A multi-channel collagen conduit with aligned Schwann cells and endothelial cells for enhanced neuronal regeneration in spinal cord injury.

Traumatic spinal cord injury (SCI) leads to Wallerian degeneration and the accompanying disruption of vasculature leads to ischemia, which damages motor and sensory function. Therefore, understanding the biological environment during regeneration is essential to promote neuronal regeneration and overcome this phenomenon. The band of Büngner is a structure of an aligned Schwann cell (SC) band that guides axon elongation providing a natural recovery environment. During axon elongation, SCs promote axon elongation while migrating along neovessels (endothelial cells [ECs]). To model this, we used extrusion 3D bioprinting to develop a multi-channel conduit (MCC) using collagen for the matrix region and sacrificial alginate to make the channel. The MCC was fabricated with a structure in which SCs and ECs were longitudinally aligned to mimic the sophisticated recovering SCI conditions. Also, we produced an MCC with different numbers of channels. The aligned SCs and ECs in the 9-channel conduit (9MCC-SE) were more biocompatible and led to more proliferation than the 5-channel conduit (5MCC-SE) in vitro. Also, the 9MCC-SE resulted in a greater healing effect than the 5MCC-SE with respect to neuronal regeneration, remyelination, inflammation, and angiogenesis in vivo. The above tissue recovery results led to motor function repair. Our results show that our 9MCC-SE model represents a new therapeutic strategy for SCI.

FeS2-incorporated 3D PCL scaffold improves new bone formation and neovascularization in a rat calvarial defect model.

199Three-dimensional (3D) scaffolds composed of various biomaterials, including metals, ceramics, and synthetic polymers, have been widely used to regenerate bone defects. However, these materials possess clear downsides, which prevent bone regeneration. Therefore, composite scaffolds have been developed to compensate these disadvantages and achieve synergetic effects. In this study, a naturally occurring biomineral, FeS2, was incorporated in PCL scaffolds to enhance the mechanical properties, which would in turn influence the biological characteristics. The composite scaffolds consisting of different weight fractions of FeS2 were 3D printed and compared to pure PCL scaffold. The surface roughness (5.77-fold) and the compressive strength (3.38-fold) of the PCL scaffold was remarkably enhanced in a dose-dependent manner. The in vivo results showed that the group with PCL/ FeS2 scaffold implanted had increased neovascularization and bone formation (2.9-fold). These results demonstrated that the FeS2 incorporated PCL scaffold might be an effective bioimplant for bone tissue regeneration.

Advances in the development of tubular structures using extrusion-based 3D cell-printing technology for vascular tissue regenerative applications.

Until recent, there are no ideal small diameter vascular grafts available on the market. Most of the commercialized vascular grafts are used for medium to large-sized blood vessels. As a solution, vascular tissue engineering has been introduced and shown promising outcomes. Despite these optimistic results, there are limitations to commercialization. This review will cover the need for extrusion-based 3D cell-printing technique capable of mimicking the natural structure of the blood vessel. First, we will highlight the physiological structure of the blood vessel as well as the requirements for an ideal vascular graft. Then, the essential factors of 3D cell-printing including bioink, and cell-printing system will be discussed. Afterwards, we will mention their applications in the fabrication of tissue engineered vascular grafts. Finally, conclusions and future perspectives will be discussed.

Overcome the barriers of the skin: exosome therapy.

Exosomes are nano-sized cargos with a lipid bilayer structure carrying diverse biomolecules including lipids, proteins, and nucleic acids. These small vesicles are secreted by most types of cells to communicate with each other. Since exosomes circulate through bodily fluids, they can transfer information not only to local cells but also to remote cells. Therefore, exosomes are considered potential biomarkers for various treatments. Recently, studies have shown the efficacy of exosomes in skin defects such as aging, atopic dermatitis, and wounds. Also, exosomes are being studied to be used as ingredients in commercialized skin treatment products. In this review, we discussed the need for exosomes in skin therapy together with the current challenges. Moreover, the functional roles of exosomes in terms of skin treatment and regeneration are overviewed. Finally, we highlighted the major limitations and the future perspective in exosome engineering.

3D-printed gelatin methacrylate (GelMA)/silanated silica scaffold assisted by two-stage cooling system for hard tissue regeneration.

Among many biomaterials, gelatin methacrylate (GelMA), a photocurable protein, has been widely used in 3D bioprinting process owing to its excellent cellular responses, biocompatibility and biodegradability. However, GelMA still shows a low processability due to the severe temperature dependence of viscosity. To overcome this obstacle, we propose a two-stage temperature control system to effectively control the viscosity of GelMA. To optimize the process conditions, we evaluated the temperature of the cooling system (jacket and stage). Using the established system, three GelMA scaffolds were fabricated in which different concentrations (0, 3 and 10 wt%) of silanated silica particles were embedded. To evaluate the performances of the prepared scaffolds suitable for hard tissue regeneration, we analyzed the physical (viscoelasticity, surface roughness, compressive modulus and wettability) and biological (human mesenchymal stem cells growth, western blotting and osteogenic differentiation) properties. Consequently, the composite scaffold with greater silica contents (10 wt%) showed enhanced physical and biological performances including mechanical strength, cell initial attachment, cell proliferation and osteogenic differentiation compared with those of the controls. Our results indicate that the GelMA/silanated silica composite scaffold can be potentially used for hard tissue regeneration.

Improved spectral resolution of the femtosecond stimulated Raman spectroscopy achieved by the use of the 2nd-order diffraction method.

Prolongation of the picosecond Raman pump laser pulse in the femtosecond stimulated Raman spectroscopy (FSRS) setup is essential for achieving the high spectral resolution of the time-resolved vibrational Raman spectra. In this work, the 2nd-order diffraction has been firstly employed in the double-pass grating filter technique for realizing the FSRS setup with the sub-5 cm-1 spectral resolution. It has been experimentally demonstrated that our new FSRS setup gives rise to a highly-resolved Raman spectrum of the excited trans-stilbene, which is much improved from those reported in the literatures. The spectral resolution of the present FSRS system has been estimated to be the lowest value ever reported to date, giving Δν = 2.5 cm-1.

Role of coherent nuclear motion in the ultrafast intersystem crossing of ruthenium complexes.

Ultrafast intersystem crossing (ISC) in transition metal complexes leads to a long-lived active state with a high yield, which leads to efficient light energy conversion. The detailed mechanism of ISC may lead to a rational molecular design of superior transition metal complexes. Coherent nuclear wave packets observed in femtosecond time-resolved spectroscopies provide important information on the excited-state dynamics. In particular, analyzing the nuclear wave packets in both the reactant and the product may unveil the molecular dynamics of an ultrafast reaction. In this study, experimental evidence proving the reaction coordinates of the ultrafast ISC of ruthenium(ii) complexes is presented using coherent vibrational spectroscopy with a quantum chemical simulation of coherent vibrational motion. We observed vibrational modes strongly coupled to the ISC, whose vibrational coherences undergo remarkable attenuation after the ISC. The coupled modes contain metal-ligand stretching or symmetry breaking components, and the faster ISC rates of lower-symmetry ruthenium(ii) complexes support the significance of the latter.

Bioprinting of Multiscaled Hepatic Lobules within a Highly Vascularized Construct.

Highly vascularized complex liver tissue is generally divided into lobes, lobules, hepatocytes, and sinusoids, which can be viewed under different types of lens from the micro- to macro-scale. To engineer multiscaled heterogeneous tissues, a sophisticated and rapid tissue engineering approach is required, such as advanced 3D bioprinting. In this study, a preset extrusion bioprinting technique, which can create heterogeneous, multicellular, and multimaterial structures simultaneously, is utilized for creating a hepatic lobule (≈1 mm) array. The fabricated hepatic lobules include hepatic cells, endothelial cells, and a lumen. The endothelial cells surround the hepatic cells, the exterior of the lobules, the lumen, and finally, become interconnected with each other. Compared to hepatic cell/endothelial cell mixtures, the fabricated hepatic lobule shows higher albumin secretion, urea production, and albumin, MRP2, and CD31 protein levels, as well as, cytochrome P450 enzyme activity. It is found that each cell type with spatial cell patterning in bioink accelerates cellular organization, which could preserve structural integrity and improve cellular functions. In conclusion, preset extruded hepatic lobules within a highly vascularized construct are successfully constructed, enabling both micro- and macro-scale tissue fabrication, which can support the creation of large 3D tissue constructs for multiscale tissue engineering.

Pre-set extrusion bioprinting for multiscale heterogeneous tissue structure fabrication.

Recent advances in three-dimensional bioprinting technology have led to various attempts in fabricating human tissue-like structures. However, current bioprinting technologies have limitations for creating native tissue-like structures. To resolve these issues, we developed a new pre-set extrusion bioprinting technique that can create heterogeneous, multicellular, and multimaterial structures simultaneously. The key to this ability lies in the use of a precursor cartridge that can stably preserve a multimaterial with a pre-defined configuration that can be simply embedded in a syringe-based printer head. The multimaterial can be printed and miniaturized through a micro-nozzle without conspicuous deformation according to the pre-defined configuration of the precursor cartridge. Using this system, we fabricated heterogeneous tissue-like structures such as spinal cords, hepatic lobule, blood vessels, and capillaries. We further obtained a heterogeneous patterned model that embeds HepG2 cells with endothelial cells in a hepatic lobule-like structure. In comparison with homogeneous and heterogeneous cell printing, the heterogeneous patterned model showed a well-organized hepatic lobule structure and higher enzyme activity of CYP3A4. Therefore, this pre-set extrusion bioprinting method could be widely used in the fabrication of a variety of artificial and functional tissues or organs.

Three-Dimensional Hepatocellular Carcinoma/Fibroblast Model on a Nanofibrous Membrane Mimics Tumor Cell Phenotypic Changes and Anticancer Drug Resistance.

Three-dimensional (3D) in vitro tissue or organ models can effectively mimic the complex microenvironment of many types of human tissues for medical applications. Unfortunately, development of 3D cancer models, which involve cancer/stromal cells in a 3D environment, has remained elusive due to the extreme complexity of the tumor microenvironment (TME) and the stepwise progression of human cancer. Here, we developed hepatocellular carcinoma (HCC) models, which consist of fibroblasts as stromal cells, HCC cells, and a nanofibrous membrane to mimic the complex TME. The 3D HCC models were fabricated using three distinct culture methods: cancer cells grown directly on the nanofibrous membrane (mono model), fibroblasts covering the nanofibrous membrane (layer model), and both cancer cells and fibroblasts grown on the nanofibrous membrane (mixed model). Interestingly, the mono model and layer model showed similar tissue structures, whereas the mixed model resulted in phenotypic changes to the cancer cells. Further analysis demonstrated that the mixed models promoted the expression of fibronectin and vimentin, and showed higher resistance to anticancer drugs compared with the other models. Thus, our 3D HCC model could be utilized for testing efficient anticancer therapies at various stages of cancer, with potential application to different tumor types.

Femtosecond-Resolved Excited State Relaxation Dynamics of Copper (II) Tetraphenylporphyrin (CuTPP) After Soret Band Excitation.

Excited state relaxation dynamics of Copper (II) tetraphenylporphyrin (CuTPP) after Soret band excitation have been investigated in various solvents by femtosecond broadband transient absorption spectroscopy. Significant role of charge transfer state has been confirmed from fast relaxation of triplet CuTPP in pyridine, giving τ ~ 26.5 ps. In piperidine, the transient measured at 480 nm shows biexponential behavior with distinct time constants of 300 fs and 27.4 ps. The fast component with τ ~ 300 fs is attributed to relaxation of the CuTPP-piperidine adduct populated in the ground state, giving the intrinsic relaxation rate of the CuTPP exciplex for the first time. For CuTPP in O-coordinating solvents of 1,4-dioxane and tetrahydrofuran (THF), a completely new relaxation channel via the 2[dz2, dx2-y2] state is opened. As the exciplex formation is diffusion controlled, triplet CuTPP lifetimes in pure solvents employed here are all measured to be more or less same to give ~30 ps, whereas the 2[dz2, dx2-y2] exciplex formed by the ligation with O-coordinating solvents is found to relax much slowly to the ground state, giving lifetimes of ~360 and ~270 ps in 1,4-dioxane and THF, respectively.

Fabrication of In Vitro Cancer Microtissue Array on Fibroblast-Layered Nanofibrous Membrane by Inkjet Printing.

In general, a drug candidate is evaluated using 2D-cultured cancer cells followed by an animal model. Despite successful preclinical testing, however, most drugs that enter human clinical trials fail. The high failure rates are mainly caused by incompatibility between the responses of the current models and humans. Here, we fabricated a cancer microtissue array in a multi-well format that exhibits heterogeneous and batch-to-batch structure by continuous deposition of collagen-suspended Hela cells on a fibroblast-layered nanofibrous membrane via inkjet printing. Expression of both Matrix Metalloproteinase 2 (MMP2) and Matrix Metalloproteinase 9 (MMP9) was higher in cancer microtissues than in fibroblast-free microtissues. The fabricated microtissues were treated with an anticancer drug, and high drug resistance to doxorubicin occurred in cancer microtissues but not in fibroblast-free microtissues. These results introduce an inkjet printing fabrication method for cancer microtissue arrays, which can be used for various applications such as early drug screening and gradual 3D cancer studies.

Precise stacking of decellularized extracellular matrix based 3D cell-laden constructs by a 3D cell printing system equipped with heating modules.

Three-dimensional (3D) cell printing systems allow the controlled and precise deposition of multiple cells in 3D constructs. Hydrogel materials have been used extensively as printable bioinks owing to their ability to safely encapsulate living cells. However, hydrogel-based bioinks have drawbacks for cell printing, e.g. inappropriate crosslinking and liquid-like rheological properties, which hinder precise 3D shaping. Therefore, in this study, we investigated the influence of various factors (e.g. bioink concentration, viscosity, and extent of crosslinking) on cell printing and established a new 3D cell printing system equipped with heating modules for the precise stacking of decellularized extracellular matrix (dECM)-based 3D cell-laden constructs. Because the pH-adjusted bioink isolated from native tissue is safely gelled at 37 °C, our heating system facilitated the precise stacking of dECM bioinks by enabling simultaneous gelation during printing. We observed greater printability compared with that of a non-heating system. These results were confirmed by mechanical testing and 3D construct stacking analyses. We also confirmed that our heating system did not elicit negative effects, such as cell death, in the printed cells. Conclusively, these results hold promise for the application of 3D bioprinting to tissue engineering and drug development.

Engineering Reaction Kinetics by Tailoring the Metal Tips of Metal-Semiconductor Nanodumbbells.

Semiconductor-metal hybrid nanostructures are one of the best model catalysts for understanding photocatalytic hydrogen generation. To investigate the optimal structure of metal cocatalysts, metal-CdSe-metal nanodumbbells were synthesized with three distinct sets of metal tips, Pt-CdSe-Pt, Au-CdSe-Au, and Au-CdSe-Pt. Photoelectrochemical responses and transient absorption spectra showed that the competition between the charge recombination at the metal-CdSe interface and the water reduction on the metal surface is a detrimental factor for the apparent hydrogen evolution rate. For instance, a large recombination rate (krec) at the Pt-CdSe interface limits the quantum yield of hydrogen generation despite a superior water reduction rate (kWR) on the Pt surface. To suppress the recombination process, Pt was selectively deposited onto the Au tips of Au-CdSe-Au nanodumbbells in which the krec was diminished at the Au-CdSe interface, and the large kWR was maintained on the Pt surface. As a result, the optimal structure of the Pt-coated Au-CdSe-Au nanodumbbells reached a quantum yield of 4.84%. These findings successfully demonstrate that the rational design of a metal cocatalyst and metal-semiconductor interface can additionally enhance the catalytic performance of the photochemical hydrogen generation reactions.

Endothelial monolayers on collagen-coated nanofibrous membranes: cell-cell and cell-ECM interactions.

Endothelial cells (ECs) form a monolayer lining over the entire vascular wall and play an important role in maintaining vascular homeostasis and cancer metastasis. Loss of proper endothelial function can lead to vascular diseases. Therefore, the endothelial monolayer is particularly important in tissue regeneration and mimicking vascular tissue in vitro. Numerous studies have described the effects of ECs on nanofibers made from a variety of synthetic polymer materials designed to mimic the extracellular matrix (ECM). However, little is known about maintaining the integrity of ECs in in vitro systems. Here we describe polycaprolactone nanofibrous membranes coated with collagen gel that overcome many limitations of conventional nanofibers used for engineering endothelia. We investigated cell-cell and cell-ECM junctional complexes using collagen-coated and conventional nanofibrous membranes. Conventional nanofibrous membranes alone did not form a monolayer with ECs, whereas collagen-coated nanofibrous membranes did. Several concentrations of collagen in the gel coating promoted the formation of cell-cell junctional complexes, facilitated the deposition of laminin, and increased the focal contact organization of ECs. These results suggest the possible use of collagen-coated nanofibrous membranes for vascular tissue engineering applications and a vascular platform for organ-on-a-chip systems.

Effect of myocardial protection of intracoronary adenosine and nicorandil injection in patients undergoing non-urgent percutaneous coronary intervention: a randomized controlled trial.

BACKGROUND: Several studies have demonstrated that adenosine and nicorandil protect the myocardium against angioplasty-related myocardial injury. We conducted a prospective study to investigate the myocardial protective effects of combination therapy with intracoronary adenosine and nicorandil. METHODS: We enrolled 213 consecutive patients with stable or unstable angina who were scheduled for non-urgent PCI for de-novo coronary lesions. Patients were randomized into group I (control saline, n=55), group II (adenosine 50 µg, n=54), group III (nicorandil 4 mg, n=54), or group IV (adenosine-nicorandil combination, n=50). Serial assessments of CK-MB were used to assess myocardial necrosis before and after PCI. The primary endpoint was the incidence of myocardial necrosis (elevation of CK-MB), and the secondary endpoints were the changes in serum CK-MB and cTnI levels and the incidence of post-procedural myocardial infarction (MI). RESULTS: No significant differences were observed among the four groups with regard to baseline or angiographic characteristics. No major adverse events related to adenosine and nicorandil were observed. There were no significant differences in the incidence of post-procedural myocardial necrosis among the four groups (10.9, 14.8, 14.8, and 14.0%, respectively, p=0.9). There were no significant differences in the incidence of post-procedural MI among groups (p=0.6). In multivariate regression analysis, multivessel stenting, median stent length, and the presence of a compromised side branch were independent predictors of myonecrosis. CONCLUSIONS: Pretreatment with intracoronary adenosine, nicorandil, or the combination of the two drugs did not reduce the incidences of myocardial necrosis or MI after non-urgent PCI in patients with low-risk angina pectoris.

[Distribution of hepatitis C virus genotypes in Jeju Island].

BACKGROUNDS/AIMS: The hepatitis C virus (HCV) genotype affects clinical outcomes of HCV infection, in terms of the response to antiviral therapy and progression of chronic liver diseases, and shows geographic differences in distribution. The aim of this study was to elucidate the HCV genotypes in patients with chronic HCV infection in Jeju, which is an island off the Korean peninsula. METHODS: The study population consisted of 162 patients with anti-HCV antibodies and HCV-RNA. HCV genotypes were determined using genotype specific primers. RESULTS: HCV genotype 2a predominated (62.3%), followed by genotype 1b (34.0%) and 2b (3.7%). The prevalence of genotypes differed significantly with age, with HCV genotypes 1 and 2 being more frequent in older and younger subjects (P=0.035), respectively. HCV-RNA levels were higher in patients with genotype 1 than in those with genotype 2 (P=0.001). HCV genotype was not significantly related to sex, clinical diagnosis and potential risk factors. CONCLUSIONS: HCV genotype 2a is most common in Jeju, followed by genotype 1b. Our results suggest that the distribution of the HCV genotype differs between regions in Korea.