RESUMEN

ß-detected NMR (ß-NMR) has been used to study the molecular-scale dynamics of lithium ions in thin films of poly(ethylene oxide) (PEO) containing either lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) or lithium trifluoroacetate (LiTFA) salts at monomer-to-salt ratios (EO/Li) of 8.3. The results are compared with previous ß-NMR measurements on pure PEO and PEO with lithium triflate (LiOTf) at the same loading [McKenzie et al., J. Am. Chem. Soc. 136, 7833 (2014)]. Activated hopping of 8Li+ was observed in all of the films above ∼250 K, with the hopping parameters strongly correlated with the ionicity of the lithium salt rather than the polymer glass transition temperature. The pre-exponential factor increases exponentially with ionicity, while the activation energy for hopping increases approximately linearly, going from 6.3±0.2 kJ mol-1 in PEO:LiTFA to 17.8±0.2 kJ mol-1 in PEO:LiTFSI. The more rapid increase in the pre-exponential factor outweighs the effect of the larger activation energy and results in 8Li+ hopping being fastest in PEO followed by PEO:LiTFSI, PEO:LiOTf, and PEO:LiTFA.

RESUMEN

ß-Detected nuclear spin relaxation of (8)Li(+) has been used to study the microscopic diffusion of lithium ions in thin films of poly(ethylene oxide) (PEO), where the implanted lithium ions are present in extremely low concentration, and PEO with 30 wt % LiCF3SO3 over a wide range of temperatures both above and below the glass transition temperature. Recent measurements by Do et al. [Phys. Rev. Lett. 2013, 111, 018301] found that the temperature dependence of the Li(+) conductivity was identical to that of the dielectric α relaxation and was well described by the Vogel-Fulcher-Tammann relation, implying the α relaxation dominates the Li(+) transport process. In contrast, we find the hopping of Li(+) in both samples in the high temperature viscoelastic phase follows an Arrhenius law and depends significantly on the salt content. We propose that the hopping of Li(+) between cages involves motion of the polymer but that it is only for long-range diffusion where the α relaxation plays an important role.

RESUMEN

Porous bulk composites were produced by depositing silver nanoparticles of diameter 11.0 +/- 3.2 nm on hydroxyapatite of micrometer sizes. Adsorption of bovine serum albumin (BSA) and lysozyme (LSZ) on the composite material was observed in 2 and 10 mol m(-3) phosphate buffer solutions. More BSA than LSZ was adsorbed in 2 mol m(-3) phosphate buffer and this was attributed to a larger a-face surface area present in the plate- and rod-shaped hydroxyapatite compared with the c-face surface area. Peak shifts in localized surface plasmon resonance (LSPR) spectra were clearly related to adsorbed amounts of BSA and LSZ after exposure of the porous bulk composites to protein solutions. The sensing capability of the porous bulk composite results from changes in the dielectric constant of the surface fluid surrounding the silver nanoparticles. Adsorption/desorption cycles of BSA were applied to the porous bulk composite, confirming the reversibility of the sensing capability.

Asunto(s)

Técnicas Biosensibles/instrumentación , Durapatita/química , Nanopartículas del Metal/química , Nanocompuestos/química , Proteínas/farmacocinética , Plata/química , Adsorción , Animales , Bovinos , Materiales Biocompatibles Revestidos/química , Modelos Biológicos , Muramidasa/metabolismo , Muramidasa/farmacocinética , Tamaño de la Partícula , Porosidad , Unión Proteica , Proteínas/metabolismo , Albúmina Sérica Bovina/metabolismo , Albúmina Sérica Bovina/farmacocinética , Resonancia por Plasmón de Superficie , Propiedades de Superficie

RESUMEN

The photodynamic effect of the glycoconjugated photosensitizer library containing 16 glycoconjugated 5,10,15,20-tetraphenylporphyrins and 8 glycoconjugated 5,10,15,20-tetraphenylchlorins were examined in HeLa cells, and analyzed by two approaches, namely, physiological properties (cellular uptake and reactive oxygen species (ROS)) and structural features of glycoconjugated photosensitizers. All glycoconjugated photosensitizers showed no cytotoxicity in the dark at a concentration of 5 muM. The photocytotoxicity profiles poorly related to the amount of cellular uptake of the photosensitizers. Photocytotoxicities of the glycoconjugated photosensitizers were inhibited by the ROS inhibitor, sodium azide. The result clearly suggests that singlet oxygen is a dominant species in all cases. The glycoconjugated photosensitizers examined have three structural features, namely, (1) the kind of sugar moieties, (2) the kind of light-absorbing moiety and (3) the substitution position of the sugar moiety. In regard to the sugar moieties, the photosensitizers bearing D-xylose tend to show higher photocytotoxicity than other photosensitizers, while those bearing D-arabinose tend to show lower photocytotoxicity. The photocytotoxicity with respect to the light-absorbing moiety tends to increase in the order of zinc porphyrin

Asunto(s)

Fármacos Fotosensibilizantes/farmacología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta a Droga , Colorantes Fluorescentes , Células HeLa , Humanos , Indoles , Fármacos Fotosensibilizantes/metabolismo , Especies Reactivas de Oxígeno/química , Especies Reactivas de Oxígeno/metabolismo , Espectrofotometría Atómica , Relación Estructura-Actividad

RESUMEN

Wnt signaling cascades play a crucial role in the maintenance of stem cell niches in many tissues as well as in embryonic patterning and cell-fate determination. Wnt signaling pathways have been well studied; however, the precise binding mechanism of Wnt protein to its receptor has not yet been clarified. Here we show the design and synthesis of seven novel peptide candidates for a receptor-binding site of human Wnt-1 based on its hydrophilicity and beta-turn profiles. Among these Wnt-derived peptides, only WP7, which corresponds to residues 301-320 of human Wnt-1, bound to the soluble receptor for Wnt-1, mouse Frizzled-1/Fc chimera, promoted PC12 cell adherence, increased level of cytosolic beta-catenin in PC12 cells, and induced adhesion and neuronal differentiation of hippocampal neural precursor cells. These results suggest that residues 301-320 of human Wnt-1 is one of the receptor-binding sites and that WP7 may activate the canonical Wnt pathway. When combined with an appropriate matrix, the action of this Wnt-derived peptide, WP7, can be limited to within a location, and therefore could be useful in the regeneration of many tissues, without fear of tumor generation.

Asunto(s)

Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Proteína Wnt1/fisiología , Animales , Hipocampo/citología , Humanos , Células PC12 , Ratas , beta Catenina/metabolismo

RESUMEN

Collagens are widely used in medical applications, but animal-derived collagens have several drawbacks, such as low thermal stability, nonspecific cell attachment, and susceptibility to contamination by infectious pathogens, such as prions, which may transfect humans. We have previously reported the chemical synthesis of polypeptides consisting of a Pro-Hyp-Gly sequence and the high thermostability of their triple-helical structure. To clarify the biomaterial characteristics of the poly(Pro-Hyp-Gly) polypeptide, we assessed its biodegradability and its capability for skin regeneration. Eight weeks after implantation, a poly(Pro-Hyp-Gly) freeze-dried sponge embedded subcutaneously into a rat dorsal area degraded at the same rate as Terudermis, which is made from bovine type I atelocollagen and is used as an artificial dermis. Surprisingly, compared with Terudermis, the poly(Pro-Hyp-Gly) sponge significantly promoted epithelialization of a full-thickness wound on a rabbit's ear pad. This chemically synthesized polypeptide may be useful as a scaffold for tissue engineering and tissue regeneration.

Asunto(s)

Materiales Biocompatibles/metabolismo , Péptidos/metabolismo , Fenómenos Fisiológicos de la Piel , Cicatrización de Heridas/fisiología , Implantes Absorbibles , Animales , Bovinos , Modelos Animales de Enfermedad , Masculino , Conejos , Ratas , Ratas Sprague-Dawley

RESUMEN

Deposition of a hydroxyapatite layer with similar structure to bone mineral is an attractive approach to the fabrication of bioactive coating layers to achieve direct bonding to living bone. To get successful coating of a hydroxyapatite layer on an organic polymer using a biomimetic solution, it is essential to find organic substrates that can effectively induce heterogeneous nucleation of hydroxyapatite after exposure to the body environment. Our previous study showed that sericin, a type of silk protein, has the ability to induce hydroxyapatite nucleation in a biomimetic solution when the sericin has a beta sheet structure. To confirm the effectiveness of the beta sheet structure in hydroxyapatite nucleation, we focused on investigating hydroxyapatite deposition on a synthetic polypeptide with a beta sheet structure in a biomimetic solution. The beta sheet forming polypeptides with and without carboxyl groups, poly(FE)(3)FG, poly(FQ)(3)FG, poly(LE)(3)LG and poly(LQ)(3)LG, were synthesized in this study. All the polypeptides had mainly beta sheet structure. After soaking the polypeptide films in 1.5SBF, which has 1.5 times the inorganic ion concentrations of human blood plasma, hydroxyapatite formed on the surfaces of the polypeptides with carboxyl groups, poly(FE)(3)FG and poly(LE)(3)LG, within 2 days, but not on those without carboxyl groups, poly(FQ)(3)FG and poly(LQ)(3)LG. We confirmed that the beta sheet structure was effective for hydroxyapatite nucleation even in the synthetic polypeptide. This finding is useful for the future design of organic polymers that can effectively induce nucleation of hydroxyapatite.

Asunto(s)

Materiales Biocompatibles/química , Biomimética , Líquidos Corporales/metabolismo , Durapatita/química , Péptidos/química , Humanos , Leucina/química , Ensayo de Materiales , Peso Molecular , Polímeros/química , Estructura Secundaria de Proteína , Propiedades de Superficie , Ingeniería de Tejidos/métodos , Difracción de Rayos X

RESUMEN

The photocytotoxicity of four glycoconjugated porphyrins, namely 5,10,15,20-tetrakis[4-(beta-D-glucopyranosyloxy)phenyl]porphyrin (p-1a), 5,10,15,20-tetrakis[4-(beta-D-galactopyranosyloxy)phenyl]porphyrin (p-1b), 5,10,15,20-tetrakis[4-(beta-D-xylopyranosyloxy)phenyl]porphyrin (p-1c) and 5,10,15,20-tetrakis[4-(beta-D-arabinopyranosyloxy)phenyl]porphyrin (p-1d), was evaluated in HeLa cells in the concentration range from 1 to 7 microM using a light dose of 16 J x cm(-2) with a wavelength greater than 500 nm. The photocytotoxicity depends on the sugar moieties, and increases in the order of p-1d

Asunto(s)

Carbohidratos/fisiología , Glicoconjugados/química , Porfirinas/metabolismo , Porfirinas/toxicidad , Albúmina Sérica Bovina/metabolismo , Animales , Tampones (Química) , Carbohidratos/química , Carbohidratos/farmacología , Bovinos , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Fluorometría , Glicoconjugados/farmacología , Células HeLa , Humanos , Luz , Estructura Molecular , Fosfatos/química , Fotoquímica , Fotoquimioterapia/métodos , Porfirinas/química , Cloruro de Sodio/química , Soluciones/química , Espectrometría de Fluorescencia , Volumetría

RESUMEN

In previous studies, we have reported that the BMP-2-derived peptide KIPKASSVPTELSAISTLYL, corresponding to BMP-2 residues 73-92, binds to a BMP-2-specific receptor, and elevates both alkaline phosphatase activity and osteocalcin mRNA in the murine mesenchymal cell line, C3H10T1/2. This 73-92 peptide conjugated to a covalently crosslinked alginate gel induced ectopic bone formation in rat calf muscle, and activated osteoblasts to promote the repair of rat tibial bone defects. Here, we report repair of 20-mm long rabbit radial bone defects using the 73-92 peptide combined with a porous alpha-tricalcium phosphate (TCP) scaffold. In vitro, the 73-92 peptide was released from the porous alpha-TCP scaffold over more than one week. In vivo, radiomorphometric analysis showed that the 73-92 peptide combined with the porous alpha-TCP scaffold promoted calcification in the implanted area in a dose-dependent manner, and that 5 mg of the 73-92 peptide induced connection of 20-mm long defects, defects of critical size, 12 weeks after implantation. Histological examination revealed newly formed bone and a marrow cavity in the implanted area. The area of bone denser than 690 mg/cm(3) induced by the 73-92 peptide was nearly equal to that of the contralateral radius.

Asunto(s)

Proteínas Morfogenéticas Óseas/genética , Sustitutos de Huesos , Fosfatos de Calcio , Péptidos , Radio (Anatomía)/cirugía , Secuencia de Aminoácidos/genética , Animales , Masculino , Datos de Secuencia Molecular , Conejos

RESUMEN

In order to explore the influence of the sugar moieties of glycoconjugated chlorins on the photocytotoxicity, we studied the photochemical properties of four glycoconjugated chlorins in aqueous media such as cytoplasm and the concentration dependence of photocytotoxicity in HeLa cells. In phosphate-buffered saline, the fluorescence intensities of 5,10,15,20-tetrakis[3-(beta-D-glucopyranosyloxy)phenyl]chlorin (m-1a) and 5,10,15,20-tetrakis[3-(beta-D-galactopyranosyloxy)phenyl]chlorin (m-1b), i.e., chlorins having hexose groups, were about 2-fold greater than those of 5,10,15,20-tetrakis[3-(beta-d-xylopyranosyloxy)phenyl]chlorin (m-1c) and 5,10,15,20-tetrakis[3-(beta-d-arabinopyranosyloxy)phenyl]chlorin (m-1d), i.e., chlorins having pentose groups, owing to a sugar-dependent difference of aggregation behavior. While no cytotoxicity was found in the dark, the highest photocytotoxicity was shown by m-1a (82% inhibition) in HeLa cells. This was higher than those of m-1b, m-1c, m-1d and tetraphenylporphyrin tetrasulfonic acid. The glycoconjugated chlorins except for m-1b appeared to be distributed diffusely throughout the cytoplasm. Among the four photosensitizers, m-1a showed the highest intensity in confocal fluorescence images, in agreement with the in vitro photocytotoxicity results. For m-1c, no photocytotoxicity was found at drug concentrations from 0.2 to 0.04 microM. Hence, sugar-dependent aggregation is not the major reason for the unexpected lack of efficacy of m-1c, which is uptaken efficiently by HeLa cells. For the glycoconjugated chlorins, these results suggest the biological aspects of sugar moiety play much crucial role rather than chemical aspects.

Asunto(s)

Carbohidratos/fisiología , Glicoconjugados/química , Fármacos Fotosensibilizantes/química , Porfirinas/química , Neoplasias del Cuello Uterino/tratamiento farmacológico , Transporte Biológico , Carbohidratos/química , Carbohidratos/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Glicoconjugados/farmacología , Glicosilación , Células HeLa , Humanos , Microscopía Confocal , Estructura Molecular , Fotoquímica , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Soluciones/química , Análisis Espectral , Neoplasias del Cuello Uterino/metabolismo , Agua/química

RESUMEN

Pro-Hyp-Gly is a characteristic amino acid sequence found in fibrous collagens, and (Pro-Hyp-Gly)(10), which has been widely used as a collagen-model peptide, forms a stable triple-helical structure. Here, we synthesized polypeptides consisting of the Pro-Hyp-Gly sequence by direct poly-condensation of (Pro-Hyp-Gly)(n), where n=1, 5, and 10, using 1-hydroxybenzotriazole and 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride in both phosphate buffer (pH=7.4) and dimethylsulfoxide (DMSO) solutions for 48 h at 20 degrees C. The reaction of (Pro-Hyp-Gly)(5) and (Pro-Hyp-Gly)(10) in DMSO successfully gave polypeptides with molecular weights over 10,000, whereas low molecular weight products were obtained by reaction in phosphate buffer (pH=7.4). In contrast, Pro-Hyp-Gly at a concentration of 50 mg/mL in phosphate buffer (pH=7.4) gave polypeptides with molecular weights over 10,000. The Fourier transform infrared (FTIR) and (1)H nuclear magnetic resonance (NMR) spectra of poly(Pro-Hyp-Gly)(10) revealed that the polymerization of (Pro-Hyp-Gly)(10) described in this report had no side reactions. Each polypeptide obtained shows a collagen-like triple-helical structure, and the triple-helical structures of poly(Pro-Hyp-Gly) and poly(Pro-Hyp-Gly)(10) were stable up to T=80 degrees C, which suggests that the high molecular weight promotes stability of the triple-helical structure, in addition to the high Hyp content. Furthermore, transmission electron microscopy (TEM) observations show that poly(Pro-Hyp-Gly)(10) aggregates to form nanofiber-like structures about 10 nm in width, which suggests that a Pro-Hyp-Gly repeating sequence contains enough information for triple-helix formation, and for subsequent nanofiber-like structure formation.

Asunto(s)

Oligopéptidos/síntesis química , Péptidos/química , Péptidos/síntesis química , Secuencia de Aminoácidos , Tampones (Química) , Cromatografía en Gel , Dicroismo Circular , Dimetilsulfóxido/química , Estabilidad de Medicamentos , Etildimetilaminopropil Carbodiimida/análogos & derivados , Etildimetilaminopropil Carbodiimida/farmacología , Etilenodiaminas/química , Concentración de Iones de Hidrógeno , Microscopía Electrónica , Peso Molecular , Resonancia Magnética Nuclear Biomolecular , Tamaño de la Partícula , Conformación Proteica , Estructura Secundaria de Proteína , Temperatura , Triazoles/farmacología

RESUMEN

Eight 5,10,15,20-tetrakis[3- or 4-(beta-D-glycopyranosyloxy)phenyl]chlorins were synthesized by means of the Whitlock method with diimide reduction and purified by reversed-phase thin layer chromatography (RP-TLC). All compounds were characterized by (1)H NMR spectroscopy, electron-spray ionization time-of-flight mass spectrometry (ESI-TOF MS), and UV-Vis spectroscopy. ESI-TOF MS could detect the 2H difference in molecular weight between a glycoconjugated chlorin and its corresponding porphyrin (i.e., 5,10,15,20-tetrakis[3- or 4-(beta-D-glycopyranosyloxy)phenyl]porphyrin). The cellular uptake of the eight chlorins was evaluated in HeLa cells. All glycoconjugated chlorins showed higher cellular uptake than tetraphenylporphyrin tetrasulfonic acid (TPPS), and 5,10,15,20-tetrakis[3-(beta-D-xylopyranosyloxy)phenyl]chlorin showed 50-fold higher uptake than TPPS. The photocytotoxicity of 5,10,15,20-tetrakis[3-(beta-D-glucopyranosyloxy)phenyl]chlorin, 5,10,15,20-tetrakis[3-(beta-D-xylopyranosyloxy)phenyl]chlorin and TPPS towards HeLa cells was examined at the concentration of 2x10(-7) M (mol/dm(3)). These photosensitizers had no cytotoxicity in the dark, but their photocytotoxicity decreased in the order of 5,10,15,20-tetrakis[3-(beta-D-glucopyranosyloxy)phenyl]chlorin>5,10,15,20-tetrakis[3-(beta-D-xylopyranosyloxy)phenyl]chlorin>TPPS. The results indicate that the photocytotoxicity is not related simply to cellular uptake.

Asunto(s)

Porfirinas/metabolismo , Porfirinas/toxicidad , Transporte Biológico , Glicosilación , Células HeLa , Humanos , Estructura Molecular , Fotoquímica , Porfirinas/síntesis química , Porfirinas/química , Análisis Espectral

RESUMEN

Cytokines such as tumor necrosis factor-α (TNF-α), FasL, and TNF-related apoptosis-inducing ligand (TRAIL) induce apoptosis or inflammation through binding to their specific receptors, TNFR1, Fas, and DR5, respectively. We have previously reported ligand-binding and cell death-inhibiting synthetic peptides, which were designed based on the crystal structure of a ligand-receptor complex and the homology of the amino acid sequence among the death receptor family members. Here we show that, among these death receptor-derived peptides, the TNFR1-derived peptide specifically arrested cell proliferation and promoted cell adhesion of fetal rat (E16) hippocampal cells, and promoted neurite outgrowth from hippocampus-derived neurospheres cultured with the addition of the peptide or cultured on a peptide-coated surface. Furthermore, among these death receptor-derived peptides, marked neurite outgrowth was observed only when the neurospheres were cultured on a TNFR1-derived peptide-conjugated covalently cross-linked alginate gel. The neurites from the neurospheres positively immunostained with an antibody against neurofilaments. These results suggest that the TNFR1-derived peptide promotes neuronal differentiation of the hippocampal neural stem cells and the TNFR1-derived peptide-conjugated covalently cross-linked alginate gel may be a useful material for assisting neural stem cell transplantation.

RESUMEN

Cytokines such as tumor necrosis factor-alpha (TNF-alpha), FasL, and TNF-related apoptosis-inducing ligand (TRAIL) induce apoptosis or inflammation through binding to their specific receptors, TNFR1, Fas, and DR5, respectively. We have previously reported ligand-binding and cell death-inhibiting synthetic peptides, which were designed based on the crystal structure of a ligand-receptor complex and the homology of the amino acid sequence among the death receptor family members. Here we show that, among these death receptor-derived peptides, the TNFR1-derived peptide specifically arrested cell proliferation and promoted cell adhesion of fetal rat (E16) hippocampal cells, and promoted neurite outgrowth from hippocampus-derived neurospheres cultured with the addition of the peptide or cultured on a peptide-coated surface. Furthermore, among these death receptor-derived peptides, marked neurite outgrowth was observed only when the neurospheres were cultured on a TNFR1-derived peptide-conjugated covalently cross-linked alginate gel. The neurites from the neurospheres positively immunostained with an antibody against neurofilaments. These results suggest that the TNFR1-derived peptide promotes neuronal differentiation of the hippocampal neural stem cells and the TNFR1-derived peptide-conjugated covalently cross-linked alginate gel may be a useful material for assisting neural stem cell transplantation.

Asunto(s)

Hipocampo/citología , Hipocampo/efectos de los fármacos , Neuritas/fisiología , Fragmentos de Péptidos/farmacología , Receptores Tipo I de Factores de Necrosis Tumoral/química , Células Madre/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Femenino , Humanos , Inmunohistoquímica , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Fragmentos de Péptidos/síntesis química , Embarazo , Ratas , Ratas Wistar , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

A novel synthetic peptide corresponding to BMP-2 residues 73-92 that can induce bone formation and can form a conjugate with a carrier to localize its effect has been reported previously. The synthetic peptide was bound to a BMP-2-specific receptor, and it elevated both the alkaline phosphatase activity and the osteocalcin mRNA in the murine multipotent mesenchymal cell line, C3H10T1/2. The 73-92 peptide also induced ectopic bone formation when conjugated to a covalently crosslinked alginate gel and implanted into a rat's calf muscle. Here, it is reported that the 73-92 peptide-conjugated alginate gel particles significantly promoted the repair of rat tibial bone defects, whereas the alginate gel sponge that the peptide was conjugated with was less effective. Further acceleration and denser bone regeneration was achieved when the 73-92 peptide-conjugated alginate gel particles were coimplanted with syngeneic rat bone-marrow stromal cells. Therefore, the 73-92 peptide can induce differentiation of osteoblast precursor cells into osteoblasts, and can activate osteoblasts to promote the repair of bone defects.

Asunto(s)

Proteínas Morfogenéticas Óseas/farmacología , Regeneración Ósea/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Células del Estroma/fisiología , Factor de Crecimiento Transformador beta/farmacología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Proteína Morfogenética Ósea 2 , Diferenciación Celular , Línea Celular , Portadores de Fármacos , Masculino , Osteoblastos/citología , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/síntesis química , Ratas , Ratas Wistar , Células del Estroma/efectos de los fármacos , Células del Estroma/trasplante

RESUMEN

Acidic proteins play an important role during mineral formation in biological systems, but the mechanism of mineral formation is far from understood. In this paper, we report on the relationship between the structure of a protein and hydroxyapatite deposition under biomimetic conditions. Sericin, a type of silk protein, was adopted as a suitable protein for studying structural effect on hydroxyapatite deposition, since it forms a hydroxyapatite layer on its surface in a metastable calcium phosphate solution, and its structure has been reported. Sericin effectively induced hydroxyapatite nucleation when it has high molecular weight and a beta sheet structure. This indicates that the specific structure of a protein can effectively induce heterogeneous nucleation of hydroxyapatite in a biomimetic solution, i.e. a metastable calcium phosphate solution. This finding is useful in understanding biomineralization, as well as for the design of organic polymers that can effectively induce hydroxyapatite nucleation.

Asunto(s)

Sustitutos de Huesos/química , Materiales Biocompatibles Revestidos/química , Cristalización/métodos , Durapatita/química , Sericinas/química , Sericinas/ultraestructura , Anisotropía , Ensayo de Materiales , Conformación Proteica , Relación Estructura-Actividad , Propiedades de Superficie

RESUMEN

Tricalcium phosphate (TCP) ceramic is known in orthopedics to be a bioresorbable bone substitute. A porous TCP ceramic body also has high potential as a drug delivery system in bony defects. Porous alpha-TCP ceramic can be easily fabricated using conventional sintering of beta-TCP, since alpha-TCP is the thermodynamically stable phase at temperatures above 1 100 degrees C. However, the solubility of alpha-TCP is much higher than that of beta-TCP. Therefore, the dissolution of porous alpha-TCP progresses at a higher rate than bone repair. In the present study, we attempted to reduce the dissolution rate of porous alpha-TCP by employing an organic polymer coating. We fabricated porous alpha-TCP ceramic with a continuous 10-50 microm diameter pore structure by sintering a body made from a beta-TCP and potato starch slurry. The porous body obtained was coated with hydroxypropylcellulose (HPC), and then subjected to heat treatment. The chemical durability and mechanical properties of the body were examined before and after coating with the HPC. The dissolution of porous alpha-TCP in buffered solutions was reduced by coating with HPC and drying at 60 degrees C. The compressive strength of the porous alpha-TCP was also improved by coating with HPC. The results of in vivo experiments showed that some parts of the porous alpha-TCP ceramic coated with HPC remained in the canal of the tibia of a rabbit four weeks after implantation, whereas no residual was observed in a non-coated alpha-TCP ceramic. Coating with HPC was found to be effective for controlling bioresorption and improving the workability of porous alpha-TCP ceramic. The prepared porous alpha-TCP ceramic is expected to be useful as a novel material for bone fillers by incorporating it with drugs or osteoinductive factors.

Asunto(s)

Materiales Biocompatibles , Sustitutos de Huesos , Fosfatos de Calcio/química , Celulosa/análogos & derivados , Celulosa/química , Animales , Tampones (Química) , Calcio/química , Cerámica/química , Ensayo de Materiales , Microscopía , Microscopía Electrónica de Rastreo , Conejos , Temperatura , Resistencia a la Tracción , Termogravimetría , Tibia/patología , Factores de Tiempo , Tomografía Computarizada por Rayos X , Rayos X

RESUMEN

Hybrids consisting of bone-like apatite and biodegradable polymers are attractive materials for bone repair. We have shown that an alginate gel crosslinked covalently with ethylenediamine (EDA) enhances the repair of skin and nerves. In this study, we report a novel method for fabrication of an apatite-alginate nanohybrid using a simulated body fluid (SBF). Alginate was reacted with 3-aminopropyltriethoxysilane (APES), which gives silanol groups after hydrolysis, and/or EDA, by dehydration condensation using water-soluble carbodiimide to form gels. Modification of alginate with APES alone also gave a gel, because the alginate could be crosslinked by dehydration of silanol groups derived from APES. The gels obtained were soaked in a 1 mol/L CaCl2 solution and subsequently soaked in SBF. Apatite was formed on and inside the alginate gels modified with APES, whereas it was not formed on the gels without APES. Modification of alginate with silanol groups induced not only gel formation but also the apatite-forming ability on and inside the alginate gel in SBF. Consequently, a hydroxyapatite-alginate hybrid can be produced by modification of alginate with silanol groups and subsequent soaking in CaCl2 solution and SBF. Such a material is expected to be useful in bone repair.

Asunto(s)

Alginatos/química , Reactivos de Enlaces Cruzados/química , Silanos/química , Líquidos Corporales/química , Calcio/química , Cloruro de Calcio/química , Geles , Microscopía Electrónica de Rastreo , Soluciones , Espectroscopía Infrarroja por Transformada de Fourier , Difracción de Rayos X

RESUMEN

Thirty-two glycoconjugated porphyrins were synthesized by a modification of Lindsey method in the presence of Zn(OAc)(2).2H(2)O as a template. The Zn(2+) ion template strategy improved the yield about three-fold in the case of meta-substituted tetraphenylporphyrins. In addition, free-base porphyrins were obtained almost quantitatively by demetalation with 4 M HCl. Sixteen deacetylated glycoconjugated porphyrins were tested as candidate photodynamic therapy (PDT) drugs using HeLa cells. Most of the deacetylated glycoconjugated porphyrins showed higher cellular uptake than tetraphenylporphyrin tetrasulfonic acid (TPPS), and 5,10,15,20-tetrakis[4-(beta-D-arabinopyranosyloxy)phenyl]porphyrin (p-5d) in particular showed 18.5-fold higher uptake than TPPS. The photocytotoxicity of 5,10,15,20-tetrakis[4-(beta-D-glucopyranosyloxy)phenyl]porphyrin (p-5a), p-5d and TPPS was examined with HeLa cells, using a light dose of 16 J/cm(2). These photosensitizers had no cytotoxicity in the dark, but their photocytotoxicity increased in the order of TPPS < p-5a < p-5d. These results suggest p-5d is a good candidate for a PDT drug.

Asunto(s)

Porfirinas/metabolismo , Porfirinas/toxicidad , Transporte Biológico , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Glicosilación , Células HeLa , Humanos , Estructura Molecular , Fármacos Fotosensibilizantes/sangre , Fármacos Fotosensibilizantes/síntesis química , Fármacos Fotosensibilizantes/metabolismo , Fármacos Fotosensibilizantes/toxicidad , Porfirinas/sangre , Porfirinas/química

RESUMEN

Bone morphogenetic protein-2 (BMP-2) promotes the formation and regeneration of bone and cartilage, and therefore constitutes the most promising candidate for a bone repair material. However, it also has a wide range of functions, such as in organogenesis and apoptosis. Therefore, we investigated a novel synthetic peptide corresponding to residues 73-92 of BMP-2. This peptide bound to a BMP-2-specific receptor and elevated both alkaline phosphatase activity and osteocalcin mRNA in the murine cell line, C3H10T1/2. The 73-92 peptide also induced ectopic calcification when conjugated to a covalently crosslinked alginate gel. Here we report that the 73-92 peptide-conjugated alginate gel showed prolonged ectopic calcification for up to 7 weeks in rat calf muscle. In contrast, rhBMP-2-impregnated collagen gel showed maximum ectopic calcification at 3 weeks, and the calcified products that had formed disappeared after 5 weeks. Histological examination showed that the 73-92 peptide-conjugated alginate gel induced many osteoblast-like cells and few osteoclasts. In contrast, rhBMP-2-impregnated collagen gel induced many osteoclasts. These results suggest that the 73-92 peptide on alginate gel remains active at the implanted site, continuously induces differentiation of osteoblast precursor cells into osteoblasts, and activates osteoblasts to promote ectopic calcification.

Asunto(s)

Proteínas Morfogenéticas Óseas/farmacología , Osificación Heterotópica/inducido químicamente , Fragmentos de Péptidos/farmacología , Factor de Crecimiento Transformador beta/farmacología , Alginatos/química , Alginatos/farmacología , Animales , Proteína Morfogenética Ósea 2 , Reactivos de Enlaces Cruzados , Geles , Cinética , Masculino , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Músculo Esquelético/patología , Osteoblastos/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Ratas , Ratas Wistar , Difracción de Rayos X