Gelatin/Hyaluronic Acid Content in Hydrogels Obtained through Blue Light-Induced Gelation Affects Hydrogel Properties and Adipose Stem Cell Behaviors.

Composite hydrogels of hyaluronic acid and gelatin attract great attention in biomedical fields. In particular, the composite hydrogels obtained through processes that are mild for cells are useful in tissue engineering. In this study, hyaluronic acid/gelatin composite hydrogels obtained through a blue light-induced gelation that is mild for mammalian cells were studied for the effect of the content of each polymer in the precursor solution on gelation, properties of resultant hydrogels, and behaviors of human adipose stem cells laden in the hydrogels. Control of the content enabled gelation in less than 20 s, and also enabled hydrogels to be obtained with 0.5-1.2 kPa Young's modulus. Human adipose stem cells were more elongated in hydrogels with a higher rather than lower content of hyaluronic acid. Stem cell marker genes, Nanog, Oct4, and Sox2, were expressed more in the cells in the composite hydrogels with a higher content of hyaluronic acid compared with those in the hydrogel composed of gelatin alone and on tissue culture dishes. These results are useful for designing conditions for using gelatin/hyaluronic acid composite hydrogels obtained through blue light-induced gelation suitable for tissue engineering applications.

Visible Light-Induced Hydrogelation of an Alginate Derivative and Application to Stereolithographic Bioprinting Using a Visible Light Projector and Acid Red.

Visible light-induced hydrogelation is attractive for various biomedical applications. In this study, hydrogels of alginate with phenolic hydroxyl groups (Alg-Ph) were obtained by irradiating a solution containing the polymer, ruthenium II trisbipyridyl chloride ([Ru(bpy)3]2+) and sodium persulfate (SPS), with visible light. The hydrogelation kinetics and the mechanical properties of the resultant hydrogels were tunable by controlling the intensity of the light and the concentrations of [Ru(bpy)3]2+ and SPS. With appropriate concentrations of [Ru(bpy)3]2+ and SPS, the hydrogel could be obtained following approximately 10 s of irradiation using a normal desktop lamp. The hydrogelation process and the resultant hydrogel were cytocompatible; mouse fibroblast cells enclosed in the Alg-Ph hydrogel maintained more than 90% viability for 1 week. The solution containing Alg-Ph, [Ru(bpy)3]2+ and SPS was useful as a bioink for stereolithographic bioprinting. Cell-laden hydrogel constructs could be printed using the bioprinting system equipped with a visible light projector without a significant decrease in cell viability in the presence of photoabsorbent Acid Red 18. The hydrogel construct including a perfusable helical lumen of 1 mm in diameter could be fabricated using the printing system. These results demonstrate the significant potential of this visible light-induced hydrogelation system and the stereolithographic bioprinting using the hydrogelation system for tissue engineering and regenerative medicine.

Differentiation potential of human adipose stem cells bioprinted with hyaluronic acid/gelatin-based bioink through microextrusion and visible light-initiated crosslinking.

Bioprinting has a great potential to fabricate three-dimensional (3D) functional tissues and organs. In particular, the technique enables fabrication of 3D constructs containing stem cells while maintaining cell proliferation and differentiation abilities, which is believed to be promising in the fields of tissue engineering and regenerative medicine. We aimed to demonstrate the utility of the bioprinting technique to create hydrogel constructs consisting of hyaluronic acid (HA) and gelatin derivatives through irradiation by visible light to fabricate 3D constructs containing human adipose stem cells (hADSCs). The hydrogel was obtained from a solution of HA and gelatin derivatives possessing phenolic hydroxyl moieties in the presence of ruthenium(II) tris-bipyridyl dication and sodium ammonium persulfate. hADSCs enclosed in the bioprinted hydrogel construct elongated and proliferated in the hydrogel. In addition, their differentiation potential was confirmed by examining the expression of pluripotency marker genes and cell surface marker proteins, and differentiation to adipocytes in adipogenic differentiation medium. Our results demonstrate the great potential of the bioprinting method and the resultant hADSC-laden HA/gelatin constructs for applications in tissue engineering and regenerative medicine.

SOX10-Nano-Lantern Reporter Human iPS Cells; A Versatile Tool for Neural Crest Research.

The neural crest is a source to produce multipotent neural crest stem cells that have a potential to differentiate into diverse cell types. The transcription factor SOX10 is expressed through early neural crest progenitors and stem cells in vertebrates. Here we report the generation of SOX10-Nano-lantern (NL) reporter human induced pluripotent stem cells (hiPS) by using CRISPR/Cas9 systems, that are beneficial to investigate the generation and maintenance of neural crest progenitor cells. SOX10-NL positive cells are produced transiently from hiPS cells by treatment with TGFß inhibitor SB431542 and GSK3 inhibitor CHIR99021. We found that all SOX10-NL-positive cells expressed an early neural crest marker NGFR, however SOX10-NL-positive cells purified from differentiated hiPS cells progressively attenuate their NL-expression under proliferation. We therefore attempted to maintain SOX10-NL-positive cells with additional signaling on the plane and sphere culture conditions. These SOX10-NL cells provide us to investigate mass culture with neural crest cells for stem cell research.

Antitumor effects in gastrointestinal stromal tumors using photodynamic therapy with a novel glucose-conjugated chlorin.

Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract. Except for surgical resection, no effective treatment strategies have been established. Photodynamic therapy (PDT) consists of intravenous administration of a photosensitizer, activated by a specific wavelength of light, which produces reactive oxygen species that directly kill tumor cells. We analyzed the efficacy of PDT using a newly developed photosensitizer, 5,10,15,20-tetrakis [4-[ß-d-glucopyranosylthio-2,3,5,6-tetrafluorophenyl]-2,3,[methano[N-methyl] iminomethano] chlorin (H(2)TFPC-SGlc), for the GIST treatment. Various photosensitizers were administered in vitro to GIST (GIST-T1) and fibroblast (WI-38) cells, followed by irradiation, after which cell death was compared. We additionally established xenograft mouse models with GIST-T1 tumors and examined the accumulation and antitumor effects of these photosensitizers in vivo. In vitro, the expression of the glucose transporters GLUT1, GLUT3, and GLUT4, the cellular uptake of H(2)TFPC-SGlc, and apoptosis mediated by PDT with H(2)TFPC-SGlc were significantly higher in GIST-T1 than in WI-38 cells. In vivo, H(2)TFPC-SGlc accumulation was higher in xenograft tumors of GIST-T1 cells than in the adjacent normal tissue, and tumor growth was significantly suppressed following PDT. PDT with novel H(2)TFPC-SGlc is potentially useful for clinical applications about the treatment of GIST.

Anti-cancer effects of newly developed chemotherapeutic agent, glycoconjugated palladium (II) complex, against cisplatin-resistant gastric cancer cells.

BACKGROUND: Cisplatin (CDDP) is the most frequently used chemotherapeutic agent for various types of advanced cancer, including gastric cancer. However, almost all cancer cells acquire resistance against CDDP, and this phenomenon adversely affects prognosis. Thus, new chemotherapeutic agents that can overcome the CDDP-resistant cancer cells will improve the survival of advanced cancer patients. METHODS: We synthesized new glycoconjugated platinum (II) and palladium (II) complexes, [PtCl2 (L)] and [PdCl2 (L)]. CDDP-resistant gastric cancer cell lines were established by continuous exposure to CDDP, and gene expression in the CDDP-resistant gastric cancer cells was analyzed. The cytotoxicity and apoptosis induced by [PtCl2 (L)] and [PdCl2 (L)] in CDDP-sensitive and CDDP-resistant gastric cancer cells were evaluated. DNA double-strand breaks by drugs were assessed by evaluating phosphorylated histone H2AX. Xenograft tumor mouse models were established and antitumor effects were also examined in vivo. RESULTS: CDDP-resistant gastric cancer cells exhibit ABCB1 and CDKN2A gene up-regulation, as compared with CDDP-sensitive gastric cancer cells. In the analyses of CDDP-resistant gastric cancer cells, [PdCl2 (L)] overcame cross-resistance to CDDP in vitro and in vivo. [PdCl2 (L)] induced DNA double-strand breaks. CONCLUSION: These results indicate that [PdCl2 (L)] is a potent chemotherapeutic agent for CDDP-resistant gastric cancer and may have clinical applications.

Syntheses, characterization, and antitumor activities of platinum(II) and palladium(II) complexes with sugar-conjugated triazole ligands.

Four platinum(II) and palladium(II) complexes with sugar-conjugated bipyridine-type triazole ligands, [Pt(II)Cl(2)(AcGlc-pyta)] (3), [Pd(II)Cl(2)(AcGlc-pyta)] (4), [Pt(II)Cl(2)(Glc-pyta)] (5), and [Pd(II)Cl(2)(Glc-pyta)] (6), were prepared and characterized by mass spectrometry, elemental analysis, (1)H- and (13)C-NMR, IR as well as UV/VIS spectroscopy, where AcGlc-pyta and Glc-pyta denote 2-[4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl]ethyl 2,3,4,6-tetra-O-acetyl-ß-D-glucopyranoside (1) and 2-[4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl]ethyl ß-D-glucopyranoside (2), respectively. The solid-state structure of complex 6 was determined by single-crystal X-ray-diffraction analysis. These complexes exhibited in vitro cytotoxicity against human cervix tumor cells (HeLa) though weaker than that of cisplatin.

Anticancer effects of novel photodynamic therapy with glycoconjugated chlorin for gastric and colon cancer.

BACKGROUND/AIM: Photodynamic therapy (PDT) is an attractive, minimally invasive modality for cancer therapy that utilizes the interaction of light and photosensitizer. To improve the efficacy of PDT, development of cancer specificity of the photosensitizer is needed. Cancer cells consume more glucose than normal cells. In this study, the efficacy of PDT using a newly developed photosensitizer, glycoconjugated chlorin (H2TFPC-SGlc), was compared with Talaporfin, which is clinically used in Japan. MATERIALS AND METHODS: Photosensitizers were administered to gastric and colon cancer cell lines, followed by irradiation of light, and the cell death-inducing effects were compared. Xenograft tumor mouse models were established and photosensitizer accumulation was assessed and antitumor effects analyzed. RESULTS: In vitro, H(2)TFPC-SGlc was 30 times more cytotoxic to cancer cells than was Talaporfin. In vivo, H(2)TFPC-SGlc accumulation was higher in xenograft tumors and significantly suppressed tumor growth when compared with Talaporfin. CONCLUSION: This novel glycoconjugated chlorin is potentially useful in PDT.

[Measures taken for Influenza by organizing a relief unit in the nursing department].

Nurses have the most frequent contact with patients, and it makes their risk of infection very high. At the same time, they have a lot of opportunities to control patient's behavior to prevent infection. So it takes important part of precaution that all nurses sufficiently acknowledge their roles and responsibilities. Also, it's necessary to establish organization which can arrange competent nurses for influenza (A/H1N1) epidemic so that they can perform efficiently.

Effect and mechanism of a new photodynamic therapy with glycoconjugated fullerene.

When irradiated, fullerene efficiently generates reactive oxygen species (ROS) and is an attractive photosensitizer for photodynamic therapy (PDT). Ideally, photosensitizers for PDT should be water-soluble and tumor-specific. Because cancer cells endocytose glucose more effectively than normal cells, the characteristics of fullerene as a photosensitizer were improved by combining it with glucose. The cytotoxicity of PDT was studied in several cancer cell lines cultured with C(60)-(Glc)1 (D-glucose residue pendant fullerene) and C(60)-(6Glc)1 (a maltohexaose residue pendant fullerene) subsequently irradiated with UVA(1). PDT alone induced significant cytotoxicity. In contrast, PDT with the glycoconjugated fullerene exhibited no significant cytotoxicity against normal fibroblasts, indicating that PDT with these compounds targeted cancer cells. To investigate whether the effects of PDT with glycoconjugated fullerene were because of the generation of singlet oxygen ((1)O(2)), NaN(3) was added to cancer cells during irradiation. NaN(3) extensively blocked PDT-induced apoptosis, suggesting that PDT-induced cell death was a result of the generation of (1)O(2). Finally, to investigate the effect of PDT in vivo, melanoma-bearing mice were injected intratumorally with C(60)-(Glc)1 and irradiated with UVA(1). PDT with C(60)-(Glc)1 suppressed tumor growth. These findings indicate that PDT with glycoconjugated fullerene exhibits tumor-specific cytotoxicity both in vivo and in vitro via the induction of (1)O(2).

Extended X-ray absorption fine structure study on reaction of anti-tumor platinum complexes with reduced glutathione.

Reactions of cis-diamminedichloroplatinum(II) (cisplatin) and 1,1-cyclobutanedicarboxylatodiammineplatinum(II) (carboplatin) with reduced glutathione, a tripeptide that is abundant in cells, were studied by means of X-ray absorption spectroscopy. Back-scattering amplitudes F(i)(k) and phase shifts Phi(i)(k) were theoretically derived, and validated by applying them to calculate extended X-ray absorption fine structure (EXAFS) oscillations of cisplatin and K(2)[Pt(SCN)(4)] in the solid state. EXAFS oscillations of reaction mixtures of cisplatin or carboplatin with reduced glutathione were fitted to the standard EXAFS equation using the F(i)(k) and Phi(i)(k) functions to give the coordination numbers of N or O atoms (N(N/O)) and of Cl or S atoms (N(Cl/S)). For both cisplatin and carboplatin, the N(N/O) value decreased and the N(Cl/S) values increased monotonically as the reaction proceeded. However, the reaction rate for carboplatin was significantly slower than that for cisplatin.

Antioxidant action of sugar-pendant C60 fullerenes.

The action of C60 fullerene and its derivatives as a radical-scavenging antioxidant has received much attention, but their reactivity toward free radicals and antioxidant capacity have not been well elucidated yet. In the present study, the reactivity of the two types of water-soluble, sugar-pendant C60 fullerenes, C60-1S and C60-2S, toward peroxyl radical and their effect against human plasma lipid peroxidation were measured. The rate constants for the reaction of C60-1S and C60-2S with peroxyl radicals were obtained from their effect on the bleaching of beta-carotene in lipid-SDS micelle system as 4.6 x 10(3) and 8.0 x 10(3) M(-1) s(-1) at 37 degrees C, respectively. They inhibited the free radical-induced lipid peroxidation in human plasma in a concentration-dependent manner. These results suggest that the sugar-pendant fullerenes C60-1S and C60-2S act as a radical-scavenging antioxidant with the activity similar to the phenolic antioxidants.

Poly[[{µ(3)-tris-[2-(4-phenyl-1,2,3-triazol-1-yl)eth-yl]amine}silver(I)] hexa-fluorido-phosphate].

The title compound, {[Ag(L)]PF(6))(n) {L is tris-[2-(4-phenyl-1,2,3-triazol-1-yl)eth-yl]amine, C(30)H(30)N(10)}, consists of alternating two-dimensional cationic layers of [Ag(L)](+) and anionic PF(6) (-) layers. Each Ag(I) atom is three coordinated in a T-shaped geometry by three N atoms from three ligands. Each ligand links three Ag(I) atoms, generating a two-dimensional network structure with two different metallacycles, A and B. In A, eight coordination units from four ligands connect four Ag(I) atoms, forming a 48-membered ring. In B, four coordination units from two ligands link two Ag(I) atoms, forming a 24-membered ring. Each B ring is surrounded by four A rings, and each A ring has four A and four B rings as neighbours. This cationic layer thus generates a 4.8(2) topology network, with each Ag(I) centre and ligand acting as a three-connected topological node.

In situ generation of oxo-sulfidobis(dithiolene)tungsten(VI) complexes: active-site models for the aldehyde ferredoxin oxidoreductase family of tungsten enzymes.

Oxo-sulfidobis(dithiolene)tungsten(VI) complexes were prepared in situ by the reaction of oxobis(dithiolene)tungsten(V) precursors with hydrosulfide (SH-). The complexes, characterized by UV-vis, electrospray ionization mass spectrometry, IR, and resonance Raman spectroscopies, model the proposed coordination environment and observed hydrolytic reactions of members of the aldehyde ferredoxin oxidoreductase family of tungsten enzymes.

Modeling the mononuclear, dinuclear, and trinuclear copper(I) reaction centers of copper proteins using pyridylalkylamine ligands connected to 1,3,5-triethylbenzene spacer.

The structure and O2-reactivity of copper(I) complexes supported by novel ligands, Pye2 (1,3,5-triethyl-2,4-bis((N-benzyl-N-(2-(pyridin-2-yl)ethyl)-)aminomethyl)benzene), Pye3 (1,3,5-triethyl-2,4,6-tris((N-benzyl-N-(2-(pyridin-2-yl)ethyl))aminomethyl)benzene), MePym2 (1,3,5-triethyl-2,4-bis((N-benzyl-N-(6-methylpyridin-2-ylmethyl))aminomethyl)benzene), and MePym3 (1,3,5-triethyl-2,4,6-tris((N-benzyl-N-(6-methylpyridin-2-ylmethyl))aminomethyl)benzene) have been examined. The ligands are designed to construct mono-, di-, and trinuclear copper(I) complexes by connecting two or three pyridylalkylamine metal-binding sites to a 1,3,5-triethylbenzene spacer. Thus, the reaction of the ligands with [CuI(CH3CN)4]X (X = PF6, CF3SO3) or CuICl gave the expected mononuclear copper(I) complexes [CuI(Pye2)(CF3SO3)] (1) and [CuI(Pye3)](CF3SO3) (2), dinuclear copper(I) complex [CuI2(MePym2)(Cl)]CuICl2 (3), and trinuclear copper(I) complex [CuI3(MePym3)(CH3CN)3](CF3SO3)3 (4), the structures of which were determined by X-ray crystallographic analysis. The mononuclear copper(I) complexes, 1 and 2, exhibit a distorted three-coordinate T-shape structure and a trigonal planar structure, respectively, which are very close to the coordination geometry of the CuA site of PHM (peptidylglycine alpha-hydroxylating monooxygenase) and the CuB site of CcO (cytochrome c oxidase). Notably, 1 and 2 showed a significantly high oxidation potential (990 mV vs SCE), thus showing virtually no reactivity toward O2. On the other hand, the metal centers of the dinuclear and trinuclear copper(I) complexes, 3 and 4, exhibit a distorted trigonal planar geometry and a trigonal pyramidal geometry, respectively. In contrast to the mononuclear copper(I) complexes, these dinuclear and trinuclear copper(I) complexes reacted with O2 to induce an aromatic ligand hydroxylation reaction involving an NIH-shift of one of the ethyl substituents on the benzene spacer. The NIH-shift of the alkyl substituent on the aromatic ring is strong evidence of the electrophilic aromatic substitution mechanism, although the active oxygen intermediate could not be directly detected during the course of the reaction. The biological relevance of the copper(I) complexes is also discussed on the basis of structure and O2-reactivity.

Structure and photoluminescence property of two-dimensional coordination polymer complexes involving Cu(I)(6)X(6)(X = Cl, Br, I) hexagon prism cluster supported by a tripodal tripyridine ligand with 1,3,5-triethylbenzene spacer.

Treatment of cuprous halide (Cu(I)X, X = Cl, Br, and I) and a tripodal tripyridine ligand (L) consisting of a 1,3,5-triethylbenzene spacer gave a unique two-dimensional (2D) polymer sheet structure involving a rare Cu(I)(6)X(6) hexagon prism cluster unit, which exhibits intense fluorescence around 448-476 nm.

Supramolecular and coordination polymer complexes supported by a tripodal tripyridine ligand containing a 1,3,5-triethylbenzene spacer.

By By combining a tripodal tripyridine ligand containing a 1,3,5-triethylbenzene spacer (L) with several divalent transition-metal chlorides, we have selectively prepared a capsule-type supramolecular complex, [PdII3(L)2Cl6] x 2H2O, and one-dimensional (1D) coordination polymer complexes, ([CuII(L)Cl2] x C2H5OH)n, ([CoII3(L)2Cl6] x 2CH2Cl2)n, and ([ZnII3(L)2Cl6] x 2H2O)n, with a zigzag polymer chain, a linear polymer chain, and a ladder polymer chain structure, respectively. All the structures were established in detail by single-crystal X-ray diffraction analysis, and the factors inducing the structural differences among the complexes are discussed by taking account of the differences in coordination geometry (square planar vs tetrahedral) as well as metal-ligand binding strength in the complexes.