Stretchable zein-coated alginate fiber for aligning muscle cells to artificially produce cultivated meat.

Numerous studies have explored the cultivation of muscle cells using non-animal materials for cultivated meat production. Achieving muscle cell proliferation and alignment using 3D scaffolds made from plant-based materials remains challenging. This study introduces a technique to culture and align muscle cells using only plant-based materials, avoiding toxic chemical modifications. Zein-alginate fibers (ZA fibers) were fabricated by coating zein protein onto alginate fibers (A fibers). Zein's excellent cell compatibility and biodegradability enable high cell adhesion and proliferation rates, and the good ductility of the ZA fibers enable a high strain rate (>75%). We demonstrate mature and aligned myotube formation in ZA fibers, providing a simple way to align muscle cells using plant-based materials. Additionally, cultivated meat was constructed by assembling muscle, fat, and vessel fibers. This method holds promise for the future mass production of cultivated meat.

4D-printed microneedles from dual-sensitive chitosan for non-transdermal drug delivery.

Microneedles are a promising micro-scale drug delivery platform that has been under development for over two decades. While 3D printing technology has been applied to fabricate these systems, the challenge of achieving needle sharpness remains. In this study, we present an innovative approach for microneedle fabrication using digital light processing (DLP) 3D printing and smart chitosan biomaterial. For the first time, we used hydroxybutyl methacrylated chitosan (HBCMA), which possesses dual temperature- and photo-sensitive properties, to create microneedles. The DLP approach enabled a quick generation of HBCMA-based microneedles with a high resolution. The microneedles exhibited 4D properties with a change in needle dimensions upon exposure to temperature, which enhances resolution, sharpens needles, and improves mechanical strength. We demonstrated the ability of these microneedles to load, deliver, sustained release small molecular drugs and penetrate soft tissue. Overall, the HBCMA-based microneedles show promising potential in non-dermal drug delivery applications.

Development of cultivable alginate fibers for an ideal cell-cultivated meat scaffold and production of hybrid cultured meat.

Slaughtering animals for meat pose several challenges, including environmental pollution and ethical concerns. Scaffold-based cell-cultivated meat has been proposed as a solution to these problems, however, the utilization of animal-derived materials for scaffolding or the high cost of production remains a significant challenge. Alginate is an ideal material for cell-cultivated meat scaffolds but has poor cell adhesion properties. To address this issue, we achieved 82 % cell adhesion coverage by controlling the specific structure generated during the ionic crosslinking process of alginate. Post 11 days of culture; we evaluated cell adhesion, differentiation, and aligned cell networks. The cell growth increased by 12.7 % compared to the initial seeding concentration. Finally, we created hybrid cell-cultivated meat by combining single-cell protein from mycelium and cell-cultivated meat. This is non-animal based, edible, cost-effective, and has a desirable texture by blending cell-cultivated meat with a meat analogue. In summary, the creation of improved alginate fibers can effectively tackle various obstacles encountered in the manufacturing of cell-cultivated meat. This includes enhancing cell adhesion, reducing costs, and streamlining the production procedure.

Molecularly Imprinted Polymer-Based Sensors for the Detection of Skeletal- and Cardiac-Muscle-Related Analytes.

Molecularly imprinted polymers (MIPs) are synthetic polymers with specific binding sites that present high affinity and spatial and chemical complementarities to a targeted analyte. They mimic the molecular recognition seen naturally in the antibody/antigen complementarity. Because of their specificity, MIPs can be included in sensors as a recognition element coupled to a transducer part that converts the interaction of MIP/analyte into a quantifiable signal. Such sensors have important applications in the biomedical field in diagnosis and drug discovery, and are a necessary complement of tissue engineering for analyzing the functionalities of the engineered tissues. Therefore, in this review, we provide an overview of MIP sensors that have been used for the detection of skeletal- and cardiac-muscle-related analytes. We organized this review by targeted analytes in alphabetical order. Thus, after an introduction to the fabrication of MIPs, we highlight different types of MIP sensors with an emphasis on recent works and show their great diversity, their fabrication, their linear range for a given analyte, their limit of detection (LOD), specificity, and reproducibility. We conclude the review with future developments and perspectives.

Bioprinting and biomaterials for dental alveolar tissue regeneration.

Three dimensional (3D) bioprinting is a powerful tool, that was recently applied to tissue engineering. This technique allows the precise deposition of cells encapsulated in supportive bioinks to fabricate complex scaffolds, which are used to repair targeted tissues. Here, we review the recent developments in the application of 3D bioprinting to dental tissue engineering. These tissues, including teeth, periodontal ligament, alveolar bones, and dental pulp, present cell types and mechanical properties with great heterogeneity, which is challenging to reproduce in vitro. After highlighting the different bioprinting methods used in regenerative dentistry, we reviewed the great variety of bioink formulations and their effects on cells, which have been established to support the development of these tissues. We discussed the different advances achieved in the fabrication of each dental tissue to provide an overview of the current state of the methods. We conclude with the remaining challenges and future needs.

Alterations and Co-Occurrence of C-MYC, N-MYC, and L-MYC Expression are Related to Clinical Outcomes in Various Cancers.

Background and Objectives: MYC, also known as an oncogenic reprogramming factor, is a multifunctional transcription factor that maintains induced pluripotent stem cells (iPSCs). Although MYC is frequently upregulated in various cancers and is correlated with a poor prognosis, MYC is downregulated and correlated with a good prognosis in lung adenocarcinoma. MYC and two other MYC family genes, MYCN and MYCL, have similar structures and could contribute to tumorigenic conversion both in vitro and in vivo. Methods and Results: We systematically investigated whether MYC family genes act as prognostic factors in various human cancers. We first evaluated alterations in the expression of MYC family genes in various cancers using the Oncomine and The Cancer Genome Atlas (TCGA) database and their mutation and copy number alterations using the TCGA database with cBioPortal. Then, we investigated the association between the expression of MYC family genes and the prognosis of cancer patients using various prognosis databases. Multivariate analysis also confirmed that co-expression of MYC/MYCL/MYCN was significantly associated with the prognosis of lung, gastric, liver, and breast cancers. Conclusions: Taken together, our results demonstrate that the MYC family can function not only as an oncogene but also as a tumor suppressor gene in various cancers, which could be used to develop a novel approach to cancer treatment.

State-of-the-art techniques for promoting tissue regeneration: Combination of three-dimensional bioprinting and carbon nanomaterials.

181Biofabrication approaches, such as three-dimensional (3D) bioprinting of hydrogels, have recently garnered increasing attention, especially in the construction of 3D structures that mimic the complexity of tissues and organs with the capacity for cytocompatibility and post-printing cellular development. However, some printed gels show poor stability and maintain less shape fidelity if parameters such as polymer nature, viscosity, shear-thinning behavior, and crosslinking are affected. Therefore, researchers have incorporated various nanomaterials as bioactive fillers into polymeric hydrogels to address these limitations. Carbon-family nanomaterials (CFNs), hydroxyapatites, nanosilicates, and strontium carbonates have been incorporated into printed gels for application in various biomedical fields. In this review, following the compilation of research publications on CFNs-containing printable gels in various tissue engineering applications, we discuss the types of bioprinters, the prerequisites of bioink and biomaterial ink, as well as the progress and challenges of CFNs-containing printable gels in this field.

Latest developments in engineered skeletal muscle tissues for drug discovery and development.

INTRODUCTION: With the advances in skeletal muscle tissue engineering, new platforms have arisen with important applications in biology studies, disease modeling, and drug testing. Current developments highlight the quest for engineering skeletal muscle tissues with higher complexity . These new human skeletal muscle tissue models will be powerful tools for drug discovery and development and disease modeling. AREAS COVERED: The authors review the latest advances in in vitro models of engineered skeletal muscle tissues used for testing drugs with a focus on the use of four main cell culture techniques: Cell cultures in well plates, in microfluidics, in organoids, and in bioprinted constructs. Additional information is provided on the satellite cell niche. EXPERT OPINION: In recent years, more sophisticated in vitro models of skeletal muscle tissues have been fabricated. Important developments have been made in stem cell research and in the engineering of human skeletal muscle tissue. Some platforms have already started to be used for drug testing, notably those based on the parameters of hypertrophy/atrophy and the contractibility of myotubes. More developments are expected through the use of multicellular types and multi-materials as matrices . The validation and use of these models in drug testing should now increase.

Triple-conjugated photo-/temperature-/pH-sensitive chitosan with an intelligent response for bioengineering applications.

Hybrid-crosslinked systems, which can be formed using heat and visible light, are significant for improving the stability of hydrogels under physiological conditions. However, several challenges for their practical application remain, such as shrinking under culture medium conditions or the neutral pH in the small intestine. Therefore, a multi-sensitive hydrogel with response to external conditions has been designed and prepared, which could be employed as a biopolymer ink formulation for three-dimensional printing in bioengineering applications. When exposed to body temperature and visible light, the N-succinyl hydroxybutyl methacrylated chitosan (NS-HBC-MA) undergoes a sol-gel phase transition. The NS-HBC-MA hydrogel exhibits pH-responsive swelling, effectively preventing shrinkage at a neutral pH. Furthermore, NS-HBC-MA hydrogel demonstrates excellent biocompatibility and biodegradability. This study demonstrates that the NS-HBC-MA hydrogel has significant potential for various applications, including wound healing, delivery systems, and tissue engineering.

Efficient Myogenic/Adipogenic Transdifferentiation of Bovine Fibroblasts in a 3D Bioprinting System for Steak-Type Cultured Meat Production.

The interest in cultured meat is increasing because of the problems with conventional livestock industry. Recently, many studies related to cultured meat have been conducted, but producing large-sized cultured meat remains a challenge. It is aimed to introduce 3D bioprinting for producing large cell aggregates for cultured meat production. A hydrogel scaffold is produced at the centimeter scale using a bioink consisting of photocrosslinkable materials for digital light processing-based (DLP) printing, which has high printing accuracy and can produce geometrically complex structures. The light exposure time for hydrogel photopolymerization by DLP bioprinting is optimized based on photorheometry and cell viability assays. Naturally immortalized bovine embryonic fibroblast cells transformed with MyoD and PPARγ2 instead of primary cells are used as the latter have difficulties in maintaining stemness and are associated with animal ethics issues. The cells are mixed into the hydrogel for printing. Myogenesis and adipogenesis are induced simply by changing the medium after printing. Scaffolds are obtained successfully with living cells and large microchannels. The cooked cultured meat maintains its size and shape upon cutting. The overall dimensions are 3.43 cm × 5.53 cm × 0.96 cm. This study provides proof-of-concept for producing 3D cultured meat using bioinks.

Improving Printability of Digital-Light-Processing 3D Bioprinting via Photoabsorber Pigment Adjustment.

Digital-light-processing (DLP) three-dimensional (3D) bioprinting, which has a rapid printing speed and high precision, requires optimized biomaterial ink to ensure photocrosslinking for successful printing. However, optimization studies on DLP bioprinting have yet to sufficiently explore the measurement of light exposure energy and biomaterial ink absorbance controls to improve the printability. In this study, we synchronized the light wavelength of the projection base printer with the absorption wavelength of the biomaterial ink. In this paper, we provide a stepwise explanation of the challenges associated with unsynchronized absorption wavelengths and provide appropriate examples. In addition to biomaterial ink wavelength synchronization, we introduce photorheological measurements, which can provide optimized light exposure conditions. The photorheological measurements provide precise numerical data on light exposure time and, therefore, are an effective alternative to the expendable and inaccurate conventional measurement methods for light exposure energy. Using both photorheological measurements and bioink wavelength synchronization, we identified essential printability optimization conditions for DLP bioprinting that can be applied to various fields of biological sciences.

Self-Oxygenation of Tissues Orchestrates Full-Thickness Vascularization of Living Implants.

Bioengineering of tissues and organs has the potential to generate functional replacement organs. However, achieving the full-thickness vascularization that is required for long-term survival of living implants has remained a grand challenge, especially for clinically sized implants. During the pre-vascular phase, implanted engineered tissues are forced to metabolically rely on the diffusion of nutrients from adjacent host-tissue, which for larger living implants results in anoxia, cell death, and ultimately implant failure. Here it is reported that this challenge can be addressed by engineering self-oxygenating tissues, which is achieved via the incorporation of hydrophobic oxygen-generating micromaterials into engineered tissues. Self-oxygenation of tissues transforms anoxic stresses into hypoxic stimulation in a homogenous and tissue size-independent manner. The in situ elevation of oxygen tension enables the sustained production of high quantities of angiogenic factors by implanted cells, which are offered a metabolically protected pro-angiogenic microenvironment. Numerical simulations predict that self-oxygenation of living tissues will effectively orchestrate rapid full-thickness vascularization of implanted tissues, which is empirically confirmed via in vivo experimentation. Self-oxygenation of tissues thus represents a novel, effective, and widely applicable strategy to enable the vascularization living implants, which is expected to advance organ transplantation and regenerative medicine applications.

Photo-Cross-Linkable Human Albumin Colloidal Gels Facilitate In Vivo Vascular Integration for Regenerative Medicine.

Biodegradable cellular and acellular scaffolds have great potential to regenerate damaged tissues or organs by creating a proper extracellular matrix (ECM) capable of recruiting endogenous cells to support cellular ingrowth. However, since hydrogel-based scaffolds normally degrade through surface erosion, cell migration and ingrowth into scaffolds might be inhibited early in the implantation. This could result in insufficient de novo tissue formation in the injured area. To address these challenges, continuous and microsized strand-like networks could be incorporated into scaffolds to guide and recruit endogenous cells in rapid manner. Fabrication of such microarchitectures in scaffolds is often a laborious and time-consuming process and could compromise the structural integrity of the scaffold or impact cell viability. Here, we have developed a fast single-step approach to fabricate colloidal hydrogels, which are made up of randomly packed human serum albumin-based photo-cross-linkable microparticles with continuous internal networks of microscale voids. The human serum albumin conjugated with methacrylic groups were assembled to microsized aggregates for achieving unique porous structures inside the colloidal gels. The albumin hydrogels showed tunable mechanical properties such as elastic modulus, porosity, and biodegradability, providing a suitable ECM for various cells such as cardiomyoblasts and endothelial cells. In addition, the encapsulated cells within the hydrogel showed improved cell retention and increased survivability in vitro. Microporous structures of the colloidal gels can serve as a guide for the infiltration of host cells upon implantation, achieving rapid recruitment of hematopoietic cells and, ultimately, enhancing the tissue regeneration capacity of implanted scaffolds.

Optimization of Polysaccharide Hydrocolloid for the Development of Bioink with High Printability/Biocompatibility for Coextrusion 3D Bioprinting.

Bioink is the main component of 3D bioprinting process and is crucial for the generation of sophisticated 3D structures through precise spatial control. Therefore, bioink's core material must have characteristics that support good printability as well as biocompatibility. However, there is a lack of bioinks developed that satisfy these characteristics at the same time. In this work, our aim was to develop a bioink that satisfies the needs for both printability and biocompatibility through effectively utilizing hydrocolloid materials. To do so, carboxymethyl cellulose (CMC) and xanthan gum (XG) were used to maintain proper shear properties at high pressure and increase the mechanical properties of bioink without excessively affecting the viscosity, and thus enhance printability and biocompatibility. Various bioink formulations were applied to 3D printing process and the printability optimization was carried out through adjusting the hydrocolloid contents in connection with different cross-linking methods. Through utilization of hydrocolloids, the printability and rheological analysis showed that the bioink has improved mechanical properties and confirmed that the printability could be adjusted by controlling the CMC and XG ratio. Moreover, cell viability and immunocytochemical staining analyses showed cell compatibility with enhanced stability. The proposed convenient method to control the printability with improved biocompatibility suggests more appropriate use of bioink for co-axial 3D bioprinting.

Injectable hydrogel derived from chitosan with tunable mechanical properties via hybrid-crosslinking system.

Thermo-sensitive injectable hydrogels that spontaneously react to physiological temperature have been widely studied to be used in biomedical fields. However, several challenges on their unstable structures with large-sized pores and low mechanical strength under physiological conditions must be addressed to enable their practical applications. We synthesized the hydroxybutyl methacrylated chitosan (HBC-MA) hydrogel that possesses both thermo-sensitive and photo-crosslinkable properties. The HBC-MA showed effective sol-gel transition under physiological temperature as well as a sensitive photo-crosslinkable property with visible light capable of skin penetration. The co-nonsolvency property and thermo-sensitivity of HBC-MA prevented unintended loss of the hydrogel graft after being subcutaneously injected in mice. Subsequently applied visible light on the skin beneath which the hydrogel was injected significantly improved the mechanical strength and stability of the graft. The injectable HBC-MA hydrogel developed in this study can be applicable to a wide range of biomedical fields such as drug delivery system and tissue engineering.

Cell-Laden Gelatin Methacryloyl Bioink for the Fabrication of Z-Stacked Hydrogel Scaffolds for Tissue Engineering.

Hydrogel-based scaffolds have been widely used to fabricate artificial tissues capable of replacing tissues and organs. However, several challenges inherent in fabricating tissues of large size and complex morphology using such scaffolds while ensuring cell viability remain. To address this problem, we synthesized gelatin methacryloyl (GelMA) based bioink with cells for fabricating a scaffold with superior characteristics. The bioink was grafted onto a Z-stacking bioprinter that maintained the cells at physiological temperature during the printing process, without exerting any physical pressure on the cells. Various parameters, such as the bioink composition and light exposure time, were optimized. The printing accuracy of the scaffolds was evaluated using photorheological studies. The internal morphology of the scaffolds at different time points was analyzed using electron microscopy. The Z-stacked scaffolds were fabricated using high-speed printing, with the conditions optimized to achieve high model reproducibility. Stable adhesion and high proliferation of cells encapsulated within the scaffold were confirmed. We introduced various strategies to improve the accuracy and reproducibility of Z-stack GelMA bioprinting while ensuring that the scaffolds facilitated cell adhesion, encapsulation, and proliferation. Our results demonstrate the potential of the present method for various applications in tissue engineering.

Kappa-Carrageenan-Based Dual Crosslinkable Bioink for Extrusion Type Bioprinting.

Bioink based 3D bioprinting is a promising new technology that enables fabrication of complex tissue structures with living cells. The printability of the bioink depends on the physical properties such as viscosity. However, the high viscosity bioink puts shear stress on the cells and low viscosity bioink cannot maintain complex tissue structure firmly after the printing. In this work, we applied dual crosslinkable bioink using Kappa-carrageenan (κ-CA) to overcome existing shortcomings. κ-CA has properties such as biocompatibility, biodegradability, shear-thinning and ionic gelation but the difficulty of controlling gelation properties makes it unsuitable for application in 3D bioprinting. This problem was solved by synthesizing methacrylated Kappa-carrageenan (MA-κ-CA), which can be dual crosslinked through ionic and UV (Ultraviolet) crosslinking to form hydrogel using NIH-3T3 cells. Through MA substitutions, the rheological properties of the gel could be controlled to reduce the shear stress. Moreover, bioprinting using the cell-laden MA-κ-CA showed cell compatibility with enhanced shape retention capability. The potential to control the physical properties through dual crosslinking of MA-κ-CA hydrogel is expected to be widely applied in 3D bioprinting applications.

Hydrogel Production Platform with Dynamic Movement Using Photo-Crosslinkable/Temperature Reversible Chitosan Polymer and Stereolithography 4D Printing Technology.

BACKGROUND: Three-dimensional (3D) printing using hydrogel has made great strides when it comes to mimicking 3D artificial tissue in the medical field. However, most structures do not mimic the dynamic movement of the tissues. Without imitating dynamic movements, there are limitations on the extent to which the proper implementation of the tissue's own functions can be achieved. METHOD: In this study, we intend to present an approach to solving this problem using hydroxybutyl methacrylated chitosan (HBC-MA), a photo-crosslinkable/temperature reversible chitosan polymer. In addition, stereolithography-3D (SLA-3D) printing technology was used, which is more likely to mimic the complex microstructure. As a control, a 3D structure made with pristine poly(ethylene glycol) dimethacrylate (PEG-DMA) was created, and a 4D structure was prepared by adding HBC-MA to poly(ethylene glycol) dimethacrylate (PEG-DMAP) resin. RESULTS: HBC-MA caused the expansion of water into the polymer matrix at low temperature, and the 4D structure resulted in expansion of the polymer volume, generating dynamic movement due to the expansion of water. Conversely, as the temperature rose, deswelling occurred, followed by a decrease in the volume, showing a shape memory property of returning to the existing structure. Morphological, swelling, and mechanical analysis further confirmed the principle of dynamic movement. In addition, parameters were provided through calculation of the bending ratio angle (θ). CONCLUSION: Through this, it is suggested that HBC-MA can be applied as a core polymer for SLA-4D printing, and has high potential for realizing the dynamic movement of tissue.

Nanogels Derived from Fish Gelatin: Application to Drug Delivery System.

The gelatin extracted from mammals of porcine and bovine has been prominently used in pharmaceutical, medical, and cosmetic products. However, there have been some concerns for their usage due to religious, social and cultural objections, and animal-to-human infectious disease. Recently, gelatin from marine by-products has received growing attention as an alternative to mammalian gelatin. In this study, we demonstrate the formation of nanogels (NGs) using fish gelatin methacryloyl (GelMA) and their application possibility to the drug delivery system. The fabrication of fish GelMA NGs is carried out by crosslinking through the photopolymerization of the methacryloyl substituent present in the nanoemulsion droplets, followed by purification and redispersion. There were different characteristics depending on the aqueous phase in the emulsion and the type of solvent used in redispersion. The PBS-NGs/D.W., which was prepared using PBS for the aqueous phase and D.W. for the final dispersion solution, had a desirable particle size (<200 nm), low PdI (0.16), and high drug loading efficiency (77%). Spherical NGs particles were observed without aggregation in TEM images. In vitro release tests of doxorubicin (DOX)-GelMA NGs showed the pH-dependent release behavior of DOX. Also, the MTT experiments demonstrated that DOX-GelMA NGs effectively inhibited cell growth, while only GelMA NGs exhibit higher percentages of cell viability. Therefore, the results suggest that fish GelMA NGs have a potential for nano-carrier as fine individual particles without the aggregation and cytotoxicity to deliver small-molecule drugs.

Effects of vitamin D2-fortified shiitake mushroom on bioavailability and bone structure.

Bioavailability and bone loss inhibitory effects of vitamin D2 derived from UV-irradiated shiitake mushroom were determined in vivo. The effect of the absence of ovaries on the bioavailability of vitamin D2 and bone structure was also investigated. Sham operated (sham) and ovariectomized (OVX) rats were divided in 3 groups according to their diets, i.e. control: only vitamin D-deficient diets; UV(X): vitamin D-deficient diets with non-irradiated mushroom powder; UV(O): vitamin D-deficient diets with irradiated mushroom powder. The obtained results showed that vitamin D2 from shiitake mushroom was able to increase bone mineral density and trabecular bone structure of femur bone as well as its bioavailability. The absence of estrogen induced adverse effects not only on bioavailability of vitamin D2 but also on trabecular bone. In conclusion, vitamin D2-fortified shiitake mushroom might help postmenopausal women increase vitamin D2 bioavailability and retard trabecular bone loss. Abbreviations: OVX: ovariectomized; 25(OH)D: 25-hydroxyvitamin D; 1,25(OH)2D: 1,25-dihydroxyvitamin D; BMD: bone mineral density; micro-CT: micro computed tomography; RSM: response surface methodology; RP-HPLC: Reverse phase-high performance liquid chromatography; MS/MS: tandem mass spectrometry; E2: estradiol; NTx: N-terminal telopeptide of type I collagen; BV/TV: bone volume/total volume; BS/BV: bone surface/bone volume; Tb.Th: trabecular thickness; Tb.Sp: trabecular separation.