Ferritin Nanoshuttle for Long-Lasting Self-Healing of Phenolic Hydrogels.

Herein, we highlight a novel finding that ferritin can play a crucial role in the "self-healing lifetime" of soft phenolic materials. Ferritin interacts with a catechol-functionalized polymer to form a self-healable and adhesive hydrogel bidirectionally by providing and retrieving Fe3+. As a result of its unique role as a nanoshuttle to store and release iron, ferritin significantly increases the self-healing lifetime of the hydrogel compared with that afforded by catechol-Fe3+ coordination through direct Fe3+ addition without ferritin. Ferritin also induces stable oxidative coupling between catechol moieties following metal coordination, which contributes to double cross-linking networks of catechol-catechol adducts and catechol-Fe3+ coordination. Thus, ferritin-mediated cross-linking can provide phenolic hydrogels with the advantages of hydrogels prepared by both metal coordination and oxidative coupling, thereby overcoming the limitations of the current cross-linking methods of phenolic hydrogels and broadening their versatility in biomedical applications.

Decellularized Tissue Matrix for Stem Cell and Tissue Engineering.

Decellularization is a technique to remove cellular components from native tissues, which could reduce immune reactions to the scaffolds. Decellularized matrices are valuable for tissue engineering, as they preserve tissue-specific structural, mechanical, and biochemical microenvironments, while promoting cellular engraftment and functions in the matrix. So far, various tissues have been decellularized by combinations of mechanical, chemical, and enzymatic processes and utilized in preparing bioscaffolds to provide tissue-specific environments for various cell types, including primary cells, progenitor cells, and stem cells. In addition, decellularized matrices could be manipulated into several formats according to the final application, such as tissue-engineering scaffolds, artificial organs, cell culture matrices, and transplantation carriers. In this chapter, we describe various types of decellularized tissue matrices and their extensive use in regenerative medicine, including reconstruction of artificial organs and regeneration of damaged tissues.

Protective effect of fermented Cyclopia intermedia against UVB-induced damage in HaCaT human keratinocytes.

BACKGROUND: The fermented leaves and stems of Cyclopia intermedia are used to brew honeybush tea, a herbal tea indigenous to South Africa. The aim of this study was to evaluate the protective effect of fermented honeybush extracts (FH ex) and scale-up fermented honeybush extracts (SFH ex) against ultraviolet B (UVB)-induced damage in HaCaT keratinocytes. To this end, we examined UVB-induced cell viability, antioxidant enzymes, and inflammatory mediators in HaCaT cells. METHODS: UVB significantly decreased HaCaT cell viability, whereas FH ex and SFH ex did not exhibit cytotoxic effects and increased the viability of the HaCaT cells. To further investigate the protective effects of FH ex on UVB-induced oxidative stress in HaCaT cells, the activities of superoxide dismutase (SOD), catalase (CAT), matrix metalloproteinases (MMPs), pro-inflammatory cytokines and skin barrier function in terms of involucrin, filaggrin, and loricrin were analyzed. RESULTS: UVB-induced treatment reduced the activity of antioxidant enzymes and skin barrier function, while FH ex or SFH ex increased their activity. These results suggest that FH ex exerted cytoprotective activity against UVB-induced oxidative stress in HaCaT cells through stimulation of antioxidant enzymes activities. Furthermore, FH ex and SFH ex suppressed the UVB-induced expression of inflammatory mediators, such as IL-1ß, IL-6, and IL-8, at mRNA level together with down regulation of matrix metalloproteinase (MMPs). In addition, FH ex and SFH ex reversed the phosphorylation of mitogen-activated protein kinase (MAPK) induced by UVB-irradiation. Notably, FH ex and SFH ex markedly inhibited UVB-induced activation of ERK, p38, and JNK. Thus, this agent exhibits anti-oxidative and -inflammatory effects via lowering ROS production, suppressing p38, ERK, and JNK activation, and down-regulating expression of MMPs. CONCLUSIONS: These findings suggest that FH ex and SFH ex can be used as a skin anti-photoaging agent.

Liver extracellular matrix providing dual functions of two-dimensional substrate coating and three-dimensional injectable hydrogel platform for liver tissue engineering.

Decellularization of tissues or organs can provide an efficient strategy for preparing functional scaffolds for tissue engineering. Microstructures of native extracellular matrices and their biochemical compositions can be retained in the decellularized matrices, providing tissue-specific microenvironments for efficient tissue regeneration. Here, we report the versatility of liver extracellular matrix (LEM) that can be used for two-dimensional (2D) coating and three-dimensional (3D) hydrogel platforms for culture and transplantation of primary hepatocytes. Collagen type I (Col I) has typically been used for hepatocyte culture and transplantation. In this study, LEM was compared with Col I in terms of biophysical and mechanical characteristics and biological performance for enhancing cell viability, differentiation, and hepatic functions. Surface properties of LEM coating and mechanical properties and gelation kinetics of LEM hydrogel could be manipulated by adjusting the LEM concentration. In addition, LEM hydrogel exhibited improved elastic properties, rapid gelation, and volume maintenance compared to Col I hydrogel. LEM coating significantly improved hepatocyte functions such as albumin secretion and urea synthesis. More interestingly, LEM coating upregulated hepatic gene expression of human adipose-derived stem cells, indicating enhanced hepatic differentiation of these stem cells. The viability and hepatic functions of primary hepatocytes were also significantly improved in LEM hydrogel compared to Col I hydrogel both in vitro and in vivo. Albumin and hepatocyte transcription factor expression was upregulated in hepatocytes transplanted in LEM hydrogels. In conclusion, LEM can provide functional biomaterial platforms for diverse applications in liver tissue engineering by promoting survival and maturation of hepatocytes and hepatic commitment of stem cells. This study demonstrates the feasibility of decellularized matrix for both 2D coating and 3D hydrogel in liver tissue engineering.

Polymeric DNase-I nanozymes targeting neutrophil extracellular traps for the treatment of bowel inflammation.

Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is a family of chronic disorders along the gastrointestinal tract. Because of its idiopathic nature, IBD does not have a fundamental cure; current available therapies for IBD are limited to prolonged doses of immunomodulatory agents. While these treatments may reduce inflammation, limited therapeutic efficacy, inconsistency across patients, and adverse side effects from aggressive medications remain as major drawbacks. Recently, excessive production and accumulation of neutrophil extracellular traps (NETs) also known as NETosis have been identified to exacerbate inflammatory responses and induce further tissue damage in IBD. Such discovery invited many researchers to investigate NETs as a potential therapeutic target. DNase-I is a natural agent that can effectively destroy NETs and, therefore, potentially reduce NETs-induced inflammations even without the use of aggressive drugs. However, low stability and rapid clearance of DNase-I remain as major limitations for further therapeutic applications. In this research, polymeric nanozymes were fabricated to increase the delivery and therapeutic efficacy of DNase-I. DNase-I was immobilized on the surface of polymeric nanoparticles to maintain its enzymatic properties while extending its activity in the colon. Delivery of DNase-I using this platform allowed enhanced stability and prolonged activity of DNase-I with minimal toxicity. When administered to animal models of IBD, DNase-I nanozymes successfully alleviated various pathophysiological symptoms of IBD. More importantly, DNase-I nanozyme administration successfully attenuated neutrophil infiltration and NETosis in the colon compared to free DNase-I or mesalamine.

Bioinspired, calcium-free alginate hydrogels with tunable physical and mechanical properties and improved biocompatibility.

Alginate hydrogels are for various biomedical applications including tissue engineering, cell therapy, and drug delivery. However, it is not easy to control swelling or viscoelastic and biophysical properties of alginate hydrogels prepared by conventional cross-linking methods (ionic interaction using divalent cations). In this study, we describe a bioinspired approach for preparing divalent ion-free alginate hydrogels that exhibit tunable physical and mechanical properties and improved biocompatibility due to the absence of cations in the gel matrices. We conjugated dopamine, a minimalized adhesive motif found in the holdfast pads of mussels, to alginate backbones (alginate-catechol) and the tethered catechols underwent oxidative cross-linking. This resulted in divalent cation-free alginate hydrogels. The swelling ratios and moduli of the alginate-catechol hydrogels are readily tunable, which is difficult to achieve in ionic bond-based alginate hydrogels. Furthermore, alginate-catechol hydrogels enhanced the survival of various human primary cells including stem cells in the three-dimensional gel matrix, indicating that intrinsic cytotoxicity caused by divalent cations becomes negligible when employing catechol oxidation for alginate cross-linking. The inflammatory response in vivo was also significantly attenuated compared to conventional alginate hydrogels with calcium cross-linking. This biomimetic approach for the preparation of alginate hydrogels may provide a novel platform technology to develop tunable, functional, biocompatible, three-dimensional scaffolds for tissue engineering and cell therapy.

Stretchable Gold Nanomembrane Electrode with Ionic Hydrogel Skin-Adhesive Properties.

Skin has a dynamic surface and offers essential information through biological signals originating from internal organs, blood vessels, and muscles. Soft and stretchable bioelectronics can be used in wearable machines for long-term stability and to continuously obtain distinct bio-signals in conjunction with repeated expansion and contraction with physical activities. While monitoring bio-signals, the electrode and skin must be firmly attached for high signal quality. Furthermore, the signal-to-noise ratio (SNR) should be high enough, and accordingly, the ionic conductivity of an adhesive hydrogel needs to be improved. Here, we used a chitosan-alginate-chitosan (CAC) triple hydrogel layer as an interface between the electrodes and the skin to enhance ionic conductivity and skin adhesiveness and to minimize the mechanical mismatch. For development, thermoplastic elastomer Styrene-Ethylene-Butylene-Styrene (SEBS) dissolved in toluene was used as a substrate, and gold nanomembranes were thermally evaporated on SEBS. Subsequently, CAC triple layers were drop-casted onto the gold surface one by one and dried successively. Lastly, to demonstrate the performance of our electrodes, a human electrocardiogram signal was monitored. The electrodes coupled with our CAC triple hydrogel layer showed high SNR with clear PQRST peaks.

Enzyme-Triggered Intestine-Specific Targeting Adhesive Platform for Universal Oral Drug Delivery.

Patient adherence to chronic therapies can be suboptimal, leading to poor therapeutic outcomes. Dosage forms that enable reduction in dosing frequency stand to improve patient adherence. Variation in gastrointestinal transit time, inter-individual differences in gastrointestinal physiology and differences in physicochemical properties of drugs represent challenges to the development of such systems. To this end, a small intestine-targeted drug delivery system is developed, where prolonged gastrointestinal retention and sustained release are achieved through tissue adhesion of drug pills mediated by an essential intestinal enzyme catalase. Here proof-of-concept pharmacokinetics is demonstrated in the swine model for two drugs, hydrophilic amoxicillin and hydrophobic levodopa. It is anticipated that this system can be applicable for many drugs with a diverse of physicochemical characteristics.

Engineering of Immune Microenvironment for Enhanced Tissue Remodeling.

The capability to restore the structure and function of tissues damaged by fatal diseases and trauma is essential for living organisms. Various tissue engineering approaches have been applied in lesions to enhance tissue regeneration after injuries and diseases in living organisms. However, unforeseen immune reactions by the treatments interfere with successful healing and reduce the therapeutic efficacy of the strategies. The immune system is known to play essential roles in the regulation of the microenvironment and recruitment of cells that directly or indirectly participate in tissue remodeling in defects. Accordingly, regenerative immune engineering has emerged as a novel approach toward efficiently inducing regeneration using engineering techniques that modulate the immune system. It is aimed at providing a favorable immune microenvironment based on the controlled balance between pro-inflammation and anti-inflammation. In this review, we introduce recent developments in immune engineering therapeutics based on various cell types and biomaterials. These developments could potentially overcome the therapeutic limitations of tissue remodeling.

Biomaterials as therapeutic drug carriers for inflammatory bowel disease treatment.

Inflammatory bowel diseases (IBDs) are idiopathic gastrointestinal inflammatory disorders featuring chronic intestinal inflammation. Although IBDs are increasingly becoming globally prevalent, the exact etiology of IBD remains obscure. Recently, the ability of various drugs for mucosal healing such as corticosteroids, antibiotics, and immunosuppressants has been proven. However, the delivery of free drugs is insufficient and inadequate since some patients have experienced reduced efficacy due to repeated administration and others have suffered side effects. In this regard, novel platforms based on biomaterials are required to deliver pharmaceutical agents to the damaged site with increased efficacy and reduced side effects. In this review, we summarize the most recent status of numerous biomaterials in treating IBD. This review addresses various nanoparticles, microparticles, and hydrogels recently prepared from natural polymers, lipids, synthetic polymers, and inorganic materials. These diverse biomaterials can be used as effective drug-delivery systems to promote colon-specific delivery and for the stable release of drugs in IBD treatments.

Delivery of therapeutic carbon monoxide by gas-entrapping materials.

Carbon monoxide (CO) has long been considered a toxic gas but is now a recognized bioactive gasotransmitter with potent immunomodulatory effects. Although inhaled CO is currently under investigation for use in patients with lung disease, this mode of administration can present clinical challenges. The capacity to deliver CO directly and safely to the gastrointestinal (GI) tract could transform the management of diseases affecting the GI mucosa such as inflammatory bowel disease or radiation injury. To address this unmet need, inspired by molecular gastronomy techniques, we have developed a family of gas-entrapping materials (GEMs) for delivery of CO to the GI tract. We show highly tunable and potent delivery of CO, achieving clinically relevant CO concentrations in vivo in rodent and swine models. To support the potential range of applications of foam GEMs, we evaluated the system in three distinct disease models. We show that a GEM containing CO dose-dependently reduced acetaminophen-induced hepatocellular injury, dampened colitis-associated inflammation and oxidative tissue injury, and mitigated radiation-induced gut epithelial damage in rodents. Collectively, foam GEMs have potential paradigm-shifting implications for the safe therapeutic use of CO across a range of indications.

Immunomodulatory Scaffolds Derived from Lymph Node Extracellular Matrices.

Immunomodulation in the local tissue microenvironment is pivotal for the determination of macrophage phenotypes and regulation of functions necessary for pro-healing effects. Herein, we demonstrate that a lymph node extracellular matrix (LNEM) prepared by the decellularization of lymph node tissues can mimic lymph node microenvironments for immunomodulation in two-dimensional (2D) and three-dimensional (3D) formats. The LNEM exhibits strengthened immunomodulatory effects in comparison to conventional collagen-based platforms. A 3D LNEM hydrogel is more effective than the 2D LNEM coating in inducing M2 macrophage polarization. The 3D LNEM induces macrophage elongation and enhances the M2-type marker expression and the secretion of anti-inflammatory cytokines. Additionally, the phagocytic function of macrophages is improved upon exposure to the intricate 3D LNEM environment. We demonstrate the reduced susceptibility of liver organoids to a hepatotoxic drug when co-cultured with macrophages in a 3D LNEM. This effect could be attributed to the enhanced anti-inflammatory functions and indicates its potential as a drug-testing platform that enables drug responses similar to those observed in vivo. Finally, the implantation of an LNEM hydrogel in a mouse volumetric muscle loss model facilitates the recruitment of host macrophages to the site of injury and enhances macrophage polarization toward the M2 phenotype for tissue healing in vivo. Therefore, 3D immune system-mimicking biomaterials could serve as useful platforms for tissue modeling and regenerative medicine development.

Implantable system for chronotherapy.

Diurnal variation in enzymes, hormones, and other biological mediators has long been recognized in mammalian physiology. Developments in pharmacobiology over the past few decades have shown that timing drug delivery can enhance drug efficacy. Here, we report the development of a battery-free, refillable, subcutaneous, and trocar-compatible implantable system that facilitates chronotherapy by enabling tight control over the timing of drug administration in response to external mechanical actuation. The external wearable system is coupled to a mobile app to facilitate control over dosing time. Using this system, we show the efficacy of bromocriptine on glycemic control in a diabetic rat model. We also demonstrate that antihypertensives can be delivered through this device, which could have clinical applications given the recognized diurnal variation of hypertension-related complications. We anticipate that implants capable of chronotherapy will have a substantial impact on our capacity to enhance treatment effectiveness for a broad range of chronic conditions.

Microfluidic device with brain extracellular matrix promotes structural and functional maturation of human brain organoids.

Brain organoids derived from human pluripotent stem cells provide a highly valuable in vitro model to recapitulate human brain development and neurological diseases. However, the current systems for brain organoid culture require further improvement for the reliable production of high-quality organoids. Here, we demonstrate two engineering elements to improve human brain organoid culture, (1) a human brain extracellular matrix to provide brain-specific cues and (2) a microfluidic device with periodic flow to improve the survival and reduce the variability of organoids. A three-dimensional culture modified with brain extracellular matrix significantly enhanced neurogenesis in developing brain organoids from human induced pluripotent stem cells. Cortical layer development, volumetric augmentation, and electrophysiological function of human brain organoids were further improved in a reproducible manner by dynamic culture in microfluidic chamber devices. Our engineering concept of reconstituting brain-mimetic microenvironments facilitates the development of a reliable culture platform for brain organoids, enabling effective modeling and drug development for human brain diseases.

Gastrointestinal synthetic epithelial linings.

Epithelial tissues line the organs of the body, providing an initial protective barrier as well as a surface for nutrient and drug absorption. Here, we identified enzymatic components present in the gastrointestinal epithelium that can serve as selective means for tissue-directed polymerization. We focused on the small intestine, given its role in drug and nutrient absorption and identified catalase as an essential enzyme with the potential to catalyze polymerization and growth of synthetic biomaterial layers. We demonstrated that the polymerization of dopamine by catalase yields strong tissue adhesion. We characterized the mechanism and specificity of the polymerization in segments of the gastrointestinal tracts of pigs and humans ex vivo. Moreover, we demonstrated proof of concept for application of these gastrointestinal synthetic epithelial linings for drug delivery, enzymatic immobilization for digestive supplementation, and nutritional modulation through transient barrier formation in pigs. This catalase-based approach to in situ biomaterial generation may have broad indications for gastrointestinal applications.

Ferritin nanoparticles for improved self-renewal and differentiation of human neural stem cells.

BACKGROUND: Biomaterials that promote the self-renewal ability and differentiation capacity of neural stem cells (NSCs) are desirable for improving stem cell therapy to treat neurodegenerative diseases. Incorporation of micro- and nanoparticles into stem cell culture has gained great attention for the control of stem cell behaviors, including proliferation and differentiation. METHOD: In this study, ferritin, an iron-containing natural protein nanoparticle, was applied as a biomaterial to improve the self-renewal and differentiation of NSCs and neural progenitor cells (NPCs). Ferritin nanoparticles were added to NSC or NPC culture during cell growth, allowing for incorporation of ferritin nanoparticles during neurosphere formation. RESULTS: Compared to neurospheres without ferritin treatment, neurospheres with ferritin nanoparticles showed significantly promoted self-renewal and cell-cell interactions. When spontaneous differentiation of neurospheres was induced during culture without mitogenic factors, neuronal differentiation was enhanced in the ferritin-treated neurospheres. CONCLUSIONS: In conclusion, we found that natural nanoparticles can be used to improve the self-renewal ability and differentiation potential of NSCs and NPCs, which can be applied in neural tissue engineering and cell therapy for neurodegenerative diseases.

Highly durable and biocompatible periodical Si/DLC nanocomposite coatings.

Functional nanocomposite coatings comprised of periodically stacked nanolayers of diamond-like carbon (DLC) and amorphous silicon were developed for biomedical applications. The periodical nanocomposite structure provided high surface durability while silicon aided in reducing the residual stress. The structural, mechanical, tribological, and biomedical properties of the Si/DLC coatings deposited by magnetron sputtering were investigated systematically. The effect of the negative substrate bias on the structure and properties of the coatings was also assessed. The coatings demonstrated high durability and high biocompatibility. The bias voltage and bias mode affected both the hardness and residual stress of the Si/DLC coatings. Particularly, application of 60 V negative bias during the DLC layer deposition resulted in the lowest wear rate. FEM simulations showed that the wear resistance of the coatings was dictated by the hardness as well as the adhesion between the coatings and a chromium sub-layer. The periodical alternation of Si and DLC nanolayers led to a significant improvement of MC3T3 cell adhesion compared to the previously published data for Si-DLC composites.

Microchannel system for rate-controlled, sequential, and pH-responsive drug delivery.

Controlled delivery of drug at a constant rate, in a sequential order, or responsive to environment conditions has been pursued for a long time to enhance the efficacy of therapeutic molecules and to minimize side effects of highly potent drugs. However, achieving such delicately-controlled delivery of a drug molecule is non-trivial and still remains a challenge. We propose the use of microchannels to control the rate, sequence, and pH-responsiveness of drug delivery for high precision and predictability. In this study, we introduce elementary drug delivery units consisting of micro-reservoirs and microchannels that have variations in their lengths, widths, numbers, and straightness. The release study demonstrates that the release rates of model drugs can be modulated by the design of microchannels. Finite element modeling of drug release predicts the performance of the drug delivery units with high accuracy. The possibility of sequential drug delivery is also demonstrated using biodegradable polymer plug in microchannels. Finally, pH-responsive delivery of drugs in microfluidic units is also discussed and demonstrated via cell viability tests. STATEMENT OF SIGNIFICANCE: In this work, we developed microchannel-based drug delivery devices whose release rate could be accurately calculated and controlled by design of microchannel geometry. Although there have been many advances in microfabricated drug delivery systems, in particular, reservoir-based systems, no systematic investigation has been made to utilize the release channels. In our work, an equivalent electrical circuit concept was applied to the microfluidic systems for more detailed design and analysis. A microfluidic channel was regarded as an electrical resistor; their diffusion/electrical flux could be tuned with geometric factors such as length, width, a number of channel/resistor and their connections. Furthermore, from delivery rate control using channel geometry, multifunctional channel-based release systems for sequential and pH-responsive were demonstrated.

Bio-artificial tongue with tongue extracellular matrix and primary taste cells.

Artificial taste devices for tastant sensing and taste information standardization are attracting increasing attention with the exponential growth of the food and beverage industries. Despite recent developments in artificial taste sensors incorporating polymers, lipid membranes, and synthetic vesicles, current devices have limited functionality and sensitivity, and are complex to manufacture. Moreover, such synthetic systems cannot simulate the taste signal transmissions that are critical for complicated taste perception. The current document describes a primary taste cell-based artificial tongue that can mimic taste sensing. To maintain viable and functional taste cells required for in vitro tastant sensing, a tongue extracellular matrix (TEM) prepared by decellularization of tongue tissue was applied to two- and three-dimensional taste cell cultures. The TEM-based system recreates the tongue's microenvironment and significantly improves the functionality of taste cells for sensing tastant molecules by enhancing cellular adhesion and gustatory gene expression compared with conventional collagen-based systems. The TEM-based platform simulates signal transmission from tastant-treated taste cells to adjacent neuronal cells, which was impossible with previous artificial taste sensors. The artificial tongue device may provide highly efficient, functional sensors for tastant detection and in vitro organ models that mimic the tongue allowing elucidation of the mechanisms of taste.

Single-Droplet Multiplex Bioassay on a Robust and Stretchable Extreme Wetting Substrate through Vacuum-Based Droplet Manipulation.

Herein, a droplet manipulation system with a superamphiphobic (SPO)-superamphiphilic (SPI) patterned polydimethylsiloxane (PDMS) substrate is developed for a multiplex bioassay from single-droplet samples. The SPO substrate is fabricated by sequential spraying of adhesive and fluorinated silica nanoparticles onto a PDMS substrate. It is subsequently subjected to oxygen plasma with a patterned mask to form SPI patterns. The SPO layer exhibits extreme liquid repellency with a high contact angle (>150°) toward low surface tension and viscous biofluidic droplets (e.g., ethylene glycol, blood, dimethyl sulfoxide, and alginate hydrogel). In contrast, the SPI exhibits liquid adhesion with a near zero contact angle. Using the droplet manipulation system, various liquid droplets can be precisely manipulated and dispensed onto the predefined SPI patterns on the SPO PDMS substrate. This system enables a multiplex colorimetric bioassay, capable of detecting multiple analytes, including glucose, uric acid, and lactate, from a single sample droplet. In addition, the detection of glucose concentrations in a plasma droplet of diabetic and healthy mice are performed to demonstrate the feasibility of the proposed system for efficient clinical diagnostic applications.