Dual crosslinking silk fibroin/pectin-based bioink development and the application on neural stem/progenitor cells spheroid laden 3D bioprinting.

The human nervous system is an incredibly intricate physiological network, and neural cells lack the ability to repair and regenerate after a brain injury. 3-dimensional (3D) bioprinting technology offers a promising strategy for constructing biomimetic organ constructs and in vitro brain/disease models. The bioink serves as a pivotal component that emulates the microenvironment of biomimetic construct and exerts a profound influence on cellular behaviors. In this study, a series of mechanically adjustable and dual crosslinking bioinks were developed using photocrosslinkable methacrylated silk fibroin (SilMA) in combination with the ionic crosslinking material, pectin, or pectin methacryloyl (PecMA) with silk fibroin (SF) supplementation. SilMA/pectin exhibited superior properties, with SilMA providing biocompatibility and adjustable mechanical properties, while the addition of pectin enhanced printability. The porous structure supported neural cell growth, and 15 % SilMA/0.5 % pectin bioinks displayed excellent printability and shape fidelity. Neural stem/progenitor cells (NSPCs)-loaded bioinks were used to construct a 3D brain model, demonstrating sustained vitality and high neuronal differentiation without the need for growth factors. The SilMA/pectin bioinks demonstrated adjustable mechanical properties, favorable biocompatibility, and an environment highly conducive to neural induction, offering an alternative approach for neural tissue engineering applications or in vitro brain models.

Recent advances using MXenes in biomedical applications.

An MXene is a novel two-dimensional transition metal carbide or nitride, with a typical formula of Mn+1XnTx (M = transition metals, X = carbon or nitrogen, and T = functional groups). MXenes have found wide application in biomedicine and biosensing, owing to their high biocompatibility, abundant reactive surface groups, good conductivity, and photothermal properties. Applications include photo- and electrochemical sensors, energy storage, and electronics. This review will highlight recent applications of MXene and MXene-derived materials in drug delivery, tissue engineering, antimicrobial activity, and biosensors (optical and electrochemical). We further elaborate on recent developments in utilizing MXenes for photothermal cancer therapy, and we explore multimodal treatments, including the integration of chemotherapeutic agents or magnetic nanoparticles for enhanced therapeutic efficacy. The high surface area and reactivity of MXenes provide an interface to respond to the changes in the environment, allowing MXene-based drug carriers to respond to changes in pH, reactive oxygen species (ROS), and electrical signals for controlled release applications. Furthermore, the conductivity of MXene enables it to provide electrical stimulation for cultured cells and endows it with photocatalytic capabilities that can be used in antibiotic applications. Wearable and in situ sensors incorporating MXenes are also included. Major challenges and future development directions of MXenes in biomedical applications are also discussed. The remarkable properties of MXenes will undoubtedly lead to their increasing use in the applications discussed here, as well as many others.

Machine Learning Integrated Workflow for Predicting Schwann Cell Viability on Conductive MXene Biointerfaces.

Severe injuries to the peripheral nervous system (PNS) require Schwann cells to aid in neuronal regeneration. Low-frequency electrical stimulation is known to induce the cogrowth of neurons and Schwann cells in an injured PNS. However, the correlations between electrical stimulation and Schwann cell viability are complex and not well understood. In this work, we develop a machine learning (ML)-integrated workflow that uses conductive hydrogel biointerfaces to evaluate the impacts of fabrication parameters and electrical stimulation on the Schwann cell viability. First, a hydrogel array with varying MXene and peptide loadings is fabricated, which serves as conductive biointerfaces to incubate Schwann cells and introduce various electrical stimulation (at different voltages and frequencies). Upon specific fabrication parameters and stimulation, the cell viability is evaluated and input into an artificial neural network model to train the model. Additionally, a data augmentation method is applied to synthesize 1000-fold virtual data points, enabling the construction of a high-accuracy prediction model (with a testing mean absolute error ≤11%). By harnessing the model's predictive power, we can accurately predict Schwann cell viability based on a given set of fabrication/stimulation parameters. Finally, the SHapley Additive exPlanations model interpretation provides several data-scientific insights that are validated by microscopic cellular observations. Our hybrid approach, involving conductive biointerface fabrication, ML algorithms, and data analysis, offers an unconventional platform to construct a preclinical prediction model at the cellular level.

Activation of Insulin Gene Expression via Transfection of a CRISPR/dCas9a System Using Magnetic Peptide-Imprinted Nanoparticles.

A CRISPRa transcription activation system was used to upregulate insulin expression in HEK293T cells. To increase the delivery of the targeted CRISPR/dCas9a, magnetic chitosan nanoparticles, imprinted with a peptide from the Cas9 protein, were developed, characterized, and then bound to dCas9a that was complexed with a guide RNA (gRNA). The adsorption of dCas9 proteins conjugated with activators (SunTag, VPR, and p300) to the nanoparticles was monitored using both ELISA kits and Cas9 staining. Finally, the nanoparticles were used to deliver dCas9a that was complexed with a synthetic gRNA into HEK293T cells to activate their insulin gene expression. Delivery and gene expression were examined using quantitative real-time polymerase chain reaction (qRT-PCR) and staining of insulin. Finally, the long-term release of insulin and the cellular pathway related to stimulation by glucose were also investigated.

Nanoparticle-mediated CRISPR/dCas9a activation of multiple transcription factors to engineer insulin-producing cells.

Insulin may help to control blood glucose levels in diabetes; however, the long-term release of insulin is important for therapy. In this work, four guide RNAs (gRNA) for factors that promote specification and maturation of insulin-producing cells were synthesized: pancreatic and duodenal homeobox 1 (PDX1), protoendocrine factor (neurogenin 3, NGN3), NK6 homeobox 1 (NKX6.1), and musculoaponeurotic fibrosarcoma oncogene family A (MAFA). These gRNAs were used to form ribonucleoproteins (RNPs) with tracRNA and dCas9-VPR, and were then immobilized on magnetic peptide-imprinted chitosan nanoparticles, which enhanced transfection. The production and release of insulin from transfected cells were then measured using ELISA and staining with anti-insulin antibodies. The expression of the genes was evaluated using qRT-PCR; this was also used to investigate the cascade of additional transcriptional regulators. The magnitude and duration of insulin production were evaluated for single and repeated transfections (using different transfection schedules) to identify the most promising protocol.

Cation-Induced Assembly of Conductive MXene Fibers for Wearable Heater, Wireless Communication, and Stem Cell Differentiation.

Emerging wearable electronics, wireless communication, and tissue engineering require the development of conductive fiber-shaped electrodes and biointerfaces. Ti3C2Tx MXene nanosheets serve as promising building block units for the construction of highly conductive fibers with integrated functionalities, yet a facile and scalable fabrication scheme is highly required. Herein, a cation-induced assembly process is developed for the scalable fabrication of conductive fibers with MXene sheaths and alginate cores (abbreviated as MXene@A). The fabrication scheme of MXene@A fibers includes the fast extrusion of alginate fibers followed by electrostatic assembly of MXene nanosheets, enabling high-speed fiber production. When multiple fabrication parameters are optimized, the MXene@A fibers exhibit a superior electrical conductivity of 1083 S cm-1, which can be integrated as Joule heaters into textiles for wearable thermal management. By triggering reversible de/hydration of alginate cores upon heating, the MXene@A fibers can be repeatedly contracted and generate large contraction stress that is >40 times higher than the ones of mammalian skeletal muscle. Furthermore, the MXene@A springs demonstrate large contraction strains up to 65.5% and are then fabricated into a reconfigurable dipole antenna to wirelessly monitor the surrounding heat sources. In the end, with the biocompatibility of MXene nanosheets, the MXene@A fibers enable the guidance of neural stem/progenitor cells differentiation and the promotion of neurite outgrowth. With a cation-induced assembly process, our multifunctional MXene@A fibers exhibit high scalability for future manufacturing and hold the prospect to inspire other applications.

A Printable Magnetic-Responsive Iron Oxide Nanoparticle (ION)-Gelatin Methacryloyl (GelMA) Ink for Soft Bioactuator/Robot Applications.

The features or actuation behaviors of nature's creatures provide concepts for the development of biomimetic soft bioactuators/robots with stimuli-responsive capabilities, design convenience, and environmental adaptivity in various fields. Mimosa pudica is a mechanically responsive plant that can convert pressure to the motion of leaves. When the leaves receive pressure, the occurrence of asymmetric turgor in the extensor and flexor sides of the pulvinus from redistributing the water in the pulvinus causes the bending of the pulvinus. Inspired by the actuation of Mimosa pudica, designing soft bioactuators can convert external stimulations to driving forces for the actuation of constructs which has been receiving increased attention and has potential applications in many fields. 4D printing technology has emerged as a new strategy for creating versatile soft bioactuators/robots by integrating printing technologies with stimuli-responsive materials. In this study, we developed a hybrid ink by combining gelatin methacryloyl (GelMA) polymers with iron oxide nanoparticles (IONs). This hybrid ION-GelMA ink exhibits tunable rheology, controllable mechanical properties, magnetic-responsive behaviors, and printability by integrating the internal metal ion-polymeric chain interactions and photo-crosslinking chemistries. This design offers the inks a dual crosslink mechanism combining the advantages of photocrosslinking and ionic crosslinking to rapidly form the construct within 60 s of UV exposure time. In addition, the magnetic-responsive actuation of ION-GelMA constructs can be regulated by different ION concentrations (0-10%). Furthermore, we used the ION-GelMA inks to fabricate a Mimosa pudica-like soft bioactuator through a mold casting method and a direct-ink-writing (DIW) printing technology. Obviously, the pinnule leaf structure of printed constructs presents a continuous reversible shape transformation in an air phase without any liquid as a medium, which can mimic the motion characteristics of natural creatures. At the same time, compared to the model casting process, the DIW printed bioactuators show a more refined and biomimetic transformation shape that closely resembles the movement of the pinnule leaf of Mimosa pudica in response to stimulation. Overall, this study indicates the proof of concept and the potential prospect of magnetic-responsive ION-GelMA inks for the rapid prototyping of biomimetic soft bioactuators/robots with untethered non-contact magneto-actuations.

A Real-Time Sensing System for Monitoring Neural Network Degeneration in an Alzheimer's Disease-on-a-Chip Model.

Stem cell-based in vitro models may provide potential therapeutic strategies and allow drug screening for neurodegenerative diseases, including Alzheimer's disease (AD). Herein, we develop a neural stem cell (NSC) spheroid-based biochip that is characterized by a brain-like structure, well-defined neural differentiation, and neural network formation, representing a brain-on-a-chip. This system consisted of microelectrode arrays with a multichannel platform and allowed the real-time monitoring of network formation and degeneration by impedance analysis. The parameters of this platform for the real-time tracking of network development and organization were established based on our previous study. Subsequently, ß-amyloid (Aß) was added into the brain-on-a-chip system to generate an AD-on-a-chip model, and toxic effects on neurons and the degeneration of synapses were observed. The AD-on-a-chip model may help us to investigate the neurotoxicity of Aß on neurons and neural networks in real time. Aß causes neural damage and accumulates around neurites or inside neurospheroids, as observed by immunostaining and scanning electron microscopy (SEM). After incubation with Aß, reactive oxygen species (ROS) increased, synapse function decreased, and the neurotransmitter-acetylcholine (ACh) concentration decreased were observed. Most importantly, the real-time analysis system monitored the impedance value variation in the system with Aß incubation, providing consecutive network disconnection data that are consistent with biological data. This platform provides simple, real-time, and convenient sensing to monitor the network microenvironment. The proposed AD-on-a-chip model enhances the understanding of neurological pathology, and the development of this model provides an alternative for the study of drug discovery and cell-protein interactions in the brain.

Design and Synthesis of Stem Cell-Laden Keratin/Glycol Chitosan Methacrylate Bioinks for 3D Bioprinting.

With the advancements in tissue engineering and three-dimensional (3D) bioprinting, physiologically relevant three-dimensional structures with suitable mechanical and bioactive properties that mimic the biological tissue can be designed and fabricated. However, the available bioinks are less than demanded. In this research, the readily available biomass sources, keratin and glycol chitosan, were selected to develop a UV-curable hydrogel that is feasible for the 3D bioprinting process. Keratin methacrylate and glycol chitosan methacrylate were synthesized, and a hybrid bioink was created by combining this protein-polysaccharide cross-linked hydrogel. While human hair keratin could provide biological functions, the other composition, glycol chitosan, could further enhance the mechanical strength of the construct. The mechanical properties, degradation profile, swelling behavior, cell viability, and proliferation were investigated with various ratios of keratin methacrylate to glycol chitosan methacrylate. The composition of 2% (w/v) keratin methacrylate and 2% (w/v) chitosan methacrylate showed a significantly higher cell number and swelling percentage than other compositions and was designated as the bioink for 3D printing afterward. The feasibility of stem cell loading in the selected formula was examined with an extrusion-based bioprinter. The cells and spheroids can be successfully printed with the synthesized bioink into a specific shape and cultured. This work provides a potential option for bioinks and delivers insights into personalization research on stem cell-laden biofabricated hydrogels in the future.

Remodeling Effects of the Combination of GGT Scaffolds, Percutaneous Electrical Stimulation, and Acupuncture on Large Bone Defects in Rats.

The regeneration defect of bone is a long-term physiological process after bone injuries. To accelerate the bone remodeling process, the combination of chemical and physical stimulations provides an efficient strategy to allow maturation and to functionalize osteoclasts and osteoblasts. This study aims to investigate the dual effects of a tricalcium phosphate (TCP)-based gelatin scaffold (GGT) in combination with electroacupuncture stimulation on the activation of osteoclasts and osteoblasts, as well as new bone regrowth in vitro and in vivo. We demonstrated that electrical stimulation changes the pH of a culture medium and activates osteoblasts and osteoclasts in an in vitro co-culture system. Furthermore, we showed that electroacupuncture stimulation can enhance osteogenesis and new bone regrowth in vivo and can upregulate the mechanism among parathyroid hormone intact (PTH-i), calcium, osteoclasts, and osteoblasts in the bone-defected rats. Those results showed the potential interest to combine the electroacupuncture technique with GGT scaffolds to improve bone remodeling after injury.

An Injectable Hybrid Gelatin Methacryloyl (GelMA)/Phenyl Isothiocyanate-Modified Gelatin (Gel-Phe) Bioadhesive for Oral/Dental Hemostasis Applications.

Biomaterials are widely used for effectively controlling bleeding in oral/dental surgical procedures. Here, gelatin methacryloyl (GelMA) was synthesized by grafting methacrylic anhydride on gelatin backbone, and phenyl isothiocyanate-modified gelatin (Gel-Phe) was synthesized by conjugating different gelatin/phenyl isothiocyanate molar ratios (G/P ratios) (i.e., 1:1, 1:5, 1:10, 1:15, 1:25, 1:50, 1:100, and 1:150) with gelatin polymer chains. Afterward, we combined GelMA and Gel-Phe as an injectable and photo-crosslinkable bioadhesive. This hybrid material system combines photo-crosslinking chemistry and supramolecular interactions for the design of bioadhesives exhibiting a highly porous structure, injectability, and regulable mechanical properties. By simply regulating the G/P ratio (1:1-1:15) and UV exposure times (15-60 s), it was possible to modulate the injectability and mechanical properties of the GelMA/Gel-Phe bioadhesive. Moreover, we demonstrated that the GelMA/Gel-Phe bioadhesive showed low cytotoxicity, a highly porous network, and the phenyl-isothiourea and amine residues on Gel-Phe and GelMA polymers with synergized hemostatic properties towards fast blood absorption and rapid clotting effect. An in vitro porcine skin bleeding and an in vitro dental bleeding model confirmed that the bioadhesive could be directly extruded into the bleeding site, rapidly photo-crosslinked, and reduced blood clotting time by 45%. Moreover, the in situ crosslinked bioadhesive could be easily removed from the bleeding site after clotting, avoiding secondary wound injury. Overall, this injectable GelMA/Gel-Phe bioadhesive stands as a promising hemostatic material in oral/dental surgical procedures.

An injectable, dual crosslinkable hybrid pectin methacrylate (PECMA)/gelatin methacryloyl (GelMA) hydrogel for skin hemostasis applications.

Biomaterials for effective hemorrhage control are urgently needed in clinics as uncontrolled bleeding is associated with high mortality. Herein, we developed an injectable and in situ photo-crosslinkable hybrid hemostatic hydrogel by combining pectin methacrylate (PECMA) and gelatin methacryloyl (GelMA). This modular material system combines ionic- and photo-crosslinking chemistries to design interpenetrating networks (IPN) exhibiting tunable rheology, highly porous structure, and controllable swelling and mechanical properties. By simply changing the calcium (0-15 mM) and polymer (1.5-7%) content used for the sequential crosslinking of hydrogels via calcium gelation and UV-photopolymerization, it was possible to precisely modulate the injectability, degradation, and swelling ratio. Moreover, it is demonstrated that PECMA/GelMA hydrogels present good cytocompatibility and uniquely synergize the hemostatic properties of calcium ions on PECMA, the amine residues on GelMA, and the highly porous network toward rapid blood absorption and fast coagulation effect. An in vitro porcine skin bleeding model confirmed that the hydrogel could be directly injected into the wound and rapidly photo-crosslinked, circumventing the bleeding and decreasing the coagulation time by 39%. Importantly, the crosslinked hydrogel could be easily removed to prevent secondary wound injury. Overall, this injectable hybrid PECMA/GelMA hydrogel stands as a promising hemostatic material.

The Role of Alcohol, LPS Toxicity, and ALDH2 in Dental Bony Defects.

It is estimated that 560 million people carry an East Asian-specific ALDH2*2 dominant-negative mutation which leads to enzyme inactivation. This common ALDH2 polymorphism has a significant association with osteoporosis. We hypothesized that the ALDH2*2 mutation in conjunction with periodontal Porphyromonas gingivalis bacterial infection and alcohol drinking had an inhibitory effect on osteoblasts and bone regeneration. We examined the prospective association of ALDH2 activity with the proliferation and mineralization potential of human osteoblasts in vitro. The ALDH2 knockdown experiments showed that the ALDH2 knockdown osteoblasts lost their proliferation and mineralization capability. To mimic dental bacterial infection, we compared the dental bony defects in wild-type mice and ALDH2*2 knockin mice after injection with purified lipopolysaccharides (LPS), derived from P. gingivalis which is a bacterial species known to cause periodontitis. Micro-computed tomography (micro-CT) scan results indicated that bone regeneration was significantly affected in the ALDH2*2 knockin mice with about 20% more dental bony defects after LPS injection than the wild-type mice. Moreover, the ALDH2*2 knockin mutant mice had decreased osteoblast growth and more dental bone loss in the upper left jaw region after LPS injection. In conclusion, these results indicated that the ALDH2*2 mutation with alcohol drinking and chronic exposure to dental bacterial-derived toxin increased the risk of dental bone loss.

Toward a neurospheroid niche model: optimizing embedded 3D bioprinting for fabrication of neurospheroid brain-like co-culture constructs.

A crucial step in creating reliablein vitroplatforms for neural development and disorder studies is the reproduction of the multicellular three-dimensional (3D) brain microenvironment and the capturing of cell-cell interactions within the model. The power of self-organization of diverse cell types into brain spheroids could be harnessed to study mechanisms underlying brain development trajectory and diseases. A challenge of current 3D organoid and spheroid models grown in petri-dishes is the lack of control over cellular localization and diversity. To overcome this limitation, neural spheroids can be patterned into customizable 3D structures using microfabrication. We developed a 3D brain-like co-culture construct using embedded 3D bioprinting as a flexible solution for composing heterogenous neural populations with neurospheroids and glia. Specifically, neurospheroid-laden free-standing 3D structures were fabricated in an engineered astrocyte-laden support bath resembling a neural stem cell niche environment. A photo-crosslinkable bioink and a thermal-healing supporting bath were engineered to mimic the mechanical modulus of soft tissue while supporting the formation of self-organizing neurospheroids within elaborate 3D networks. Moreover, bioprinted neurospheroid-laden structures exhibited the capability to differentiate into neuronal cells. These brain-like co-cultures could provide a reproducible platform for modeling neurological diseases, neural regeneration, and drug development and repurposing.

The Current Trends of Biosensors in Tissue Engineering.

Biosensors constitute selective, sensitive, and rapid tools for disease diagnosis in tissue engineering applications. Compared to standard enzyme-linked immunosorbent assay (ELISA) analytical technology, biosensors provide a strategy to real-time and on-site monitor micro biophysiological signals via a combination of biological, chemical, and physical technologies. This review summarizes the recent and significant advances made in various biosensor technologies for different applications of biological and biomedical interest, especially on tissue engineering applications. Different fabrication techniques utilized for tissue engineering purposes, such as computer numeric control (CNC), photolithographic, casting, and 3D printing technologies are also discussed. Key developments in the cell/tissue-based biosensors, biomolecular sensing strategies, and the expansion of several biochip approaches such as organs-on-chips, paper based-biochips, and flexible biosensors are available. Cell polarity and cell behaviors such as proliferation, differentiation, stimulation response, and metabolism detection are included. Biosensors for diagnosing tissue disease modes such as brain, heart, lung, and liver systems and for bioimaging are discussed. Finally, we discuss the challenges faced by current biosensing techniques and highlight future prospects of biosensors for tissue engineering applications.

PCL-Blended Chitosan Substrates for Patterning the Heterotypic Cell Distribution in an Epithelial and Mesenchymal Coculture System.

Cell-cell and cell-substrate interactions in coculture systems are very important to the context of biomaterial scaffolds for tissue engineering applications. Understanding the cellular interactions and distribution of epithelial-mesenchymal microtissues on the controllable biomaterial surfaces is useful to study the organoid applications. The aim of the present study is to investigate the effects of chitosan/poly(ε-caprolactone) (PCL)-blended biomaterials on the distribution and spheroid formation of HaCaT and Hs68 cells in a coculture system. In this study, we demonstrated that the cocultured cells gradually changed their pattern from core/shell spheroid to monolayered morphology as the PCL content increased in the blended substrates. This indicates that the chitosan/PCL-blended substrates are able to regulate cell-substrate and cell-cell interactions to modify the distribution of HaCaT and Hs68 cells similar to various mesenchymal-epithelial organizations in biological tissues. Moreover, we also developed a two-dimension lattice model to elaborate the dependence of cell spheroid development on complex cell-cell interactions. This information may be helpful to develop appropriate biomaterials with appropriate properties to the applications of engineered epithelial-mesenchymal organoids.

Sustainable Biomass Materials for Biomedical Applications.

Renewable resources are abundant worldwide in the form of raw materials, which come from terrestrial/marine animals, agricultural plants, microorganisms, and their residues. Sustainable biomass materials derived from these raw materials have provided an opening for the development of new alternatives to replace traditional petro-materials for a variety of purposes, including green energy, paint, food packaging, and biomedical applications. In this Review, we highlight the potential use of various sustainable biomass materials in three-dimensional/four-dimensional bioprinting, drug delivery/controlled release, tissue engineering, and biosensing applications. We then discuss the features of sustainable biomass materials in association with the basic requirements of these applications, such as printability, sensitivity, mechanical properties, and modifiability. We finally conclude with future perspectives.

Brain-on-a-chip Devices for Drug Screening and Disease Modeling Applications.

Neurodegenerative disorders are related to the progressive functional loss of the brain, often connected to emotional and physical disability and, ultimately, to death. These disorders, strongly connected to the aging process, are becoming increasingly more relevant due to the increase of life expectancy. Current pharmaceutical treatments poorly tackle these diseases, mainly acting only on their symptomology. One of the main reasons of this is the current drug development process, which is not only expensive and time-consuming but, also, still strongly relies on animal models at the preclinical stage. Organ-on-a-chip platforms have the potential to strongly impact and improve the drug screening process by recreating in vitro the functionality of human organs. Patient-derived neurons from different regions of the brain can be directly grown and differentiated on a brain-on-a-chip device where the disease development, progression and pharmacological treatments can be studied and monitored in real time. The model reliability is strongly improved by using human-derived cells, more relevant than animal models for pharmacological screening and disease monitoring. The selected cells will be then capable of proliferating and organizing themselves in the in vivo environment thanks to the device architecture, materials selection and bio-chemical functionalization. In this review, we start by presenting the fundamental strategies adopted for brain-on-a-chip devices fabrication including e.g., photolithography, micromachining and 3D printing technology. Then, we discuss the state-of-theart of brain-on-a-chip platforms including their role in the study of the functional architecture of the brain e.g., blood-brain barrier, or of the most diffuse neurodegenerative diseases like Alzheimer's and Parkinson's. At last, the current limitations and future perspectives of this approach for the development of new drugs and neurodegenerative diseases modeling will be discussed.