Investigation of the Dysfunction Caused by High Glucose, Advanced Glycation End Products, and Interleukin-1 Beta and the Effects of Therapeutic Agents on the Microphysiological Artery Model.

Diabetes mellitus (DM) has substantial global implications and contributes to vascular inflammation and the onset of atherosclerotic cardiovascular diseases. However, translating the findings from animal models to humans has inherent limitations, necessitating a novel platform. Therefore, herein, an arterial model is established using a microphysiological system. This model successfully replicates the stratified characteristics of human arteries by integrating collagen, endothelial cells (ECs), and vascular smooth muscle cells (VSMCs). Perfusion via a peristaltic pump shows dynamic characteristics distinct from those of static culture models. High glucose, advanced glycation end products (AGEs), and interleukin-1 beta are employed to stimulate diabetic conditions, resulting in notable cellular changes and different levels of cytokines and nitric oxide. Additionally, the interactions between the disease models and oxidized low-density lipoproteins (LDL) are examined. Finally, the potential therapeutic effects of metformin, atorvastatin, and diphenyleneiodonium are investigated. Metformin and diphenyleneiodonium mitigate high-glucose- and AGE-associated pathological changes, whereas atorvastatin affects only the morphology of ECs. Altogether, the arterial model represents a pivotal advancement, offering a robust and insightful platform for investigating cardiovascular diseases and their corresponding drug development.

Plant-Based Decellularization: A Novel Approach for Perfusion-Compatible Tissue Engineering Structures.

This study explores the potential of plant-based decellularization in regenerative medicine, a pivotal development in tissue engineering focusing on scaffold development, modification, and vascularization. Plant decellularization involves removing cellular components from plant structures, offering an eco-friendly and cost-effective alternative to traditional scaffold materials. The use of plant-derived polymers is critical, presenting both benefits and challenges, notably in mechanical properties. Integration of plant vascular networks represents a significant bioengineering breakthrough, aligning with natural design principles. The paper provides an in-depth analysis of development protocols, scaffold fabrication considerations, and illustrative case studies showcasing plant-based decellularization applications. This technique is transformative, offering sustainable scaffold design solutions with readily available plant materials capable of forming perfusable structures. Ongoing research aims to refine protocols, assess long-term implications, and adapt the process for clinical use, indicating a path toward widespread adoption. Plant-based decellularization holds promise for regenerative medicine, bridging biological sciences with engineering through eco-friendly approaches. Future perspectives include protocol optimization, understanding long-term impacts, clinical scalability, addressing mechanical limitations, fostering collaboration, exploring new research areas, and enhancing education. Collectively, these efforts envision a regenerative future where nature and scientific innovation converge to create sustainable solutions, offering hope for generations to come.

Laminin-Augmented Decellularized Extracellular Matrix Ameliorating Neural Differentiation and Neuroinflammation in Human Mini-Brains.

Non-neural extracellular matrix (ECM) has limited application in humanized physiological neural modeling due to insufficient brain-specificity and safety concerns. Although brain-derived ECM contains enriched neural components, certain essential components are partially lost during the decellularization process, necessitating augmentation. Here, it is demonstrated that the laminin-augmented porcine brain-decellularized ECM (P-BdECM) is xenogeneic factor-depleted as well as favorable for the regulation of human neurons, astrocytes, and microglia. P-BdECM composition is comparable to human BdECM regarding brain-specificity through the matrisome and gene ontology-biological process analysis. As augmenting strategy, laminin 111 supplement promotes neural function by synergic effect with laminin 521 in P-BdECM. Annexin A1(ANXA1) and Peroxiredoxin(PRDX) in P-BdECM stabilized microglial and astrocytic behavior under normal while promoting active neuroinflammation in response to neuropathological factors. Further, supplementation of the brain-specific molecule to non-neural matrix also ameliorated glial cell inflammation as in P-BdECM. In conclusion, P-BdECM-augmentation strategy can be used to recapitulate humanized pathophysiological cerebral environments for neurological study.

Rapid acoustofluidic mixing by ultrasonic surface acoustic wave-induced acoustic streaming flow.

Ultrasonic surface acoustic wave (SAW)-induced acoustic streaming flow (ASF) has been utilized for microfluidic flow control, patterning, and mixing. Most previous research employed cross-type SAW acousto-microfluidic mixers, in which the SAWs propagated perpendicular to the flow direction. In this configuration, the flow mixing was induced predominantly by the horizontal component of the acoustic force, which was usually much smaller than the vertical component, leading to energy inefficiency and limited controllability. Here, we propose a vertical-type ultrasonic SAW acousto-microfluidic mixer to achieve rapid flow mixing with improved efficiency and controllability. We conducted in-depth numerical and experimental investigations of the vertical-type SAW-induced ASF to elucidate the acousto-hydrodynamic phenomenon under varying conditions of total flow rate, acoustic wave amplitude, and fluid viscosity conditions. We conducted computational fluid dynamics simulations for numerical flow visualization and utilized micro-prism-embedded microchannels for experimental flow visualization for the vertical SAW-induced ASF. We found that the SAW-induced vortices served as a hydrodynamic barrier for the co-flow streams for controlled flow mixing in the proposed device. For proof-of-concept application, we performed chemical additive-free rapid red blood cell lysis and achieved rapid cell lysis with high lysis efficiency based on the physical interactions of the suspended cells with the SAW-induced acoustic vortical flows. We believe that the proposed vertical-type ultrasonic SAW-based mixer can be broadly utilized for various microfluidic applications that require rapid, controlled flow mixing.

Bioprinting Methods for Fabricating In Vitro Tubular Blood Vessel Models.

Dysfunctional blood vessels are implicated in various diseases, including cardiovascular diseases, neurodegenerative diseases, and cancer. Several studies have attempted to prevent and treat vascular diseases and understand interactions between these diseases and blood vessels across different organs and tissues. Initial studies were conducted using 2-dimensional (2D) in vitro and animal models. However, these models have difficulties in mimicking the 3D microenvironment in human, simulating kinetics related to cell activities, and replicating human pathophysiology; in addition, 3D models involve remarkably high costs. Thus, in vitro bioengineered models (BMs) have recently gained attention. BMs created through biofabrication based on tissue engineering and regenerative medicine are breakthrough models that can overcome limitations of 2D and animal models. They can also simulate the natural microenvironment in a patient- and target-specific manner. In this review, we will introduce 3D bioprinting methods for fabricating bioengineered blood vessel models, which can serve as the basis for treating and preventing various vascular diseases. Additionally, we will describe possible advancements from tubular to vascular models. Last, we will discuss specific applications, limitations, and future perspectives of fabricated BMs.

A Review on Bioinks and their Application in Plant Bioprinting.

In recent years, the characterization and fabrication methods concerning new bioinks have received much attention, largely because the absence of bioprintable materials has been identified as one of the most rudimentary challenges for rapid advancement in the field of three-dimensional (3D) printing. Bioinks for printing mammalian organs have been rapidly produced, but bioinks in the field of plant science remain sparse. Thus, 3D fabrication of plant parts is still in its infancy due to the lack of appropriate bioink materials, and aside from that, the difficulty in recreating sophisticated microarchitectures that accurately and safely mimic natural biological activities is a concern. Therefore, this review article is designed to emphasize the significance of bioinks and their applications in plant bioprinting.

3D Bioprinting of an In Vitro Model of a Biomimetic Urinary Bladder with a Contract-Release System.

The development of curative therapy for bladder dysfunction is usually hampered owing to the lack of reliable ex vivo human models that can mimic the complexity of the human bladder. To overcome this issue, 3D in vitro model systems offering unique opportunities to engineer realistic human tissues/organs have been developed. However, existing in vitro models still cannot entirely reflect the key structural and physiological characteristics of the native human bladder. In this study, we propose an in vitro model of the urinary bladder that can create 3D biomimetic tissue structures and dynamic microenvironments to replicate the smooth muscle functions of an actual human urinary bladder. In other words, the proposed biomimetic model system, developed using a 3D bioprinting approach, can recreate the physiological motion of the urinary bladder by incorporating decellularized extracellular matrix from the bladder tissue and introducing cyclic mechanical stimuli. The results showed that the developed bladder tissue models exhibited high cell viability and proliferation rate and promoted myogenic differentiation potential given dynamic mechanical cues. We envision the developed in vitro bladder mimicry model can serve as a research platform for fundamental studies on human disease modeling and pharmaceutical testing.

Neural stem cell delivery using brain-derived tissue-specific bioink for recovering from traumatic brain injury.

Traumatic brain injury is one of the leading causes of accidental death and disability. The loss of parts in a severely injured brain induces edema, neuronal apoptosis, and neuroinflammation. Recently, stem cell transplantation demonstrated regenerative efficacy in an injured brain. However, the efficacy of current stem cell therapy needs improvement to resolve issues such as low survival of implanted stem cells and low efficacy of differentiation into respective cells. We developed brain-derived decellularized extracellular matrix (BdECM) bioink that is printable and has native brain-like stiffness. This study aimed to fabricate injured cavity-fit scaffold with BdECM bioink and assessed the utility of BdECM bioink for stem cell delivery to a traumatically injured brain. Our BdECM bioink had shear thinning property for three-dimensional (3D)-cell-printing and physical properties and fiber structures comparable to those of the native brain, which is important for tissue integration after implantation. The human neural stem cells (NSCs) (F3 cells) laden with BdECM bioink were found to be fully differentiated to neurons; the levels of markers for mature differentiated neurons were higher than those observed with collagen bioinkin vitro. Moreover, the BdECM bioink demonstrated potential in defect-fit carrier fabrication with 3D cell-printing, based on the rheological properties and shape fidelity of the material. As F3 cell-laden BdECM bioink was transplanted into the motor cortex of a rat brain, high efficacy of differentiation into mature neurons was observed in the transplanted NSCs; notably increased level of MAP2, a marker of neuronal differentiation, was observed. Furthermore, the transplanted-cell bioink suppressed reactive astrogliosis and microglial activation that may impede regeneration of the injured brain. The brain-specific material reported here is favorable for NSC differentiation and suppression of neuroinflammation and is expected to successfully support regeneration of a traumatically injured brain.

Promoting Long-Term Cultivation of Motor Neurons for 3D Neuromuscular Junction Formation of 3D In Vitro Using Central-Nervous-Tissue-Derived Bioink.

3D cell printing technology is in the spotlight for producing 3D tissue or organ constructs useful for various medical applications. In printing of neuromuscular tissue, a bioink satisfying all the requirements is a challenging issue. Gel integrity and motor neuron activity are two major characters because a harmonious combination of extracellular materials essential to motor neuron activity consists of disadvantages in mechanical properties. Here, a method for fabrication of 3D neuromuscular tissue is presented using a porcine central nervous system tissue decellularized extracellular matrix (CNSdECM) bioink. CNSdECM retains CNS tissue-specific extracellular molecules, provides rheological properties crucial for extrusion-based 3D cell printing, and reveals positive effects on the growth and maturity of axons of motor neurons compared with Matrigel. It also allows long-term cultivation of human-induced-pluripotent-stem-cell-derived lower motor neurons and sufficiently supports their cellular behavior to carry motor signals to muscle fibers. CNSdECM bioink holds great promise for producing a tissue-engineered motor system using 3D cell printing.

Application of 3D bioprinting in the prevention and the therapy for human diseases.

Rapid development of vaccines and therapeutics is necessary to tackle the emergence of new pathogens and infectious diseases. To speed up the drug discovery process, the conventional development pipeline can be retooled by introducing advanced in vitro models as alternatives to conventional infectious disease models and by employing advanced technology for the production of medicine and cell/drug delivery systems. In this regard, layer-by-layer construction with a 3D bioprinting system or other technologies provides a beneficial method for developing highly biomimetic and reliable in vitro models for infectious disease research. In addition, the high flexibility and versatility of 3D bioprinting offer advantages in the effective production of vaccines, therapeutics, and relevant delivery systems. Herein, we discuss the potential of 3D bioprinting technologies for the control of infectious diseases. We also suggest that 3D bioprinting in infectious disease research and drug development could be a significant platform technology for the rapid and automated production of tissue/organ models and medicines in the near future.

Introduction to bioprinting of in vitro cancer models.

Cancer models are essential in cancer research and for new drug development pipelines. However, conventional cancer tissue models have failed to capture the human cancer physiology, thus hindering drug discovery. The major challenge is the establishment of physiologically relevant cancer models that reflect the complexity of the tumor microenvironment (TME). The TME is a highly complex milieu composed of diverse factors that are associated with cancer progression and metastasis, as well as with the development of cancer resistance to therapeutics. To emulate the TME, 3D bioprinting has emerged as a way to create engineered cancer tissue models. Bioprinted cancer tissue models have the potential to recapitulate cancer pathology and increased drug resistance in an organ-mimicking 3D environment. This review overviews the bioprinting technologies used for the engineering of cancer tissue models and provides a future perspective on bioprinting to further advance cancer research.

3D Bioprinting of In Vitro Models Using Hydrogel-Based Bioinks.

Coronavirus disease 2019 (COVID-19), which has recently emerged as a global pandemic, has caused a serious economic crisis due to the social disconnection and physical distancing in human society. To rapidly respond to the emergence of new diseases, a reliable in vitro model needs to be established expeditiously for the identification of appropriate therapeutic agents. Such models can be of great help in validating the pathological behavior of pathogens and therapeutic agents. Recently, in vitro models representing human organs and tissues and biological functions have been developed based on high-precision 3D bioprinting. In this paper, we delineate an in-depth assessment of the recently developed 3D bioprinting technology and bioinks. In particular, we discuss the latest achievements and future aspects of the use of 3D bioprinting for in vitro modeling.

3D Cell-Printed Hypoxic Cancer-on-a-Chip for Recapitulating Pathologic Progression of Solid Cancer.

Cancer microenvironment has a significant impact on the progression of the disease. In particular, hypoxia is the key driver of cancer survival, invasion, and chemoresistance. Although several in vitro models have been developed to study hypoxia-related cancer pathology, the complex interplay of the cancer microenvironment observed in vivo has not been reproduced yet owing to the lack of precise spatial control. Instead, 3D biofabrication approaches have been proposed to create microphysiological systems for better emulation of cancer ecology and accurate anticancer treatment evaluation. Herein, we propose a 3D cell-printing approach to fabricate a hypoxic cancer-on-a-chip. The hypoxia-inducing components in the chip were determined based on a computer simulation of the oxygen distribution. Cancer-stroma concentric rings were printed using bioinks containing glioblastoma cells and endothelial cells to recapitulate a type of solid cancer. The resulting chip realized central hypoxia and aggravated malignancy in cancer with the formation of representative pathophysiological markers. Overall, the proposed approach for creating a solid-cancer-mimetic microphysiological system is expected to bridge the gap between in vivo and in vitro models for cancer research.

Microphysiological Systems for Neurodegenerative Diseases in Central Nervous System.

Neurodegenerative diseases are among the most severe problems in aging societies. Various conventional experimental models, including 2D and animal models, have been used to investigate the pathogenesis of (and therapeutic mechanisms for) neurodegenerative diseases. However, the physiological gap between humans and the current models remains a hurdle to determining the complexity of an irreversible dysfunction in a neurodegenerative disease. Therefore, preclinical research requires advanced experimental models, i.e., those more physiologically relevant to the native nervous system, to bridge the gap between preclinical stages and patients. The neural microphysiological system (neural MPS) has emerged as an approach to summarizing the anatomical, biochemical, and pathological physiology of the nervous system for investigation of neurodegenerative diseases. This review introduces the components (such as cells and materials) and fabrication methods for designing a neural MPS. Moreover, the review discusses future perspectives for improving the physiological relevance to native neural systems.

A bioprinted human-glioblastoma-on-a-chip for the identification of patient-specific responses to chemoradiotherapy.

Patient-specific ex vivo models of human tumours that recapitulate the pathological characteristics and complex ecology of native tumours could help determine the most appropriate cancer treatment for individual patients. Here, we show that bioprinted reconstituted glioblastoma tumours consisting of patient-derived tumour cells, vascular endothelial cells and decellularized extracellular matrix from brain tissue in a compartmentalized cancer-stroma concentric-ring structure that sustains a radial oxygen gradient, recapitulate the structural, biochemical and biophysical properties of the native tumours. We also show that the glioblastoma-on-a-chip reproduces clinically observed patient-specific resistances to treatment with concurrent chemoradiation and temozolomide, and that the model can be used to determine drug combinations associated with superior tumour killing. The patient-specific tumour-on-a-chip model might be useful for the identification of effective treatments for glioblastoma patients resistant to the standard first-line treatment.

A 3D cell printed muscle construct with tissue-derived bioink for the treatment of volumetric muscle loss.

Volumetric muscle loss (VML) is an irrecoverable injury associated with muscle loss greater than 20%. Although hydrogel-based 3D engineered muscles and the decellularized extracellular matrix (dECM) have been considered for VML treatment, they have shown limited efficacy. We established a novel VML treatment with dECM bioink using 3D cell printing technology. Volumetric muscle constructs composed of cell-laden dECM bioinks were generated with a granule-based printing reservoir. The 3D cell printed muscle constructs exhibited high cell viability without generating hypoxia and enhanced de novo muscle formation in a VML rat model. To improve functional recovery, prevascularized muscle constructs that mimic the hierarchical architecture of vascularized muscles were fabricated through coaxial nozzle printing with muscle and vascular dECM bioinks. Spatially printing tissue-specific dECM bioinks offers organized microenvironmental cues for the differentiation of each cell and improves vascularization, innervation, and functional recovery. Our present results suggest that a 3D cell printing and tissue-derived bioink-based approach could effectively generate biomimetic engineered muscles to improve the treatment of VML injuries.

Three-dimensional printing of a patient-specific engineered nasal cartilage for augmentative rhinoplasty.

Autologous cartilages or synthetic nasal implants have been utilized in augmentative rhinoplasty to reconstruct the nasal shape for therapeutic and cosmetic purposes. Autologous cartilage is considered to be an ideal graft, but has drawbacks, such as limited cartilage source, requirements of additional surgery for obtaining autologous cartilage, and donor site morbidity. In contrast, synthetic nasal implants are abundantly available but have low biocompatibility than the autologous cartilages. Moreover, the currently used nasal cartilage grafts involve additional reshaping processes, by meticulous manual carving during surgery to fit the diverse nose shape of each patient. The final shapes of the manually tailored implants are highly dependent on the surgeons' proficiency and often result in patient dissatisfaction and even undesired separation of the implant. This study describes a new process of rhinoplasty, which integrates three-dimensional printing and tissue engineering approaches. We established a serial procedure based on computer-aided design to generate a three-dimensional model of customized nasal implant, and the model was fabricated through three-dimensional printing. An engineered nasal cartilage implant was generated by injecting cartilage-derived hydrogel containing human adipose-derived stem cells into the implant containing the octahedral interior architecture. We observed remarkable expression levels of chondrogenic markers from the human adipose-derived stem cells grown in the engineered nasal cartilage with the cartilage-derived hydrogel. In addition, the engineered nasal cartilage, which was implanted into mouse subcutaneous region, exhibited maintenance of the exquisite shape and structure, and striking formation of the cartilaginous tissues for 12 weeks. We expect that the developed process, which combines computer-aided design, three-dimensional printing, and tissue-derived hydrogel, would be beneficial in generating implants of other types of tissue.

Effect of an oxygen-generating scaffold on the viability and insulin secretion function of porcine neonatal pancreatic cell clusters.

BACKGROUND: Islet encapsulation techniques have shown limited success in maintaining islet survival and function because encapsulation decreases oxygen supply. In this study, an oxygen-generating scaffold was fabricated to prevent hypoxic cell damage and improve the viability and insulin secretion of islets. METHODS: We fabricated an oxygen-generating scaffold by mixing calcium peroxide (CaO2 ) with polydimethylsiloxane (PDMS). We evaluated the effects of the oxygen-generating PDMS + CaO2 scaffold on viability, caspase-3 and caspase-7 activity, oxygen consumption rate (OCR), glucose-stimulated insulin secretion (GSIS), hypoxic cell marker expression, and reactive oxygen species (ROS) levels in porcine neonatal pancreatic cell clusters (NPCCs). We also fabricated a microfluidic device that allowed measuring the effects of the oxygen-generating scaffold on viability. RESULTS: Oxygen generation by the PDMS + CaO2 scaffold was sustained for more than 24 hours in vitro. NPCCs encapsulated in PDMS + CaO2 showed higher viability than NPCCs in PDMS scaffolds and control NPCCs grown without a scaffold. PDMS + CaO2 -encapsulated NPCCs showed lower caspase-3 and caspase-7 activity, hypoxic cell expression, and ROS levels, and higher OCR and GSIS than those in PDMS or control cells. Using the microfluidic device, we observed that the viability of PDMS + CaO2 -encapsulated NPCCs was higher than that of PDMS-encapsulated NPCCs. CONCLUSIONS: NPCCs in PDMS + CaO2 scaffolds show higher viability and insulin secretion than do NPCCs in PDMS scaffolds and control cells. Therefore, this oxygen-generating scaffold has potential for application in future islet transplantation studies.

Muscle-derived extracellular matrix on sinusoidal wavy surfaces synergistically promotes myogenic differentiation and maturation.

The generation of physiologically aligned multinucleated myotubes is critical in the fabrication of functional engineered skeletal muscle. Although micro-/nano-topographical contact guidance, such as groove/ridge structures, has induced the alignment of muscle fibers by providing cells with extracellular matrix (ECM) topography, the complex biochemical microenvironment of the ECM cannot be recapitulated. Here, we report the enhancement of myogenic differentiation and maturation using muscle decellularized ECM (mdECM) and sinusoidal wavy surfaces, which provided a biochemical microenvironment and microscale contact guidance, respectively. Sinusoidal wavy polystyrene surfaces with wavelengths of 20, 40, and 80 µm were fabricated by a deep X-ray lithography-based process. The mdECM was prepared by decellularization of porcine tibialis anterior skeletal muscle. An mdECM coating significantly improved the surface wettability of polystyrene substrates and exhibited higher seeding efficiency, cell viability, and proliferation compared with collagen- and non-coating cases. The sinusoidal wavy surfaces induced well-aligned myotubes and showed significantly enhanced formation of myotubes and myogenic differentiation when the surface was coated with mdECM. Particularly, there was an approximately 1.5-2 fold improvement in morphological analysis and gene expression for mdECM-compared to non-coated sinusoidal wavy surfaces. These results suggest that the consideration of both topographical and biochemical environmental cues can generate a highly mimicked ECM environment, thereby providing cells with a synergistic effect on myogenic differentiation and maturation. The outcome of this study will be useful in developing of functional engineered muscle for application in tissue regeneration or a high-throughput in vitro model for drug screening.

A cell-laden hybrid fiber/hydrogel composite for ligament regeneration with improved cell delivery and infiltration.

Ligament, a fibrous connective tissue between bones, is a unique tissue in human anatomy because it has complex viscoelastic properties and is very tough. Moreover, it is an important tissue for regeneration because frequent injuries occur, but there are limited types of substitutes that can be used as a tissue replacement. In this study, we present a stem cell-laden fiber/hydrogel composite structure with a layered fibrous structure, which can enhance cell infiltration, topographical cue and mechanical properties. It can promote cell viability, proliferation, and differentiation of the ligament phenotype with the help of a growth factor. The mechanical properties of the developed structure were experimentally identified using tensile tests, while cell viability and various functionalities were verified through culture tests using mesenchymal stem cells.