Methods of ex vivo analysis of tissue status in vascularized composite allografts.

Vascularized composite allotransplantation can improve quality of life and restore functionality. However, the complex tissue composition of vascularized composite allografts (VCAs) presents unique clinical challenges that increase the likelihood of transplant rejection. Under prolonged static cold storage, highly damage-susceptible tissues such as muscle and nerve undergo irreversible degradation that may render allografts non-functional. Skin-containing VCA elicits an immunogenic response that increases the risk of recipient allograft rejection. The development of quantitative metrics to evaluate VCAs prior to and following transplantation are key to mitigating allograft rejection. Correspondingly, a broad range of bioanalytical methods have emerged to assess the progression of VCA rejection and characterize transplantation outcomes. To consolidate the current range of relevant technologies and expand on potential for development, methods to evaluate ex vivo VCA status are herein reviewed and comparatively assessed. The use of implantable physiological status monitoring biochips, non-invasive bioimpedance monitoring to assess edema, and deep learning algorithms to fuse disparate inputs to stratify VCAs are identified.

Electrically stimulated gene expression under exogenously applied electric fields.

Introduction: Electrical stimulation, the application of an electric field to cells and tissues grown in culture to accelerate growth and tight junction formation among endothelial cells, could be impactful in cardiovascular tissue engineering, allotransplantation, and wound healing. Methods: Using Electrical Cell Stimulation And Recording Apparatus (ECSARA), the exploration of the stimulatory influences of electric fields of different magnitude and frequencies on growth and proliferation, trans endothelial electrical resistance (TEER) and gene expression of human endothelia cells (HUVECs) were explored. Results: Within the range of endogenous electrical pulses studied, frequency was found to be more significant (p = 0.05) than voltage in influencing HUVEC gene expression. Localization of Yes Associated Protein (YAP) and expression of CD-144 are shown to be consistent with temporal manifestations of TEER. Discussion: This work introduces the field of electromics, the study of cellular gene expression profiles and their implications under the influence of exogenously applied electric fields. Homology of electrobiology and mechanobiology suggests use of such exogenous cues in tissue and regenerative engineering.

Real-Time Monitoring Using Multiplexed Multi-Electrode Bioelectrical Impedance Spectroscopy for the Stratification of Vascularized Composite Allografts: A Perspective on Predictive Analytics.

Vascularized composite allotransplantation addresses injuries to complex anatomical structures such as the face, hand, and abdominal wall. Prolonged static cold storage of vascularized composite allografts (VCA) incurs damage and imposes transportation limits to their viability and availability. Tissue ischemia, the major clinical indication, is strongly correlated with negative transplantation outcomes. Machine perfusion and normothermia can extend preservation times. This perspective introduces multiplexed multi-electrode bioimpedance spectroscopy (MMBIS), an established bioanalytical method to quantify the interaction of the electrical current with tissue components, capable of measuring tissue edema, as a quantitative, noninvasive, real-time, continuous monitoring technique to provide crucially needed assessment of graft preservation efficacy and viability. MMBIS must be developed, and appropriate models explored to address the highly complex multi-tissue structures and time-temperature changes of VCA. Combined with artificial intelligence (AI), MMBIS can serve to stratify allografts for improvement in transplantation outcomes.

Nanoengineered Ink for Designing 3D Printable Flexible Bioelectronics.

Flexible electronics require elastomeric and conductive biointerfaces with native tissue-like mechanical properties. The conventional approaches to engineer such a biointerface often utilize conductive nanomaterials in combination with polymeric hydrogels that are cross-linked using toxic photoinitiators. Moreover, these systems frequently demonstrate poor biocompatibility and face trade-offs between conductivity and mechanical stiffness under physiological conditions. To address these challenges, we developed a class of shear-thinning hydrogels as biomaterial inks for 3D printing flexible bioelectronics. These hydrogels are engineered through a facile vacancy-driven gelation of MoS2 nanoassemblies with naturally derived polymer-thiolated gelatin. Due to shear-thinning properties, these nanoengineered hydrogels can be printed into complex shapes that can respond to mechanical deformation. The chemically cross-linked nanoengineered hydrogels demonstrate a 20-fold rise in compressive moduli and can withstand up to 80% strain without permanent deformation, meeting human anatomical flexibility. The nanoengineered network exhibits high conductivity, compressive modulus, pseudocapacitance, and biocompatibility. The 3D-printed cross-linked structure demonstrates excellent strain sensitivity and can be used as wearable electronics to detect various motion dynamics. Overall, the results suggest that these nanoengineered hydrogels offer improved mechanical, electronic, and biological characteristics for various emerging biomedical applications including 3D-printed flexible biosensors, actuators, optoelectronics, and therapeutic delivery devices.

Electrospun electroconductive constructs of aligned fibers for cardiac tissue engineering.

Myocardial infarction remains the leading cause of death in the western world. Since the heart has limited regenerative capabilities, several cardiac tissue engineering (CTE) strategies have been proposed to repair the damaged myocardium. A novel electrospun construct with aligned and electroconductive fibers combining gelatin, poly(lactic-co-glycolic) acid and polypyrrole that may serve as a cardiac patch is presented. Constructs were characterized for fiber alignment, surface wettability, shrinkage and swelling behavior, porosity, degradation rate, mechanical properties, and electrical properties. Cell-biomaterial interactions were studied using three different types of cells, Neonatal Rat Ventricular Myocytes (NRVM), human lung fibroblasts (MRC-5) and induced pluripotent stem cells (iPSCs). All cell types showed good viability and unique organization on construct surfaces depending on their phenotype. Finally, we assessed the maturation status of NRVMs after 14 days by confocal images and qRT-PCR. Overall evidence supports a proof-of-concept that this novel biomaterial construct could be a good candidate patch for CTE applications.

Bioelectrical Impedance Spectroscopy for Monitoring Mammalian Cells and Tissues under Different Frequency Domains: A Review.

Bioelectrical impedance analysis and bioelectrical impedance spectroscopy (BIA/BIS) of tissues reveal important information on molecular composition and physical structure that is useful in diagnostics and prognostics. The heterogeneity in structural elements of cells, tissues, organs, and the whole human body, the variability in molecular composition arising from the dynamics of biochemical reactions, and the contributions of inherently electroresponsive components, such as ions, proteins, and polarized membranes, have rendered bioimpedance challenging to interpret but also a powerful evaluation and monitoring technique in biomedicine. BIA/BIS has thus become the basis for a wide range of diagnostic and monitoring systems such as plethysmography and tomography. The use of BIA/BIS arises from (i) being a noninvasive and safe measurement modality, (ii) its ease of miniaturization, and (iii) multiple technological formats for its biomedical implementation. Considering the dependency of the absolute and relative values of impedance on frequency, and the uniqueness of the origins of the α-, ß-, δ-, and γ-dispersions, this targeted review discusses biological events and underlying principles that are employed to analyze the impedance data based on the frequency range. The emergence of BIA/BIS in wearable devices and its relevance to the Internet of Medical Things (IoMT) are introduced and discussed.

On the intersection of molecular bioelectronics and biosensors: 20 Years of C3B.

Formed in 2000 at Virginia Commonwealth University, the Center for Bioelectronics, Biosensors and Biochips (C3B®) has subsequently been located at Clemson University and at Texas A&M University. Established as an industry-university collaborative center of excellence, the C3B has contributed new knowledge and technology in the areas of i) molecular bioelectronics, ii) responsive polymers, iii) multiplexed biosensor systems, and iv) bioelectronic biosensors. Noteworthy contributions in these areas include i) being the first to report direct electron transfer of oxidoreductase enzymes enabled by single walled carbon nanotubes and colloidal clays, ii) the molecular level integration of inherently conductive polymers with bioactive hydrogels using bi-functional monomers such as poly(pyrrole-co-3-pyrrolylbutyrate-conj-aminoethylmethacrylate) [PyBA-conj-AEMA] and 3-(1-ethyl methacryloylate)aniline to yield hetero-ladder electroconductive hydrogels, iii) the development of a multi-analyte physiological status monitoring biochip, and iv) the development of a bioanalytical Wien-bridge oscillator for the fused measurement to lactate and glucose. The present review takes a critical look of these contributions over the past 20 years and offers some perspective on the future of bioelectronics-based biosensors and systems. Particular attention is given to multiplexed biosensor systems and data fusion for rapid decision making.

Microfabricated and 3-D printed electroconductive hydrogels of PEDOT:PSS and their application in bioelectronics.

Biofabrication techniques such as microlithography and 3-D bioprinting have emerged in recent years as technologies capable of rendering complex, biocompatible constructs for biosensors, tissue and regenerative engineering and bioelectronics. While instruments and processes have been the subject of immense advancement, multifunctional bioinks have received less attention. A novel photocrosslinkable, hybrid bioactive and inherently conductive bioink formed from poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) nanomaterials within poly(2-hydroxyethyl methacrylate-co-polyethyleneglycol methacrylate) p(HEMA-co-EGMA) was used to render complex hydrogel constructs through microlithographic fabrication and 3-D printing. Constructs were directly compared through established metrics of acuity and fidelity, using side-by-side comparison of microarray grids, triangles incorporating angles 15-90°, and a multi-ink hydrogel disk array. Compositional variation from 0.01 to 1.00 wt% PEDOT:PSS produced hydrogels of varying and tunable electrical and electrochemical properties, while maintaining similar rheological properties (up to 0.50 wt% PEDOT:PSS). Furthermore, hydrogel membrane resistances extracted from equivalent circuit modeling of electrical impedance spectroscopy data varied only according to the included wt% of PEDOT:PSS and were agnostic of fabrication method. An in-silico variable frequency active low-pass filter was developed using a microlithographically fabricated Individually Addressable Microband Electrode (IAME) as the filtering capacitor, wherein 3-D printed lines of varying wt% of PEDOT:PSS hydrogels were shown to alter the cutoff frequency of the analog filter, indicating a potential use as tunable 3-D printed organic electronic analog filtering elements for biosensors. Bioinks of different PEDOT:PSS (0.0, 0.1, and 0.5 wt%) manufactured into hydrogel disks using the two methods were shown to yield similarly cytocompatible substrates for attachment and differentiation of PC-12 neural progenitor cells.

Design, fabrication and testing of an electrical cell stimulation and recording apparatus (ECSARA) for cells in electroculture.

A new dual-function electrical cell stimulation and recording apparatus (ECSARA) for simultaneously electrically stimulating cellular behavior within programmed stand-off electric fields (EFs) and monitoring cellular responses via AC electrical impedance spectroscopy (EIS) is reported. ECSARA is designed to have a footprint similar to that of a common 24-well cell culture plate within which each well is electrified via a pair of opposing planar titanium electrodes, within the cover (0.10 cm2) and base (0.50 cm2) of each well. Porous cell culture inserts established a 3-D milieu for bathing cells while keeping them away from unfavorable fields and forces in the vicinity of the electrodes. ECSARA was tested for its temporal stability, well-to-well variability, and responses in different media. EF modeling showed the field strength to be uniform in the subtending plane of the insert and the magnitude to be influenced by the porosity of the insert membrane. HUVECs were exposed to EF (162 mV/mm at 1.2 Hz) and monitored with standard viability Blue assay and EIS with equivalent circuit modeling. During the first 24 h, the viability (population) of EF-stimulated cells was smaller than non-stimulated control (0.8) but after 72 h they outnumbered the control (1.2) indicating that stimulation initially inhibited growth but resulted in eventual adaptive proliferation. EIS monitoring showed an increase in RCell of EF stimulated and control HUVECs after 54 h and 78 h, respectively. This was in accord with viability data that showed faster growth of EF-stimulated HUVECSs. Confluence was confirmed by VE-cadherin staining. The potential to explore the stimulatory influences of electric fields on cellular processes in tissue and regenerative engineering is now easily possible.

Chitosan-based advanced materials for docetaxel and paclitaxel delivery: Recent advances and future directions in cancer theranostics.

Paclitaxel (PTX) and docetaxel (DTX) are key members of taxanes with high anti-tumor activity against various cancer cells. These chemotherapeutic agents suffer from a number of drawbacks and it seems that low solubility in water is the most important one. Although much effort has been made in improving the bioavailability of PTX and DTX, the low bioavailability and minimal accumulation at tumor sites are still the challenges faced in PTX and DTX therapy. As a consequence, bio-based nanoparticles (NPs) have attracted much attention due to unique properties. Among them, chitosan (CS) is of interest due to its great biocompatibility. CS is a positively charged polysaccharide with the capability of interaction with negatively charged biomolecules. Besides, it can be processed into the sheet, micro/nano-particles, scaffold, and is dissolvable in mildly acidic pH similar to the pH of the tumor microenvironment. Keeping in mind the different applications of CS in the preparation of nanocarriers for delivery of PTX and DTX, in the present review, we demonstrate that how CS functionalized-nanocarriers and CS modification can be beneficial in enhancing the bioavailability of PTX and DTX, targeted delivery at tumor site, image-guided delivery and co-delivery with other anti-tumor drugs or genes.

Physiochemical and morphological dependent growth of NIH/3T3 and PC-12 on polyaniline-chloride/chitosan bionanocomposites.

Biomimetic scaffolds inspired by fields and forces of the natural environment of cells is essential in tissue engineering. This study reports on controlled growth of two model cell lines, NIH/3T3 (promiscuous, fibroblast) and PC-12 (electroresponsive, neural progenitor) cells, given electrical and topographical cues that were delivered from a bionanocomposite of polyaniline-chloride and chitosan (PAn-Cl/CHI). The conductivity and morphology of the scaffold were controlled by varying the wt% of PAn-Cl (0-50 wt%) in CHI and processing methods, air-drying (nanofeatured) versus lyophilization (microporous-reticulated), respectively. Bionanocomposites supported the growth of both cell types independent of the availability of receptor-mediated ligands (laminin). NIH/3T3 cells were less elongated on lyophilized (microporous-reticulated) and more conductive (higher wt% PAn-Cl) composites. PC-12 cells had higher viability and less aggregation when grown on conductive substrates. Air-dried bionanocomposites were more supportive of growth but not attachment of PC-12 cells, suggesting that processing of composites could provide an additional level of engineering control to alter the PC-12 cell attachment and growth. In general, PC-12 cells responded more distinctly and dramatically to the substrate properties than NIH/3T3 cells, supporting a clear role for electrical conductivity on neural cell behavior. Nerve growth factor(NGF)-induced differentiation of PC-12 cells resulted in extensive neurite extension in the presence of adsorbed laminin. In a substrate composition-dependent manner, extension and rate of neurite outgrowth were higher when cultured on the conductive substrates. Overall, this study demonstrates the suitability of conductive PAn-Cl/CHI scaffold to host different cell types and support their responses.

Biotechnical Properties of Poly(HEMA-co-HPMA) Hydrogels Are Governed by Distribution among Water States.

Controlling the biotechnical properties of synthetic hydrogels allows their application in a wide range of biomedical fields. Cross-linker concentration and monomer mole ratio of poly(2-hydroxyethylmethacrylate-co-N-(2-hydroxypropyl) methacrylamide) [poly(HEMA-co-HPMA)]-based hydrogels were used to control the degree of hydration and water distribution within constructs. Cross-linker concentrations corresponding to 0.1, 0.5, 1.0, and 3.0 mol % tetraethylene glycol (TEGDA) with HEMA/HPMA mole ratios of 1:0 and 4:1, and poly(HEMA-co-HPMA) of cross-linker concentration corresponding to 1.0 mol % TEGDA with a HEMA/HPMA ratio of 1:1 were investigated for their degree of hydration, water distribution, and corresponding physiochemical and mechanical properties. Copolymerization of HEMA and HPMA was confirmed by Fourier-transform infrared spectroscopy. Both cross-linker concentration and chemical composition (HEMA/HPMA) systematically changed the water content and free/bound water distribution in the polymer, which resulted in different biochemical and transport properties. The addition of 20% HPMA (poly(HEMA-co-HPMA) (4:1)) increased total hydration (25%) and glass-transition temperature (9%) and decreased elastic modulus (31%) and nonfreezable bound water (33%) of the hydrogel. Increasing cross-linker concentration resulted in a stiffer hydrogel with less total water but larger nonfreezable water content. Evaluation of poly(HEMA-co-HPMA) (1:1) revealed that further increase of HPMA content increased the degree of hydration by 25% and decreased nonfreezable water content and elastic modulus by 33 and 16%, respectively, compared to that of poly(HEMA-co-HPMA) (4:1). The hydrogel correspondingly had a higher void fraction and rougher freeze-fractured surface. The diffusion-related processes depended more on water distribution within the hydrogel. The poly(HEMA) showed the fastest swelling kinetics with a concomitant burst release profile of fluorescein isothiocyanate-dextran (a drug surrogate), while the compositions containing HPMA showed a sustained release pattern. The biotechnical properties are illustrative examples of key properties that are influenced by the water distribution rather than the absolute water content of hydrogels.

Microfabricated and 3-D Printed Soft Bioelectronic Constructs from PAn-PAAMPSA-Containing Hydrogels.

The formation of hybrid bioactive and inherently conductive constructs of composites formed from polyaniline-polyacrylamidomethylpropane sulfonic acid (PAn-PAAMPSA) nanomaterials (0.00⁻10.0 wt%) within poly(2-hydroxy ethyl methacrylate-co-N-{Tris(hydroxymethyl)methyl} acrylamide)-co-polyethyleneglycol methacrylate) p(HEMA-co-HMMA-co-PEGMA) hydrogels was made possible using microlithographic fabrication and 3-D printing. Hybrid constructs formed by combining a non-conductive base (0.00 wt% PAn-PAAMPSA) and electroconductive (ECH) (varying wt% PAn-PAAMPSA) hydrogels using these two production techniques were directly compared. Hydrogels were electrically characterized using two-point probe resistivity and electrochemical impedance spectroscopy. Results show that incorporation of >0.10 wt% PAn-PAAMPSA within the base hydrogel matrices was enough to achieve percolation and high conductivity with a membrane resistance (RM) of 2140 Ω and 87.9 Ω for base (0.00 wt%) and ECH (10.0 wt%), respectively. UV-vis spectroscopy of electroconductive hydrogels indicated a bandgap of 2.8 eV that was measurable at concentrations of >0.10 wt% PAn-PAAMPSA. Both base and electroconductive hydrogels supported the attachment and growth of NIH/3T3 fibroblast cells. When the base hydrogel was rendered bioactive by the inclusion of collagen (>200 µg/mL), it also supported the attachment, but not the differentiation, of PC-12 neural progenitor cells.