Effect of Morphological Characteristics and Biomineralization of 3D-Printed Gelatin/Hyaluronic Acid/Hydroxyapatite Composite Scaffolds on Bone Tissue Regeneration.

The use of porous three-dimensional (3D) composite scaffolds has attracted great attention in bone tissue engineering applications because they closely simulate the major features of the natural extracellular matrix (ECM) of bone. This study aimed to prepare biomimetic composite scaffolds via a simple 3D printing of gelatin/hyaluronic acid (HA)/hydroxyapatite (HAp) and subsequent biomineralization for improved bone tissue regeneration. The resulting scaffolds exhibited uniform structure and homogeneous pore distribution. In addition, the microstructures of the composite scaffolds showed an ECM-mimetic structure with a wrinkled internal surface and a porous hierarchical architecture. The results of bioactivity assays proved that the morphological characteristics and biomineralization of the composite scaffolds influenced cell proliferation and osteogenic differentiation. In particular, the biomineralized gelatin/HA/HAp composite scaffolds with double-layer staggered orthogonal (GEHA20-ZZS) and double-layer alternative structure (GEHA20-45S) showed higher bioactivity than other scaffolds. According to these results, biomineralization has a great influence on the biological activity of cells. Hence, the biomineralized composite scaffolds can be used as new bone scaffolds in bone regeneration.

Study on the High Functional Contact Lens Material with Superior Oxygen Transmissibility and Antibacterial Properties.

The silicone monomer (SiH2) used in this study contains acrylate monomer, DMA (n,n-dimethylacrylamide). For the polymerization, PtOEP (platinum octaethylporphyrin) was added to the synthesized silicone, DMA (n,n-dimethyl acrylamide), HPMA (hydroxy propyl methacrylate), PEGMEMA [poly(ethylene glycol) methyl ether methylacrylate], EGDMA (ethylene glycol dimethacrylate) and the initiator AIBN (azobisisobutyronitrile) with various concentrations. Using the polymer produced through the thermal polymerization process, the optical and physical characteristics of high performance silicone hydrogel lenses were measured. The water content of sample containing PtOEP was in the range of 65.21∼66.56%. And also, oxygen permeability (Dk) of sample containing PtOEP was in the range of 30.20∼30.85 × 10-11 cm²/sec × mlO2/ml × mmHg. In case of the sample with nanoparticle, the oxygen permeability was higher than that of Ref. sample without nanoparticle. Silicone hydrogel monomer containing PtOEP was expected to be used usefully as a material for red tinted chromagen optical lens with high oxygen permeability for color amblyopia.

Conformations of adducts formed between the genotoxic benzo[a]pyrene-7,8-dione and 2'-deoxycytidine.

Benzo[a]pyrene-7,8-dione (BPQ) is formed by the activation of benzo[a]pyrene(B[a]P), which is an environmental toxic substance that is easily exposed in daily life, due to P450/epoxide hydrolase, and is a substance that induces DNA deformation by forming adducts with DNA. In this study, to investigate the form of bonding between BPQ and DNA, the structures of adducts between BPQ and 2'-deoxycytidine were examined. To examine BPQ-dC adduct conformation, geometry optimization of a total of 16 structural isomers was performed using the density functional theory method. In the structures of BPQ-dC adducts, for the cis-form, the angle between BPQ and dC is nearly perpendicular; but for the trans-form, the bending angle is small. The trans-form had a larger energy gap between ground state and excited state than the cis-form, and had a smaller HOMO-LUMO gap than the cis-form. Therefore, it was found that the trans-form absorbs stronger light and has higher reactivity than the cis-form. Molecular electrostatic potential was calculated and analyzed. The calculated ESP contour map shows the electrophilic and nucleophilic regions of the molecule.

Conformational analysis of genotoxic benzo[a]pyrene-7,8-dione-duplex DNA adducts using a molecular dynamics method (II).

The conformations of the benzo[a]pyrene-7,8-quinone (BPQ) modified oligonucleotide were investigated using molecular dynamic simulation. In the initial structures, the central guanine base was modified with BPQ resulting in the formation of four structurally distinguishable 10-(N2-deoxyguanosyl)-9,10-dihydro-9-hydroxy benzo[a]pyrene-7,8-dione adducts (BPQ-G3,4). Each of the oligonucleotide adduct consisted of two conformers, namely syn and anti conformations, depending on the rotation around the glycosidic bond between BPQ and the guanine base. The results revealed that the BPQ moiety was located in the major groove for all four syn conformers. The relative energies of these conformers were high, and the backbone largely deviated from the B-form. On the other hand, BPQ was located in the minor groove with relatively low energies, and backbone was retained in all of the anti conformer cases. The most conceivable BPQ-modified double stranded oligonucleotide structure was proposed from the energy calculation and the structural analysis.

Conformational analysis of genotoxic benzo[a]pyrene- 7,8-dione-duplex DNA adducts using a molecular dynamics method.

Benzo[a]pyrene-7,8-quinone (BPQ), a metabolite of the wide spread carcinogen benzo[a]pyrene, has been known to form adducts with DNA bases, namely guanine and adenine. The adducts include stereoisomers of the eight BPQ-G(1,2) and products of the formation of the cyclic five-member ring between BPQ and the G base. The stereochemical characteristics, structural properties, and thermodynamic aspects of the formation of the BPQ-G modified duplexes were investigated in this work using molecular modeling and a molecular dynamics (MD) simulation technique. Three conformations for the BPQ- oligonucleotides adduct showed the most favorable free energy (DeltaG). The molecular plane of the BPQ is nearly perpendicular to the DNA base plane for all three stable adducts. In two adducts, BPQ is located in the minor groove with either anti or syn conformations around the glycosidic bond. One of adducts in which BPQ locates into the major groove with the anti conformation around the glycosidic bond was also energetically possible. The resulting detailed structures of the three conceivable BPQ-oligonucleotide adducts are elucidated in this work.

A gel-forming poly-L: -guluronic acid produced from no guluronate-rich marine algae using new hydrolysis method: test for endovascular embolization.

To prepare a gel-forming poly-L-guluronic acid (Poly-G) from no guluronate-rich Laminaria japonica, a new hydrolysis method was employed with a lower HCl concentration (0.025-0.15 M) and a shorter treatment time (5 min). The Poly-Gs were set to measure purity, presence of poly-L-guluronic block, molecular weight distribution, polymer yield, viscosity, and compressive gel strength. Finally, the Poly-G was tested to embolize the renal vascular system by using a rabbit model and angiography. Optimized Poly-G could be selected with respect to wt% concentration, polymer yield, gel-forming stability, viscosity, and gel strength as an endovascular embolizing agent. Overall, 0.4-0.6% of 0.03 M-Poly-G obtained from acid treatment with 0.03 M of HCl had molecular weights greater than 80 kDa, and the best gelling capacity with an injectable viscosity (30-120 cP). It was successfully delivered into the vascular bed of a rabbit kidney and was shown angiographically to embolize the renal vascular system.

Two-port total laparoscopic hysterectomy with a multichannel port.

Total laparoscopic hysterectomy (TLH) is often performed in many countries. TLH has been shown to involve shorter hospital stays, as well as less bleeding and postoperative pain, than conventional abdominal hysterectomy. The skin incision for the laparoscopy is smaller than that of the laparotomy, but multiple incisions may decrease the cosmetic satisfaction of patients. The multiple puncture sites also increase the cost of trocars and trocar-associated complications, such as bleeding, hernias, and wound infection. Therefore, in this paper we introduce a two-port TLH technique with a multichannel port employing the transumbilical one-port technique performed by Ryu et al. To perform a two-port TLH, the ancillary 5-mm trocar was inserted at the left iliac fossa under laparoscopic view. The inserted umbilical trocar was removed and the skin incision was extended about 1.5 cm. An Alexis wound retractor XS (Applied Medical) was inserted through the extended umbilical wound. The wrist portion of a 6 (1/2) rubber glove covered the wound retractor, and two trocars were inserted into the fingers of the glove and ligated with rubber bands. A 5- or 10-mm laparoscope and atraumatic forceps were inserted through the umbilical port. TLH was performed in the usual manner. This two-port method would be cost-effective considering the conventional laparoscopic instruments. The development of multichannel trocars and minimized laparoscopic devices may confer less invasive operative techniques that also cause less scarring.

Molecular modeling study on the enantioselective binding of S- and R-ofloxacin to various DNA sequences.

The complex formation of S- and R-ofloxacin with the self-complementary oligonucleotides, namely d[ATAGCGCTAT](2), d[GCGATATCGC](2) and d[ATAICICTAT](2), were investigated by the molecular dynamics (MD) simulation. Four starting positions, including two intercalation positions with different insertion directions and two minor groove binding positions, were considered. The total energy of both S- and R-ofloxacin-d[ATAGCGCTAT](2) complex, in which ofloxacin binds in the minor groove of the oligonucleotide, were lower than any intercalation binding mode. For both enantiomers, formation of the complex with GC oligonucleotide is more favorable than AT and IC oligonucleotides. When S- and R-ofloxacin are compared, the S-enantiomer exhibits more favorable total energy and torsion angles in the complex formation. This result is in agreement with the experimental observation [Hwangbo et al., Eur J Pharm Sci 18, 197 (2003)]. In the complex, both enantiomers form two hydrogen bonds: one between the carbonyl group of ofloxacin and the amine group of G16 and the other between the fluorine group and the G6 amine for S-ofloxacin. However, only one hydrogen bond is formed between endocyclic hydrogen atom at the C2 position of adenine and inosine base and carbonyl group of ofloxacin, which may be the reason for the GC preferentiality of ofloxacin.

The light switch effect upon the association of [Ru(1,10-phenanthroline)2dipyrido[3,2-a:2',3'-c]phenazine]2+ with single stranded poly(dA) and poly(dT).

Large enhancement in the luminescence intensity of the Delta- and Lambda-Ru(phenanthroline)(2)dipyrido[3,2-a:2',3'-c]phenazine](2+) ([Ru(phen)(2)DPPZ](2+)) complexes upon their association with single stranded poly(dA) and poly(dT) is reported in this work. As the mixing ratio ([[Ru(phen)(2)DPPZ](2+)]/[DNA base]) increases, the luminescence intensity increase in a sigmoidal manner, indicating that the enhancement involves some cooperativity. At a high mixing ratio, the luminescence properties are affected by the nature of the DNA bases and not by the absolute configuration of the [Ru(phen)(2)DPPZ](2+) complex, indicating that the single stranded poly(dA) and poly(dT) do not recognize the configuration of the metal complex. In the case of the Lambda-[Ru(phen)(2)DPPZ](2+)-poly(dT) complex, the manner of the enhancement is somewhat different from the other Ru(II) complex-polynucelotide combinations: the luminescence intensity reached a maximum at an intermediate mixing ratio of 0.32, and gradually decreased as the mixing ratio increased. In contrast to other complexes at high mixing ratios, an upward bending curve was found in the Stern-Volmer plot, which indicates that the micro-environment of the Lambda-[Ru(phen)(2)DPPZ](2+) is heterogeneous. In the Delta-[Ru(phen)(2)DPPZ](2+)-poly(dT) complex case, formation of this highly luminescent species at an intermediate mixing ratio is far less effective.

Conformations of adducts formed between the genotoxic benzo[a]pyrene-7,8-dione and nucleosides studied by density functional theory.

Benzo[a]pyrene (BP) is a widely distributed environmental pollutant that is metabolized by mammalian cells to a variety of genotoxic and carcinogenic intermediates that form covalent adducts with cellular DNA. One such pathway involves the metabolic activation of BP by members of the aldo-keto-reductase (AKR) family of enzymes to the highly reactive ortho-quinone, benzo[a]pyrene-7,8-dione (BPQ). This compound has been reported to react with the 2'-deoxynucleosides, dA and dG, under physiological conditions. Four BPQ-dG adducts and two -dA adducts were identified by mass spectrometry and NMR methods [Balu et al. (2004) Chem. Res. Toxicol. 17, 827-838]. However, the detailed conformations and absolute configurations around the linkage site have not been resolved. In order to determine the full conformations of these purine adducts, we carried out quantum mechanical geometry optimization using density functional theory. In the case of the BPQ-guanine adducts, six possible structures, each of which consists of two isomers, were identified. However, in the case of the adenine adducts, only four isomers were identified. The results suggest that stereoisomeric adduct pairs are expected to adopt opposite orientations with respect to the 5'-->3' direction of the modified DNA strands. The stereochemistry-dependent variations in adduct orientation may produce different biological effects, as has been observed in the case of DNA adducts derived from other metabolites of polycyclic aromatic hydrocarbons.

Formation of Cu(II)-brazilin complex in the presence of DNA and its activities as chemical nuclease.

Brazilin, a traditional medicine for the treatment of pain and inflammation, forms a complex with Cu(II) in the presence as well as the absence of DNA. The Cu(II)-brazilin complex exhibited the strand cleavage activity for the pBR322 supercoiled DNA, converting supercoiled form to nicked form. The presence of various scavengers for the oxygen species suppresses or reduces the cleavage activity of the complex, indicating that the DNA cleavage is oxidative. The binding mode of the Cu(II)-brazilin complex was studied by absorption and CD spectroscopy. While a large metal-to-ligand charge transfer (MLCT) band was apparent when Cu(II) and brazilin was mixed in the presence and absence of DNA, the CD did not show any signal in the same region in the presence of DNA, suggesting a weak interaction between the Cu(II)-brazilin complex and DNA bases.

Simultaneous binding of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin and 4',6-diamidino-2-phenylindole at the minor grooves of poly(dA).poly(dT) and poly[d(A-T)(2)]: fluorescence resonance energy transfer between DNA bound drugs.

The spectral properties of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP) bound to poly(dA).poly(dT) and poly[d(A-T)(2)] in the presence and in the absence of 4',6-diamidino-2-phenylindole (DAPI) have been studied. DAPI fits deeply into the minor groove of both poly(dA).poly(dT) and poly[d(A-T)(2)], and TMPyP is also situated at the minor groove. The nature of the absorption, circular dichroism (CD), and flow linear dichroism (LD) spectra of the TMPyP-poly(dA).poly(dT) and -poly[d(A-T)(2)] complexes in the Soret band is essentially unaffected whether the minor groove is blocked by DAPI or not, although small variations been noticed in the presence of DAPI. Furthermore, a close analysis of the reduced LD spectrum in the Soret band results in angles of approximately 80 degrees and 55 degrees between transition moments of the TMPyP and DNA helix axes in the absence of DAPI. All these observations indicate that the side of TMPyP whose structure resembles that of classical minor groove binding drugs does not fit deeply into the minor groove. This suggests that TMPyP binds across the minor groove: two positively charged pyridiniumyl rings interact electrostatically with negatively charged phosphate groups of DNA. When DAPI and TMPyP are simultaneously bound to poly(dA).poly(dT) or poly[d(A-T)(2)], the fluorescence intensity of DAPI decreases as TMPyP concentration increases, indicating that the excited energy of DAPI is transferred to TMPyP.

In vivo 1H-MRS evaluation of malignant and benign breast diseases.

This study was purposed to evaluate malignant and benign breast diseases on the basis of detecting choline compounds by in vivo localized 1H-MR spectroscopy. Thirty-five patients, prior to surgical treatments, including 19 cancers and 16 benign diseases were examined with breast imaging coil and inversion recovery 1H-PRESS (TR/TE/TI: 2000 ms/288 ms/160 ms) MR spectroscopy. Detection of choline was compared with the results of biopsy in each case. Choline was observed in all 19 cancer patients, while it was not detected in all other benign diseases. Detecting sensitivity of choline widely varied over the cancer cases in the range of 2.4-12.7 (average SNR value of 5.4) depending on the water/fat suppression and voxel size. Localized 1H-MRS using breast imaging coil can provide excellent sensitivity and spectral resolution to detect choline compounds present in reasonably small voxel (<1.5 cm3) of breast cancer lesion. Substantially this finding would be useful for differential diagnosis between malignant and benign breast diseases on the basis of choline observation by 1H-MRS when combined use with MR imaging.

Anomalous excitonic CD of meso-tetrakis(3-N-methylpyridiniumyl)porphyrin bound to poly[d(A-T)(2)].

When meso-tetrakis(3-N-methylpyridiniumyl)porphyrin (m-TMPyP) formed a complex with poly[d(A-T)(2)], an intense bisignate excitonic CD in the Soret absorption region was observed. The excitonic CD of the m-TMPyP-poly[d(A-T)(2)] complex is unique in that no other combination of the related porphyrin, namely, meso-tetrakis(n-N-methylpyridiniumyl)porphyrin (where n = 2, 4), and polynucleotide including calf thymus DNA, poly[d(G-C)(2)], poly[d(I-C)(2)], and poly(dA).poly(dT), exhibits a comparable CD spectrum. From the [drug]/[DNA] ratio-dependence of the intensity and the shape of the CD spectrum, this porphyrin species is assigned to an extensively aggregated form. The extensively aggregated porphyrin disperses in 1 h after mixing to form moderately stacked porphyrin at a low mixing ratio. The magnitude of linear dichroism of the extensively aggregated porphyrin was small and the sign was negative in the Soret band, which indicated that the molecular plane of porphyrin in the complex is strongly tilted. On the other hand, the molecular plane of porphyrin is almost parallel to the DNA base plane (perpendicular to the DNA helix axis) in the moderately stacked form.

H-NMR studies of duplex DNA decamer containing a uracil cyclobutane dimer: implications regarding the high UV mutagenecity of CC photolesions.

To determine the origin of the UV-specific CC to TT tandem mutation at the CC site, we made a duplex DNA decamer containing a uracil cis-syn cyclobutane dimer (CBD) as the deaminated model of a cytosine dimer. Two-dimensional 1H-NMR spectroscopy studies were performed on this sequence where two adenines (Ade) were opposite to the uracil dimer. Two imino protons of the uracil dimer were found to retain Watson-Crick hydrogen bonding with the opposite Ade, although the 5'-U(NH) of the dimer site showed unusual upfield shift like that of the 5'-T(NH) of the TT dimer, which seemed to be associated with deshielding by the flanking base rather than with reduced hydrogen bonding. (McAteer et al. 1998, J. Mol. Biol. 282:1013-1032). Hydrogen bondings at the dimer site were also supported by detecting typical strong nuclear Overhauser effects (NOE) between two imino protons and the opposite Ade H2 or NH2. But sequential NOE interactions of base protons with sugar protons were absent at the two flanking nucleotides of the 5' side of the uracil dimer and at the intradimer site, contrasting with its thymine analog where sequential NOE was absent only at the A4-T5 step. In addition, NOE cross peak for U5(NH) <--> A4(H2) was detected, although the NOE interactions of U6(NH) with A7(H2) and A17(H2) were not observed in contrast to the thymine dimer duplex. This different local structural alteration may be affected by the induced right-hand twisted puckering mode of cis-syn cyclobutane ring of the uracil dimer in the B-DNA duplex, even though the isolated uracil dimer had left-hand twisted puckering rigidly. In parallel, these observations may be correlated with observed differences in mutagenic properties between cis-syn UU dimer and cis-syn TT dimer.

Rotation of periphery methylpyridine of meso-tetrakis(n-N-methylpyridiniumyl)porphyrin (n = 2, 3, 4) and its selective binding to native and synthetic DNAs.

By utilizing circular and linear dichroism, the binding mode of meso-tetrakis(n-N-methylpyridiniumyl)porphyrin (n = 2, 3, 4) to various DNAs was studied in this work. 2-N-(methylpyridiniumyl)porphyrin(o-TMPyP), in which rotation of the periphery pyridinium ring is prevented, exhibits similar spectral properties when bound to DNA, poly[d(G-C)(2)] and poly[d(A-T)(2)], suggesting a similar binding mode. Close analysis of the spectral properties led us to conclude that o-TMPyP sits in the major groove. However, both 3-N- and 4-N-(methylpyridiniumyl)porphyrin (m- and p-TMPyP), of which the periphery pyridinium ring is free to rotate, intercalate between the basepairs of DNA and poly[d(G-C)(2)]. In the presence of poly[d(A-T)(2)], m-TMPyP exhibits a typical bisignate excitonic CD spectrum in the Soret band, while p-TMPyP shows two positive CD bands. The excitonic CD spectrum of the m-TMPyP-poly[d(A-T)(2)] complex and the positive CD band of the o-TMPyP-poly[d(A-T)(2)] complex were not affected by the presence of the minor groove binding drug, 4',6-diamidino-2-phenylindole (DAPI), indicating that this porphyrin is bound in the major groove. In contrast, two positive CD bands of the p-TMPyP-poly[d(A-T)(2)] complex altered in the presence of DAPI. From the changes in CD spectrum and other spectral properties, a few possible binding modes for p-TMPyP to poly[d(A-T)(2)] are suggested.