3D Porous Edible Scaffolds from Rye Secalin for Cell-Based Pork Fat Tissue Culturing.

Cellular agriculture holds hope for a sustainable alternative to conventional meat, yet multiple technical challenges remain. These include the large-scale production of edible scaffolds and culturing methods for fat tissues, which are key to meat texture, flavor, and nutritional values. Herein. we disclose our method in the facile fabrication of sponge-like plant protein scaffolds by applying commercial sugar cubes as highly permeable templates. The prepared secalin scaffolds feature a high porosity of 85-90%, fully interconnected pores, and high water stability. The mechanical properties of scaffolds could be tuned by varying sugar-to-protein weight ratio and post-water annealing treatment. Moreover, murine preadipocytes (3T3-L1) and porcine adipose-derived stem cells (ADSCs) readily infiltrate, adhere, proliferate, and differentiate on the secalin scaffolds to develop a fat tissue morphology. A cultured fat tissue was produced by culturing porcine ADSCs for 12 days, which remarkably resembles conventional porcine subcutaneous adipose tissue in appearance, texture, flavor, and fatty acid profiles. The research demonstrates the great potential of sponge-like secalin scaffolds for cultured fat tissue production.

Authentication of ten distinctive triterpenoids in Antrodia cinnamomea serves as a crucial aspect for ensuring the quality control of associated nutraceutical products.

Edible mushroom Antrodia cinnamomea is distinctive for its use in many health supplement products in relieving of diverse health-related conditions. A. cinnamomea is known for its rich array of bioactive secondary metabolites, predominantly terpenoids, that possess anti-inflammatory properties. Despite the abundance of these compounds, only some compounds have demonstrated notable anti-inflammatory activity. Moreover, there is a lack of established quality control methods specifically tailored to the active constituents of these products. Consequently, there is a great need for the development of precise and effective quality control methods for A. cinnamomea-based products, targeting their active components to ensure the consistency and reliability of these products in harnessing their anti-inflammatory potential. Herein we report a quantitative HPLC method for better evaluating the quality of A. cinnamomea based dietary supplements. Based on their bioactivities, we selected ten benchmark compounds, i. e. antcin K, (25S)-antcin H, (25R)-antcin H, (25R)-antcin C, (25S)-antcin C, (25R)-antcin A, 15α-acetyl-dehydrosulphurenic acid, versisponic acid D, dehydroeburicoic acid, and eburicoic acid and developed and validated a HPLC-UV method for quantification of these compounds simultaneously with high sensitivity, linearity and range, precision, and accuracy. Furthermore, we applied our method to quantify the commercially available A. cinnamomea containing supplements and found that the quality of these supplements varies greatly with only one product containing good amount of the active compounds. Our method provides a needed solution to quality control problem of the highly priced A. cinnamomea food and nutraceutical products that show great variety and inconsistency.

Machine learning and 3D bioprinting.

48With the growing number of biomaterials and printing technologies, bioprinting has brought about tremendous potential to fabricate biomimetic architectures or living tissue constructs. To make bioprinting and bioprinted constructs more powerful, machine learning (ML) is introduced to optimize the relevant processes, applied materials, and mechanical/biological performances. The objectives of this work were to collate, analyze, categorize, and summarize published articles and papers pertaining to ML applications in bioprinting and their impact on bioprinted constructs, as well as the directions of potential development. From the available references, both traditional ML and deep learning (DL) have been applied to optimize the printing process, structural parameters, material properties, and biological/mechanical performance of bioprinted constructs. The former uses features extracted from image or numerical data as inputs in prediction model building, and the latter uses the image directly for segmentation or classification model building. All of these studies present advanced bioprinting with a stable and reliable printing process, desirable fiber/droplet diameter, and precise layer stacking, and also enhance the bioprinted constructs with better design and cell performance. The current challenges and outlooks in developing process-material-performance models are highlighted, which may pave the way for revolutionizing bioprinting technologies and bioprinted construct design.

3D-Printed Prolamin Scaffolds for Cell-Based Meat Culture.

Cultivating meat from muscle stem cells in vitro requires 3D edible scaffolds as the supporting matrix. Electrohydrodynamic (EHD) printing is an emerging 3D-printing technology for fabricating ultrafine fibrous scaffolds with high precision microstructures for biomedical applications. However, edible EHD-printed scaffolds remain scarce in cultured meat (CM) production partly due to special requirements with regard to the printability of ink. Here, hordein or secalin is mixed, which are cereal prolamins extracted from barley or rye, with zein to produce pure prolamin-based inks, which exhibit favorable printability similar to common polycaprolactone ink. Zein/hordein and zein/secalin scaffolds with highly ordered tessellated structures are successfully fabricated after optimizing printing conditions. The prolamin scaffolds demonstrated good water stability and in vitro degradability due to the porous fiber surface, which is spontaneously generated by culturing muscle cells for 1 week. Moreover, mouse skeletal myoblasts (C2C12) and porcine skeletal muscle satellite cells (PSCs) can adhere and proliferate on the fibrous matrix, and a CM slice is produced by culturing PSCs on prolamin scaffolds with high tissue similarity. The upregulation of myogenic proteins shows that the differentiation process is triggered in the 3D culture, demonstrating the great potential of prolamin scaffolds in CM production.

Plant proteins as the functional building block of edible microcarriers for cell-based meat culture application.

Edible microcarriers are essential for developing cell-based meat in large-scale cell cultures. As they are required to be embedded in the final products, the microcarriers should be edible, biocompatible, cost-effective, and pathogen-free. The invention of edible animal-free microcarriers would be a breakthrough for cell-based meat culture. We reviewed the fabrication techniques and the materials of microcarriers, and found that plant proteins, having diverse structures and composition, could possess the active domains that are hypnotized to replace the animal-based extracellular matrix (ECM) for meat culture applications. In addition, the bioactive peptides in plants have been reviewed and most of them were resulted from enzyme hydrolysis. Therefore, plant proteins with rich bioactive peptides have the potential in the development microcarriers. Our work provided some new trains of thought for developing plant-based biomaterials as ECM materials and advances the fabrication of microcarriers for meat culture.

Electrohydrodynamic Jet-Printed Ultrathin Polycaprolactone Scaffolds Mimicking Bruch's Membrane for Retinal Pigment Epithelial Tissue Engineering.

Age-related macular degeneration (AMD) is the leading cause of visual loss and affects millions of people worldwide. Dysfunction of the retinal pigment epithelium (RPE) is associated with the pathogenesis of AMD. The purpose of this work is to build and evaluate the performance of ultrathin scaffolds with an electrohydrodynamic jet (EHDJ) printing method for RPE cell culture. We printed two types of ultrathin (around 7 µm) polycaprolactone scaffolds with 20 µm and 50 µm pores, which possess mechanical properties resembling that of native human Bruch's membrane and are biodegradable. Light microscopy and cell proliferation assay showed that adult human retinal pigment epithelial (ARPE-19) cells adhered and proliferated to form a monolayer on the scaffolds. The progress of culture matured on the scaffolds was demonstrated by immunofluorescence (actin, ZO-1, and Na+/K+-ATPase) and Western blot analysis of the respective proteins. The RPE cells cultured on EHDJ-printed scaffolds with 20 µm pores presented higher permeability, higher transepithelial potential difference, and higher expression level of Na+/K+-ATPase than those cultured on Transwell inserts. These findings suggest that the EHDJ printing can fabricate scaffolds that mimic Bruch's membrane by promoting maturation of RPE cells to form a polarized and functional monolayered epithelium with potential as an in vitro model for studying retinal diseases and treatment methods.

Three-Dimensional Quantitative Structure and Activity Relationship of Flavones on Their Hypochlorite Scavenging Capacity.

Flavonoids, a class of polyphenolic substances widely present in the plant realm, are considered as ideal hypochlorite scavengers. However, to our knowledge, little study has focused on the structure-activity relationship between flavonoids and hypochlorite scavenging capacity. Herein, we report for the first time the three-dimensional quantitative structure and activity relationship (3D-QSAR) combined with comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). Four models derived from CoMFA and CoMSIA with different combinations of descriptors were built and compared; the CoMFA model, which included both steric and electrostatic fields, showed great potential (R2 = 0.989; Q2 = 0.818) in predictive quality according to both internal and external validation criteria. Additionally, the average local ionization energy (ALIE), electrostatic potential (ESP), and orbital weighted dual descriptor (OWDD) were determined to identify the key structural moiety for scavenging capacity of flavonoids against hypochlorite. The computational results indicated that hypochlorous acid (HClO) serves as an electrophile undergoing electrophilic addition to the C6 carbon, which has the highest negative charge density, which are influenced by the functional groups on the flavones. The DFT calculated mechanism revealed the catalytic role of water of mono- and di-chlorination reactions, characterized by low activation barriers, and the involvement of neutral, instead of high-energy carbocation, intermediates.

Quantitative Determination of Ethylene Using a Smartphone-Based Optical Fiber Sensor (SOFS) Coupled with Pyrene-Tagged Grubbs Catalyst.

For rapid and portable detection of ethylene in commercial fruit ripening storage rooms, we designed a smartphone-based optical fiber sensor (SOFS), which is composed of a 15 mW 365 nm laser for fluorescence signal excitation and a bifurcated fiber system for signal flow direction from probe to smartphone. Paired with a pyrene-tagged Grubbs catalyst (PYG) probe, our SOFS showed a wide linearity range up to 350 ppm with a detection limit of 0.6 ppm. The common gases in the warehouse had no significant interference with the results. The device is portable (18 cm × 8 cm × 6 cm) with an inbuilt power supply and replaceable optical fiber sensor tip. The images are processed with a dedicated smartphone application for RGB analysis and ethylene concentration. The device was applied in detection of ethylene generated from apples, avocados, and bananas. The linear correlation data showed agreement with data generated from a fluorometer. The SOFS provides a rapid, compact, cost-effective solution for determination of the fruit ethylene concentration dynamic during ripening for better fruit harvest timing and postharvest management to minimize wastage.

Analyzing Cell-Scaffold Interaction through Unsupervised 3D Nuclei Segmentation.

Fibrous scaffolds have been extensively used in three-dimensional (3D) cell culture systems to establish in vitro models in cell biology, tissue engineering, and drug screening. It is a common practice to characterize cell behaviors on such scaffolds using confocal laser scanning microscopy (CLSM). As a noninvasive technology, CLSM images can be utilized to describe cell-scaffold interaction under varied morphological features, biomaterial composition, and internal structure. Unfortunately, such information has not been fully translated and delivered to researchers due to the lack of effective cell segmentation methods. We developed herein an end-to-end model called Aligned Disentangled Generative Adversarial Network (AD-GAN) for 3D unsupervised nuclei segmentation of CLSM images. AD-GAN utilizes representation disentanglement to separate content representation (the underlying nuclei spatial structure) from style representation (the rendering of the structure) and align the disentangled content in the latent space. The CLSM images collected from fibrous scaffold-based culturing A549, 3T3, and HeLa cells were utilized for nuclei segmentation study. Compared with existing commercial methods such as Squassh and CellProfiler, our AD-GAN can effectively and efficiently distinguish nuclei with the preserved shape and location information. Building on such information, we can rapidly screen cell-scaffold interaction in terms of adhesion, migration and proliferation, so as to improve scaffold design.

Using Plant Proteins to Develop Composite Scaffolds for Cell Culture Applications.

Electrohydrodynamic printing (EHDP) is capable of fabricating scaffolds that consist of micro/nano scale orientated fibers for three-dimensional (3D) cell culture models and drug screening applications. One of the major hurdles that limit the widespread application of EHDP is the lack of diverse biomaterial inks with appropriate printability and desired mechanical and biological properties. In this work, we blended plant proteins with synthetic biopolymer poly(ε-caprolactone) (PCL) to develop composite biomaterial inks, such as PCL/gliadin and PCL/zein for scaffold fabrication through EHDP. The tensile test results showed that the composite materials with a relatively small amount of plant protein portions, such as PCL/gliadin-10 and PCL/zein-10, can significantly improve tensile properties of the fabricated scaffolds such as Young's modulus and yield stress. These scaffolds were further evaluated by culturing mouse embryonic fibroblasts (NIH/3T3) cells and proven to enhance cell adhesion and proliferation, apart from temporary inhibition effects for PCL/gliadin-20 scaffold at the initial growth stage. After these plant protein nanoparticles were gradually released into culture medium, the generated nanoporous structures on the scaffold fiber surfaces became favorable for cellular attachment, migration, and proliferation. As competent candidates that regulate cell behaviors in 3D microenvironment, such composite scaffolds manifest a great potential in drug screening and 3D in vitro model development.

Chitosan and Whey Protein Bio-Inks for 3D and 4D Printing Applications with Particular Focus on Food Industry.

The application of chitosan (CS) and whey protein (WP) alone or in combination in 3D/4D printing has been well considered in previous studies. Although several excellent reviews on additive manufacturing discussed the properties and biomedical applications of CS and WP, there is a lack of a systemic review about CS and WP bio-inks for 3D/4D printing applications. Easily modified bio-ink with optimal printability is a key for additive manufacturing. CS, WP, and WP-CS complex hydrogel possess great potential in making bio-ink that can be broadly used for future 3D/4D printing, because CS is a functional polysaccharide with good biodegradability, biocompatibility, non-immunogenicity, and non-carcinogenicity, while CS-WP complex hydrogel has better printability and drug-delivery effectivity than WP hydrogel. The review summarizes the current advances of bio-ink preparation employing CS and/or WP to satisfy the requirements of 3D/4D printing and post-treatment of materials. The applications of CS/WP bio-ink mainly focus on 3D food printing with a few applications in cosmetics. The review also highlights the trends of CS/WP bio-inks as potential candidates in 4D printing. Some promising strategies for developing novel bio-inks based on CS and/or WP are introduced, aiming to provide new insights into the value-added development and commercial CS and WP utilization.

Three-Dimensional RAW264.7 Cell Model on Electrohydrodynamic Printed Poly(ε-Caprolactone) Scaffolds for In Vitro Study of Anti-Inflammatory Compounds.

Inflammation plays an essential role in the human immune system, and anti-inflammatory compounds are important to promote health. However, the in vitro screening of these compounds is largely dependent on flat biology. Herein, we report our efforts in establishing a 3D inflammation murine macrophage model. Murine macrophage RAW 264.7 cells were cultured on poly(ε-caprolactone) (PCL) scaffolds fabricated through an electrohydrodynamic jetting 3D printer and their behavior were examined. Cells on PCL scaffolds showed a 3D shape and morphology with multilayers and a lower proliferation rate. Moreover, macrophages were not activated by scaffold material PCL and 3D microenvironment. The 3D cells showed greater sensitivity to lipopolysaccharide stimulation with higher production activity of nitric oxide (NO), nitric oxide synthases (iNOS), and cyclooxygenase-2 (COX-2). Additionally, the 3D macrophage model showed lower drug sensitivity to commercial anti-inflammatory drugs including aspirin, ibuprofen, and dexamethasone, and natural flavones apigenin and luteolin with higher IC50 for NO production and lower iNOS and COX-2 inhibition efficacy. Overall, the 3D macrophage model showed promise for higher accurate screening of anti-inflammatory compounds. We developed, for the first time, a 3D macrophage model based on a 3D-printed PCL scaffold that provides an extracellular matrix environment for cells to grow in the 3D dimension. 3D-grown RAW 264.7 cells showed different sensitivities and responses to anti-inflammatory compounds from its 2D model. The 3D cells have lower sensitivity to both commercial and natural anti-inflammatory compounds. Consequently, our 3D macrophage model could be applied to screen anti-inflammatory compounds more accurately and thus holds great potential in next-generation drug screening applications.

Noninvasive In Vivo Imaging and Monitoring of 3D-Printed Polycaprolactone Scaffolds Labeled with an NIR Region II Fluorescent Dye.

Significant progress has been made in fabricating porous scaffolds with ultrafine fibers for tissue regeneration. However, the lack of noninvasive tracking methods in vivo makes it impossible to track the fate of such scaffolds in situ. The development of near-infrared region II (NIR-II, 1000-1700 nm) dyes provides the possibility of performing noninvasive visualization with deep-tissue penetration and high spatial resolution in vivo. Herein, we developed a polycaprolactone (PCL) ink containing the small organic NIR-II dye SY-1030 and the fluorescently labeled macromolecular dye SY-COO-PCL and fabricated high-resolution NIR-II active scaffolds via electrohydrodynamic jet (EHDJ) printing. All printed scaffolds subcutaneously implanted in mice were clearly imaged one week after the operation. Compared with scaffolds containing SY-1030, the fluorescence intensity emitted from scaffolds containing SY-COO-PCL can be tracked for up to three weeks. Moreover, the image quality can be optimized by adjusting the dye concentration, laser power, and exposure time. The advantage of such NIR-II active scaffolds is evidenced by the lower dye concentration, longer tracking period, and better in vivo stability. We also demonstrated the biocompatibility and biodegradability of the scaffolds containing SY-COO-PCL over a 3-month period. The developed NIR-II active scaffolds have potential applications in biopolymer implant tracking, tissue reconstruction monitoring, and target-position-based drug delivery.

Engineered Nanotopography on the Microfibers of 3D-Printed PCL Scaffolds to Modulate Cellular Responses and Establish an In Vitro Tumor Model.

Scaffold-based three-dimensional (3D) cell culture systems have gained increased interest in cell biology, tissue engineering, and drug screening fields as a replacement of two-dimensional (2D) monolayer cell culture and as a way to provide biomimetic extracellular matrix environments. In this study, microscale fibrous scaffolds were fabricated via electrohydrodynamic printing, and nanoscale features were created on the fiber surface by simply leaching gliadin of poly(ε-caprolactone) (PCL)/gliadin composites in ethanol solution. The microstructure of the printed scaffolds could be precisely controlled by printing parameters, and the surface nanotopography of the printed fiber could be tuned by varying the PCL/gliadin ratios. By seeding mouse embryonic fibroblast (NIH/3T3) cells and human nonsmall cell lung cancer (A549) cells on the printed scaffolds, the cellular responses showed that the fiber nanotopography on printed scaffolds efficiently favored cell adhesion, migration, proliferation, and tissue formation. Quantitative analysis of the transcript expression levels of A549 cells seeded on nanoporous scaffolds further revealed the upregulation of integrin-ß1, focal adhesion kinase, Ki-67, E-cadherin, and epithelial growth factor receptors over what was observed in the cells grown on the pure PCL scaffold. Furthermore, a significant difference was found in the relevant biomarker expression on the developed scaffolds compared with that in the monolayer culture, demonstrating the potential of cancer cell-seeded scaffolds as 3D in vitro tumor models for cancer research and drug screening.

Electrohydrodynamic printing process monitoring by microscopic image identification.

Electrohydrodynamic printing (EHDP) is able to precisely manipulate the position, size, and morphology of micro-/nano-fibers and fabricate high-resolution scaffolds using viscous biopolymer solutions. However, less attention has been paid to the influence of EHDP jet characteristics and key process parameters on deposited fiber patterns. To ensure the printing quality, it is very necessary to establish the relationship between the cone shapes and the stability of scaffold fabrication process. In this work, we used a digital microscopic imaging technique to monitor EHDP cones during printing, with subsequent image processing algorithms to extract related features, and a recognition algorithm to determine the suitability of Taylor cones for EHDP scaffold fabrication. Based on the experimental data, it has been concluded that the images of EHDP cone modes and the extracted features (centroid, jet diameter) are affected by their process parameters such as nozzle-substrate distance, the applied voltage, and stage moving speed. A convolutional neural network is then developed to classify these EHDP cone modes with the consideration of training time consumption and testing accuracy. A control algorithm will be developed to regulate the process parameters at the next stage for effective scaffold fabrication.

Zein Increases the Cytoaffinity and Biodegradability of Scaffolds 3D-Printed with Zein and Poly(ε-caprolactone) Composite Ink.

Electrohydrodynamic printing (EHDP) has attracted extensive interests as a powerful technology to fabricate micro- to nano-scale fibrous scaffolds in a custom-tailored manner for biomedical applications. A few synthetic biopolymer inks are applicable to this EHDP technology, but the fabricated scaffolds suffered from low mechanical strength, biocompatibility, and biodegradability. In this study, a series of poly(ε-caprolactone) (PCL)/zein composite inks were developed and their printability was examined on a solution-based EHDP system for scaffold fabrication. Multilayer grid scaffolds were manufactured by PCL, PCL/zein-10, and PCL/zein-20 inks, respectively and characterized. The mechanical strength of scaffolds printed by PCL/zein composite inks was remarkably enhanced in terms of Young's modulus and yield stress. The enzyme-accelerated in vitro degradation study demonstrated that zein-containing scaffolds exhibited dose-responsive improvement on the degradation rate as evidenced by surface morphological change of fibers. Moreover, the biocompatibility of PCL/zein scaffolds, tested on mice embryonic fibroblast (NIH/3T3) and human nonsmall lung cancer cell (H1299), manifested better cell affinity. Our findings suggest that scaffolds fabricated by the solution-based EHDP with PCL/zein composite inks can significantly improve Young's modulus, yield stress, biocompatibility, and biodegradability and have potential applications in drug delivery systems, 3D cell culture modeling, or tissue engineering.

Influence of electrohydrodynamic jetting parameters on the morphology of PCL scaffolds.

One of the important constituents in tissue engineering is scaffold, which provides structural support and suitable microenvironment for the cell attachment, growth and proliferation. To fabricate micro/nano structures for soft tissue repair and three-dimensional (3D) cell culture, the key is to improve fibre-based scaffold fabrication. Electrohydrodynamic (EHD) jetting is capable of producing and orientating submicron fibres for 3D scaffold fabrication. In this work, an EHD-jetting system was developed to explore the relationship between vital processing parameters and fibre characteristics. In this study, polycaprolactone (PCL) solution prepared by dissolving PCL pellets in acetic acid was used to fabricate the scaffolds. The influence of voltage, motorized stage speed, solution feed rate, and solution concentration on fibre characteristics and scaffold pattern were studied. Morphology of the EHD-jetted PCL fibres and scaffolds were analysed using optical microscope images and scanning electron microscope (SEM) images. Multi-layer scaffolds with the varied coiled pattern were fabricated and analysed. Cell attachment and proliferation have to be investigated in the future by further cell culture studies on these multi-layer coiled scaffolds.

Benzo-thia-fused [n]thienoacenequinodimethanes with small to moderate diradical characters: the role of pro-aromaticity versus anti-aromaticity.

Open-shell singlet diradicaloids have recently received much attention due to their unique optical, electronic and magnetic properties and promising applications in materials science. Among various diradicaloids, quinoidal π-conjugated molecules have become the prevailing design. However, the need for a fundamental understanding on how the fusion mode and pro-aromaticity/anti-aromaticity affect their diradical character and physical properties remains unaddressed. In this work, a series of pro-aromatic benzo-thia-fused [n]thienoacenequinodimethanes (Thn-TIPS (n = 1-3) and BDTh-TIPS) were synthesized and compared with the previously reported anti-aromatic bisindeno-[n]thienoacenes (Sn-TIPS, n = 1-4). The ground-state geometric and electronic structures of these new quinoidal molecules were systematically investigated by X-ray crystallographic analysis, variable temperature NMR, ESR, SQUID, Raman, and electronic absorption spectroscopy, assisted by DFT calculations. It was found that the diradical character index (y0) increased from nearly zero for Th1-TIPS to 2.4% for Th2-TIPS, 18.2% for Th3-TIPS, and 38.2% for BDTh-TIPS, due to the enhanced aromatic stabilization. Consequently, with the extension of molecular size, the one-photon absorption spectra are gradually red-shifted, the two-photon absorption (TPA) cross section values increase, and the singlet excited state lifetimes decrease. By comparison with the corresponding anti-aromatic analogues Sn-TIPS (n = 1-3), the pro-aromatic Thn-TIPS (n = 1-3) exhibit larger diradical character, longer singlet excited state lifetime and larger TPA cross section value. At the same time, they display distinctively different electronic absorption spectra and improved electrochemical amphotericity. Spectroelectrochemical studies revealed a good linear relationship between the optical energy gaps and the molecular length in the neutral, radical cationic and dicationic forms. Our research work discloses a significant difference between the pro-aromatic and anti-aromatic quinoidal compounds and provides guidance for the design of new diradicaloids with desirable properties.

Pro-aromatic bisphenaleno-thieno[3,2-b]thiophene versus anti-aromatic bisindeno-thieno[3,2-b]thiophene: different ground-state properties and applications in field-effect transistors.

A pro-aromatic bisphenaleno-thieno[3,2-b]thiophene (BPT-TIPS) was synthesized and compared with an anti-aromatic bisindeno-thieno[3,2-b]thiophene (S2-TIPS). BPT-TIPS showed larger diradical character, stronger absorption, longer excited state lifetime and better redox amphotericity than S2-TIPS.