Use of blends of bioabsorbable poly(L-lactic acid)/poly(hydroxybutyrate-co-hydroxyvalerate) as surfaces for Vero cell culture

Vero cells, a cell line established from the kidney of the African green monkey (Cercopithecus aethiops), were cultured in F-10 Ham medium supplemented with 10% fetal calf serum at 37 degrees C on membranes of poly(L-lactic acid) (PLLA), poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and their blends in different proportions (100/0, 60/40, 50/50, and 0/100). The present study evaluated morphology of cells 40/60, grown on different polymeric substrates after 24 h of culture by scanning electron microscopy. Cell adhesion was also analyzed after 2 h of inoculation. For cell growth evaluation, the cells were maintained in culture for 48, 120, 240, and 360 h. For cytochemical study, the cells were cultured for 120 or 240 h, fixed, processed for histological analysis, and stained with Toluidine blue, pH 4.0, and Xylidine ponceau, pH 2.5. Our results showed that cell adhesion was better when 60/40 and 50/50 blends were used although cells were able to grow and proliferate on all blends tested. When using PLLA/PHBV (50150) slightly flattened cells were observed on porous and smooth areas. PLLA/PHBV (40/60) blends presented flattened cells on smooth areas. PLLA/PHBV (0/100), which presented no pores, also supported spreading cells interconnected by thin filaments. Histological sections showed that cells grew as a confluent monolayer on different substrates. Cytochemical analysis showed basophilic cells, indicating a large amount of RNA and proteins. Hence, we detected changes in cell morphology induced by alterations in blend proportions. This suggests that the cells changed their differentiation pattern when on various PLLA/PHBV blend surfaces.38111623163

Uso do Doppler vascular para detectar o efeito agudo do estradiol em mulheres na pós-menopausa

OBJECTIVES: To report on a simple practical test for assessing acute estradiol vascular effects on healthy and unhealthy postmenopausal women. INTRODUCTION: Estradiol acts in the endothelium to promote vasodilatation through genomic and non-genomic mechanisms, but its vascular action may be impaired in diabetes mellitus, hypertension, smoking and obesity. METHODS: Nineteen postmenopausal women (nine healthy and 10 with two or more of the above factors) of similar age and time since menopause were examined with vascular Doppler ultrasound. Resistance indexes and systolic and diastolic flow velocities were determined for the brachial and internal carotid arteries at baseline and 20 minutes after administration of a nasal estradiol formulation, available on the market, which reaches 1,200-1,500 pg/ml in the serum in 10-30 minutes. Estradiol blood levels were measured at 30 minutes. RESULTS: The carotid resistance index increased 14.2% (vasoconstriction) in the unhealthy group after estradiol, from a mean ± S.E. of 0.56 ± 0.016 at baseline to 0.64 ± 0.05 (p=0.033), and remained unchanged in healthy women. Brachial diastolic flow velocity increased 19.7% (vasodilatation) in healthy women, from 16.2 ± 1.93 to 19.4 ± 0.64 cm/s (p=0.046), and did not change in the unhealthy subjects. Estradiol levels were similar in both groups. DISCUSSION: Healthy postmenopausal women showed brachial vasodilatation while unhealthy postmenopausal women displayed vasoconstriction at the carotid artery. Vascular responses to estradiol were divergent between the groups. CONCLUSIONS: The acute estradiol test, coupled with Doppler ultrasound, seemed to be able to differentiate women with normal and abnormal endothelial function in a simple, non-invasive manner.OBJETIVO: Descrever um teste simples e prático para avaliar o efeito vascular agudo do estradiol em mulheres saudáveis e não-saudáveis na menopausa. INTRODUÇÃO: O estradiol atua no endotélio promovendo vasodilatação através de mecanismos genômicos e não-genômicos e esta ação pode estar prejudicada em morbidades como diabetes mellitus, hipertensão, tabagismo e obesidade. MÉTODOS: Dezenove mulheres na pós-menopausa (9 saudáveis e 10 com dois ou mais dos fatores acima), com idade e tempo de menopausa semelhantes foram examinadas por Doppler vascular. O índice de resistência e as velocidades de fluxo sistólico e diastólico foram medidos nas artérias braquial e carótida, em condições basais e 20 min após a administração de uma preparação nasal de estradiol, disponível comercialmente, que atinge 1200 a 1500 pg/ml no soro, entre 10 e 30 min após a aplicação. Os níveis séricos de estradiol foram determinados 30 min após a aplicação nasal. RESULTADOS: O índice de resistência da artéria carótida aumentou em 14,2% (vasoconstricção) após o estradiol no grupo não-saudável, partindo da média ± SE de 0,56 ± 0,016 para 0,64 ± 0,05 (p=0,033) e não se modificou nas mulheres saudáveis. A velocidade de fluxo diastólico da artéria braquial aumentou 19,7% (vasodilatação) nas mulheres saudáveis, partindo de 16,2 ± 1,93 para 19,4 ± 0,64 cm/s (p=0,046) e não apresentou alteração nas não saudáveis. Os níveis de estradiol foram semelhantes nos dois grupos. DISCUSSÃO: Nas mulheres saudáveis na menopausa houve vasodilatação da artéria braquial e nas não-saudáveis vasoconstricção na artéria carótida. A resposta vascular ao estradiol foi divergente entre os grupos estudados. CONCLUSÃO: O teste com estradiol agudo, associado ao Doppler vascular, parecem diferenciar, de forma simples e não-invasiva, mulheres com função endotelial normal e anormal

Use of vascular Doppler ultrasound to detect acute estradiol vascular effect in postmenopausal women

OBJECTIVES: To report on a simple practical test for assessing acute estradiol vascular effects on healthy and unhealthy postmenopausal women. INTRODUCTION: Estradiol acts in the endothelium to promote vasodilatation through genomic and non-genomic mechanisms, but its vascular action may be impaired in diabetes mellitus, hypertension, smoking and obesity. METHODS: Nineteen postmenopausal women (nine healthy and 10 with two or more of the above factors) of similar age and time since menopause were examined with vascular Doppler ultrasound. Resistance indexes and systolic and diastolic flow velocities were determined for the brachial and internal carotid arteries at baseline and 20 minutes after administration of a nasal estradiol formulation, available on the market, which reaches 1,200-1,500 pg/ml in the serum in 10-30 minutes. Estradiol blood levels were measured at 30 minutes. RESULTS: The carotid resistance index increased 14.2% (vasoconstriction) in the unhealthy group after estradiol, from a mean ± S.E. of 0.56 ± 0.016 at baseline to 0.64 ± 0.05 (p=0.033), and remained unchanged in healthy women. Brachial diastolic flow velocity increased 19.7% (vasodilatation) in healthy women, from 16.2 ± 1.93 to 19.4 ± 0.64 cm/s (p=0.046), and did not change in the unhealthy subjects. Estradiol levels were similar in both groups. DISCUSSION: Healthy postmenopausal women showed brachial vasodilatation while unhealthy postmenopausal women displayed vasoconstriction at the carotid artery. Vascular responses to estradiol were divergent between the groups. CONCLUSIONS: The acute estradiol test, coupled with Doppler ultrasound, seemed to be able to differentiate women with normal and abnormal endothelial function in a simple, non-invasive manner.OBJETIVO: Descrever um teste simples e prático para avaliar o efeito vascular agudo do estradiol em mulheres saudáveis e não-saudáveis na menopausa. INTRODUÇÃO: O estradiol atua no endotélio promovendo vasodilatação através de mecanismos genômicos e não-genômicos e esta ação pode estar prejudicada em morbidades como diabetes mellitus, hipertensão, tabagismo e obesidade. MÉTODOS: Dezenove mulheres na pós-menopausa (9 saudáveis e 10 com dois ou mais dos fatores acima), com idade e tempo de menopausa semelhantes foram examinadas por Doppler vascular. O índice de resistência e as velocidades de fluxo sistólico e diastólico foram medidos nas artérias braquial e carótida, em condições basais e 20 min após a administração de uma preparação nasal de estradiol, disponível comercialmente, que atinge 1200 a 1500 pg/ml no soro, entre 10 e 30 min após a aplicação. Os níveis séricos de estradiol foram determinados 30 min após a aplicação nasal. RESULTADOS: O índice de resistência da artéria carótida aumentou em 14,2% (vasoconstricção) após o estradiol no grupo não-saudável, partindo da média ± SE de 0,56 ± 0,016 para 0,64 ± 0,05 (p=0,033) e não se modificou nas mulheres saudáveis. A velocidade de fluxo diastólico da artéria braquial aumentou 19,7% (vasodilatação) nas mulheres saudáveis, partindo de 16,2 ± 1,93 para 19,4 ± 0,64 cm/s (p=0,046) e não apresentou alteração nas não saudáveis. Os níveis de estradiol foram semelhantes nos dois grupos. DISCUSSÃO: Nas mulheres saudáveis na menopausa houve vasodilatação da artéria braquial e nas não-saudáveis vasoconstricção na artéria carótida. A resposta vascular ao estradiol foi divergente entre os grupos estudados. CONCLUSÃO: O teste com estradiol agudo, associado ao Doppler vascular, parecem diferenciar, de forma simples e não-invasiva, mulheres com função endotelial normal e anormal

Molecular detection of Ehrlichia canisand Anaplasma platys in dogs in Southern Brazil

The aims of this study were to determine the occurrence of Anaplasma platys and Ehrlichia canis infection in dogs in Porto Alegre, Southern Brazil; and to investigate their association with hematological abnormalities. Serum samples from 196 dogs were first tested using dot-ELISA for antibodies against Anaplasma spp. and Ehrlichia canis. Peripheral blood samples from 199 dogs were subjected to 16S rRNA nested PCR (nPCR) for A. platys and E. canis, followed by DNA sequencing to ensure pathogen identity. A total of 19/196 samples (9.69%) were positive for Anaplasma spp. using ELISA and 28/199 (14.07%) samples were positive for A. platys by nested PCR. All the dog samples were negative for E. canis, both in anti-E. canis antibody tests and in nested PCR. There were no significant differences in hematological parameters between A. platys-PCR positive and negative dogs and Anaplasma spp. serologically positive dogs, except for basophil counts, which were higher in nPCR-positive dogs. This is the first report showing A. platys presence in dogs in Southern Brazil. In conclusion, hematological parameters may not be sufficient to diagnose A. platys infection in dogs in Southern Brazil, probably due either to low pathogenicity or to chronic infection. On the other hand, E. canis may either have very low occurrence or be absent in dogs in Porto Alegre.O objetivo deste estudo foi determinar a ocorrência de Anaplasma platys e Ehrlichia canis em cães de Porto Alegre, sul do Brasil, sua detecção molecular e associação com anormalidades hematológicas. Amostras séricas de 196 cães foram inicialmente triadas por dot-ELISA para a presença de anticorpos contra Anaplasma spp. e Ehrlichia canis. Amostras de sangue periférico de 199 cães foram submetidas à nested PCR (16S rRNA) para A. platys e E. canis, seguido de sequenciamento do DNA para confirmar a identidade do agente. Do total, 19/196 (9,69%) amostras foram positivas para Anaplasma spp. por dot-ELISA e 28/199 (14,07%) por nPCR. Todas as amostras dos cães foram negativas para E. canis no teste sorológico anti-E. canis e também na nPCR. Não houve diferença significativa nos parâmetros hematológicos, exceto a contagem de basófilos, que apresentou valores mais altos em cães positivos na nPCR para A. platys. Este é o primeiro relato da presença de A. platys no Rio Grande do Sul, e a primeira detecção molecular do agente no sul do Brasil. Em conclusão, parâmetros hematológicos não são suficientes para diagnosticar a infecção por A. platys em cães, provavelmente devido sua baixa patogenicidade ou infecção crônica. Por outro lado, E. canis parece ter ocorrência baixa ou mesmo nula em cães de Porto Alegre

Films of PLLA/PHBV: Thermal, morphological, and mechanical characterization

Blends Of poly(L-lactic acid)/poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PLLA/PHBV), both semicrystalline polymers, were prepared in different compositions (100/0, 80/20, 60/40, 50/50, 40/60, 20/80, and 0/100) and characterized by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA), scanning electron microscopy (SEM), polarized light microscopy (PLM), and tensile tests. Although PLLA/PHBV blends do not present clear phase separation by SEM, the analyses by TGA, DSC, and DMA showed that the PLLA/PHBV blends are immiscible. The cross sections observed by SEM showed that the morphology of the blends changes from porous to dense, due to the composition. DSC and DMA data showed two distinct glass transition and melting temperatures. However, the DMA analysis related to frequency variation showed partial molecular interactions between PHBV and PLLA. (C) 2002 Wiley Periodicals, Inc.86112898290

Increased response of Vero cells to PHBV matrices treated by plasma

The copolymers poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) are being intensely studied as a tissue engineering substrate. It is known that poly 3-hydroxybutyric acids (PHBs) and their copolymers are quite hydrophobic polyesters. Plasma-surface modification is an effective and economical surface treatment technique for many materials and of growing interest in biomedical engineering. In this study we investigate the advantages of oxygen and nitrogen plasma treatment to modify the PHBV surface to enable the acceleration of Vero cell adhesion and proliferation. PHBV was dissolved in methylene chloride at room temperature. The PHBV membranes were modified by oxygen or nitrogen-plasma treatments using a plasma generator. The membranes were sterilized by UV irradiation for 30 min and placed in 96-well plates. Vero cells were seeded onto the membranes and their proliferation onto the matrices was also determined by cytotoxicity and cell adhesion assay. After 2, 24, 48 and 120 h of incubation, growth of fibroblasts on matrices was observed by scanning electron microscopy (SEM). The analyses of the membranes indicated that the plasma treatment decreased the contact angle and increased the surface roughness; it also changed surface morphology, and consequently, enhanced the hydrophilic behavior of PHBV polymers. SEM analysis of Vero cells adhered to PHBV treated by plasma showed that the modified surface had allowed better cell attachment, spreading and growth than the untreated membrane. This combination of surface treatment and polymer chemistry is a valuable guide to prepare an appropriate surface for tissue engineering application.19263564

Differentiation pattern of Vero cells cultured on poly(L-lactic acid)/poly(hydroxybutyrate-co-hydroxyvalerate) blends

This study evaluates the effect of poly(L-lactic acid) (PLLA) and poly(hydroxybutyrate-cohydroxyvalerate) (PHBV) bioabsorbable polymers and their blends on the induction of alteration of cell growth pattern in vitro. Vero cells were cultured on PLLA, PHBV, and different blends (100/0, 60/40, 50/50, 40/60, and 0/100). The cell adhesion assay showed that the best results were obtained with the (60/40, 50/50) blends. Scanning electron microscopy showed that the cells on (100/0) and (60/40) samples grew with a round morphology preferentially in the porous areas. The (50/50) blends had cells in the porous and smooth areas in a similar way. The (40/60) blends showed spreading cells on the smooth areas. The (0/100) sample, which had no pores, had spreading cells interconnected by filaments. Histological sections showed a confluent cell monolayer and the immunocytochemistry showed that the cells produced collagen IV and fibronectin on all substrates. Thus, we conclude that PLLA/PHBV blends were efficient in maintaining cell growth and producing an extracellular matrix on them.28438138

PHBV/PLLA-based composite scaffolds containing nano-sized hydroxyapatite particles for bone tissue engineering

Composite scaffolds made of PHBV/PLLA blends and nano-sized hydroxyapatite (HA) particles can be useful for bone tissue engineering as different blends of PHBV with PLLA should have higher degradation rates than PHBV itself and scaffolds containing bioactive HA particles will provide desired osteoconductivity. In this investigation, composite scaffolds were produced via an emulsion freezing/freeze-drying process and subsequently characterised using several techniques. Highly porous and interconnected scaffolds with porosity greater than 70% could be made. It was found that the crystallinity of PHBV in the scaffolds was decreased due to blending with the PLLA polymer, which is beneficial for the faster degradation of the scaffolds in vitro and in vivo. It was shown that the incorporation of HA nanoparticles enhanced compressive mechanical properties of scaffolds.link_to_subscribed_fulltex