Pseudo-3D Topological Alignments Regulate Mechanotransduction and Maturation of Smooth Muscle Cells.

Smooth muscle cells (SMCs) sense and respond to mechanical stimuli in their extracellular microenvironments (ECMs), playing a crucial role in muscle tissue engineering. Increasing evidence from topological cues-mediated mechanotransduction of SMCs in ECMs has suggested some potential underlying mechanisms of how SMC functions and maturation are regulated by their mechanosensing leading to transduction. However, how the expression of yes-associated protein 1 (YAP) influences the phenotypic shift from synthetic to contractile is still controversial. Here, pseudo-3D topological alignments mimicking native muscle tissues are generated using laser-cutter engraving to explore the influence of topological cues on SMC mechanotransduction and maturation. The analysis of topological cue-mediated mechanotransduction and maturation marker expression revealed YAP is involved in mechanotransduction for SMCs cultured on cross-patterned substrates in the presence of cell-cell interactions. Moreover, these SMCs with YAP-linked mechanosensing showed higher expression of calponin, indicating a shift toward contractile phenotypes in vitro and in vivo. Furthermore, it showed skeletal muscle cells has different mechanosensing and maturation mechanisms compared to SMCs, revealing muscle type-dependent different sensing of topological cues, and converting into maturation-associated signaling cascades. This study provides insights into the regulation of SMC mechanotransduction and maturation by topological cues, suggesting the involvement of YAP-linked signaling pathways in this process.

Engineering large-scale hiPSC-derived vessel-integrated muscle-like lattices for enhanced volumetric muscle regeneration.

Engineering biomimetic tissue implants with human induced pluripotent stem cells (hiPSCs) holds promise for repairing volumetric tissue loss. However, these implants face challenges in regenerative capability, survival, and geometric scalability at large-scale injury sites. Here, we present scalable vessel-integrated muscle-like lattices (VMLs), containing dense and aligned hiPSC-derived myofibers alongside passively perfusable vessel-like microchannels inside an endomysium-like supporting matrix using an embedded multimaterial bioprinting technology. The contractile and millimeter-long myofibers are created in mechanically tailored and nanofibrous extracellular matrix-based hydrogels. Incorporating vessel-like lattice enhances myofiber maturation in vitro and guides host vessel invasion in vivo, improving implant integration. Consequently, we demonstrate successful de novo muscle formation and muscle function restoration through a combinatorial effect between improved graft-host integration and its increased release of paracrine factors within volumetric muscle loss injury models. The proposed modular bioprinting technology enables scaling up to centimeter-sized prevascularized hiPSC-derived muscle tissues with custom geometries for next-generation muscle regenerative therapies.

Conductive PEDOT-Dominant Surface of Transparent Electrode Patch via Selective Phase Transfer for Efficient Flexible Photoelectronic Devices.

In this study, a conductive patch for a flexible organic optoelectronic device is proposed and implemented using a poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) polymer electrode based on a transfer process to achieve its high conductivity with an efficient conductive pathway. This PEDOT-dominant surface is induced by phase inversion during the transfer process owing to the solvent affinity of the PSS phase. The PEDOT:PSS patch formed by the transfer process minimizes the power loss in a flexible optoelectronic device due to the improved charge collection and suppressed leakage current responses. In addition, the bending stability of the flexible photoelectronic device is also enhanced by maintaining performance for 1000 bending cycles. Therefore, in the fabrication of a transparent flexible conductive PEDOT:PSS patch, the transfer process of a conducting polymer constitutes an effective strategy that can improve conductivity and embellished morphology.

Gelatin methacryloyl granular scaffolds for localized mRNA delivery.

mRNA therapy is the intracellular delivery of messenger RNA (mRNA) to produce desired therapeutic proteins. Developing strategies for local mRNA delivery is still required where direct intra-articular injections are inappropriate for targeting a specific tissue. The mRNA delivery efficiency depends on protecting nucleic acids against nuclease-mediated degradation and safe site-specific intracellular delivery. Herein, we report novel mRNA-releasing matrices based on RGD-moiety-rich gelatin methacryloyl (GelMA) microporous annealed particle (MAP) scaffolds. GelMA concentration in aerogel-based microgels (µgels) produced through a microfluidic process, MAP stiffnesses, and microporosity are crucial parameters for cell adhesion, spreading, and proliferation. After being loaded with mRNA complexes, MAP scaffolds composed of 10 % GelMA µgels display excellent cell viability with increasing cell infiltration, adhesion, proliferation, and gene transfer. The intracellular delivery is achieved by the sustained release of mRNA complexes from MAP scaffolds and cell adhesion on mRNA-releasing scaffolds. These findings highlight that hybrid systems can achieve efficient protein expression by delivering mRNA complexes, making them promising mRNA-releasing biomaterials for tissue engineering.

Enhancing the soluble expression of α-1,2-fucosyltransferase in E. coli using high-throughput flow cytometry screening coupled with a split-GFP.

2'-Fucosyllactose (2'-FL), one of the major human milk oligosaccharides, was produced in several engineered microorganisms. However, the low solubility of α-1,2-fucosyltransferase (α1,2-FucT) often becomes a bottleneck to produce maximum amount of 2'-FL in the microorganisms. To overcome this solubility issue, the following studies were conducted to improve the soluble expression of α1,2-FucT. Initially, hydrophobic amino acids in the hydrophilic region of the 6 α-helices were mutated, adhering to the α-helix rule. Subsequently, gfp11 was fused to the C-terminal of futC gene encoding α1,2-FucT (FutC), enabling selection of high-fluorescence mutants through split-GFP. Each mutant library was screened via fluorescence activated cell sorting (FACS) to separate soluble mutants for high-throughput screening. As a result, L80C single mutant and A121D/P124A/L125R triple mutant were found, and a combined quadruple mutant was created. Furthermore, we combined mutations of conserved sequences (Q150H/C151R/Q239S) of FutC, which showed positive effects in the previous studies from our lab, with the above quadruple mutants (L80C/A121D/P124A/L125R). The resulting strain produced approximately 3.4-fold higher 2'-FL titer than that of the wild-type, suggesting that the conserved sequence mutations are an independent subset of the mutations that further improve the solubility of the target protein acquired by random mutagenesis using split-GFP.

Injectable Nanoengineered Adhesive Hydrogel for Treating Enterocutaneous Fistulas.

Enterocutaneous fistula (ECF) is a severe medical condition where an abnormal connection forms between the gastrointestinal tract and skin. ECFs are, in most cases, a result of surgical complications such as missed enterotomies or anastomotic leaks. The constant leakage of enteric and fecal contents from the fistula site leads to skin breakdown and increases the risk of infection. Despite advances in surgical techniques and postoperative management, ECF accounts for significant mortality rates, estimated between 15-20%, and causes debilitating morbidity. Therefore, there is a critical need for a simple and effective method to seal and heal ECF. Injectable hydrogels with combined properties of robust mechanical properties and cell infiltration/proliferation have the potential to block and heal ECF. Herein, we report the development of an injectable nanoengineered adhesive hydrogel (INAH) composed of a synthetic nanosilicate (Laponite®) and a gelatin-dopamine conjugate for treating ECF. The hydrogel undergoes fast cross-linking using a co-injection method, resulting in a matrix with improved mechanical and adhesive properties. INAH demonstrates appreciable blood clotting abilities and is cytocompatible with fibroblasts. The adhesive properties of the hydrogel are demonstrated in ex vivo adhesion models with skin and arteries, where the volume stability in the hydrated internal environment facilitates maintaining strong adhesion. In vivo assessments reveal that the INAH is biocompatible, supporting cell infiltration and extracellular matrix deposition while not forming fibrotic tissue. These findings suggest that this INAH holds promising translational potential for sealing and healing ECF.

Hydrogel design to overcome thermal resistance and ROS detoxification in photothermal and photodynamic therapy of cancer.

Responsive heat resistance (by heat shock protein upregulation) and spontaneous reactive oxygen species (ROS) detoxification have been regarded as the major obstacles for photothermal/photodynamic therapy of cancer. To overcome the thermal resistance and improve ROS susceptibility in breast cancer therapy, Au ion-crosslinked hydrogels including indocyanine green (ICG) and polyphenol are devised. Au ion has been introduced for gel crosslinking (by catechol-Au3+ coordination), cellular glutathione depletion, and O2 production from cellular H2O2. ICG can generate singlet oxygen from O2 (for photodynamic therapy) and induce hyperthermia (for photothermal therapy) under the near-infrared laser exposure. (-)-Epigallocatechin gallate downregulates heat shock protein to overcome heat resistance during hyperthermia and exerts multiple anticancer functions in spite of its ironical antioxidant features. Those molecules are concinnously engaged in the hydrogel structure to offer fast gel transformation, syringe injection, self-restoration, and rheological tuning for augmented photo/chemotherapy of cancer. Intratumoral injection of multifunctional hydrogel efficiently suppressed the growth of primary breast cancer and completely eliminated the residual tumor mass. Proposed hydrogel system can be applied to tumor size reduction prior to surgery of breast cancer and the complete remission after its surgery.

Engineered organoids for biomedical applications.

As miniaturized and simplified stem cell-derived 3D organ-like structures, organoids are rapidly emerging as powerful tools for biomedical applications. With their potential for personalized therapeutic interventions and high-throughput drug screening, organoids have gained significant attention recently. In this review, we discuss the latest developments in engineering organoids and using materials engineering, biochemical modifications, and advanced manufacturing technologies to improve organoid culture and replicate vital anatomical structures and functions of human tissues. We then explore the diverse biomedical applications of organoids, including drug development and disease modeling, and highlight the tools and analytical techniques used to investigate organoids and their microenvironments. We also examine the latest clinical trials and patents related to organoids that show promise for future clinical translation. Finally, we discuss the challenges and future perspectives of using organoids to advance biomedical research and potentially transform personalized medicine.

Simple Point-of-Care Nucleic Acid Amplification Test for Rapid SARS-CoV-2 Infection Diagnosis.

After three years of the SARS-CoV-2 pandemic, the demand for developing field-deployable point-of-care (PoC) molecular diagnostic tests has increased. Although RT-qPCR is the molecular diagnostic gold standard and is accurate, it is not readily applied to point-of-care testing (POCT). Meanwhile, rapid diagnostic kits have the disadvantage of low sensitivity. Recently, rapid isothermal nucleic acid amplification technology has emerged as an alternative for rapid diagnosis. Here, we developed a rapid SARS-CoV-2 reverse transcription loop-mediated isothermal amplification (RT-LAMP)-lateral flow assay (LFA) kit. This kit includes a Chelex-100/boiling nucleic acid extraction device and a one-step amplification detection apparatus capable of performing the entire process, from RNA extraction to detection, and diagnosing SARS-CoV-2 infection within 40 min without contamination. The detection limits of the rapid SARS-CoV-2 RT-LAMP-LFA kit were 100 plaque-forming units (PFUs) mL-1 and 10-1 PFU mL-1 for RNA samples extracted using the Chelex-100/boiling nucleic acid extraction device and commercial AdvansureTM E3 system, respectively. The sensitivity and specificity of the rapid SARS-CoV-2 RT-LAMP-LFA kit were 97.8% and 100%, respectively. Our SARS-CoV-2 RT-LAMP-LFA kit exhibited high sensitivity and specificity within 40 min without requiring laboratory instruments, suggesting that the kit could be used as a rapid POC molecular diagnostic test for SARS-CoV-2.

Nanorod/nanodisk-integrated liquid crystalline systems for starvation, chemodynamic, and photothermal therapy of cancer.

Indocyanine green (ICG), glucose oxidase (GOx), and copper(II) sulfate (Cu)-installed hybrid gel based on organic nanorod (cellulose nanocrystal [CNC]) and inorganic nanodisk (Laponite [LAP]) was developed to perform a combination of starvation therapy (ST), chemodynamic therapy (CDT), and photothermal therapy (PTT) for localized cancers. A hybrid CNC/LAP network with a nematic phase was designed to enable instant gelation, controlled viscoelasticity, syringe injectability, and longer in vivo retention. Moreover, ICG was introduced into the CNC/LAP gel system to induce hyperthermia of tumor tissue, amplifying the CDT effect; GOx was used for glucose deprivation (related to the Warburg effect); and Cu was introduced for hydroxyl radical generation (based on Fenton-like chemistry) and cellular glutathione (GSH) degradation in cancer cells. The ICG/GOx/Cu-installed CNC/LAP gel in combination with near-infrared (NIR) laser realized improved antiproliferation, cellular reactive oxygen species (ROS) generation, cellular GSH degradation, and apoptosis induction in colorectal cancer (CT-26) cells. In addition, local injection of the CNC/ICG/GOx/Cu/LAP gel into the implanted CT-26 tumor while irradiating it with NIR laser provided strong tumor growth suppression effects. In conclusion, the designed hybrid nanorod/nanodisk gel network can be efficiently applied to the local PTT/ST/CDT of cancer cells.

Production of S-methyl-methionine using engineered Saccharomyces cerevisiae sake K6.

S-methyl-methionine (SMM), also known as vitamin U, is an important food supplement produced by various plants. In this study, we attempted to produce it in an engineered microorganism, Saccharomyces cerevisiae, by introducing an MMT gene encoding a methionine S-methyltransferase from Arabidopsis thaliana. The S. cerevisiae sake K6 strain, which is a Generally Recognized as Safe (GRAS) strain, was chosen as the host because it produces a significant amount of S-adenosylmethionine (SAM), a precursor of SMM. To increase SMM production in the host, MHT1 and SAM4 genes encoding homocysteine S-methyltransferase were knocked out to prevent SMM degradation. Additionally, MMP1, which encodes S-methyl-methionine permease, was deleted to prevent SMM from being imported into the cell. Finally, ACS2 gene encoding acetyl-CoA synthase was overexpressed, and MLS1 gene encoding malate synthase was deleted to increase SAM availability. Using the engineered strain, 1.92 g/L of SMM was produced by fed-batch fermentation. ONE-SENTENCE SUMMARY: Introducing a plant-derived MMT gene encoding methionine S-methyltransferase into engineered Saccharomyces cerevisiae sake K6 allowed microbial production of S-methyl-methionine (SMM).

Enhanced Maturation of 3D Bioprinted Skeletal Muscle Tissue Constructs Encapsulating Soluble Factor-Releasing Microparticles.

Several microfabrication technologies have been used to engineer native-like skeletal muscle tissues. However, the successful development of muscle remains a significant challenge in the tissue engineering field. Muscle tissue engineering aims to combine muscle precursor cells aligned within a highly organized 3D structure and biological factors crucial to support cell differentiation and maturation into functional myotubes and myofibers. In this study, the use of 3D bioprinting is proposed for the fabrication of muscle tissues using gelatin methacryloyl (GelMA) incorporating sustained insulin-like growth factor-1 (IGF-1)-releasing microparticles and myoblast cells. This study hypothesizes that functional and mature myotubes will be obtained more efficiently using a bioink that can release IGF-1 sustainably for in vitro muscle engineering. Synthesized microfluidic-assisted polymeric microparticles demonstrate successful adsorption of IGF-1 and sustained release of IGF-1 at physiological pH for at least 21 days. Incorporating the IGF-1-releasing microparticles in the GelMA bioink assisted in promoting the alignment of myoblasts and differentiation into myotubes. Furthermore, the myotubes show spontaneous contraction in the muscle constructs bioprinted with IGF-1-releasing bioink. The proposed bioprinting strategy aims to improve the development of new therapies applied to the regeneration and maturation of muscle tissues.

Comparative Clinical Evaluation of a Novel FluA/FluB/SARS-CoV-2 Multiplex LAMP and Commercial FluA/FluB/SARS-CoV-2/RSV RT-qPCR Assays.

Influenza and coronaviruses cause highly contagious respiratory diseases that cause millions of deaths worldwide. Public health measures implemented during the current coronavirus disease (COVID-19) pandemic have gradually reduced influenza circulation worldwide. As COVID-19 measures have relaxed, it is necessary to monitor and control seasonal influenza during this COVID-19 pandemic. In particular, the development of rapid and accurate diagnostic methods for influenza and COVID-19 is of paramount importance because both diseases have significant public health and economic impacts. To address this, we developed a multi-loop-mediated isothermal amplification (LAMP) kit capable of simultaneously detecting influenza A/B and SARS-CoV-2. The kit was optimized by testing various ratios of primer sets for influenza A/B (FluA/FluB) and SARS-CoV-2 and internal control (IC). The FluA/FluB/SARS-CoV-2 multiplex LAMP assay showed 100% specificity for uninfected clinical samples and sensitivities of 90.6%, 86.89%, and 98.96% for LAMP kits against influenza A, influenza B, and SARS-CoV-2 clinical samples, respectively. Finally, the attribute agreement analysis for clinical tests indicated substantial agreement between the multiplex FluA/FluB/SARS-CoV-2/IC LAMP and commercial AllplexTM SARS-CoV-2/FluA/FluB/RSV assays.

Rapid Detection of Mycobacterium Tuberculosis Using a Novel Point-of-Care BZ TB/NTM NALF Assay: Integrating LAMP and LFIA Technologies.

Tuberculosis (TB) is one of the leading causes of infectious mortality from a single infectious agent, Mycobacterium tuberculosis (MTB). This study evaluated the performance of the newly developed BZ TB/NTM NALF assay, which integrated loop-mediated isothermal amplification and lateral flow immunochromatographic assay technologies, for the detection of MTB. A total of 80 MTB-positive samples and 115 MTB-negative samples were collected, all of which were confirmed by TB real-time PCR (RT-PCR) using either AdvanSureTM TB/NTM RT-PCR Kit or Xpert® MTB/RIF Assay. The performance of the BZ TB/NTM NALF assay was evaluated by calculating its sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) in comparison to those of the RT-PCR methods. Compared to the RT-PCR, the sensitivity, specificity, PPV, and NPV of BZ TB/NTM NALF assay were 98.7%, 99.1%, 98.7%, and 99.1%, respectively. The concordance rate between BZ TB/NTM NALF and RT-PCR was 99.0%. Rapid and simple detection of MTB is essential for global case detection and further elimination of TB. The performance of the BZ TB/NTM NALF Assay is acceptable with a high concordance with RT-PCR, indicating that it is reliable for use in a low-resource environment.

Aerogel-Based Biomaterials for Biomedical Applications: From Fabrication Methods to Disease-Targeting Applications.

Aerogel-based biomaterials are increasingly being considered for biomedical applications due to their unique properties such as high porosity, hierarchical porous network, and large specific pore surface area. Depending on the pore size of the aerogel, biological effects such as cell adhesion, fluid absorption, oxygen permeability, and metabolite exchange can be altered. Based on the diverse potential of aerogels in biomedical applications, this paper provides a comprehensive review of fabrication processes including sol-gel, aging, drying, and self-assembly along with the materials that can be used to form aerogels. In addition to the technology utilizing aerogel itself, it also provides insight into the applicability of aerogel based on additive manufacturing technology. To this end, how microfluidic-based technologies and 3D printing can be combined with aerogel-based materials for biomedical applications is discussed. Furthermore, previously reported examples of aerogels for regenerative medicine and biomedical applications are thoroughly reviewed. A wide range of applications with aerogels including wound healing, drug delivery, tissue engineering, and diagnostics are demonstrated. Finally, the prospects for aerogel-based biomedical applications are presented. The understanding of the fabrication, modification, and applicability of aerogels through this study is expected to shed light on the biomedical utilization of aerogels.

Production of 2'-fucosyllactose using ⍺1,2-fucosyltransferase from a GRAS bacterial strain.

2'-Fucosyllactose (2'-FL) is a major oligosaccharide found in human breast milk. It is produced from GDP-L-fucose and D-lactose by ⍺1,2-fucosyltransferase (⍺1,2-fucT), but the enzyme has been identified mostly in pathogens. In this study, an ⍺1,2-fucT was isolated from a Generally Recognized as Safe (GRAS) Bacillus megaterium strain. The enzyme was successfully expressed in metabolically-engineered Escherichia coli. Furthermore, replacement of non-conserved amino acid residues with conserved ones in the protein led to an increase in the rate of 2'-FL production. As a result, fed-batch fermentation of E. coli produced 30 g/L of 2'-FL from glucose and lactose. Thus, the overproduction of 2'-FL using a novel enzyme from a GRAS bacteria strain was successfully demonstrated.

Tunable hybrid hydrogels with multicellular spheroids for modeling desmoplastic pancreatic cancer.

The tumor microenvironment consists of diverse, complex etiological factors. The matrix component of pancreatic ductal adenocarcinoma (PDAC) plays an important role not only in physical properties such as tissue rigidity but also in cancer progression and therapeutic responsiveness. Although significant efforts have been made to model desmoplastic PDAC, existing models could not fully recapitulate the etiology to mimic and understand the progression of PDAC. Here, two major components in desmoplastic pancreatic matrices, hyaluronic acid- and gelatin-based hydrogels, are engineered to provide matrices for tumor spheroids composed of PDAC and cancer-associated fibroblasts (CAF). Shape analysis profiles reveals that incorporating CAF contributes to a more compact tissue formation. Higher expression levels of markers associated with proliferation, epithelial to mesenchymal transition, mechanotransduction, and progression are observed for cancer-CAF spheroids cultured in hyper desmoplastic matrix-mimicking hydrogels, while the trend can be observed when those are cultured in desmoplastic matrix-mimicking hydrogels with the presence of transforming growth factor-ß1 (TGF-ß1). The proposed multicellular pancreatic tumor model, in combination with proper mechanical properties and TGF-ß1 supplement, makes strides in developing advanced pancreatic models for resembling and monitoring the progression of pancreatic tumors, which could be potentially applicable for realizing personalized medicine and drug testing applications.

Performance Evaluation of Microscanner Plus, an Automated Image-Based Cell Counter, for Counting CD4+ T Lymphocytes in HIV Patients.

Counting CD4+ T lymphocytes using flow cytometry is a standard method for monitoring patients with HIV infections. Simpler and cheaper alternatives to flow cytometry are in high demand because getting access to flow cytometers is difficult or impossible in resource-limited settings. We evaluated the performance of the Microscanner Plus, a simple and automated image-based cell counter, in determining CD4 counts against a flow cytometer. CD4 count results of the Microscanner Plus and flow cytometer were compared using samples from 47 HIV-infected patients and 87 healthy individuals. All CV% for precision and reproducibility tests were less than 10%. The Microscanner Plus's lowest detectable CD4 count was determined to be 15.27 cells/µL of whole blood samples. The correlation coefficient (R) between Microscanner Plus and flow cytometry for CD4 counting in 134 clinical samples was very high, at 0.9906 (p < 0.0001). The automated Microscanner Plus showed acceptable analytical performance for counting CD4+ T lymphocytes and may be particularly useful for monitoring HIV patients in resource-limited settings.

Engineered Biomimetic Membranes for Organ-on-a-Chip.

Organ-on-a-chip (OOC) systems are engineered nanobiosystems to mimic the physiochemical environment of a specific organ in the body. Among various components of OOC systems, biomimetic membranes have been regarded as one of the most important key components to develop controllable biomimetic bioanalysis systems. Here, we review the preparation and characterization of biomimetic membranes in comparison with the features of the extracellular matrix. After that, we review and discuss the latest applications of engineered biomimetic membranes to fabricate various organs on a chip, such as liver, kidney, intestine, lung, skin, heart, vasculature and blood vessels, brain, and multiorgans with perspectives for further biomedical applications.

Photoswitchable Microgels for Dynamic Macrophage Modulation.

Dynamic manipulation of supramolecular self-assembled structures is achieved irreversibly or under non-physiological conditions, thereby limiting their biomedical, environmental, and catalysis applicability. In this study, microgels composed of azobenzene derivatives stacked via π-cation and π-π interactions are developed that are electrostatically stabilized with Arg-Gly-Asp (RGD)-bearing anionic polymers. Lateral swelling of RGD-bearing microgels occurs via cis-azobenzene formation mediated by near-infrared-light-upconverted ultraviolet light, which disrupts intermolecular interactions on the visible-light-absorbing upconversion-nanoparticle-coated materials. Real-time imaging and molecular dynamics simulations demonstrate the deswelling of RGD-bearing microgels via visible-light-mediated trans-azobenzene formation. Near-infrared light can induce in situ swelling of RGD-bearing microgels to increase RGD availability and trigger release of loaded interleukin-4, which facilitates the adhesion structure assembly linked with pro-regenerative polarization of host macrophages. In contrast, visible light can induce deswelling of RGD-bearing microgels to decrease RGD availability that suppresses macrophage adhesion that yields pro-inflammatory polarization. These microgels exhibit high stability and non-toxicity. Versatile use of ligands and protein delivery can offer cytocompatible and photoswitchable manipulability of diverse host cells.