Lyoprotectant Constituents Suited for Lyophilization and Reconstitution of Stem-Cell-Derived Extracellular Vesicles.

Stem-cell-derived extracellular vesicles (EVs) are emerging as an alternative approach to stem cell therapy. Successful lyophilization of EVs could enable convenient storage and distribution of EV medicinal products at room temperature for long periods, thus considerably increasing the accessibility of EV therapeutics to patients. In this study, we aimed to identify an appropriate lyoprotectant composition for the lyophilization and reconstitution of stem-cell-derived EVs. MSC-derived EVs were lyophilized using different lyoprotectants, such as dimethyl sulfoxide, mannitol, trehalose, and sucrose, at varying concentrations. Our results revealed that a mixture of trehalose and sucrose at high concentrations could support the formation of amorphous ice by enriching the amorphous phase of the solution, which successfully inhibited the acceleration of buffer component crystallization during lyophilization. Lyophilized and reconstituted EVs were thoroughly evaluated for concentration and size, morphology, and protein and RNA content. The therapeutic effects of the reconstituted EVs were examined using a tube formation assay with human umbilical vein endothelial cells. After rehydration of the lyophilized EVs, most of their generic characteristics were well-maintained, and their therapeutic capacity recovered to levels similar to those of freshly collected EVs. The concentrations and morphologies of the lyophilized EVs were similar to the initial features of the fresh EV group until day 30 at room temperature, although their therapeutic capacity appeared to decrease after 7 days. Our study suggests an appropriate composition of lyoprotectants, particularly for EV lyophilization, which could encourage the applications of stem-cell-derived EV therapeutics in the health industry.

Mesenchymal Stem Cell-Extracellular Vesicle Therapy for Stroke: Scalable Production and Imaging Biomarker Studies.

A major clinical hurdle to translate MSC-derived extracellular vesicles (EVs) is the lack of a method to scale-up the production of EVs with customized therapeutic properties. In this study, we tested whether EV production by a scalable 3D-bioprocessing method is feasible and improves neuroplasticity in animal models of stroke using MRI study. MSCs were cultured in a 3D-spheroid using a micro-patterned well. The EVs were isolated with filter and tangential flow filtration and characterized using electron microscopy, nanoparticle tracking analysis, and small RNA sequencing. Compared to conventional 2D culture, the production-reproduction of EVs (the number/size of particles and EV purity) obtained from 3D platform were more consistent among different lots from the same donor and among different donors. Several microRNAs with molecular functions associated with neurogenesis were upregulated in EVs obtained from 3D platform. EVs induced both neurogenesis and neuritogenesis via microRNAs (especially, miR-27a-3p and miR-132-3p)-mediated actions. EV therapy improved functional recovery on behavioral tests and reduced infarct volume on MRI in stroke models. The dose of MSC-EVs of 1/30 cell dose had similar therapeutic effects. In addition, the EV group had better anatomical and functional connectivity on diffusion tensor imaging and resting-state functional MRI in a mouse stroke model. This study shows that clinical-scale MSC-EV therapeutics are feasible, cost-effective, and improve functional recovery following experimental stroke, with a likely contribution from enhanced neurogenesis and neuroplasticity.

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

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

Preclinical Study of Human Bone Marrow-Derived Mesenchymal Stem Cells Using a 3-Dimensional Manufacturing Setting for Enhancing Spinal Fusion.

Spinal fusion surgery is a surgical technique that connects one or more vertebrae at the same time to prevent movement between the vertebrae. Although synthetic bone substitutes or osteogenesis-inducing recombinant proteins were introduced to promote bone union, the rate of revision surgery is still high due to pseudarthrosis. To promote successful fusion after surgery, stem cells with or without biomaterials were introduced; however, conventional 2D-culture environments have resulted in a considerable loss of the innate therapeutic properties of stem cells. Therefore, we conducted a preclinical study applying 3D-spheroids of human bone marrow-dewrived mesenchymal stem cells (MSCs) to a mouse spinal fusion model. First, we built a large-scale manufacturing platform for MSC spheroids, which is applicable to good manufacturing practice (GMP). Comprehensive biomolecular examinations, which include liquid chromatography-mass spectrometry and bioinformatics could suggest a framework of quality control (QC) standards for the MSC spheroid product regarding the identity, purity, viability, and potency. In our animal study, the mass-produced and quality-controlled MSC spheroids, either undifferentiated or osteogenically differentiated were well-integrated into decorticated bone of the lumbar spine, and efficiently improved angiogenesis, bone regeneration, and mechanical stability with statistical significance compared to 2D-cultured MSCs. This study proposes a GMP-applicable bioprocessing platform and QC directions of MSC spheroids aiming for their clinical application in spinal fusion surgery as a new bone graft substitute.

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

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

Development of a Novel Perfusion Rotating Wall Vessel Bioreactor with Ultrasound Stimulation for Mass-Production of Mineralized Tissue Constructs.

BACKGROUND: As stem cells are considered a promising cell source for tissue engineering, many culture strategies have been extensively studied to generate in vitro stem cell-based tissue constructs. However, most approaches using conventional tissue culture plates are limited by the lack of biological relevance in stem cell microenvironments required for neotissue formation. In this study, a novel perfusion rotating wall vessel (RWV) bioreactor was developed for mass-production of stem cell-based 3D tissue constructs. METHODS: An automated RWV bioreactor was fabricated, which is capable of controlling continuous medium perfusion, highly efficient gas exchange with surrounding air, as well as low-intensity pulsed ultrasound (LIPUS) stimulation. Embryonic stem cells encapsulated in alginate/gelatin hydrogel were cultured in the osteogenic medium by using our bioreactor system. Cellular viability, growth kinetics, and osteogenesis/mineralization were thoroughly evaluated, and culture media were profiled at real time. The in vivo efficacy was examined by a rabbit cranial defect model. RESULTS: Our bioreactor successfully maintained the optimal culture environments for stem cell proliferation, osteogenic differentiation, and mineralized tissue formation during the culture period. The mineralized tissue constructs produced by our bioreactor demonstrated higher void filling efficacy in the large bone defects compared to the group implanted with hydrogel beads only. In addition, the LIPUS modules mounted on our bioreactor successfully reached higher mineralization of the tissue constructs compared to the groups without LIPUS stimulation. CONCLUSION: This study suggests an effective biomanufacturing strategy for mass-production of implantable mineralized tissue constructs from stem cells that could be applicable to future clinical practice.

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

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

3D-Bioprinting Strategies Based on In Situ Bone-Healing Mechanism for Vascularized Bone Tissue Engineering.

Over the past decades, a number of bone tissue engineering (BTE) approaches have been developed to address substantial challenges in the management of critical size bone defects. Although the majority of BTE strategies developed in the laboratory have been limited due to lack of clinical relevance in translation, primary prerequisites for the construction of vascularized functional bone grafts have gained confidence owing to the accumulated knowledge of the osteogenic, osteoinductive, and osteoconductive properties of mesenchymal stem cells and bone-relevant biomaterials that reflect bone-healing mechanisms. In this review, we summarize the current knowledge of bone-healing mechanisms focusing on the details that should be embodied in the development of vascularized BTE, and discuss promising strategies based on 3D-bioprinting technologies that efficiently coalesce the abovementioned main features in bone-healing systems, which comprehensively interact during the bone regeneration processes.

Next-Generation Wearable Biosensors Developed with Flexible Bio-Chips.

The development of biosensors that measure various biosignals from our body is an indispensable research field for health monitoring. In recent years, as the demand to monitor the health conditions of individuals in real time have increased, wearable-type biosensors have received more attention as an alternative to laboratory equipment. These biosensors have been embedded into smart watches, clothes, and accessories to collect various biosignals in real time. Although wearable biosensors attached to the human body can conveniently collect biosignals, there are reliability issues due to noise generated in data collection. In order for wearable biosensors to be more widely used, the reliability of collected data should be improved. Research on flexible bio-chips in the field of material science and engineering might help develop new types of biosensors that resolve the issues of conventional wearable biosensors. Flexible bio-chips with higher precision can be used to collect various human data in academic research and in our daily lives. In this review, we present various types of conventional biosensors that have been used and discuss associated issues such as noise and inaccuracy. We then introduce recent studies on flexible bio-chips as a solution to these issues.

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

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

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

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

Corrigendum to "The Analysis of Biomechanical Properties of Proximal Femur after Implant Removal".

[This corrects the article DOI: 10.1155/2016/4987831.].

Preliminary Characterization of Glass/Alumina Composite Using Laser Powder Bed Fusion (L-PBF) Additive Manufacturing.

Powder bed fusion (PBF) additive manufacturing (AM) is currently used to produce high-efficiency, high-density, and high-performance products for a variety of applications. However, existing AM methods are applicable only to metal materials and not to high-melting-point ceramics. Here, we develop a composite material for PBF AM by adding Al2O3 to a glass material using laser melting. Al2O3 and a black pigment are added to a synthesized glass frit for improving the composite strength and increased laser-light absorption, respectively. Our sample analysis shows that the glass melts to form a composite when the mixture is laser-irradiated. To improve the sintering density, we heat-treat the sample at 750 °C to synthesize a high-density glass frit composite. As per our X-ray diffraction (XRD) analysis to confirm the reactivity of the glass frit and Al2O3, we find that no reactions occur between glass and crystalline Al2O3. Moreover, we obtain a high sample density of ≥95% of the theoretical density. We also evaluate the composite's mechanical properties as a function of the Al2O3 content. Our approach facilitates the manufacturing of ceramic 3D structures using glass materials through PBF AM and affords the benefits of reduced process cost, improved performance, newer functionalities, and increased value addition.

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

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

Application of Mesenchymal Stem Cell-Derived Extracellular Vesicles for Stroke: Biodistribution and MicroRNA Study.

Mesenchymal stem cells (MSCs) exert their therapeutic capability through a variety of bioactive substances, including trophic factors, microRNAs, and extracellular vesicles (EVs) in infarcted tissues. We therefore hypothesized that MSC-derived EVs (MSC-EVs) possess therapeutic molecules similar to MSCs. Moreover, given their nature as nanosized and lipid-shielded particles, the intravenous infusion of MSC-EVs would be advantageous over MSCs as a safer therapeutic approach. In this study, we investigated the biodistribution, therapeutic efficacy, and mode of action of MSC-EVs in a rat stroke model. MSC-EVs successfully stimulated neurogenesis and angiogenesis in vivo. When compared to the MSC-treated group, rats treated with MSC-EVs exhibited greater behavioral improvements than the control group (p < 0.05). Our biodistribution study using fluorescence-labeled MSC-EVs and MSCs demonstrated that the amounts of MSC-EVs in the infarcted hemisphere increased in a dose-dependent manner, and were rarely found in the lung and liver. In addition, MSC-EVs were highly inclusive of various proteins and microRNAs (miRNAs) associated with neurogenesis and/or angiogenesis compared to fibro-EVs. We further analyzed those miRNAs and found that miRNA-184 and miRNA-210 were essential for promoting neurogenesis and angiogenesis of MSC-EVs, respectively. MSC-EVs represent an ideal alternative to MSCs for stroke treatment, with similar medicinal capacity but an improved safety profile that overcomes cell-associated limitations in stem cell therapy.

Water Resistances of Ag2O-, BaO-, and CuO-Doped V2O5-P2O5-TeO2 Glass Sealants.

V2O5-P2O5-TeO2, a low-temperature vanadate-based glass sealant, was doped with metal oxides (MO = Ag2O, BaO, or CuO), which generate Ag, Ba, and Cu ions, respectively, to strengthen the glass structure and improve its water resistance. These ions reduce the number of nonbridging oxygen atoms in the glass structure by forming V-O-M or P-O-M crosslinks in the V2O5-P2O5 glass system. Structural analysis using Fourier-transform infrared spectroscopy indicated that the numbers of P-O-P, V═O, and V-O-V bonds decreased with increasing metal oxide content. Thermal property analyses revealed that the glass transition temperatures increased by approximately 2-30 °C and that the coefficients of thermal expansion only varied within approximately ±10×10-7 K-1 among all the glass samples. The contact angles were measured to quantify the wetting properties of the doped glasses. The contact angle increased from 11 to 36° with increasing metal oxide content at 410 °C. As an indication of the water resistances of the doped glasses, the dissolution rates of the 9 mol% Ag2O-doped and pure glasses were 0.078 and 0.523 g cm-2, respectively.

Production of Cu@SiO2 Core-Shell Nanoparticles with Antibacterial Properties.

Antimicrobial agents based on organic materials have limited use owing to their low heat resistance and short lifetimes. Therefore, various studies on antibacterial agents that are based on inorganic material systems are increasingly being performed to supplement them. In this study, Cu@SiO2 core-shells are fabricated using Cu cores and SiO2 shells, and are known to have antimicrobial effects. The core-shell was coated with SiO2 using the sol-gel method. Experiments were conducted using X-ray diffraction and the shaking flask method (KS J 4206) to evaluate the characteristics of the core-shell. In the case of X-ray diffraction, both the Cu core and the core-shell fabricated using the sol-gel process were characterized. Escherichia coli and Staphylococcus aureus were evaluated using the KS J 4206 method for their antibacterial properties. Through this study, it is confirmed that a Cu@SiO2 core-shell can be fabricated via the sol-gel method, and that Cu with a core-shell structure has antibacterial effects.

Effects of Melting Conditions on Cerium Oxidation State and Catalytic Properties of CeO2-P2O5 Glass Systems.

As with any solvent, stabilizing a multivalent element at a given oxidation state in glass depends on the thermodynamic conditions. The effects of temperature on the oxidation-reduction equilibrium have been previously noted with higher temperatures being more conducive to reduced states. Herein, 30CeO2-70P2O5 binary system glasses were prepared. The melting temperature and time dependency on Ce4+ and Ce3+ ion concentrations were studied using X-ray photoelectron spectroscopy. Different melting conditions were investigated at temperatures ranging from 1300 °C to 1500 °C for 60 min, and at 1400 °C for durations ranging from 30 min to 90 min. The changes in the catalytic properties of the glasses as a function of Ce4+ and Ce3+ ion concentrations were confirmed based on the changes in the decomposition starting temperatures using thermogravimetric analysis. The main changes in the oxidation states according to melting conditions were discussed in terms of the catalytic properties of CeO2-P2O5 glass systems.

Marine Biomaterial-Based Bioinks for Generating 3D Printed Tissue Constructs.

Biologically active materials from marine sources have been receiving increasing attention as they are free from the transmissible diseases and religious restrictions associated with the use of mammalian resources. Among various other biomaterials from marine sources, alginate and fish gelatin (f-gelatin), with their inherent bioactivity and physicochemical tunability, have been studied extensively and applied in various biomedical fields such as regenerative medicine, tissue engineering, and pharmaceutical products. In this study, by using alginate and f-gelatin's chemical derivatives, we developed a marine-based interpenetrating polymer network (IPN) hydrogel consisting of alginate and f-gelatin methacryloyl (f-GelMA) networks via physical and chemical crosslinking methods, respectively. We then evaluated their physical properties (mechanical strength, swelling degree, and degradation rate) and cell behavior in hydrogels. Our results showed that the alginate/f-GelMA hydrogel displayed unique physical properties compared to when alginate and f-GelMA were used separately. These properties included high mechanical strength, low swelling and degradation rate, and an increase in cell adhesive ability. Moreover, for the first time, we introduced and optimized the application of alginate/f-GelMA hydrogel in a three-dimensional (3D) bioprinting system with high cell viability, which breaks the restriction of their utilization in tissue engineering applications and suggests that alginate/f-GelMA can be utilized as a novel bioink to broaden the uses of marine products in biomedical fields.

Author Correction: Closed-type pre-treatment device for point-of-care testing of sputum.

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