RESUMO
Composites of biodegradable polymers and calcium phosphate are bioactive and flexible, and have been proposed for use in tissue engineering and bone regeneration. When associated with the broad-spectrum antibiotic doxycycline (DOX), they could favor antimicrobial action and enhance the action of osteogenic composites. Composites of polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and a bioceramic of biphasic calcium phosphate Osteosynt® (BCP) were loaded with DOX encapsulated in ß-cyclodextrin (ßCD) and were evaluated for effects on osteoblastic cell cultures. The DOX/ßCD composite was prepared with a double mixing method. Osteoblast viability was assessed with methyl tetrazolium (MTT) assays after 1day, 7day, and 14days of composite exposure; alkaline phosphatase (AP) activity and collagen production were evaluated after 7days and 14days, and mineral nodule formation after 14days. Composite structures were evaluated by scanning electron microscopy (SEM). Osteoblasts exposed to the composite containing 25µg/mL DOX/ßCD had increased cell proliferation (p<0.05) compared to control osteoblast cultures at all experimental time points, reaching a maximum in the second week. AP activity and collagen secretion levels were also elevated in osteoblasts exposed to the DOX/ßCD composite (p<0.05 vs. controls) and reached a maximum after 14days. These results were corroborated by Von Kossa test results, which showed strong formation of mineralization nodules during the same time period. SEM of the composite material revealed a surface topography with pore sizes suitable for growing osteoblasts. Together, these results suggest that osteoblasts are viable, proliferative, and osteogenic in the presence of a DOX/ßCD-containing BCP ceramic composite.
Assuntos
Fosfatos de Cálcio , Ciclodextrinas , Doxiciclina , Portadores de Fármacos , Ácido Láctico , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Poliésteres , Ácido Poliglicólico , Animais , Fosfatos de Cálcio/química , Fosfatos de Cálcio/farmacocinética , Fosfatos de Cálcio/farmacologia , Células Cultivadas , Ciclodextrinas/química , Ciclodextrinas/farmacocinética , Ciclodextrinas/farmacologia , Doxiciclina/química , Doxiciclina/farmacocinética , Doxiciclina/farmacologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacologia , Ácido Láctico/química , Ácido Láctico/farmacocinética , Ácido Láctico/farmacologia , Masculino , Osteoblastos/citologia , Poliésteres/química , Poliésteres/farmacocinética , Poliésteres/farmacologia , Ácido Poliglicólico/química , Ácido Poliglicólico/farmacocinética , Ácido Poliglicólico/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos , Ratos WistarRESUMO
Although several studies have shown that chlorhexidine (Cx) has bactericidal activity and exerts toxic effects on periodontal tissues a few studies evaluated mechanisms to reduce its adverse effects maintaining the antimicrobial properties. Therefore, the aim of the present study was to investigate the in vitro antimicrobial activity and cellular cytotoxicity of Cx included on cyclodextrins (Cd), α, ß or Hp-ß-cyclodextrins (Hp-ß-Cd). The influence of Cds was determined by increasing its molar rate 1:1 to 1:4 in relation with free Cx. The minimal inhibitory concentrations (MICs) for Candida albicans, Aggregatibacter actinomycetemcomitans actinomycemcomitans and Streptococcus mutans were determined. An ergosterol solubilization assay was carried out using the C. albicans model and osteoblasts, fibroblasts and tumoral Caco-2 cells for cytotoxicity assay. The antimicrobial activity results in a significant growth inhibition of C. albicans when it was treated with Cx:α-Cd complexes, whereas Cx:ß-Cd was more effective for A. actinomycetemcomitans, and Cx:Hp-ß-Cd complexes was for S. mutans when compared to the other complexes. The cytotoxicity for fibroblasts and osteoblasts decreased in relation with each kind of Cd been ß-Cd ≤ Hp-ß-Cd ≤ α-Cd. Although the Hp-ß-Cd inclusion complexes had more severe effects on Caco-2 cells, all complexes exhibited less cytotoxicity than free Cx. The α-Cd, ß-Cd and Hp-ß-Cd increase the antimicrobial activity of Cx, but decrease its cytotoxic effects on mammalian cells. Taken together these findings suggest that cyclodextrins are a tool for modulation of effects of Cx. It could be useful to design Cx/Cd delivery systems with high efficacy and minimum cytotoxic effects.
Assuntos
Anti-Infecciosos/farmacologia , Clorexidina/farmacologia , Ciclodextrinas/farmacologia , Portadores de Fármacos/farmacologia , Aggregatibacter actinomycetemcomitans/efeitos dos fármacos , Animais , Anti-Infecciosos/administração & dosagem , Anti-Infecciosos/química , Células CACO-2 , Candida albicans/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Clorexidina/administração & dosagem , Clorexidina/química , Ciclodextrinas/química , Portadores de Fármacos/química , Fibroblastos/efeitos dos fármacos , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Osteoblastos/efeitos dos fármacos , Ratos Wistar , Streptococcus mutans/efeitos dos fármacosRESUMO
Bovine cervical mucus (BCM) is important for selection and transport of spermatozoa. When air-dried, BCM obtained at oestrus exhibits arborescent crystallizations, among other arrangements. Considering the relevant endocrine and reproductive information indirectly obtained from BCM crystallization, a morphological investigation was carried out to study its crystalline patterns. BCM samples were collected from healthy Holstein Friesian heifers at oestrus, their crystalline patterns photographed and its morphology analyzed. The majority of the crystallizations obtained showed the typical tree-like patterns reported for BCM. However, a highly symmetrical arrangement was found, characterized by a star-like morphology with six straight, highly defined axes that protrude from the same central point, forming 60º angles. In terms of current knowledge, this short report is the first to show this crystallization geometry in BCM, which, additionally, is remarkably similar to P6B mucus reported for periovulatory human cervical mucus. Even though the role of mucus presenting this type of crystallization is as yet unknown for bovines, its possible functions are also briefly discussed here.
El moco cervical bovino es importante para la selección y el transporte espermático. El moco, obtenido durante el estro y secado al aire, exhibe cristalizaciones con formas principalmente arborescentes. Considerando la importante información endocrina y reproductiva que es posible obtener a partir de la cristalización del moco cervical, se efectuó una investigación morfológica con el propósito de estudiar sus patrones cristalinos. Las muestras de moco se obtuvieron de novillas Holstein Friesian en estro; posteriormente, los patrones de cristalización del moco fueron fotografiados para finalmente analizar su morfología. Las cristalizaciones obtenidas correspondieron a típicos patrones arboriformes previamente reportados. Sin embargo, lo que llamó la atención fue el hallazgo de un arreglo altamente simétrico en una novilla, caracterizado por una morfología similar a estrella con seis ejes rectos, bien definidos, que surgen desde el mismo punto central y forman ángulos de 60º. Según nuestro conocimiento, esta comunicación breve reporta por primera vez la presencia de dicha geometría de cristalización en vaquillas, la cual es muy semejante al patrón cristalino subtipo P6 reportado para el moco cervical perioBvulatorio humano. Si bien el rol ejercido por este tipo de cristalización de moco aún se desconoce en bovinos, se discuten aquí sus posibles funciones.
RESUMO
Chlorhexidine (Cx) augmented with beta-cyclodextrin (β-cd) inclusion compounds, termed Cx:β-cd complexes, have been developed for use as antiseptic agents. The aim of this study was to examine the interactions of Cx:β-cd complexes, prepared at different molecular ratios, with sterol and yeast membranes. The Minimal Inhibitory Concentration (MIC) against the yeast Candida albicans (C.a.) was determined for each complex; the MICs were found to range from 0.5 to 2 µg/mL. To confirm the MIC data, quantitative analysis of viable cells was performed using trypan blue staining. Mechanistic characterization of the interactions that the Cx:β-cd complexes have with the yeast membrane and assessment of membrane morphology following exposure to Cx:β-cd complexes were performed using Sterol Quantification Method analysis (SQM) and scanning electron microscopy (SEM). SQM revealed that sterol extraction increased with increasing β-cd concentrations (1.71 × 10³; 1.4 × 10³; 3.45 × 10³, and 3.74 × 10³ CFU for 1:1, 1:2, 1:3, and 1:4, respectively), likely as a consequence of membrane ergosterol solubilization. SEM images demonstrated that cell membrane damage is a visible and significant mechanism that contributes to the antimicrobial effects of Cx:β-cd complexes. Cell disorganization increased significantly as the proportion of β-cyclodextrin present in the complex increased. Morphology of cells exposed to complexes with 1:3 and 1:4 molar ratios of Cx:β-cd were observed to have large aggregates mixed with yeast remains, representing more membrane disruption than that observed in cells treated with Cx alone. In conclusion, nanoaggregates of Cx:β-cd complexes block yeast growth via ergosterol extraction, permeabilizing the membrane by creating cluster-like structures within the cell membrane, possibly due to high amounts of hydrogen bonding.
Assuntos
Anti-Infecciosos Locais/análise , Candida albicans/crescimento & desenvolvimento , Clorexidina/análise , Ergosterol/análise , Corpos de Inclusão , Leveduras/crescimento & desenvolvimento , beta-Ciclodextrinas/análise , Métodos , Microscopia Eletrônica de VarreduraRESUMO
This study assess the effects of bioceramic and poly(lactic-co-glycolic acid) composite (BCP/PLGA) on the viability of cultured macrophages and human dental pulp fibroblasts, and we sought to elucidate the temporal profile of the reaction of pulp capping with a composite of bioceramic of calcium phosphate and biodegradable polymer in the progression of delayed dentine bridge after (30 and 60 days) in vivo. Histological evaluation of inflammatory infiltrate and dentin bridge formation were performed after 30 and 60 days. There was similar progressive fibroblast growth in all groups and the macrophages showed viability. The in vivo study showed that of the three experimental groups: BCP/PLGA composite, BCP and calcium hydroxide (Ca(OH)(2)) dentin bridging was the most prevalent (90 %) in the BCP/PLGA composite after 30 days, mild to moderate inflammatory response was present throughout the pulp after 30 days. After 60 days was observed dentine bridging in 60 % and necrosis in 40 %, in both groups. The results indicate that understanding BCP/PLGA composite is biocompatible and by the best tissue response as compared to calcium hydroxide in direct pulp capping may be important in the mechanism of delayed dentine bridge after 30 and 60 days.
Assuntos
Materiais Biocompatíveis , Fosfatos de Cálcio/administração & dosagem , Ácido Láctico/administração & dosagem , Ácido Poliglicólico/administração & dosagem , Células Cultivadas , Humanos , Técnicas In Vitro , Microscopia Eletrônica de Varredura , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoRESUMO
In the acrosome reaction, the spermatozoon plasma membrane fuses with the outer acrosomal membrane, resulting in the release of the acrosomal content. Several compounds, such as sex steroids, are known to modulate the acrosomal exocytosis. Testosterone regulates various functions in male reproductive physiology; however, little is known about the relationship between testosterone and the acrosome reaction. Thus, our objective was to study the effect of testosterone on the acrosome reaction of human spermatozoa. To evaluate the acrosomal exocytosis, spermatozoa were incubated with testosterone (0.2, 2.0 and 20 nmol l(-1)), progesterone and control medium for 60, 120, 240 and 1440 min. The acrosome reaction was assessed by staining with Hoechst 33258 and fluorescein isothiocyanate-conjugated P. sativum agglutinin lectin. In general, spermatozoa incubated with progesterone had the highest percentage of acrosomal exocytosis. The percentage of acrosome reaction obtained in the three treatments with testosterone differed from that observed for progesterone at 120, 240 and 1440 min (24 h). Additionally, significant differences were found between testosterone (2.0 and 20 nmol l(-1)) and progesterone after 60 min. Differences between control and the three testosterone treatments studied were obtained only at 1440 min. In general terms, these results show that testosterone exerts no inductor effects on the acrosome reaction of human spermatozoa.
Assuntos
Reação Acrossômica/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Testosterona/farmacologia , Meios de Cultura , Humanos , MasculinoRESUMO
Chlorhexidine (Cx) augmented with beta-cyclodextrin (ß-cd) inclusion compounds, termed Cx:ß-cd complexes, have been developed for use as antiseptic agents. The aim of this study was to examine the interactions of Cx:ß-cd complexes, prepared at different molecular ratios, with sterol and yeast membranes. The Minimal Inhibitory Concentration (MIC) against the yeast Candida albicans (C.a.) was determined for each complex; the MICs were found to range from 0.5 to 2 µg/mL. To confirm the MIC data, quantitative analysis of viable cells was performed using trypan blue staining. Mechanistic characterization of the interactions that the Cx:ß-cd complexes have with the yeast membrane and assessment of membrane morphology following exposure to Cx:ß-cd complexes were performed using Sterol Quantification Method analysis (SQM) and scanning electron microscopy (SEM). SQM revealed that sterol extraction increased with increasing ß-cd concentrations (1.71 ×10(3); 1.4 ×10(3); 3.45 ×10(3), and 3.74 ×10(3) CFU for 1:1, 1:2, 1:3, and 1:4, respectively), likely as a consequence of membrane ergosterol solubilization. SEM images demonstrated that cell membrane damage is a visible and significant mechanism that contributes to the antimicrobial effects of Cx:ß-cd complexes. Cell disorganization increased significantly as the proportion of ß-cyclodextrin present in the complex increased. Morphology of cells exposed to complexes with 1:3 and 1:4 molar ratios of Cx:ß-cd were observed to have large aggregates mixed with yeast remains, representing more membrane disruption than that observed in cells treated with Cx alone. In conclusion, nanoaggregates of Cx:ß-cd complexes block yeast growth via ergosterol extraction, permeabilizing the membrane by creating cluster-like structures within the cell membrane, possibly due to high amounts of hydrogen bonding.
RESUMO
Experimental paradigms conducted to assess the neurotoxic effects of ethanol exposure on hippocampus development have yielded controversial findings. Hippocampal CA1 population and some cytoarchitectural parameters of pyramidal cells were studied after exposure to ethanol during early development, in rats. Examination of 30-day-old offspring of rats exposed to moderate levels of ethanol during gestation through lactation showed an increased volume of the hippocampal CA1 field compared to untreated or pair-fed control pups, as well as a reduced number of pyramidal neurons. In addition, the number of spines from surviving CA1 pyramidal neurons was reduced. Furthermore, stubby and wide spines were proportionally increased, while the proportion of mushroom and ramified spines was reduced; no variation in the proportion of thin spines was observed. Because alcoholic women usually drink alcohol before, during, and after pregnancy, a broad-range experimental model of alcohol exposure was used in this study. The present findings show that experimental exposure to moderate levels of ethanol, resembling the human situation in alcoholic mothers, leads to loss of hippocampal CA1 pyramidal neurons, along with several pathological and plastic events in the dendritic arborization of these neurons. Some ethanol-induced excitotoxicity-related mechanisms, which may be underlying these effects, are discussed.
Assuntos
Animais Recém-Nascidos/fisiologia , Depressores do Sistema Nervoso Central/toxicidade , Etanol/toxicidade , Hipocampo/patologia , Animais , Peso Corporal/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Feminino , Tamanho da Ninhada de Vivíparos/efeitos dos fármacos , Masculino , Plasticidade Neuronal/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Ratos , Ratos Sprague-Dawley , SobrevidaRESUMO
The reaction of ammonium pyrrolidinedithiocarbamate, [NH4{S2CN(CH2)4}], with SnCl2, [Sn(C6H5)2Cl2], [Sn(C6H5)3Cl], [Sn(C4H9)2Cl2] and [Sn(C6H11)3Cl] produced in good yield the compounds [Sn{S2CN(CH2)4}2Cl2] (1), [Sn{S2CN(CH2)4}2Ph2] (2), [Sn{S2CN(CH2)4}Ph3] (3), [Sn{S2CN(CH2)4}2 n-Bu2] (4) and [Sn{S2CN(CH2)4}Cy3] (5). The complexes were characterised by infrared, multinuclear NMR (1H, 13C{1H} and 119Sn{1H}) and 119Sn Mössbauer spectroscopies. In addition, the crystal structure of 4 was determined by X-ray crystallography. The in vitro antifungal activity of the tin(IV) complexes as well of the ligand was performed on human pathogenic fungi, Candida albicans, in concentrations of 0.025; 0.050; 0.100; 0.200; 0.400; 0.800; 1.600 and 3.200 mM. The microorganism presented resistance to the dithiocarbamate ligand and all tin(IV) complexes tested were actives. The highest activity was found for compounds 1 and 4.
Assuntos
Antifúngicos , Compostos Organometálicos , Pirrolidinas/química , Tiocarbamatos/química , Estanho/química , Antifúngicos/síntese química , Antifúngicos/química , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ligantes , Testes de Sensibilidade Microbiana , Modelos Moleculares , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Relação Estrutura-AtividadeRESUMO
The involvement of the cerebellum in procedural learning is demonstrated in visuomotor-sequence tasks, as lesion of this area impedes the acquisition of new sequences. Likewise, the lateral cerebellum appears to be involved in the acquisition of new sequences, but not in the execution of learned sequences. In contrast, the dentate nucleus participates only in the execution of learned visuomotor sequences. In previous studies, disruption of the procedural elements of spatial navigation following cerebellar or dentate lesions has been reported. However, as praxic strategies (egocentric learning) are included in the procedural elements of the navigation, the participation of the cerebellar-dentate nucleus in egocentric procedural learning processes has not been evaluated. Therefore, using colchicine, bilateral lesions were made in the cerebellar-dentate nucleus of Sprague-Dawley rats, and these rats were given two tasks: egocentric-based motor sequence learning in the radial maze and egocentric navigation in the Morris water maze. The lesioned rats were unable to use the sequential information in the short term and showed delayed long-term acquisition, which was probably due to the inability to detect the sequence. No effects on the egocentric navigation task were observed. Our results indicate that the cerebellar-dentate nucleus is involved in the detection of egocentric sequential information but not in the use of this information in the navigation process. Further, they show differential involvement of the cerebellar-dentate nucleus in the execution of learned visuomotor sequences, as the dentate lesion disrupted the acquisition of new egocentric-motor-based sequences.