Multilayered Tissue Research Articles

Human airway-like multilayered tissue on 3D-TIPS printed thermoresponsive elastomer/collagen hybrid scaffolds

Developing a biologically representative complex tissue of the respiratory airway is challenging, however, beneficial for treatment of respiratory diseases, a common medical condition representing a leading cause of death in the world. This in vitro study reports a successful development of synthetic human tracheobronchial epithelium based on interpenetrated hierarchical networks composed of a reversely 3D printed porous structure of a thermoresponsive stiffness-softening elastomer nanohybrid impregnated with collagen nanofibrous hydrogel. Human bronchial epithelial cells (hBEpiCs) were able to attach and grow into an epithelial monolayer on the hybrid scaffolds co-cultured with either human bronchial fibroblasts (hBFs) or human bone-marrow derived mesenchymal stem cells (hBM-MSCs), with substantial enhancement of mucin expression, ciliation, well-constructed intercellular tight junctions and adherens junctions. The multi-layered co-culture 3D scaffolds consisting of a top monolayer of differentiated epithelium, with either hBFs or hBM-MSCs proliferating within the hyperelastic nanohybrid scaffold underneath, created a tissue analogue of the upper respiratory tract, validating these 3D printed guided scaffolds as a platform to support co-culture and cellular organization. In particular, hBM-MSCs in the co-culture system promoted an overall matured physiological tissue analogue of the respiratory system, a promising synthetic tissue for drug discovery, tracheal repair and reconstruction. Statement of SignificanceRespiratory diseases are a common medical condition and represent a leading cause of death in the world. However, the epithelium is one of the most challenging tissues to culture in vitro, and suitable tracheobronchial models, physiologically representative of the innate airway, remain largely elusive. This study presents, for the first time, a systematic approach for the development of functional multilayered epithelial synthetic tissue in vitro via co-culture on a 3D-printed thermoresponsive elastomer interpenetrated with a collagen hydrogel network. The viscoelastic nature of the scaffold with stiffness softening at body temperature provide a promising matrix for soft tissue engineering. The results presented here provide new insights about the epithelium at different surfaces and interfaces of co-culture, and pave the way to offer a customizable reproducible technology to generate physiologically relevant 3D biomimetic systems to advance our understanding of airway tissue regeneration.

RETRACTED ARTICLE: Scattering coefficients and pathgain of a multilayer tissue structure using ABCD matrix method

Numerical modeling plays an important role in the prediction of received signal strength inside the human body, in case of on-body to in-body radio wave propagation in a body area network (BAN). Since the structure of a human body is heterogeneous in nature, the Fresnel scattering coefficients could not be used to incorporate the attenuation properties of the multilayer tissue structure of the human body. This paper proposes the use of ABCD matrix method, which has been used in the analysis of multilayer dielectric slab structure, to determine the attenuation of the electro magnetic (EM) signal due to various in-body structures. In addition, variation of reflection and transmission coefficients of a four-layer body tissue structure with respect to the incident angle is presented and recommends suitable operating frequency in ultra-wide band (UWB) for BAN applications. The authors conducted measurements in body mimicking liquid phantom in UWB and included in support of their research outcome.

225 Desmoglein 1 deficiency in knockout mice impairs epidermal barrier formation and results in a psoriasis-like gene signature in E18.5 embryos

Desmoglein 1 (Dsg1) is a desmosomal cadherin expressed in concert with a commitment to stratify and differentiate in multi-layered tissues. Its importance in epidermal function is underscored by human diseases such as SAM syndrome (Severe Skin Dermatitis, Allergies and Metabolic Wasting), caused by loss of function mutations. Dsg1 promotes differentiation and normal epidermal morphogenesis in human 3D epidermal models. To determine the systemic response to loss of Dsg1, we deleted the three, tandem a, b, and g genes in the mouse cadherin cluster using CRISPR/Cas9. Dsg1 knockout (KO) mice exhibit barrier impairment accompanied by peeling, denuded skin, and postnatal lethality. The basal desmosomal cadherin, desmoglein 3, is upregulated in Dsg1 KO skin, while differentiation associated proteins loricrin and filaggrin are decreased. Disrupted ZO1 localization, increased TEWL, and increased dye penetration are consistent with a defect in barrier formation. Whole transcriptome analysis of E18.5 skin was consistent with the observed alteration in differentiation and barrier formation, but also revealed an increase in pathways linked to psoriatic processes, including IL-17 signaling, MAPK activity, and ErbB signaling. There was significant overlap between the top 100 genes increased in human psoriasis lesions and genes increased in E18.5 Dsg1 KO animals. These data not only support an important role for Dsg1 in epidermal differentiation and barrier formation, they identify a potential role for Dsg1 in regulating inflammatory responses, with loss of Dsg1 in mouse embryo skin activating inflammatory pathways similar to those observed in patients with psoriasis.

Optical volumetric projection with large NA objectives for fast high-resolution 3D imaging of neural signals.

One critical challenge in studying neural circuits of freely behaving model organisms is to record neural signals distributed within the whole brain, yet simultaneously maintaining cellular resolution. However, due to the dense packing of neuron cells in animal brains, high numerical aperture (NA) objectives are often required to differentiate neighboring neurons with the consequent need for axial scanning for whole brain imaging. Extending the depth of focus (EDoF) will be beneficial for fast 3D imaging of those neurons. However, current EDoF-enabled microscopes are primarily based on objectives with small NAs (≤0.3 ) such that the paraxial approximation can be applied. In this paper, we started from a nonparaxial approximation of the defocus aberration and derived a new phase mask that was appropriate for large NA microscopic systems. We validated the performance experimentally with a spatial light modulator (SLM) to create the designed phase mask. The performance was tested on different samples such as multilayered fluorescence beads and thick brain tissues, as well as with different objectives. Results confirmed that our design has extended the depth of focus about 10 fold and the image quality is much higher than those based on the most common EDoF method, the cubic phase method, popularly used to generate Airy beams. Meanwhile, our phase mask is rotationally symmetric and easy to fabricate. We fabricated one such phase plate and tested it on the pan-neuronal labeled Caenorhabditis elegans (C.elegans). The imaging performance demonstrated that we can capture all neurons in the whole brain with one snapshot and with cellular resolution, while the imaging speed is increased about 3 fold compared to the system using SLM. Thus we have shown that our method can not only provide the required imaging speed and resolution for studying neural activities in model animals, but also can be implemented as a low-cost, add-on module that can immediately augment existing fluorescence microscopes with only minor system modifications, and yielding substantially higher photon efficiency than SLM-based methods.

3D Bioprinting of osteochondral tissue substitutes \u2013 in vitro-chondrogenesis in multi-layered mineralized constructs

For the generation of multi-layered full thickness osteochondral tissue substitutes with an individual geometry based on clinical imaging data, combined extrusion-based 3D printing (3D plotting) of a bioink laden with primary chondrocytes and a mineralized biomaterial phase was introduced. A pasty calcium phosphate cement (CPC) and a bioink based on alginate-methylcellulose (algMC) – both are biocompatible and allow 3D plotting with high shape fidelity – were applied in monophasic and combinatory design to recreate osteochondral tissue layers. The capability of cells reacting to chondrogenic biochemical stimuli inside the algMC-based 3D hydrogel matrix was assessed. Towards combined osteochondral constructs, the chondrogenic fate in the presence of CPC in co-fabricated and biphasic mineralized pattern was evaluated. Majority of expanded and algMC-encapsulated cells survived the plotting process and the cultivation period, and were able to undergo redifferentiation in the provided environment to produce their respective extracellular matrix (ECM) components (i.e. sulphated glycosaminoglycans, collagen type II), examined after 3 weeks. The presence of a mineralized zone as located in the physiological calcified cartilage region suspected to interfere with chondrogenesis, was found to support chondrogenic ECM production by altering the ionic concentrations of calcium and phosphorus in in vitro culture conditions.

CytoCensus, mapping cell identity and division in tissues and organs using machine learning.

A major challenge in cell and developmental biology is the automated identification and quantitation of cells in complex multilayered tissues. We developed CytoCensus: an easily deployed implementation of supervised machine learning that extends convenient 2D 'point-and-click' user training to 3D detection of cells in challenging datasets with ill-defined cell boundaries. In tests on such datasets, CytoCensus outperforms other freely available image analysis software in accuracy and speed of cell detection. We used CytoCensus to count stem cells and their progeny, and to quantify individual cell divisions from time-lapse movies of explanted Drosophila larval brains, comparing wild-type and mutant phenotypes. We further illustrate the general utility and future potential of CytoCensus by analysing the 3D organisation of multiple cell classes in Zebrafish retinal organoids and cell distributions in mouse embryos. CytoCensus opens the possibility of straightforward and robust automated analysis of developmental phenotypes in complex tissues.

Ultra-Miniature Circularly Polarized CPW-Fed Implantable Antenna Design and its Validation for Biotelemetry Applications

The paper presents a coplanar waveguide (CPW)-fed ultra-miniaturized patch antenna operating in Industrial, Scientific and Medical (ISM) band (2.4–2.5 GHz) for biotelemetry applications. The proposed antenna structure is circular in shape and its ground plane is loaded with a pair of slots for obtaining circular polarization. In the proposed design, asymmetric square slots generate phase condition for right-hand circularly polarized (RHCP) radiation. And, by merely changing the position of the slots, either RHCP or left-hand circularly polarized (LHCP) radiation can be excited. In the proposed design, a meandered central strip is used for miniaturization. The simulations of the proposed antenna are carried out using Ansys HFSS software with a single-layer and multilayer human tissue models. The antenna shows good performance for different tissue properties owing to its wide axial ratio bandwidth and impedance bandwidth. The antenna is fabricated and measurements are carried out in skin mimicking phantom and pork. Simulated and measured performances of the antenna are in close agreement. The power link budget is also calculated using an exterior circularly polarized (CP) receiving antenna.

An Efficient 3-D Stochastic HIE-FDTD Algorithm for Investigation of Statistical Variation in Electromagnetic Field

We propose a stochastic hybrid implicit–explicit finite-difference time-domain method (S-HIE-FDTD) to compute the mean and variance of the electromagnetic (EM) fields using a single simulation, given those of the conductivity and permittivity in the computation domain. The mean and variance field update equations underlying the proposed method are derived from the field update equations of the “traditional” deterministic HIE-FDTD. The Courant–Friedrichs–Lewy condition of the S-HIE-FDTD depends on the spatial discretization sizes only in two dimensions; therefore, for computation domains with fine geometric features only in the remaining dimension, it uses a time step size that is larger than that of fully explicit schemes. Indeed, numerical results demonstrate that the proposed method is faster than the previously developed stochastic FDTD in computing the mean and variance of the EM fields in two different problems: wave propagation through a multilayer human tissue and transmission through a frequency selective surface.

A nanomechanical model enables comprehensive characterization of biological tissues in ultrasound imaging

Sonography, elastography, sonoelastography are ultrasound imaging techniques commonly used in the clinical practice for the diagnosis of many pathological conditions. These highly reliable, non-invasive methods use high frequency, elastic pressure waves (ultrasounds) to interrogate the internal structure of biological tissues and organs, and the continuum mechanics hypothesis to reconstruct, from the output of the system, the biophysical characteristics of the samples. Nevertheless, continuum mechanics disregards the discrete nature of tissues and organs, resulting in an inability for the model to describe some important tissue biophysical characteristics such as the cell size and their spatial layout. Here, we used the theory of doublet mechanics – a discrete nano-mechanical field theory – to model the propagation of ultrasounds in a multilayered biological tissue. We found that the output of the model exhibits a very high sensitivity to the macro and micro characteristics of the tissue, including cell size. We used results from the model to correlate the internal structure of the samples to the reflection coefficient, i.e. the continuum level response of the system. This model, and its more sophisticated evolutions that will be developed over time, can complement traditional ultrasound imaging, and provide ways to analyze non-invasively living tissues with a resolution inaccessible to conventional techniques of analysis, including positron emission tomography, computer tomography, and magnetic resonance.

Biometric Identity Based on Intra-Body Communication Channel Characteristics and Machine Learning.

In this paper, we propose and validate using the Intra-body communications channel as a biometric identity. Combining experimental measurements collected from five subjects and two multi-layer tissue mimicking materials’ phantoms, different machine learning algorithms were used and compared to test and validate using the channel characteristics and features as a biometric identity for subject identification. An accuracy of 98.5% was achieved, together with a precision and recall of 0.984 and 0.984, respectively, when testing the models against subject identification over results collected from the total samples. Using a simple and portable setup, this work shows the feasibility, reliability, and accuracy of the proposed biometric identity, which allows for continuous identification and verification.

Multi-layer stackable tissue culture platform for 3D co-culture

In vitro tools, which can enable development of models that replicate the cell microenvironment associated with complex diseases such as osteoarthritis (OA), are critically needed. In OA, catabolic and inflammatory processes orchestrated by multiple cell types lead to the eventual destruction of articular cartilage. To address this need, our group developed a device that will enable investigation of complex cell systems. Our stackable tissue culture insert was fabricated and characterized with respect to biocompatibility, ease of use, and potential for tissue culture applications. The stackable tissue culture inserts can be easily modified, fabricated, and assembled into commercially available multi-well plates. In vitro studies conducted with three different cell types demonstrated high cell viability and functional secretion when cultured in the stackable inserts. Furthermore, synergistic effects when the three cell types were cultured together were observed. This demonstrates the need to more fully interrogate in vitro culture systems, and this stackable insert can provide a tool to fill the current technological void to do so.

Modeling of Fiber Optic Probes for Selective Fluorescence Sensing of Multilayered Biological Tissues

Various probe configurations were modeled. The resulting fluorescence spectra of a human-skin tissue model were analyzed. The Monte Carlo method implemented in the TracePro software package was used to simulate light propagation in the tissue and optical fiber. The endogenous fluorophores riboflavin and protoporphyrin IX served as fluorescence sources in the skin model, had minimally overlapping emission spectra, and provided fluorescence of human skin when excited by laser radiation at 405 nm. The assumption that the fluorescence signal could be selected according to tissue depth using specific parameters of the fiber optic probes was verified. Model spectra using six configurations of fiber optic probes were obtained for the first time with selective sensing of skin auto fluorescence.

Droplet Microfluidics for the ex Vivo Expansion of Human Primary Multiple Myeloma Cells.

We previously reported a new approach for micromanipulation and encapsulation of human stem cells using a droplet-based microfluidic device We demonstrated the possibility of encapsulating and culturing difficult-to-preserve primary human hematopoietic stem cells using an engineered double layered bead composed by an inner layer of alginate and an outer layer of puramatrix constructed using a soft technology without the use of any external force. In this work, we use this micro manipulation technique to build a 3D scaffold as a biomimetic model to recapitulate the niche of patient-derived multiple myeloma cells (MM cell) using a multilayered 3D tissue scaffold constructed in a microfluidic device and cultured in 10% FBS culture medium. In the current study, we included the use of this biomimetic model comprising supporting human Mesenchymal stem cells to show the mid-term survival of MM cells in the proposed structures. We found that the generated microniches were suitable for the maintenance of MM cells with and without supporting cells. Additionally, cultured MM cells in droplets were exposed to both Bortezomib and Lenalidomide to test their toxicity in the cultured patient derived cells. Results indicate that the maintained MM cells were consistently responding to the applied medication, opening a wide field of possibilities to use the presented micro device as an ex vivo platform for drug screening.

A simple and accurate FDTD based technique to determine equivalent complex permittivity of the multi-layered human tissue in MICS band

Abstract This paper proposes a methodology to determine the equivalent electrical properties of multilayered human tissue using the Finite Difference Time Domain (FDTD) method for dispersive media. In addition, the impact of fat layer thickness on the equivalent dielectric properties has also been critically analyzed. The effect of moisture content present in the skin layer has also been studied. The main advantage of the proposed method is that it can be used for any thickness and any number of layers of human tissue. The multilayer reflection and transmission coefficients of the human tissue are first calculated using the FDTD method and then the permittivity and conductivity are extracted using the Nicholson Ross Weir (NRW) Method. The results are validated analytically using the concept of transmission line analogy for plane wave propagation. The tool used is MATLAB. In this paper, a three-layered software model of the human chest for pacemaker applications has been analyzed in the Medical Implants Communication Service band (MICS). At the frequency of 403.5 MHz in the MICS band, the equivalent permittivity of 3 layered human tissue is approximately 43 and its conductivity is 0.41 s / m . Moreover, the effective permittivity, conductivity and tan delta loss decrease with the increase in fat layer thickness. These results form the basis for the development of phantom mixtures used for designing, testing and evaluation of implantable antenna and SAR measurements. The choice of using FDTD is because it is a very powerful tool for creating a numerical mixture.

Virtual spectral histopathology of colon cancer - biomedical applications of Raman spectroscopy and imaging

Colon cancer is one of the most commonly detected pathology and is the third worldwide cause of death in the US and Europe and ranks second with regard to the incidence of malignant tumors in population. Raman spectroscopy and imaging can be used for colon cancer detection and bioanalytical characterization of noncancerous and pathologically changed tissues. These studies included Raman spectroscopy measurement of not fixed and not stained tissues samples. Presented study included patients from Poland, who were ≥18 years of age and underwent colon cancer surgery. Outcomes were measured using confocal Raman microscope, spectroscopy results were compared with traditional histopathology analysis outcomes. DNA, lipids, proteins and carotenoids can be treated as biomarkers of cancerogenesis. Ratios: 751/1156 (DNA/Carotenoids), 1228/1332 (β-sheet/α-helix proteins conformation), 1586/1004 (Phosphorylated proteins/Proteins), 2854/2935 (Lipids/Proteins) can be used to differentiate noncancerous and cancerous human sigmoid colon mucosa based on Raman spectroscopy and imaging. We have proved that Raman spectroscopy and imaging are a powerful technique to distinguish between noncancerous and cancerous human sigmoid colon mucosa and to characterize biochemical composition of multilayer sigmoid colon tissues samples.

Wristwatch-Style Wearable Dielectric Resonator Antennas for Applications on Limps

A wristwatch-style wearable dielectric resonator antenna (DRA) is proposed for the 2.45-GHz ISM band and sub-6 GHz 5G NR bands around 2.5 GHz, such as the commercial band for China Mobile Communications Corporation. The antenna is designed for applications on forearms and ankles. Practical concerns, including the aligning method, platform size, specific absorption rate, and user effects are investigated. Coplanar feeding structure is applied for alignment and isolation. Analysis of the watchband-like substrate is highlighted to avoid performance degradation. Given that platform configurations are not discretionary in actual devices, parasitic loading is introduced to attain desirable substrate effects and suppress unwanted ones. The user-device interplay is studied in light of actual application scenarios. The specific absorption rate is calculated through multilayer biological tissue models. Meanwhile, radiation variation caused by users is estimated based on human phantom models and measurements.

Numerical Simulation of High-Frequency Radio Energy Absorption in Human Head Tissues

The most relevant aspect of the mutual influence of a dipole antenna and a multilayer tissue model – the distribution of the electric field of the antenna in the layers of a spherical multilayer model – is considered. Special attention is paid to complex models providing almost perfect simulation of a human head. The obtained results are of interest not only from an engineering point of view, but from a consumer’s viewpoint as well, being related to the possible detrimental effect of cell phone radiation on health. The calculations were carried out for the most widely used digital cellular standards: GSM900 and GSM1800.

Is Pteropyrum a pathway to C4 evolution in Polygonaceae? An integrative approach to the taxonomy and anatomy of Pteropyrum (C3), an immediate relative of Calligonum (C4)

AbstractPteropyrum is a small genus of Polygonaceae with four species from the arid regions of Iran and adjacent countries. Pteropyrum spp. are not precisely delimitated and are difficult to identify because of their high plasticity in morphological characters. Pteropyrum (C3) has a close affinity to Calligonum (C4) and is therefore a suitable case for C4 evolutionary studies. We investigated the morphology and micromorphology (including pollen morphology) of Pteropyrum and elucidated the phylogenetic relationships with Atraphaxis and Calligonum using nuclear ITS sequences. Characteristics of the photosynthetic tissues such as volume and number of layers of primary carbon assimilation tissues (PCA) and photosynthetic carbon reduction tissue (PCR) were studied. In addition, the leaf and cotyledon anatomical characters of Pteropyrum (C3), Atraphaxis (C3) and Calligonum (C4), and their δ 13C values were compared to look for evolutionary changes in assimilating organs. The molecular phylogenetic tree identifies two strongly supported clades in Pteropyrum and its close relationship with Calligonum, confirming previous studies. Some morphologically similar species belong to different clades, which is probably due to convergent evolution and homoplasy. Leaf anatomical studies show that Atraphaxis has a multilayered mesophyll tissue, whereas Calligonum has one-layered mesophyll cells. The volume and layer number of mesophyll tissue cells decreases, whereas water storage tissue area significantly increases from Atraphaxis to Pteropyrum and Calligonum. This phenomenon confirms previous studies in other lineages with C4 salsoloid anatomy that have evolved through increasing of water storage tissue and succulence of assimilating organs. In the taxonomic part of the paper, a key to identification of accepted taxa of Pteropyrum, description of species and distribution maps are presented based on numerous herbarium specimens and our own rich collections from the field. Four new species are described based on a combination of morphology of seedlings and mature plants, pollen morphology and molecular data. A subspecific classification is suggested to show morpho-geographical variation of Pteropyrum aucheri s.l.

Porphyromonas gingivalis induces penetration of lipopolysaccharide and peptidoglycan through the gingival epithelium via degradation of junctional adhesion molecule 1

Porphyromonas gingivalis is a major pathogen in severe and chronic manifestations of periodontal disease, which is one of the most common infections of humans. A central feature of P. gingivalis pathogenicity is dysregulation of innate immunity at the gingival epithelial interface; however, the molecular basis underlying P. gingivalis–dependent abrogation of epithelial barrier function remains unknown. Gingival epithelial cells express junctional adhesion molecule (JAM1), a tight junction–associated protein, and JAM1 homodimers regulate epithelial barrier function. Here we show that Arg-specific or Lys-specific cysteine proteases (gingipains) secreted by P. gingivalis can specifically degrade JAM1 at K134 and R234 in gingival epithelial cells, resulting in permeability of the gingival epithelium to 40 kDa dextran, lipopolysaccharide (LPS), and proteoglycan (PGN). A P. gingivalis strain lacking gingipains was impaired in degradation of JAM1. Knockdown of JAM1 in monolayer cells and a three-dimensional multilayered tissue model also increased permeability to LPS, PGN, and gingipains. Inversely, overexpression of JAM1 in epithelial cells prevented penetration by these agents following P. gingivalis infection. Our findings strongly suggest that P. gingivalis gingipains disrupt barrier function of stratified squamous epithelium via degradation of JAM1, allowing bacterial virulence factors to penetrate into subepithelial tissues.

Cushion Mechanism of Goat Hoof Bulb Tissues

The hoof bulb sections of white goats were observed via scanning electron microscopy and stereomicroscopy in order to explore the cushion mechanism in the bulb tissue microstructures of hoofed animals. The hoof bulbs consisted of multilayer tissues, including an epidermal layer, a dermal layer, and subcutaneous tissues from outside to inside. A bionic model based on hoof bulb tissue composite structures was built with a normal model as the control. The microcosmic mechanics of the bulb tissues was analyzed via the finite element method. Simulations showed that when the bionic model was impacted by the top plates at the speed of 1-10 m/s, stress was concentrated in the epidermal layer and uniformly distributed in the dermal layer and dermal papillae, which effectively reduced the impact onto the ground. The cornified epidermal layer can resist the instant impact onto the ground, while the dermal papillae embedded in the dermal layer can store, release, and dissipate the impulsive energy, and the three parts synergically act in the cushion.