Development of tissue culture system with automated pulsation and Kalman filter control for an artificial artery model.

Tissue-engineered arterial vessels have been used as substitutes for unnecessary animal experiments to evaluate the pharmacokinetics of drugs targeting various arteriopathies caused by structural or physiological arterial defects. An arterial tissue culture system was established to simulate the mechanical characteristics of a heart-beating pump and to do online feedback control of lactate and glucose concentrations. The mechanically controlled flow pump mimicked the heart pumping inside a tissue-engineered artery composed of muscle and endothelial cells within a nanofibrous scaffold. After monitoring the pH of the culture medium online, lactate and glucose were estimated using the Kalman filter algorithm, and the set-point online control was operated to maintain glucose for artery tissue engineering. The composition of the artificial artery was confirmed by immunofluorescence staining, and its mechanical characteristics were examined. The online automated system successfully demonstrated its applicability as a standardized process for arterial tissue culture to replace animal arterial experiments.

Differential spectroscopic analysis of size-dependent phycobilisome from Spirulina maxima.

C-phycocyanin (C-Pc), a photosynthetic pigment for use as a fluorescent indicator or in pharmaceutical, food, and cosmetic products, exists in a phycobilisome complex with allophycocyanin (APC), phycoerythrin (PE), and linker polypeptides. This heterogeneity makes it difficult to quantify phycobilisome composition in an ultraviolet-visible (UV-vis) spectrum. In this study, derivative analysis of UV-vis spectra was successfully applied to display the distinct wavelengths at which C-Pc, APC, and PE have maximal peaks. In all samples, C-Pc of the largest portion had a "zero-crossing" first order, APC did not have a zero-crossing first order, and PE did not have first derivative for zero crossing or local minimum from the 500 and 700 nm, respectively. The results show that derivative analyses coupled with signal smoothing can be applied to elucidate the composition of phycobilisome under various conditions including purification and environment.

Chitosan-coated C-phycocyanin Liposome for Extending the Neuroprotective Time Window Against Ischemic Brain Stroke.

The time window for neuroprotection during ischemic brain stroke is short, and hence, development of neuroprotectants is critical to extend this time window. This study sought to verify if muco-adhesive chitosan coating improves the neuroprotective potential of the pre-proven C-Phycocyanin-pertaining liposome (C-Pc liposome). The use of chitosan-coated liposomes extended the neuroprotective time window by 6 h after occlusion, and further improved the neuroprotection efficiency of the C-Pc liposome in a rat Middle Cerebral Artery Occlusion (MCAO) model. Beneficial changes in mRNA expressions of antioxidants, inflammatory cytokines and glia scar proteoglycans were evident in the C-Pc liposomes. In addition, in the cultured astrocytes, the chitosan- coated C-Pc liposome expressed anti-oxidative activity without cytotoxicity.

A systematic correlation analysis of carotenoids, chlorophyll, non-pigmented cell mass, and cell number for the blueprint of Dunaliella salina culture in a photobioreactor.

Microalgal carotenoids are attractive health ingredients, but their production should be optimized to improve cost-effectiveness. Understanding cellular physiology centered on carotenoid synthesis is the prerequisite for this work. Therefore, systematic correlation analyses were conducted among chlorophyll, carotenoids, non-pigmented cell mass, and cell number of Dunaliella salina in a specified condition over a relatively long culture time. First, an integrated correlation was performed: a temporal profile of the carotenoids was correlated with those of other factors, including chlorophyll, non-pigmented cell mass, and cell number. Pearson and Spearman correlation analyses were performed to identify linearity and monotonicity of the correlation, respectively, and then cross-correlation was executed to determine if the correlation had a time lag. Second, to understand the cellular potential of metabolism, the procedure was repeated to provide a data set composed of the specific synthesis rates of the factors or growth rate, which additionally provided kinetic correlations among the constituting components of the cell, excluding the effect of cell number. This systematic approach could generate a blueprint model that is composed of only what it needs, which could make it possible to efficiently control and optimize the process.

Author Correction: Artificial controlled model of blood circulation system for adhesive evaluation.

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Development of coaxial alginate-PCL nanofibrous dressing for controlled release of Spirulina extract.

Spirulina has widely been highlighted as a source of bioactive material that can be impregnated into dressing materials. The aim of this study was to widen the application fields of Spirulina extract-containing nanofiber, which has been suggested as an attractive dressing material in several previous studies. The bioactivity release pattern, water absorbance, and mechanical strength must be controllable. Spirulina extract was physically impregnated inside a nanofiber without significant chemical bonding to polycaprolactone or alginate polymers. This led to an initial burst and continual release of bioactive molecules from the nanofiber. By altering the concentration of Spirulina extract, mechanical strength and water absorbance were controllable. In addition, the dressing patch showed no cytotoxicity towards human epithelial cells, not causing skin-irritation. This indicates that the coaxially fabricated patch is a controllable dressing material that can be customized to have a specific mechanical strength, water absorbance, and bioactive release pattern, making it suitable for wide applications.

A Novel Thrombolytic and Anticoagulant Serine Protease from Polychaeta, Diopatra sugokai.

Ischemic stroke can result from blockage of blood vessels, forming fibrin clots in the body and causing irreparable brain damage. Remedial thrombolytic agents or anticoagulants have been studied; however, because the FDA-approved tissue plasminogen activator has low efficacy and side effects, it is necessary to develop safer and more effective treatment candidates. This study aimed at assessing the fibrinolytic and anticoagulation features of a novel serine protease extracted and purified from Diopatra sugokai, a polychaeta that inhabits tidal flats. The purified serine protease was obtained through ammonium sulfate precipitation, affinity chromatography, and ion-exchange chromatography. Its molecular size was identified via SDS-PAGE. To characterize its enzymatic activities, the protease activity at various pH and temperatures, and in the presence of various inhibitors, was measured via azocasein assay. Its fibrinolytic activity and anticoagulant effect were assessed by fibrin zymography, fibrin plate assay, and fibrinogenolytic activity assays. The novel 38 kDa serine protease had strong indirect thrombolytic activity rather than direct activity over broad pH (4-10) and temperature (37°C-70°C) ranges. In addition, the novel serine protease exhibited anticoagulant activity by degrading the α-, ß-, and γ-chains of fibrinogen. In addition, it did not produce cytotoxicity in endothelial cells. Therefore, this newly isolated serine protease is worthy of further investigation as a novel alkaline serine protease for thrombolytic therapy against brain ischemia.

Artificial controlled model of blood circulation system for adhesive evaluation.

Since there are several casualties due to uncontrolled bleeding resulting from simple injury to surgery, effective styptic or vessel adhesives are important; however, their development is limited by the lack of standardized systems to evaluate potential compounds. The current study outlines the development of an aorta styptic evaluation system, comprising of decellularized swine aorta tissue and a heart pump-mimicking system. Although the cells in the swine aorta were removed, the structural stability of the aorta was sustained due to the maintenance of the extracellular matrix. Using a control adhesive, Cyanoacrylate, the developed model was found to have similar adhesive efficacy to intact aorta. The circulatory-mimicking system was designed to mimic the beat rate and strength of blood-flow from the heart, which was necessary to evaluate the adherent efficacy. The decellularized aorta improves instabilities of intact tissues, which occurs on account of storage and origin, thereby allowing for a more standardized system. The system was able to simulate several symptoms of circulation, according to patient age and health, by adjusting pumping frequency and intensity. Therefore, this system can be used as a standardized evaluation system for screening adhesives. Further, it would also evaluate other medical devices, such as stent or medications.

Nasal Delivery of Antioxidants by Cholesterol-incorporated Liposomes Extends the Neuroprotective Time Window in Cerebral Ischemia.

Antioxidants have the potential to prevent cerebral ischemia-reperfusion (IR)-associated secondary damage induced by reactive oxygen species (ROS); however, the short therapeutic time window of IR is a considerable obstacle. Nano-sized nasal delivery systems provide an effective means of delivering drugs through the BBB, but few such systems have been developed to extend the treatment time window in IR. In this work, a nanosized nasal delivery system for antioxidants was found to have the potential to extend the neuroprotective time window. The authors chose to use the antioxidant C-phycocyanin (C-Pc) to design a neuroprotective liposome with a long life, controllable release, and high neuronal uptake rate. Liposomes formulated with various cholesterol to phospholipid ratios were assessed thermodynamically, kinetically, and biologically. Thermodynamically stable, monodispersive, and release-controllable C-Pc liposomes were more effectively taken up by Neuro2a cells than free C-Pc and were biocompatible, maintaining the anti-oxidative properties of C-Pc. When optimal C-Pc liposomes were administered to middle cerebral artery occlusion (MCAO) rats 2 h after onset, infarct sizes were smaller and behavioral activities improved compared with the same metrics in free C-Pc-treated rats. Liposomal delivery still reduced infarct sizes and improved behavioral activity 6 h after onset, whereas free C-Pc did not.

Development of a Spirulina Extract/Alginate-Imbedded PCL Nanofibrous Cosmetic Patch.

Cosmetic patches have recently been developed as skin products for personal care owing to rapid advances in the technology of delivery of active ingredients, moisture, and adhesiveness to skin. Alginate and Spirulina are typical marine resources used in cosmetic products. This research involved the development of a Spirulina extract-impregnated alginate nanofiber cosmetic patch supported by a polycaprolactone (PCL) nanofiber cover (Spi/Alg-PCL NF patch). In addition to the ability of alginate to affect moisture and adhesiveness to skin, the impregnation of Spirulina extract strengthened those abilities as well as its own bioactive effectiveness. All fabrication processing steps were undertaken in aqueous solution. The three components (alginate, Spirulina extract, and PCL) had no detected cytotoxicity in human keratinocyte cell-based examination. In addition, wetting the pre-dried patch on the skin resulted in the Spirulina extract being released within 30 min. The results indicate the excellence of the Spi/Alg-PCL NF patch as a skin-care cosmetic device.

Purification of serine protease from polychaeta, Lumbrineris nipponica, and assessment of its fibrinolytic activity.

Ischemic stroke and cardiovascular disease can occur from blockage of blood vessels by fibrin clots formed naturally in the body. Therapeutic drugs of anticoagulant or thrombolytic agents have been studied; however, various problems have been reported such as side effects and low efficacy. Thus, development of new candidates that are more effective and safe is necessary. The objective of this study is to evaluate fibrinolytic activity, anti-coagulation, and characterization of serine protease purified from Lumbrineris nipponica, polychaeta, for new thrombolytic agents. In the present study, we isolated and identified a new fibrinolytic serine protease from L. nipponica. The N-terminal sequence of the identified serine protease was EAMMDLADQLEQSLN, which is not homologous with any known serine protease. The size of the purified serine protease was 28 kDa, and the protein purification yield was 12.7%. The optimal enzyme activity was observed at 50°C and pH 2.0. A fibrin plate assay confirmed that indirect fibrinolytic activity of the purified serine protease was higher than that of urokinase-PA, whereas direct fibrinolytic activity, which causes bleeding side effects, was relatively low. The serine protease did not induce any cytotoxicity toward the endothelial cell line. In addition, anticoagulant activity was verified by an in vivo DVT animal model system. These results suggest that serine protease purified from L. nipponica has the potential to be an alternative fibrinolytic agent for the treatment of thrombosis and use in various biomedical applications.

Assessment of Neuronal Cell-Based Cytotoxicity of Neurotoxins from an Estuarine Nemertean in the Han River Estuary.

A heteronemertean, Yininemertes pratensis, was collected in Han River Estuary, South Korea. This estuarine nemertean has been known by the local fishermen for harmful effects to the glass eels, juveniles of Japanese eel Anguilla japonica, migrating to fresh water. The present study confirmed the neurotoxic effects of this heteronemertean ribbon worm at the cellular level. Derivative types of neurotoxic tetrodotoxin (TTX), 5,11-dideoxy TTX (m/z 288) and 11-norTTX-6(S)-01 (m/z 305.97), were identified through HPLC and MALDI-TOF MS. However, significant neurotoxicity was confirmed in the fraction containing an undefined molecule corresponding to the 291.1 (m/z) peak, when tested in rat primary astrocytes and dorsal ganglion cells. This study is the first to report neurotoxins of the estuarine nemertean, fairly abundant in the Han River estuary, and suggests the long-term monitoring of population dynamics and surveillance of the toxicity in this river estuary.

Nanocellulose size regulates microalgal flocculation and lipid metabolism.

Harvesting of microalgae is a cost-consuming step for biodiesel production. Cellulose has recently been studied as a biocompatible and inexpensive flocculant for harvesting microalgae via surface modifications such as cation-modifications. In this study, we demonstrated that cellulose nanofibrils (CNF) played a role as a microalgal flocculant via its network geometry without cation modification. Sulfur acid-treated tunicate CNF flocculated microalgae, but cellulose nanocrystals (CNC) did not. In addition, desulfurization did not significantly influence the flocculation efficiency of CNF. This mechanism is likely related to encapsulation of microalgae by nanofibrous structure formation, which is derived from nanofibrils entanglement and intra-hydrogen bonding. Moreover, flocculated microalgae were subject to mechanical stress resulting in changes in metabolism induced by calcium ion influx, leading to upregulated lipid synthesis. CNF do not require surface modifications such as cation modified CNC and flocculation is derived from network geometry related to nanocellulose size; accordingly, CNF is one of the least expensive cellulose-based flocculants ever identified. If this flocculant is applied to the biodiesel process, it could decrease the cost of harvest, which is one of the most expensive steps, while increasing lipid production.

Co-stimulation of HaCaT keratinization with mechanical stress and air-exposure using a novel 3D culture device.

Artificial skin or skin equivalents have been used for clinical purpose to skin graft and as substitutes for animal experiments. The culture of cell lines such as HaCaT has the potential to produce large amounts of artificial skin at a low cost. However, there is a limit to keratinization due to the restriction of differentiation in HaCaT. In this study, a culture device that mimics the in vivo keratinization mechanism, co-stimulated by air-exposure and mechanical stimulation, was developed to construct skin equivalents. The device can reconstruct the epidermal morphology, including the cornified layer, similar to its formation in vivo. Under the condition, epidermis was differentiated in the spinous and granular layers. Formation of the stratum corneum is consistent with the mRNA and protein expressions of differentiation markers. The device is the first of its kind to combine air-exposure with mechanical stress to co-stimulate keratinization, which can facilitate the economically viable production of HaCaT-based artificial skin substitutes.

Mechanotransduction-Induced Lipid Production System with High Robustness and Controllability for Microalgae.

Microalgae lipids are a promising energy source, but current biochemical methods of lipid-inductions such as nitrogen deprivation have low process robustness and controllability. Recently, use of mechanotransduction based membrane distortion by applying compression stress in a 2D-microsystem was suggested as a way to overcome these limitations of biochemical induction. However, reproduction in large numbers of cells without cell death has been difficult to overcome because compression for direct membrane distortion reduces culture volume and leads to cell death due to nutrient deprivation. In this study, a mechanotransduction-induced lipid production (MDLP) system that redirects elastic microbeads to induce membrane distortion of microalgae with alleviating cell death was developed. This system resulted in accumulation of lipid in as little as 4 hr. Once compressed, porous microbeads absorb media and swell simultaneously while homogeneously inducing compression stress of microalgae. The absorbed media within beads could be supplied to adjacent cells and could minimize cell death from nutrient deficiency. All mechanotransduction was confirmed by measuring upregulation of calcium influx and Mat3 genes. The microbeads ensured robustness and controllability in repeated compression/de-compression processes. Overall, the MDLP system has potential for use as a fundamental biodiesel process that requires robustness and controllability.

Thermodynamic Insights and Conceptual Design of Skin-Sensitive Chitosan Coated Ceramide/PLGA Nanodrug for Regeneration of Stratum Corneum on Atopic Dermatitis.

Atopic dermatitis (AD) is a complex skin disease primarily characterized by psoriasis of the stratum corneum. AD drugs have usually been used in acidic and hydrophilic solvents to supply moisture and prevent lipid defects. Ceramide is a typical treatment agent to regenerate the stratum corneum and relieve symptoms of AD. However, ceramide has limitation on direct use for skin because of its low dispersion properties in hydrophilic phase and side effects at excessive treatment. In this study, ceramide imbedded PLGA nanoparticles were developed with chitosan coating (Chi-PLGA/Cer) to overcome this problem. The chitosan coating enhanced initial adherence to the skin and prevented the initial burst of ceramide, but was degraded by the weakly acidic nature of skin, resulting in controlled release of ceramide with additional driving force of the squeezed PLGA nanoparticles. Additionally, the coating kinetics of chitosan were controlled by manipulating the reaction conditions and then mathematically modeled. The Chi-PLGA/Cer was not found to be cytotoxic and ceramide release was controlled by pH, temperature, and chitosan coating. Finally, Chi-PLGA/Cer was demonstrated to be effective at stratum corneum regeneration in a rat AD model. Overall, the results presented herein indicated that Chi-PLGA/Cer is a novel nanodrug for treatment of AD.

Assessment of C-phycocyanin effect on astrocytes-mediated neuroprotection against oxidative brain injury using 2D and 3D astrocyte tissue model.

Drugs are currently being developed to attenuate oxidative stress as a treatment for brain injuries. C-phycocyanin (C-Pc) is an antioxidant protein of green microalgae known to exert neuroprotective effects against oxidative brain injury. Astrocytes, which compose many portions of the brain, exert various functions to overcome oxidative stress; however, little is known about how C-Pc mediates the antioxidative effects of astrocytes. In this study, we revealed that C-Pc intranasal administration to the middle cerebral artery occlusion (MCAO) rats ensures neuroprotection of ischemic brain by reducing infarct size and improving behavioral deficits. C-Pc also enhanced viability and proliferation but attenuated apoptosis and reactive oxygen species (ROS) of oxidized astrocytes, without cytotoxicity to normal astrocytes and neurons. To elucidate how C-Pc leads astrocytes to enhance neuroprotection and repair of ischemia brain, we firstly developed 3D oxidized astrocyte model. C-Pc had astrocytes upregulate antioxidant enzymes such as SOD and catalase and neurotrophic factors BDNF and NGF, while alleviating inflammatory factors IL-6 and IL-1ß and glial scar. Additionally, C-Pc improved viability of 3D oxidized neurons. In summary, C-Pc was concluded to activate oxidized astrocytes to protect and repair the ischemic brain with the combinatorial effects of improved antioxidative, neurotrophic, and anti-inflammatory mechanisms.

Regulation of astrocyte activity via control over stiffness of cellulose acetate electrospun nanofiber.

Astrocytes are involved in neuron protection following central nervous system (CNS) injury; accordingly, engineered astrocytes have been investigated for their usefulness in cell therapy for CNS injury. Nanofibers have attracted a great deal of attention in neural tissue engineering, but their mechanical properties greatly influence physiology. Cellulose acetate (CA) has been studied for use in scaffolds owing to its biocompatibility, biodegradability, and good thermal stability. In this study, stiffness of CA nanofibers controlled by heat treatment was shown to regulate astrocyte activity. Adhesion and viability increased in culture as substrate became stiffer but showed saturation at greater than 2 MPa of tensile strength. Astrocytes became more active in terms of increasing intermediate filament glial fibrillary acidic protein (GFAP). The results of this study demonstrate the effects of stiffness alone on cellular behaviors in a three-dimensional culture and highlight the efficacy of heat-treated CA for astrocyte culture in that the simple treatment enables control of astrocyte activity.

Chitosan nanoparticle/PCL nanofiber composite for wound dressing and drug delivery.

Many investigations of wound dressings equipped with drug delivery systems have recently been conducted. Chitosan is widely used not only as a material for wound dressing by the efficacy of its own, but also as a nanoparticle for drug delivery. In this study, an electrospun polycaprolactone nanofiber composite with chitosan nanoparticles (ChiNP-PCLNF) was fabricated and then evaluated for its drug release and biocompatibility to skin fibroblasts. ChiNP-PCLNF complexes showed no cytotoxicity and nanoparticles adsorbed by van der Waals force were released into aquatic environments and then penetrated into rat primary fibroblasts. Our studies demonstrate the potential for application of ChiNP-PCLNF as a wound dressing system with drug delivery for skin wound healing without side effects.

Development of a complex bone tissue culture system based on cellulose nanowhisker mechanical strain.

In bone tissue engineering, scaffolds have been investigated for their ability to support osteoblast growth and differentiation for recovery of damaged bones. Tunicate cellulose nanowhisker (CNW) film and mechanical strain were assessed for their suitability for osteoblasts. In this study, sulfuric acid hydrolysis extraction of tunicates integuments was conducted to obtain CNWs, which were found to be acceptable for adhering, growing, and differentiating osteoblasts without cytotoxicity. Mechanical stress enhanced osteoblast differentiation, and cell survival rate was recovered at around day 3, although there was a slight increase in cell death at day 1 after stimulation. We also found that intracellular flux of calcium ion was related to increased differentiation of CNWs under mechanical stress. Overall, we demonstrated the suitability of tunicate CNWs as a scaffold for bone tissue engineering and developed a complex system based on CNW for osteoblast growth and differentiation that will be useful for bone substitute fabrication.