Quantification of solution-free red blood cell staining by sorption kinetics of Romanowsky stains to agarose gels.

The imaging and quantification of stained red blood cells (RBCs) are important for identifying RBCs in hematology and for diagnosing diseased RBCs or parasites in cytopathology. Romanowsky staining has been used traditionally to produce hues in blood cells using a mixture of anionic eosin Y and cationic methylene blue and azure B. While Romanowsky stains have been widely used in cytopathology, end-users have experienced problems with varying results in staining due to the premature precipitation or evaporation of methanol, leading to the inherent inconsistency of solution-based Romanowsky staining. Herein, we demonstrate that the staining and destaining of blood smears are controllable by the contact time of agarose gel stamps. While the extent of staining and destaining is discernable by the hue values of stamped red blood cells in micrographs, the quantification of adsorbed and desorbed Romanowsky dye molecules (in particular, eosin Y, methylene blue and azure B) from and to the agarose gel stamps needs a model that can explain the sorption process. We found predictable sorption of the Romanowsky dye molecules from the pseudo-second-order kinetic model for adsorption and the one phase decay model for desorption. Thus, the method of agarose gel stamping demonstrated here could be an alternative to solution-based Romanowsky staining with the predictable quantity of sorption and timing of contact.

Injectable Antioxidant and Oxygen-Releasing Lignin Composites to Promote Wound Healing.

The application of engineered biomaterials for wound healing has been pursued since the beginning of tissue engineering. Here, we attempt to apply functionalized lignin to confer antioxidation to the extracellular microenvironments of wounds and to deliver oxygen from the dissociation of calcium peroxide for enhanced vascularization and healing responses without eliciting inflammatory responses. Elemental analysis showed 17 times higher quantity of calcium in the oxygen-releasing nanoparticles. Lignin composites including the oxygen-generating nanoparticles released around 700 ppm oxygen per day at least for 7 days. By modulating the concentration of the methacrylated gelatin, we were able to maintain the injectability of lignin composite precursors and the stiffness of lignin composites suitable for wound healing after photo-cross-linking. In situ formation of lignin composites with the oxygen-releasing nanoparticles enhanced the rate of tissue granulation, the formation of blood vessels, and the infiltration of α-smooth muscle actin+ fibroblasts into the wounds over 7 days. At 28 days after surgery, the lignin composite with oxygen-generating nanoparticles remodeled the collagen architecture, resembling the basket-weave pattern of unwounded collagen with minimal scar formation. Thus, our study shows the potential of functionalized lignin for wound-healing applications requiring balanced antioxidation and controlled release of oxygen for enhanced tissue granulation, vascularization, and maturation of collagen.

Single-Component Hydrophilic Terpolymer Thin Film Systems for Imparting Surface Chemical Versatility on Various Substrates.

We demonstrate a single-component hydrophilic photocrosslinkable copolymer system that incorporates all critical functionalities into one chain. This design allows for the creation of uniform functional organic coatings on a variety of substrates. The copolymers were composed of a poly(ethylene oxide)-containing monomer, a monomer that can release a primary amine upon UV light, and a monomer with reactive epoxide or cyclic dithiocarbonate with a primary amine. These copolymers are easily incorporated into the solution-casting process using polar solvents. Furthermore, the resulting coating can be readily stabilized through UV light-induced crosslinking, providing an advantage for controlling the surface properties of various substrates. The photocrosslinking capability further enables us to photolithographically define stable polymer domains in a desirable region. The resulting copolymer coatings were chemically versatile in immobilizing complex molecules by (i) post-crosslinking functionalization with the reactive groups on the surface and (ii) the formation of a composite coating by mixing varying amounts of a protein of interest, i.e., fish skin gelatin, which can form a uniform dual crosslinked network. The number of functionalization sites in a thin film could be controlled by tuning the composition of the copolymers. In photocrosslinking and subsequent functionalizations, we assessed the reactivity of the epoxide and cyclic dithiocarbonate with the generated primary amine. Moreover, the orthogonality of the possible reactions of the presented reactive functionalities in the crosslinked thin films with complex molecules is assessed. The resulting copolymer coatings were further utilized to define a hydrophobic surface or an active surface for the adhesion of biological objects.

Advances in non-invasive biosensing measures to monitor wound healing progression.

Impaired wound healing is a significant financial and medical burden. The synthesis and deposition of extracellular matrix (ECM) in a new wound is a dynamic process that is constantly changing and adapting to the biochemical and biomechanical signaling from the extracellular microenvironments of the wound. This drives either a regenerative or fibrotic and scar-forming healing outcome. Disruptions in ECM deposition, structure, and composition lead to impaired healing in diseased states, such as in diabetes. Valid measures of the principal determinants of successful ECM deposition and wound healing include lack of bacterial contamination, good tissue perfusion, and reduced mechanical injury and strain. These measures are used by wound-care providers to intervene upon the healing wound to steer healing toward a more functional phenotype with improved structural integrity and healing outcomes and to prevent adverse wound developments. In this review, we discuss bioengineering advances in 1) non-invasive detection of biologic and physiologic factors of the healing wound, 2) visualizing and modeling the ECM, and 3) computational tools that efficiently evaluate the complex data acquired from the wounds based on basic science, preclinical, translational and clinical studies, that would allow us to prognosticate healing outcomes and intervene effectively. We focus on bioelectronics and biologic interfaces of the sensors and actuators for real time biosensing and actuation of the tissues. We also discuss high-resolution, advanced imaging techniques, which go beyond traditional confocal and fluorescence microscopy to visualize microscopic details of the composition of the wound matrix, linearity of collagen, and live tracking of components within the wound microenvironment. Computational modeling of the wound matrix, including partial differential equation datasets as well as machine learning models that can serve as powerful tools for physicians to guide their decision-making process are discussed.

Tailoring Physical Properties of Dual-Network Acrylamide Hydrogel Composites by Engineering Molecular Structures of the Cross-linked Network.

We demonstrate the impact of engineering molecular structures of poly(acrylamide) (PAAm) and poly(N-isopropylacrylamide) (PNIPAm) hydrogel composites on several physical properties. The network structure was systematically varied by (i) the type and the concentration of difunctional cross-linkers and (ii) the type of native or chemically modified natural polymers, including sodium alginate, methacrylate/dopamine-incorporated porcine skin gelatin and fish skin gelatin, and thiol-incorporated lignosulfonate, which are attractive biopolymers generated in pulp and food industries because of their abundance, rich chemical functionalities, and environmental friendliness. First, we added cross-linking agents of varying lengths at different concentrations to assess how the cross-linking agent modulates the mechanical properties of acrylamide-based composites with alginate. After chemically modifying gelatins from fish or porcine skin with methacrylate and/or dopamine, the acrylamide-based composites were fabricated with the chemically modified gelatins and thiolated lignosulfonate to assess the stress-strain behavior. Furthermore, swelling ratios were measured with respect to temperature change. The mechanical properties were systematically modulated by the changes in the molecular structure, that is, the length of the chemical unit between two end alkene groups in the difunctional cross-linker and the types of the additive natural polymers. Overall, PAAm hydrogel composites exhibit a significant, negative correlation between toughness and the volume fraction of the swollen state and between strain at fracture and the volume fraction of the swollen state. In contrast, PNIPAm hydrogel composites showed positive, but only moderate correlations, which is attributed to the difference in the network polymer structure.

Loss of Acta2 in cardiac fibroblasts does not prevent the myofibroblast differentiation or affect the cardiac repair after myocardial infarction.

In response to myocardial infarction (MI), quiescent cardiac fibroblasts differentiate into myofibroblasts mediating tissue repair. One of the most widely accepted markers of myofibroblast differentiation is the expression of Acta2 which encodes smooth muscle alpha-actin (SMαA) that is assembled into stress fibers. However, the requirement of Acta2/SMαA in the myofibroblast differentiation of cardiac fibroblasts and its role in post-MI cardiac repair remained unknown. To answer these questions, we generated a tamoxifen-inducible cardiac fibroblast-specific Acta2 knockout mouse line. Surprisingly, mice that lacked Acta2 in cardiac fibroblasts had a normal post-MI survival rate. Moreover, Acta2 deletion did not affect the function or histology of infarcted hearts. No difference was detected in the proliferation, migration, or contractility between WT and Acta2-null cardiac myofibroblasts. Acta2-null cardiac myofibroblasts had a normal total filamentous actin level and total actin level. Acta2 deletion caused a significant compensatory increase in the transcription level of non-Acta2 actin isoforms, especially Actg2 and Acta1. Moreover, in myofibroblasts, the transcription levels of cytoplasmic actin isoforms were significantly higher than those of muscle actin isoforms. In addition, we found that myocardin-related transcription factor-A is critical for myofibroblast differentiation but is not required for the compensatory effects of non-Acta2 isoforms. In conclusion, the Acta2 deletion does not prevent the myofibroblast differentiation of cardiac fibroblasts or affect the post-MI cardiac repair, and the increased expression and stress fiber formation of non-SMαA actin isoforms and the functional redundancy between actin isoforms are able to compensate for the loss of Acta2 in cardiac myofibroblasts.

A modular surgical simulator for microlaryngoscopy using standard instruments and the carbon dioxide laser.

Objective: Build a microlaryngoscopy surgical simulator for endoscopic laryngeal surgery using standard microsurgical instruments and a CO2 laser. Study design: Anatomical modeling, CAD design and 3D printed manufacturing. Subjects and methods: We created a modular design for a microlaryngoscopy simulator in CAD software. Components include plastic and stainless-steel models of a standard operating laryngoscope and a cassette system for mounting porcine or synthetic models of the vocal folds. All simulator parts, including the metallic laryngoscope model, were manufactured using 3D printing technology. Tumors were simulated in porcine tissue models by injecting a soy protein-based tumor phantom. Residents and faculty in the Louisiana State University otolaryngology department evaluated the system. Each participant performed microlaryngoscopy with laser resection on a porcine larynx and cold instrument procedures on synthetic vocal folds. Participants scored the simulator using a 5-point Likert scale. Results: The microlaryngeal surgical simulator demonstrated in this project is realistic, economical, and easily assembled. We have included 3D printed parts files and detailed assembly instructions that will enable educators interested in surgical simulation to build the device.Participants in the simulator evaluation session felt that the simulator faithfully represented the procedure to resect vocal fold lesions using a CO2 laser. The synthetic model allows the trainee to develop hand-eye coordination while using standard laryngeal instruments. Conclusions: The simulator described herein will enable surgeons to acquire the surgical skills necessary to perform operative microlaryngoscopy prior to operating on live patients.

Stability and ocular biodistribution of topically administered PLGA nanoparticles.

Polymeric nanoparticles have been investigated as potential delivery systems for therapeutic compounds to address many ailments including eye disease. The stability and spatiotemporal distribution of polymeric nanoparticles in the eye are important regarding the practical applicability and efficacy of the delivery system in treating eye disease. We selected poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with lutein, a carotenoid antioxidant associated with eye health, as our model ophthalmic nanodelivery system and evaluated its stability when suspended in various conditions involving temperature and light exposure. We also assessed the ocular biodistribution of the fluorescently labeled nanoparticle vehicle when administered topically. Lutein-loaded nanoparticles were stable in suspension when stored at 4 °C with only 26% lutein release and no significant lutein decay or changes in nanoparticle morphology. When stored at 25 °C and 37 °C, these NPs showed signs of bulk degradation, had significant lutein decay compared to 4 °C, and released over 40% lutein after 5 weeks in suspension. Lutein-loaded nanoparticles were also more resistant to photodegradation compared to free lutein when exposed to ultraviolet (UV) light, decaying approximately 5 times slower. When applied topically in vivo, Cy5-labled nanoparticles showed high uptake in exterior eye tissues including the cornea, episcleral tissue, and sclera. The choroid was the only inner eye tissue that was significantly higher than the control group. Decreased fluorescence in all exterior eye tissues and the choroid at 1 h compared to 30 min indicated rapid elimination of nanoparticles from the eye.

Attenuating Fibrotic Markers of Patient-Derived Dermal Fibroblasts by Thiolated Lignin Composites.

We report the use of phenolic functional groups of lignosulfonate to impart antioxidant properties and the cell binding domains of gelatin to enhance cell adhesion for poly(ethylene glycol) (PEG)-based scaffolds. Chemoselective thiol-ene chemistry was utilized to form composites with thiolated lignosulfonate (TLS) and methacrylated fish gelatin (fGelMA). Antioxidant properties of TLS were not altered after thiolation and the levels of antioxidation were comparable to those of L-ascorbic acid. PEG-fGelMA-TLS composites significantly reduced the difference in COL1A1, ACTA2, TGFB1, and HIF1A genes between high-scarring and low-scarring hdFBs, providing the potential utility of TLS to attenuate fibrotic responses.

Distribution of polymeric nanoparticles in the eye: implications in ocular disease therapy.

Advantages of polymeric nanoparticles as drug delivery systems include controlled release, enhanced drug stability and bioavailability, and specific tissue targeting. Nanoparticle properties such as hydrophobicity, size, and charge, mucoadhesion, and surface ligands, as well as administration route and suspension media affect their ability to overcome ocular barriers and distribute in the eye, and must be carefully designed for specific target tissues and ocular diseases. This review seeks to discuss the available literature on the biodistribution of polymeric nanoparticles and discuss the effects of nanoparticle composition and administration method on their ocular penetration, distribution, elimination, toxicity, and efficacy, with potential impact on clinical applications.

Machine intelligence for nerve conduit design and production.

Nerve guidance conduits (NGCs) have emerged from recent advances within tissue engineering as a promising alternative to autografts for peripheral nerve repair. NGCs are tubular structures with engineered biomaterials, which guide axonal regeneration from the injured proximal nerve to the distal stump. NGC design can synergistically combine multiple properties to enhance proliferation of stem and neuronal cells, improve nerve migration, attenuate inflammation and reduce scar tissue formation. The aim of most laboratories fabricating NGCs is the development of an automated process that incorporates patient-specific features and complex tissue blueprints (e.g. neurovascular conduit) that serve as the basis for more complicated muscular and skin grafts. One of the major limitations for tissue engineering is lack of guidance for generating tissue blueprints and the absence of streamlined manufacturing processes. With the rapid expansion of machine intelligence, high dimensional image analysis, and computational scaffold design, optimized tissue templates for 3D bioprinting (3DBP) are feasible. In this review, we examine the translational challenges to peripheral nerve regeneration and where machine intelligence can innovate bottlenecks in neural tissue engineering.

Engineering Extracellular Matrix Proteins to Enhance Cardiac Regeneration After Myocardial Infarction.

Engineering microenvironments for accelerated myocardial repair is a challenging goal. Cell therapy has evolved over a few decades to engraft therapeutic cells to replenish lost cardiomyocytes in the left ventricle. However, compelling evidence supports that tailoring specific signals to endogenous cells rather than the direct integration of therapeutic cells could be an attractive strategy for better clinical outcomes. Of many possible routes to instruct endogenous cells, we reviewed recent cases that extracellular matrix (ECM) proteins contribute to enhanced cardiomyocyte proliferation from neonates to adults. In addition, the presence of ECM proteins exerts biophysical regulation in tissue, leading to the control of microenvironments and adaptation for enhanced cardiomyocyte proliferation. Finally, we also summarized recent clinical trials exclusively using ECM proteins, further supporting the notion that engineering ECM proteins would be a critical strategy to enhance myocardial repair without taking any risks or complications of applying therapeutic cardiac cells.

Development of rolled scaffold for high-density adherent cell culture.

Bioreactors for large-scale culture of mammalian cells are playing vital roles in biotechnology and bioengineering. Various bioreactors have been developed, but their capacity and efficiency are often limited by insufficient mass transfer rate and high shear stress. A rolled scaffold (RS) is a fully defined scaffold for high-density adherent culture of mammalian cells. The RS is a polymer film with spacers, that is rolled into a cylinder with a pre-determined gap between each turn. Cells are cultured on its inner surfaces, while media flows through the gap. The RS exhibits high surface-area-to-volume ratio over 100 cm2/mL and can transport nutrients and gases with significantly reduced shear stress via convection in a unidirectional laminar flow, rather than diffusion and random turbulent flow as in stirred-tank bioreactors. In this paper, we expanded Chinese Hamster Ovary cells with RS bioreactors and demonstrated cell culture density over 60 million cells/mL with a growth rate higher than conventional suspension culture. Besides, murine embryonic stem cells were successfully expanded without losing their pluripotency. The RS will provide an affordable, scalable, and reliable platform for large-scale culture of recombinant cells in biopharmaceutical industries and shear-sensitive stem cells for tissue engineering.

Directed Printing and Reconfiguration of Thermoresponsive Silica-pNIPAM Nanocomposites.

Printing of polymeric composites into desired patterns and shapes has revolutionized small-scale manufacturing processes. However, high-resolution printing of adaptive materials that change shape in response to external stimuli remains a significant technical challenge. The article presents a new approach of printing thermoresponsive poly(N-isopropylacrylamide) into macroscopic structures that dynamically reconfigure in response to heating and cooling cycles. The printing process is performed using an external laser source, which enables thermal cross-linking of the polymer ink consisting of monomer, cross-linker, initiator, and inorganic nanoparticles. It is shown that the addition of silica nanoparticles enhances the mechanical properties of poly(N-isopropylacrylamide) while maintaining its thermoresponsiveness at micrometer-scale resolution, which otherwise is not feasible by extrusion-based three-dimensional printing techniques. It is demonstrated that spatial reconfiguration of the printed monolayers upon increasing temperature is governed by the local geometry, which enables mimicking the reconfiguration of plant leaves in a natural environment. The study lays a foundation for developing a new fabrication platform to print thermoresponsive structures that may find applications in biomedical implants, sensors, and other multi-responsive materials.

Conserved pathway activation following xenogeneic, heterotypic fusion.

Fusion between cells of different organisms (i.e., xenogeneic hybrids) can occur, and for humans this may occur in the course of tissue transplantation, animal handling, and food production. Previous work shows that conferred advantages are rare in xenogeneic hybrids, whereas risks of cellular dysregulation are high. Here, we explore the transcriptome of individual xenogeneic hybrids of human mesenchymal stem cells and murine cardiomyocytes soon after fusion and ask whether the process is stochastic or involves conserved pathway activation. Toward this end, single-cell RNA sequencing was used to analyze the transcriptomes of hybrid cells with respect to the human and mouse genomes. Consistent with previous work, hybrids possessed a unique transcriptome distinct from either fusion partner but were dominated by the cardiomyocyte transcriptome. New in this work is the documentation that a few genes that were latent in both fusion partners were consistently expressed in hybrids. Specifically, human growth hormone 1, murine ribosomal protein S27, and murine ATP synthase H+ transporting, mitochondrial Fo complex subunit C2 were expressed in nearly all hybrids. The consistent activation of latent genes between hybrids suggests conserved signaling mechanisms that either cause or are the consequence of fusion of these 2 cell types and might serve as a target for limiting unwanted xenogeneic fusion in the future.-Yuan, C., Freeman, B. T., McArdle, T. J., Jung, J. P., Ogle, B. M. Conserved pathway activation following xenogeneic, heterotypic fusion.

Modulating Mechanical Properties of Collagen-Lignin Composites.

Three-dimensional matrices of collagen type I (Col I) are widely used in tissue engineering applications for its abundance in many tissues, bioactivity with many cell types, and excellent biocompatibility. Inspired by the structural role of lignin in a plant tissue, we found that sodium lignosulfonate (SLS) and an alkali-extracted lignin from switchgrass (SG) increased the stiffness of Col I gels. SLS and SG enhanced the stiffness of Col I gels from 52 to 670 Pa and 52 to 320 Pa, respectively, and attenuated shear-thinning properties, with the formulation of 1.8 mg/mL Col I and 5.0 mg/mL SLS or SG. In 2D cultures, the cytotoxicity of collagen-SLS to adipose-derived stromal cells was not observed and the cell viability was maintained over 7 days in 3D cultures. Collagen-SLS composites did not elicit immunogenicity when compared to SLS-only groups. Our collagen-SLS composites present a case that exploits lignins as an enhancer of mechanical properties of Col I without adverse cytotoxicity and immunogenicity for in vitro scaffolds or in vivo tissue repairs.

Engineering Tissue Fabrication With Machine Intelligence: Generating a Blueprint for Regeneration.

Regenerating lost or damaged tissue is the primary goal of Tissue Engineering. 3D bioprinting technologies have been widely applied in many research areas of tissue regeneration and disease modeling with unprecedented spatial resolution and tissue-like complexity. However, the extraction of tissue architecture and the generation of high-resolution blueprints are challenging tasks for tissue regeneration. Traditionally, such spatial information is obtained from a collection of microscopic images and then combined together to visualize regions of interest. To fabricate such engineered tissues, rendered microscopic images are transformed to code to inform a 3D bioprinting process. If this process is augmented with data-driven approaches and streamlined with machine intelligence, identification of an optimal blueprint can become an achievable task for functional tissue regeneration. In this review, our perspective is guided by an emerging paradigm to generate a blueprint for regeneration with machine intelligence. First, we reviewed recent articles with respect to our perspective for machine intelligence-driven information retrieval and fabrication. After briefly introducing recent trends in information retrieval methods from publicly available data, our discussion is focused on recent works that use machine intelligence to discover tissue architectures from imaging and spectral data. Then, our focus is on utilizing optimization approaches to increase print fidelity and enhance biomimicry with machine learning (ML) strategies to acquire a blueprint ready for 3D bioprinting.

A 3D in vitro model of the dermoepidermal junction amenable to mechanical testing.

Recessive dystrophic Epidermolysis Bullosa (RDEB) is caused by mutations in collagen-type VII gene critical for the dermoepidermal junction (DEJ) formation. Neither tissues of animal models nor currently available in vitro models are amenable to the quantitative assessment of mechanical adhesion between dermal and epidermal layers. Here, we created a 3D in vitro DEJ model using extracellular matrix (ECM) proteins of the DEJ anchored to a poly(ethylene glycol)-based slab (termed ECM composites) and seeded with human keratinocytes and dermal fibroblasts. Keratinocytes and fibroblasts of healthy individuals were well maintained in the ECM composite and showed the expression of collagen type VII over a 2-week period. The ECM composites with healthy keratinocytes and fibroblasts exhibited yield stress associated with the separation of the model DEJ at 0.268 ± 0.057 kPa. When we benchmarked this measure of adhesive strength with that of the model DEJ fabricated with cells of individuals with RDEB, the yield stress was significantly lower (0.153 ± 0.064 kPa) consistent with our current mechanistic understanding of RDEB. In summary, a 3D in vitro model DEJ was developed for quantification of mechanical adhesion between epidermal- and dermal-mimicking layers, which can be utilized for assessment of mechanical adhesion of the model DEJ applicable for Epidermolysis Bullosa-associated therapeutics. © 2018 The Authors. Journal Of Biomedical Materials Research Part A Published By Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3231-3238, 2018.

Engineering Breast Cancer Microenvironments and 3D Bioprinting.

The extracellular matrix (ECM) is a critical cue to direct tumorigenesis and metastasis. Although two-dimensional (2D) culture models have been widely employed to understand breast cancer microenvironments over the past several decades, the 2D models still exhibit limited success. Overwhelming evidence supports that three dimensional (3D), physiologically relevant culture models are required to better understand cancer progression and develop more effective treatment. Such platforms should include cancer-specific architectures, relevant physicochemical signals, stromal-cancer cell interactions, immune components, vascular components, and cell-ECM interactions found in patient tumors. This review briefly summarizes how cancer microenvironments (stromal component, cell-ECM interactions, and molecular modulators) are defined and what emerging technologies (perfusable scaffold, tumor stiffness, supporting cells within tumors and complex patterning) can be utilized to better mimic native-like breast cancer microenvironments. Furthermore, this review emphasizes biophysical properties that differ between primary tumor ECM and tissue sites of metastatic lesions with a focus on matrix modulation of cancer stem cells, providing a rationale for investigation of underexplored ECM proteins that could alter patient prognosis. To engineer breast cancer microenvironments, we categorized technologies into two groups: (1) biochemical factors modulating breast cancer cell-ECM interactions and (2) 3D bioprinting methods and its applications to model breast cancer microenvironments. Biochemical factors include matrix-associated proteins, soluble factors, ECMs, and synthetic biomaterials. For the application of 3D bioprinting, we discuss the transition of 2D patterning to 3D scaffolding with various bioprinting technologies to implement biophysical cues to model breast cancer microenvironments.