医用含离子嵌段聚醚氨酯的合成、精细结构与性能的研究

合成了新的扩链剂8-二(2-羟乙基)胺基-1-辛磺酸钠和新的大分子单体——含烷基磺酸钠的端烃基丙二酸二聚醚酯,并由此合成了一系列精细结构明确的、分别在硬段或软段上悬挂有正辛基磺酸钠侧链的阴离子型聚醚氨酯,通过FTIR、粘弹谱及透射电镜的研究、血液相容性的评价,阐述了精细结构、分子中的相互作用、微相分离和性能的关系。     

Effect of thermal treatment on the structure and the biomedical properties of polyetherurethane film containing dipeptides of L-serine

Prepolymers which were obtained by the reaction of 4,4'-diphenylmethane diisocyanate (MDI) with polytetramethylene glycol (PTMG) and polyethylene glycol (PEG) were chain-extended by the reaction with the linear or cyclic dipeptide of L-serine to yield a novel polyetherurethane containing dipeptide segments. In these polyetherurethanes, spherulites, fibrous structures, and needle-like crystals were formed according to the conditions used. Polyetherurethanes containing the cyclic dipeptide segments exhibit a thermally stable conformation when cast from solution at room temperature. On the other hand, polyetherurethanes containing the linear dipeptide segments exhibit a thermally stable conformation only after annealing. The properties of polyetherurethanes were elucidated by investigation of their morphology by electron microscopy, surface composition by electron spectroscopy for chemical analysis, hydrogen-bonding properties by i.r. spectroscopy, and degree of crystallinity by differential scanning calorimetry. The antithrombogenicity of polyetherurethane films containing linear dipeptides was increased by annealing. Their oxygen permeability was controlled by the linear or cyclic dipeptide segments or by annealing. The urea permeability of hydrophilic polyetherurethane film was easily controlled by the casting conditions and molecular weight of the polyether component. The relationship between the morphology and biomedical properties of these polyetherurethane films is discussed.     

Synthesis of polyetherurethanes containing L-serine dipeptide and their use as materials for biomedical films

Prepolymers, which were produced by the polyaddition reaction of polytetramethylene glycol (PTMG) or polyethylene glycol (PEG) and 4,4'-diphenylmethane diisocyanate (MDI) or hexamethylene diisocyanate (HMDI), were chain-extended with a linear dipeptide of L-serine (Z-Ser-Ser-OMe) or a cyclic dipeptide of L-serine [c-(Ser)2] to yield novel polyetherurethanes containing dipeptide segments. The relationship between the surface morphology and the biomedical properties of the film of the novel polyetherurethanes was investigated. The surface of PU(PTMG,Z-Ser-Ser-OMe,MDI) film was smooth, but fibrous structures were developed in the bulk of the film with increasing molecular weight of the PTMG segment. The antithrombogenicity of the film containing the low molecular weight PTMG segment was better than that of the usual polyetherurethane film without the dipeptide segments. The partial hydrolysis of the ester groups involved in the dipeptide segment improved the antithrombogenicity. In the surface and the bulk of PU[PTMG,c-(Ser)2,MDI] film, spherulite structures were developed when the molecular weight of the PTMG segment was high, while single crystals with a length of 3-4 microns were produced when the molecular weight of the PTMG segment was low. The antithrombogenicity of the film containing the high molecular weight PTMG segment was better than that of the usual polyetherurethane film without the dipeptide segments. PU(PTMG/PEG,Z-Ser-Ser-OMe,MDI) film and PU[PTMG/PEG,c-(Ser)2,MDI] film were permeated by uraemic toxins. The permeation was accelerated with increasing water content of the film and decreasing molecular weight of the solute. The oxygen permeability of the film of the polyetherurethane containing the linear or cyclic dipeptide segments was greater than that of polyetherurethane film which does not contain the dipeptide segments.     

Synthetic peptide segments from the Escherichia coli porin OmpF constitute leukocyte activators.

After characterization of the porin OmpF and selection of molecular structures responsible for leukocyte activation by using computer-assisted epitope analysis, the analogs OmpF (153-174) (containing amino acids 153 to 174), OmpF (157-174), and OmpF (275-285) were synthesized and tested. Like the native protein, the segments were mitogenic for BALB/c splenocytes and induced B lymphocyte differentiation into antibody-producing plasma cells and tumor cytotoxicity of macrophages against the fibroblast cell line L929. We thus demonstrated that defined peptide segments are responsible for the leukocyte-activating properties of a major bacterial surface protein.     

Liquid-crystalline poly(β-aminoester)s. Thermotropic mesomorphism and degradability in solution

Poly(β-aminoester)s 7 – 12, containing in the repeating unit ethylenebis(carbonyloxy-1,4-phenylene)carbonyloxy groups interconnected by polymethylene segments of different length and coupled by diamino spacers, were synthesized from mesomorphic diacrylates and secondary diamines. The thermal behavior and the solution properties of the polymers were investigated. Depending on the structure of the repeating unit, the polymers were found to give rise to either isotropic or anisotropic melts. The incidence and extension of the mesophase are favored in poly(β-aminoester)s based on even numbered methylene segments and 2-methylpiperazinediyl spacers. The polymer samples were found to degrade at room temperature, when dissolved in solutions containing water, an organic base or an acid. Based on a macromolecular model compound (14) a degradation process is suggested involving a selective cleavage of the benzoate ester groups assisted by the amino groups in β-position.     

Closer look at lactose-mediated support of retinal morphogenesis

We have previously shown in intact isolated eye rudiments from Xenopus laevis that lactose, but not mannose, permits the formation of organized photoreceptor outer segments in the absence of the retinal pigment epithelium (RPE). The purpose of this study was to determine, using electron microscopic analysis, the key ultrastructural differences between healthy retinas, lactose-protected retinas, and retinas that developed aberrantly to reveal which subcellular structures were exclusively present in healthy retinas. Filamentous actin was also localized in retinas to determine its distribution under the various conditions. In healthy retinas, calycal processes extending approximately three-fourths of the length of the outer segment surrounded highly organized photoreceptor outer segments. Adherens junctions were localized between adjacent photoreceptors and Müller cells at the outer limiting membrane. In addition, Müller cells possessed apical processes that extended for a short distance beyond the adherens junctions. These fine cytoarchitectural details were missing in retinas that completed differentiation in the absence of the RPE; both calycal and apical processes were no longer present and adherens junctions were sparsely intermittent. Müller cells appeared atrophic. Similarly, mannose promoted none of the fine cytoarchitectural details of the retina. Lactose, however, supported the formation of the proper subcellular cytoarchitecture of both photoreceptor and Müller cells. These results suggest that these subcellular structures may be fundamental for the proper assembly and stability of organized outer segments and are necessary to allow for normal cytogenesis of the outer retina.     

Segmented poly(esterurethane urea)s from novel urea-diol chain extenders: synthesis, characterization and in vitro biological properties

This work describes the preparation, physicochemical characterization, mechanical properties and in vitro biological properties of two bioresorbable aliphatic segmented poly(esterurethane urea)s (SPEUU) based on poly(epsilon-caprolactone) diol (PCL diol), 1,6-hexamethylene diisocyanate and two novel urea-diol chain extenders. To strengthen the interactions through hydrogen bonding in the hard segments of SPEUU, novel chain extenders containing urea groups were synthesized and used in the SPEUU formulation. The different chemical structures of the chain extenders modulated the phase separation of soft and hard segments, as demonstrated by the thermal behavior. The hard segment association was enhanced using a diurea-diol chain extender. The biological interactions between the obtained materials and blood were studied by in vitro methods. Research on the protein adsorption, platelet adhesion and thrombus formation is presented. Studies of protein adsorption onto polymeric surfaces showed that SPEUU adsorbed more albumin than fibrinogen. Studies on platelet adhesion and thrombus formation of SPEUU-coated coverslips indicated the antithrombogenic behavior of these surfaces. The synthesized SPEUU revealed no signs of cytotoxicity to Chinese hamster ovary cells, showing satisfactory cytocompatibility.     

Preparation and properties of copolyamides-6,6F and copolyamides-4,8F containing oligo(tetrafluoroethylene)segments

Two families of copolyamides-6,6F and copolyamides-4,8F containing various amounts of bis- and tris(tetrafluoroethylene) segments were prepared to study the influence of such fluorine-containing segments on bulk and surface properties. In a solution polycondensation process telechelic aliphatic diamines, obtained by reacting dimethyl α,ω-dicarboxylatooligo(tetrafluoroethylene)s with excess diamines, e. g., tetramethylenediamine and hexamethylenediamine, were polymerized with activated bis(4-nitrophenyl) adipate and bis(4-nitrophenyl) suberate. In the presence of various stoichiometric amounts of the aliphatic diamines, the fluorine content varied between 0 and 39 wt.-% and the inherent viscosities between 0,27 and 1,02 dL/g. As content and segment length of the oligo(tetrafluoroethylene) segments increased, the polymer solubilities in organic solvents and the contact angles to water increased, whereas melting temperatures, crystallinities (as measured by means of differential scanning calorimetry and wide-angle X-ray scattering (WAXS)) and thermal stabilities (as determined by means of thermogravimetric analysis of the weight loss encountered at elevated temperatures) were reduced markedly. The WAXS measurements reflected changes of crystal structures with increasing fluorine content.     

Histological and physicochemical studies of hypercalcified primear lines in the laminar bone of young calves

There are many reports on laminar bone in various young animals during their growth periods. One of the concentric laminar bone units around the long-bone marrow periphery consists of three components: bright line, woven bone, and lamellar bone in the long-bone cortex of young calves and pigs. However, the fine structure, especially the uniform bright line or 'hypercalcified line' present in the unit-center, has not been elucidated as yet. The laminar bone of young calves was found to be initially formed from the hypercalcified lines; that is, 'hypercalcified primear' reported previously by the other authors. Such primear lines containing collagen fibrils scattered deposits of rod-like structures containing fine non-collagenous fibrils and globular structures showing no fibrils and some remnants. Osteocytes occasionally existed in the lines as well as adjacent to the lines, although no cells have been reported in the line. The cell lacunae were larger than those of woven bone and lamellar bone. The hypercalcified primear lines contained higher Ca and P content, and the molar ratio (1.78) was similar to 1.75 in the woven bone and lamellar bone. However, the physical and chemical resistances were significantly lower than that of the surrounding bone. Therefore, the hypercalcified primear lines are strongly suggested to show a lower crystallization. Further, fine structural observations of the primear-line forming cells and histochemical and immuno-histochemical investigations of the primear lines will be necessary.     

FINE STRUCTURAL COMPARISON OF EWINGS SARCOMA WITH NEUROBLASTOMA

Two cases of Ewing's sarcoma and two of neuroblastoma, rosette forming and round cell type, were studied electron microscopically and their fine structures were compared. The neoplastic cells of Ewing's sarcoma were characterized by aggregated glycogen particles in the cytoplasm. They had pseudopod-like cytoplasmic processes having tight junctions, which never contained microtubules or mitochondria. Ewing's sarcoma cells exhibited several stages of cell maturation and some mature cells possessed a large amount of smooth and rough endoplasmic reticulum, large Golgi complexes and numerous phagosomes containing glycogen particles as well as cytoplasmic organelles. The neoplastic cells of neuroblastoma, rosette forming type, were characterized by synaptic junctions and numerous cytoplasmic processes with production of neurites containing microtubules, neurofibrils, mitochondria and a few catecholamine granules. A few cytoplasmic processes containing mitochondria were observed even in the round cell type.     

Fine structural comparison of Ewing's sarcoma with neuroblastoma.

Two cases of Ewing's sarcoma and two neuroblastoma, rosette forming and round cell type, were studied electron microscopically and their fine structures were compared. The neoplastic cells of Ewing's sarcoma were characterized by aggregated glycogen particles in the cytoplasm. They had pseudopod-like cytoplasmic processes having tight junctions, which never contained microtubules or mitochondria. Ewing's sarcoma cells exhibited several stages of cell maturation and some mature cells possessed a large amount of smooth and rough endoplasmic reticulum, large Golgi complexes and numerous phagosomes containing glycogen particles as well as cytoplasmic organelles. The neoplastic cells of neuroblastoma, rosette forming type, were characterized by synaptic junctions and numerous cytoplasmic processes with production of neurites containing microtubules, neurofibrils, mitochondria and a few catecholamine granules. A few cytoplasmic processes containing mitochondria were observed even in the round cell type.     

Primary B-cell response to neuropeptide Y and bovine pancreatic polypeptide.

An analysis of the murine primary response to protein epitopes has been made with two small highly structured proteins, neuropeptide Y (NPY) and bovine pancreatic polypeptide (BPP), both of 36-amino acid residue length and containing helical structures. A group of cell lines producing monoclonal IgM antibody have been prepared consisting of six anti-NPY and two anti-BPP. The VH nucleotide sequences have been determined and characterized as germ-line either by identity to established germ-line sequences or by inference from the germ-line character of the D and JH segments. The intrinsic association constants for the homologous ligands have been estimated to range from 10(4) to 10(7) M-1 based on competitive ELISA. No severe restriction in the utilization of VH families, D segments or JH segments appears to be involved in this response. Among the eight cell lines, three VH families were represented as well as all three families of D segments and all of the JH segments, although some preference for JH3 was indicated. The length of the N(D)N sequences was also not subject to restriction, ranging from 9 to 29. Two unusual features of the CRD3s were noted, one involving the utilization of an uncommon DSP2 segment and the other the apparent occurrence of a D-D fusion.     

T cell receptor diversity in alloreactive responses against HLA-B27 (B*2705) is limited by multiple-level restrictions in both α and β chains

The T cell receptors (TCR) in HLA-B27 (B*2705) alloreactivity were analyzed in cytotoxic T lymphocytes (CTL) from two individuals. Non-random usage was found in Vβ, N+Dβ, Vα, and Jα, but not in Jβ segments or Nα-regions. Vβ segments from homology subgroup 4 were predominant and not associated to a particular donor or fine specificity, suggesting involvement in recognizing the HLA-B27 molecule. In contrast, preferential Vα usage was associated with particular individuals and fine specificities, indicating distinct Vβ and Vα recruitment and contribution to allorecognition. Recurrent N+Dβ motifs and Jα segments, even from different donors, limited junctional diversity, suggesting that CDR3 usage was determined by the alloantigenic epitope independently of individuals. TCR were selected differently at various levels, as indicated by the following findings. Four clonotypes with similar fine specificity had identical β and unrelated α chains. Similar α were associated with unrelated β chains, and vice versa. CTL using Vβ subgroup 4 did not globally show concomitant predominance of other TCR elements. Vα7, one of the preferred Vα segments, was always associated with Vβ subgroups other than 4. Sometimes, a TCR showed homology in elements of one chain to a second TCR or group of TCR, and to another in the other chain. These results are best explained by differential selection of TCR elements by different epitopes, providing a key to the inner structure of allospecific TCR repertoires.     

Structure-property relationships of core-shell type waterborne polyacrylate-polyurethane microemulsions

Different cross-linking types of core-shell types waterborne polyacrylate-polyurethane (PAC-PU) microemulsions, in which polyurethane acts as the shell of the latex particles and a copolymer of acrylate monomers as the core, were synthesized. In the anionic polyurethane film. there is a certain degree of softhard phase mixing and different extents of short-range order of the hard segments. The introduction of a polyacrylate core increases the soft-hard phase separation in the polyurethane and breaks the short-range order of the hard segments in polyurethane to some extent. The hard segments in the shell and the core cause a widened tan δ peak. This peak shifts to higher frequencies as the core-shell ratio increases and also when a Type A or Type B cross-linking structures exist. The cross-linking structure, especially Type B, improves the soft-hard separation and the phase mixing of the core and the hard segments in the shell and at the same time breaks the short-range order in the hard segments. The cross-linking structure does not affect the thermogravimetric properties of the PAC-PU films.     

Synthesis and characterization of biocompatible, degradable, light-curable, polyurethane-based elastic hydrogels

A series of degradable polyurethane-based light-curable elastic hydrogels were synthesized from polycaprolactone diol, polyethylene glycol (PEG), lysine diisocyanate (LDI), and 2-hydroxyethyl methacrylate (HEMA) through UV light initiated polymerization reaction. LDI was used as hard segment and polycaprolactone (PCL) and/or PEG were used as soft segments. By changing the PCL to PEG ratio during the prepolymer synthesis, polyurethanes with different soft segmental structures, hydrophilicity, and cytophilicity were obtained after light-initiated polymerization. The chemical structures of the synthesized polymers were characterized using differential scanning calorimetry and Fourier transform infrared spectroscopy. Physical properties such as swelling, mechanical properties, and in vitro degradation were evaluated. Materials containing a higher ratio of PEG exhibit higher water absorbance, higher degradation rate in vitro, and lower mechanical strength in the hydrated state. Mouse embryonal carcinoma-derived clonal chondrocytes were used as a model cell type to study the cytocompatibility of the synthesized polymers. Chondrocyte attachment, proliferation rates, and morphologies varied with changes in the PCL/PEG ratio. With a higher PEG ratio, lower cell attachment and proliferation were observed. To improve the cell attachment and proliferation on high PEG content hydrogels, bioactive molecules, such as peptides and proteins, were conjugated or immobilized in the gel matrix during the light-curing process. In this study, a short peptide, Arg-Gly-Asp-Ser, was used as a model biomolecule and incorporated into the gels during the light-curing process and improved cell growth was observed. In summary, the use of PCL/PEG at different ratios, as well as the introduction of HEMA into polyurethane, allows the synthesis of a series of biocompatible elastic hydrogels with tunable physical and cytophilic properties through light-initiated polymerization. This series of materials also allows for controlling cell attachment and growth by incorporating bioactive molecules during the light-curing process.     

The molecular weight and packaging of dsRNAs in the mycovirus from Ustilago maydis killer strains

The mycoviruses of Ustilago maydis killer strains are isometric, 43 nm in diameter, and contain several dsRNA segments designated heavy (H), medium (M), and light (L) according to their relative size. To determine the number of dsRNA segments per virion, major sedimenting and density components were isolated, their molecular weights determined from hydrodynamic properties, and their dsRNA contents determined by electron microscopy and/or polyacrylamide gel electrophoresis. The H dsRNA segments of 2.9, 3.1, and 4.2 x 10(6) daltons are separately encapsidated in isometric capsids that band in CsCI at 1.383, 1.394, and 1.418 g/cm8, respectively. The P1 strain contains the 3.1 and 4.2 x 10(6)-dalton segments, and the 3103 strain contains the 2.9 and 4.2 x 106-dalton segments. The T-4 strain contains the 3.1 x 106-dalton H segment and two M segments of 0.67 and 0.60 x 10(6) daltons. The H segments are separately encapsidated in virions which banded at 1.394 g/cm8, whereas the M segments are encapsidated in sets of one, two, or three in virions which banded at 1.314, 1.341, and 1.370 g/cm8. Molecular weights of 9.8 and 13.0 x 106 daltons were calculated for empty capsids (pCsCl = 1.278 g/cm8) and capsids containing the 3.1 x 10(6)-dalton dsRNA segments (pCsCl = 1.394 g/cm8). Analysis of components that banded at other densities in CsCl were consistent with the hypothesis that the banding pattern is the result of the encapsidation of a finite number of dsRNA segments in a capsid of 9.8 x 106 daltons. Although one to three M dsRNA segments were encapsidated in a single virion, no particles were detected with more than one H dsRNA segment per virion.     

Effect of conditioning methods on the microtensile bond strength of phosphate monomer-based cement on zirconia ceramic in dry and aged conditions

Scaffold fabrication for regenerating functional human tissues has an important role in tissue engineering, and there has been much progress in research on scaffold fabrication. However, current methods are limited by the mechanical properties of existing biodegradable materials and the irregular structures that they produce. Recently, several promising biodegradable materials have been introduced, including poly(propylene fumarate) (PPF). The development of micro-stereolithography allows the fabrication of free-form 3D microstructures as designed. Since this technology requires a low-viscosity resin to fabricate fine structures, we reduced the viscosity of PPF by adding diethyl fumarate. Using our system, the curing characteristics and material properties of the resin were analyzed experimentally. Then, we fabricated waffle shape and 3D scaffolds containing several hundred regular micro pores. This method controlled the pore size, porosity, interconnectivity, and pore distribution. The results show that micro-stereolithography has big advantages over conventional fabrication methods. In addition, the ultimate strength and elastic modulus of the fabricated scaffolds were measured, and cell adhesion to the fabricated scaffold was observed by growing seeded cells on it. These results showed that the PPF/DEF scaffold is a potential bone scaffold for tissue engineering.     

The effect of simulated body fluid on the mechanical properties of multiblock poly(aliphatic/aromatic-ester) copolymers

The effects of simulated body fluid (SBF) on the surface and mechanical properties of poly(aliphatic/aromatic-ester) (PED) copolymers were investigated. PED copolymers containing different hard (aromatic) and soft (aliphatic) segment weight ratios were exposed to SBF for 52 days, and afterwards their mechanical properties were evaluated. A quasi-static tensile test estimating relaxation and fatigue properties from the hysteresis method was performed. The surface properties were monitored with scanning electron microscopy and energy-dispersive X-ray spectrometry. PED copolymers showed various susceptibilities to apatite layer formation on the surface. This was ascribed to the hard/soft segment ratios of PED materials. Polymers containing higher amounts of amorphous soft segments showed a better deposition with the apatite layer, which provided improved mechanical properties, especially fatigue (improved dynamic creep resistance).     

Construction of human–rodent hybrid cells containing single transferable fragments of human chromosome 10p

The introduction of chromosome 10p into human glioblastoma or prostate cancer cells has been demonstrated to suppress their malignant phenotype, suggesting the presence of glioma or prostate tumor suppressor genes on 10p. As a resource for the fine mapping of these genes, a series of human-rodent hybrid cell lines containing single transferable fragments (STFs) of 10p were constructed. Normal chromosome 10 tagged with a neomycin-resistance gene on its short arm was fragmented by gamma-irradiation of 5–10 krad, transferred into mouse L cells or Chinese hamster ovary cells by microcell-mediated chromosome transfer (MMCT), and then selected against G418. Thirty-three independent rodent-human hybrids carrying various-sized STFs were obtained. Polymerase chain reaction (PCR)-based genotyping revealed that these STFs contained the whole, or portions, of a 43-cM region on 10p14-pter and could be defined by 19 sequence-tagged-site (STS) markers. Using this panel of hybrids as donors for further MMCT, genes on the refined fragments could be transferred into other cells. This hybrid panel would therefore be a useful resource for the fine mapping of the genes on 10p14-pter to segments of about 2.4 cM by functional complementation. Received: July 28, 2000 / Accepted: September 6, 2000     

Fine structures in the light diffraction pattern of striated muscle

Summary Single skeletal muscle fibres of frog were illuminated with a He-Ne, argon-ion or rhodamine 6G dye laser. The fine structures lying within the diffraction columns moved parallel to the fibre axis without changing their pattern when either the wavelength or the incident angle of the laser beam was varied, or when the fibre was stretched slightly. However, their pattern remained nearly constant when the fibre was submerged in hypotonic or hypertonic solution. As the illumination of about 1 mm or 0.1 mm width scanned along the length of the fibre, new structures emerged while others faded away giving rise to the notion that the diffraction columns were moving in the direction of the scan. A decrease in the illumination width caused the structures lying on the periphery of the diffraction column to disappear and the width of the remaining structures to increase. Measurements rule out the existence of large diffraction planes in these muscles. In addition, they indicate that the fine structures come from the diffraction of the whole rather than independent components of the illuminated volume. The origin of the fine structures is explained by two diffraction models.