidity conditions. Therefore, this key integration of materials and resonant sensor platform could be a potential solution in the future for CO2 monitoring in smart infrastructure.

Evaluation of anhydrous processing and storage methods of the temperate bacteriophage ɸV10 for integration into foodborne pathogen detection methodologies.

Due to the nascency of bacteriophage-based pathogen detection technologies, several practical hurdles stand in the way between providing promising proof-of-concept data and development of robust detection platforms. One such hurdle, and the focus of this work, is the development of methods for transitioning laboratory stocks of bacteriophage into functional, consistent, and shelf-stable delivery methods in commercial detection kits. Research described here was undertaken to evaluate two methods for their ability to store the bacteriophage ɸV10 at ambient temperature without aqueous storage solutions while limiting loss of viability. ɸV10 is a temperate bacteriophage which solely infects the zero-tolerance food adulterant Escherichia coli O157:H7 and has been genetically modified to generate a detectable phenotype in host cells. In order to integrate this reporter bacteriophage into food-borne pathogen detection methodologies, two methods of processing phage suspensions for long-term, ambient storage were evaluated: printing solutions onto pieces of dissolvable paper and lyophilizing suspensions with sucrose. Applying phage to dissolvable paper yielded key attributes to consider when addressing phage viability, however, optimized methodology still resulted in an approximate five-log reduction in titer of viable phage. Lyophilization of ɸV10 with various concentrations of the cryoprotectant molecule, sucrose, yielded losses of approximately 0.3-log after 120 days of storage at 23°C. Liquid storage buffer samples with and without sucrose saw a reduction of viable phage of at least 3.9-log in the same period. Additionally, the ability for ɸV10 to form lysogens in an E. coli O157:H7 host was not negatively affected by lyophilization. Drying ɸV10 at ambient temperature drastically reduces the viability of the phage. However, lyophilizing ɸV10 in the presence of sucrose is an effective method for dehydration and storage of the phage in ambient environmental conditions for an extended time lending to commercial application and integration into foodborne pathogen detection methodologies.

Ionic Strength Influences on Biofunctional Au-Decorated Microparticles for Enhanced Performance in Multiplexed Colorimetric Sensors.

The rising development of biosensors offers a great potential for health, food, and environmental monitoring. However, in many colorimetric platforms, there is a performance limitation stemming from the tendency of traditional Au nanoparticles toward nonspecific aggregation in response to changing ionic strength (salt concentration). This work puts forward a new type of colorimetric aptamer-functionalized labeling of microparticles, which allows to leverage an increase in ionic strength as a positive driver of enhanced detection performance of analytical targets. The resulting device is a cost-effective, instrument-free, portable, and reliable aptasensor that serves as basis for the fabrication of universal paper-based colorimetric platforms with the capability of multiplex, multireplicates and provides quantitative colorimetric detection. A controlled fabrication process was demonstrated by keeping 90% of the signal obtained from the as-fabricated devices (n = 40) within ± 1 standard deviation (SD) (relative SD = 5.69%) and following a mesokurtic normal-like distribution (p = 0.385). We propose for the first time a salt-induced aggregation mechanism for highly stable multilayered label particles (ssDNA-PEI-Au-PS) as the basis of the detection scheme. The use of DNA aptamers as capture biomolecules and PEI as an encapsulating agent allows for a sensitive and highly specific colorimetric response. As a proof of concept, multiplexed detection of mercury (Hg2+) and arsenic (As3+) was demonstrated. In addition, we introduced a robust image analysis algorithm for testing zone segmentation and color signal quantification that allowed for analytical detection, reaching a limit of detection of 1 ppm for both targeted analytes, with enough evidence (p > 0.05) to prove the high specificity of the fabricated device versus a pool of possible interferent ions.

Inkjet Printed Nanopatterned Aptamer-Based Sensors for Improved Optical Detection of Foodborne Pathogens.

The increasing incidence of infectious outbreaks from contaminated food and water supply continues imposing a global burden for food safety, creating a market demand for on-site, disposable, easy-to-use, and cost-efficient devices. Despite of the rapid growth of biosensors field and the generation of breakthrough technologies, more than 80% of the platforms developed at lab-scale never will get to meet the market. This work aims to provide a cost-efficient, reliable, and repeatable approach for the detection of foodborne pathogens in real samples. For the first time an optimized inkjet printing platform is proposed taking advantage of a carefully controlled nanopatterning of novel carboxyl-functionalized aptameric ink on a nitrocellulose substrate for the highly efficient detection of E. coli O157:H7 (25 colony forming units (CFU) mL-1 in pure culture and 233 CFU mL-1 in ground beef) demonstrating the ability to control the variation within ±1 SD for at least 75% of the data collected even at very low concentrations. From the best of the knowledge this work reports the lowest limit of detection of the state of the art for paper-based optical detection of E. coli O157:H7, with enough evidence (p > 0.05) to prove its high specificity at genus, species, strain, and serotype level.

High-speed and high-accuracy 3D surface measurement using a mechanical projector.

This paper presents a method to achieve high-speed and high-accuracy 3D surface measurement using a custom-designed mechanical projector and two high-speed cameras. We developed a computational framework that can achieve absolute shape measurement in sub-pixel accuracy through: 1) capturing precisely phase-shifted fringe patterns by synchronizing the cameras with the projector; 2) generating a rough disparity map between two cameras by employing a standard stereo-vision method using texture images with encoded statistical patterns; and 3) utilizing the wrapped phase as a constraint to refine the disparity map. The projector can project binary patterns at a speed of up to 10,000 Hz, and the camera can capture the required number of phase-shifted fringe patterns with 1/10,000 second, and thus 3D shape measurement can be realized as high as 10,000 Hz regardless the number of phase-shifted fringe patterns required for one 3D reconstruction. Experimental results demonstrated the success of our proposed method.

Flight mechanics and control of escape manoeuvres in hummingbirds. II. Aerodynamic force production, flight control and performance limitations.

The superior manoeuvrability of hummingbirds emerges from complex interactions of specialized neural and physiological processes with the unique flight dynamics of flapping wings. Escape manoeuvring is an ecologically relevant, natural behaviour of hummingbirds, from which we can gain understanding into the functional limits of vertebrate locomotor capacity. Here, we extend our kinematic analysis of escape manoeuvres from a companion paper to assess two potential limiting factors of the manoeuvring performance of hummingbirds: (1) muscle mechanical power output and (2) delays in the neural sensing and control system. We focused on the magnificent hummingbird (Eugenes fulgens, 7.8 g) and the black-chinned hummingbird (Archilochus alexandri, 3.1 g), which represent large and small species, respectively. We first estimated the aerodynamic forces, moments and the mechanical power of escape manoeuvres using measured wing kinematics. Comparing active-manoeuvring and passive-damping aerodynamic moments, we found that pitch dynamics were lightly damped and dominated by the effect of inertia, while roll dynamics were highly damped. To achieve observed closed-loop performance, pitch manoeuvres required faster sensorimotor transduction, as hummingbirds can only tolerate half the delay allowed in roll manoeuvres. Accordingly, our results suggested that pitch control may require a more sophisticated control strategy, such as those based on prediction. For the magnificent hummingbird, we estimated that escape manoeuvres required muscle mass-specific power 4.5 times that during hovering. Therefore, in addition to the limitation imposed by sensorimotor delays, muscle power could also limit the performance of escape manoeuvres.

Flight mechanics and control of escape manoeuvres in hummingbirds. I. Flight kinematics.

Hummingbirds are nature's masters of aerobatic manoeuvres. Previous research shows that hummingbirds and insects converged evolutionarily upon similar aerodynamic mechanisms and kinematics in hovering. Herein, we use three-dimensional kinematic data to begin to test for similar convergence of kinematics used for escape flight and to explore the effects of body size upon manoeuvring. We studied four hummingbird species in North America including two large species (magnificent hummingbird, Eugenes fulgens, 7.8 g, and blue-throated hummingbird, Lampornis clemenciae, 8.0 g) and two smaller species (broad-billed hummingbird, Cynanthus latirostris, 3.4 g, and black-chinned hummingbirds Archilochus alexandri, 3.1 g). Starting from a steady hover, hummingbirds consistently manoeuvred away from perceived threats using a drastic escape response that featured body pitch and roll rotations coupled with a large linear acceleration. Hummingbirds changed their flapping frequency and wing trajectory in all three degrees of freedom on a stroke-by-stroke basis, likely causing rapid and significant alteration of the magnitude and direction of aerodynamic forces. Thus it appears that the flight control of hummingbirds does not obey the 'helicopter model' that is valid for similar escape manoeuvres in fruit flies. Except for broad-billed hummingbirds, the hummingbirds had faster reaction times than those reported for visual feedback control in insects. The two larger hummingbird species performed pitch rotations and global-yaw turns with considerably larger magnitude than the smaller species, but roll rates and cumulative roll angles were similar among the four species.

Corticosteroids and monocytosis.

Although the association between steroid administration and neutrophilia is well known, the association with monocytosis is not as common and the mechanism less clear. This report illustrates the association and provides an update of postulated mechanisms and clinical significance.

Management of oral anticoagulation in a population of children with cardiac disease using a computerised system to support decision-making.

OBJECTIVE: To assess the impact of a computerised system to support decision-making concerning the management of warfarin used in maintenance of anti-coagulation. DESIGN: Retrospective case series study comparing manual and computerised records of prescribing. SETTING: A tertiary paediatric cardiology department in a teaching hospital. PARTICIPANTS: The 26 children receiving warfarin to maintain anticoagulation at the time of introduction of a computerised system to support decision-making. INTERVENTIONS: A rules-based computerised system to support decisions, based on existing departmental guidelines, for management of anticoagulation using warfarin was introduced to aid prescribing physicians. MAIN OUTCOMES: We assessed the stability of the International Normalised Ratio, along with the number of checks made of the ratio, and the adjustments of dosage. Dosages, and recheck interval prescriptions, were compared to the guidelines established by our department. RESULTS: We compared 274 prescriptions made manually, and 608 made using the computerised system to support decision-making, covering periods of 4, and 11, months respectively. The mean proportion of time spent by the patients within their target range for the International ratio was maintained during the period studied, at 76 percent versus 79 percent (p = 0.79). The median number of checks of the ratio made for each patient over a period of 28 days was unchanged, at 1.9 versus 2.1 (p = 0.58). There was a significant change in prescribing practices, which more closely followed the departmental guidelines. CONCLUSION: The introduction of a computerised system to support decision-making maintained the stability of the International ratio using warfarin, without increasing the number of checks or adjustments of dosages, in a point-of-care service for anticoagulation in children.

Classical cadherin and catenin expression in normal myometrial tissues and uterine leiomyomas.

Uterine leiomyomas are the most common benign uterine tumors in the women of reproductive age. Previous studies have suggested that uterine leiomyomas are monoclonal tumors derived from a single neoplastic myometrial cell. However, the neoplastic transformation of myometrium to leiomyomas remains to be elucidated. The classical cadherins are a gene family of integral membrane glycoproteins that mediate calcium-dependent cell-cell adhesion in a homophilic manner. These cell adhesion molecules (CAMs) have been shown to play pivotal roles in tumorigenesis. Catenins are intracellular proteins that link the cytoplasmic domains of the cadherins to the cytoskeletons to promote the biological functions of these CAMs. In this study, we compared the expression of E-, N-, and P-cadherins and alpha-, beta-, and gamma-catenins in the uterine leiomyomas and the counterparts of normal myometrium of the same patients by using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Of these, E-cadherin (E-cad) was not detected in both uterine leiomyomas and myometrium, P-cadherin (P-cad) was similarly expressed in these two tissues, but N-cadherin (N-cad) mRNA and protein expression levels in uterine leiomyomas were significantly greater than those observed in the myometrium. Catenins were not differentially expressed in uterine myometrium and leiomyomas. The overexpression of N-cad in uterine leiomyomas suggests that this CAM may play a central role in the development of uterine leiomyomas.

Identification of the cadherin subtypes present in the human peritoneum and endometriotic lesions: potential role for P-cadherin in the development of endometriosis.

Endometriosis is defined as endometrial tissue outside of the uterine cavity. The pathogenesis of this common disease remains poorly understood. However, the implantation and invasion of the viable cells from retrograde menstruation into the peritoneum is a widely accepted theory. To date, the mechanisms by which cell adhesion molecules mediate the development of human endometriosis remain unclear. Cadherins are a family of cell adhesion molecules that mediate cell-cell adhesion in a homophilic manner. In this study, the cadherins present in the peritoneum and endometriotic lesions were identified by RT-PCR using degenerate primers. In addition, differences in the levels of the cadherin mRNA transcripts present in eutopic endometrium and endometriotic lesions of the same patients were then compared by semiquantitative RT-PCR. Multiple cadherins were detected in the peritoneum and endometriotic lesions. Of these, P-cadherin appears to be the predominant cadherin subtype present in the peritoneum. Similarly, P-cadherin mRNA levels in endometriotic lesions were significantly greater than those observed in the corresponding eutopic endometrium. The expression of P-cadherin in both the human peritoneum and endometriotic lesions suggests that this cell adhesion molecule may play a central role in the development of endometriosis by mediating endometrial-peritoneal cell interactions in a homophilic manner.

Adaptive tuning of an electrodynamically driven thermoacoustic cooler.

The commercial development of thermoacoustic coolers has been hampered in part by their low efficiencies compared to vapor compression systems. A key component of electrodynamically driven coolers is the electromechanical transducer, or driver. The driver's electroacoustic transduction efficiency, defined as the ratio of the acoustic power delivered to the working gas by the moving piston and the electrical power supplied, must be maintained near its maximum value if a high overall system efficiency is to be achieved. Modeling and experiments have shown that the electroacoustic efficiency peaks sharply near the resonance frequency of the electro-mechano-acoustic system. The optimal operating frequency changes as the loading condition changes, and as the properties of the working gas vary. The driver efficiency may thus drop significantly during continuous operation at a fixed frequency. In this study, an on-line driver efficiency measurement scheme was implemented. It was found that the frequency for maximum electroacoustic efficiency does not precisely match any particular resonance frequency, and that the efficiency at resonance can be significantly lower than the highest achievable efficiency. Therefore, a direct efficiency measurement scheme was implemented and validated using a functional thermoacoustic cooler. An adaptive frequency-tuning scheme was then implemented. Experiments were performed to investigate the effectiveness of the control scheme to maintain the maximum achievable driver efficiency for varying operating conditions.

Ly49I NK cell receptor transgene inhibition of rejection of H2b mouse bone marrow transplants.

The Ly49 family of genes encode NK cell receptors that bind class I MHC Ags and transmit negative signals if the cytoplasmic domains have immunoregulatory tyrosine-based inhibitory motifs (ITIMs). 5E6 mAbs recognize Ly49C and Ly49I receptors and depletion of 5E6+ NK cells prevents rejection of allogeneic or parental-strain H2d bone marrow cell (BMC) grafts. To determine the function of the Ly49I gene in the rejection of BMC grafts, we transfected fertilized eggs of FVB mice with a vector containing DNA for B6 strain Ly49I (Ly49IB6). Ly49IB6 is ITIM+ and is recognized by 5E6 as well as Ly49I-specific 8H7 mAbs. Normal FVB H2q mice reject H2b but not H2d BMC allografts, and the rejection of H2b BMC was inhibited partially by anti-NK1.1 and completely by anti-asialo GM1, but not by anti-CD8, Abs. In FVB mice, NK1.1 is expressed on only 60% NK cells. FVB. Ly49IB6 hosts failed to reject H2d or H2b BMC, but did reject class I-deficient TAP-1-/- BMC, indicating that NK cells were functional. Nondepleting doses of anti-Ly49I Abs reversed the acceptance of H2b BMC by FVB.Ly49IB6 mice. FVB.Ly49IB6+/- mice were crossed and back-crossed with 129 mice-H2b, 5E6-, poor responders to H2d BMC grafts. While transgene-negative H2b/q F1 or first-generation back-crossed mice rejected H2b marrow grafts (hybrid resistance), transgene-positive mice did not. Thus B6 strain Ly49I receptors transmit inhibitory signals from H2b MHC class I molecules. Moreover, Ly49IB6 has no positive influence on the rejection of H2d allografts.

Tolerance and alloreactivity of the Ly49D subset of murine NK cells.

Class I-specific stimulatory and inhibitory receptors expressed by NK cell subsets contribute to the alloreactive potential of the self-tolerant murine NK cell repertoire. In this report, we have studied potential mechanisms of tolerance to the function of the positive signaling Ly49D receptor in mice that express one of its ligands, H2-Dd. Our results demonstrate that H2-Dd-expressing mice possess a large Ly49D+ subset of NK cells that is functionally capable of rejecting bone marrow cell (BMC) allografts in vivo and lysing allogeneic Con A lymphoblasts in vitro. Also, we show that the Ly49D receptor is responsible for the ability of H2b/d F1 hybrid mice to reject H2d/d parental BMC (hybrid resistance). Thus, deletion or anergy of Ly49D+ cells in H2-Dd+ hosts cannot explain self tolerance. Our functional studies revealed that coexpression of the Dd-specific Ly49A or Ly49G2 inhibitory receptors by Ly49D+ cells resulted in tolerance to Dd+ targets, while coexpression of Kb-specific inhibitory receptors Ly49C/I resulted in tolerance to Kb+ targets. Only in H2d/d cells did Ly49C/I dominantly inhibit Ly49D-Dd stimulation. This correlated with an increased mean fluorescence intensity of Ly49C expression, as well as an increased percentage of Ly49C+ cells in the Ly49D+A/G2- compartment. Therefore, we conclude that self tolerance of the Ly49D subset can be achieved through coexpression of a sufficient level of self-specific inhibitory receptors.

Positive recognition of MHC class I molecules by the Ly49D receptor of murine NK cells.

Members of the murine Ly49 family of receptors have been shown to inhibit and activate NK cell function. Subsets of Ly49-expressing NK cells mediate the rejection of bone marrow cell allografts and the lysis of allogeneic lymphoblasts. In this report we have studied Ly49-mediated positive and negative signaling in an in vitro cytotoxicity assay using sorted NK cell subsets as effectors and a panel of 51Cr-labeled Con A lymphoblasts as targets in the presence or the absence of Abs to Ly49 and/or class I molecules. Our results demonstrate that the activating receptor Ly49D delivers stimulatory signals for target cell lysis upon interacting with H2-Dd, Dr, and Dsp2, but not H2b or H2k class I Ags. On the other hand, the inhibitory receptor Ly49G2 delivers negative signals for target cell lysis upon interacting with Dd, Dr, and H2k, but not H2b or Dsp2, class I Ags. Furthermore, Ly49-mediated negative signaling dominates Ly49D-mediated positive signaling. Thus, lysis of class I MHC-bearing targets by NK cells is not merely the consequence of the absence of an Ly49-mediated negative signal, but also requires positive recognition of class I molecules by certain Ly49 receptors. Activation of NK cells by nonself class I molecules was not predicted by the missing self hypothesis.