Preparation and characterization of a novel antimicrobial film dressing for wound healing application

Antibacterial activity and good mechanical properties are some of the characteristics required for an appropriate film dressing. A novel polymer blend was developed for wound healing application. Twenty-four formulations using the polymers chitosan, poly(vinyl alcohol) and/or ɛ-Polylysine and the plasticizer glycerol were designed using factorial design and then the films were prepared by the casting/solvent evaporation method. Seventeen films were obtained among the twenty-four proposed formulations that were characterized by Field Emission Scanning Electron Microscopy (FE-SEM) and Fourier Transform Infrared Spectroscopy (FTIR). Mechanical properties, such as tensile strength (σ), elongation at break (ɛ) and Young's modulus (Y) as well as antibacterial properties were determined. The best candidate was then further analyzed with regard to porosity, Water Vapor Transmission Rate (WVTR), swelling and cytotoxicity experiments. The results showed a film with semi-occlusive characteristics, good mechanical properties and no toxic. Incorporation of ɛ-Polylysine increased antibacterial activity against gram-negative (Escherichia coli) and gram-positive (Staphylococcus aureus) bacteria

Using allogeneic cortical graft preserved in glycerin as spacer in the advancement of tibial tuberosity in 34 dogs / Uso de enxerto cortical alogênico preservado em glicerina como espaçador no avanço da tuberosidade tibial em 34 cães

Cranial cruciate ligament is the main responsible for knee stability by preventing cranial tibial displacement regarding the femur. Deficiency in this ligament (CCLD) may cause subluxation of the tibia and dysfunction of the pelvic member due to overloading. Tibial osteotomies are among the more current surgical techniques for treating CCLD in dogs and they proportionate the dynamic stability by means of modifying bone geometry and the distribution of forces acting on the articulation. The objective of this work is to describe the use of the allogeneic cortical bone graft conserved in glycerin as a spacer on the tibial tuberosity advancement (TTA) for treating the CCLD. In order to do that, 34 dogs submitted to TTA surgery correction were evaluated, being 23 males (67.35%) and 11 females (32.35%). Surgical procedures happened from May 2011 to October 2015. Regarding the surgical procedure after osteotomy of the tibial tuberosity, a disk of allogeneic cortical disk, sawn wedge-hapsed, conserved in glycerin, proportions of 2x1mm was applied as spacer, enabling TTA. Advancements from 3 to 12 mm were executed, depending on the need of the patient. For animals with patella dislocation, trochleoplasty and TTA were executed in order to correct the deviation. The mean ± SD age of animals was 6.67±3.58 and weight was 15.16±12.97 kg. Mongrel dogs, Poodles and Yorkshire terriers were the most affected ones. From the 36 evaluated knees, 11 (30.56%) were associated with some traumatic process and in 25 (69.44%) there was no relation with previous trauma. From those wounds, 20 (55.56%) happened in the right limb and 16 (44.44%) in the left limb and two animals had CCLD bilaterally. Animals had continuous support, discreet drawer movement and negative tibial compression 15 days after surgery. At 30 days, 26 cases (72.22%) had firm support (FS); at 45 days, 24 cases (66 test at 7 and 67%) had FS and eight cases (22.22%) without claudication (WC). During subsequent radiographic evaluations the progressive incorporation of the graft and osteotomy union were observed. In this study, most of the diagnosed CCLD occurred in males diverging from results obtained by other authors that found greater frequency in females. Support without claudication it was observed in most of the cases of implants at 60 days. We concluded that the conserved allogeneic cortical bone graft was able to promote bone union in TTA of dogs with CCLD. None of the animals had signs of contamination, infection of the surgical wound or rejection related with the presence of the graft, demonstrated by the complete graft-bone incorporation observed early at 45 days in some animals. The glycerin was a good conservation medium for those fragments intended for grafting because, besides being of low cost, it kept bone fragments free of contamination, reducing antigenicity and preserving the functions of osteoinduction and osteoconduction. The possibility of molding the graft to the animal need is a characteristic favorable to executing the modified technique that could be molded according to the size of the animal, allowing perfect adaptation to the osteotomized local in different breeds. Intercurrences commonly observed in TTA with patellar dislocation, meniscal lesions, tibial crest fracture and displacement were not found in the animals of this study, probably due to the better distribution of forces between the pass screw in TT and the TTA plate confirming that it has good adaptation to the technique conferring to the modified TTA advantages regarding the conventional TTA.(AU)

O ligamento cruzado cranial é o principal responsável pela estabilidade do joelho, impedindo o deslocamento da tíbia cranial em relação ao fêmur. A deficiência neste ligamento (CCLD) pode causar subluxação da tíbia e disfunção do membro pélvico devido à sobrecarga. As osteotomias tibiais estão entre as técnicas cirúrgicas mais atuais para o tratamento de CCLD em cães e proporcionam a estabilidade dinâmica por meio da modificação da geometria óssea da distribuição das forças que atuam sobre a articulação. O objetivo desse estudo é descrever o uso do enxerto ósseo cortical alogênico conservado em glicerina como espaçador no avanço da tuberosidade tibial (TTA) para o tratamento do CCLD. Para isso, 34 cães submetidos à cirurgia de TTA foram avaliados, sendo 23 machos (67,35%) e 11 fêmeas (32,35%). Os procedimentos curúrgicos aconteceram entre maio de 2011 e outubro de 2015. Com relação ao procedimento cirúrgico após a osteotomia da tuberosidade tibial, um disco alogênico cortical, em forma de cunha serrada, conservado em glicerina com proporções de 2 x 1mm foi aplicado como espaçador possibilitando a TTA. Avanços de 3 a 12mm foram executados, dependendo da necessidade do paciente. Para animais com luxação da patela, realizou-se a trocleoplastia e a TTA para a correção do desvio. A idade média dos animais foi de 6,67±3,58 anos e pesos médios de 15,16±12,97kg. Cães sem raça definida, Poodles e Yorkshire Terriers foram os mais afetados. Dos 36 joelhos avaliados, 11 (30,56%) foram associados a algum processo traumático e em 25 (69,44%) não havia nenhuma relação com um trauma prévio. Dos ferimentos, 20 (55,56%) aconteceram no membro direito e 16 (44,44%) no esquerdo, sendo que dois animais apresentavam CCLD bilateralmente. Os animais tiveram suporte contínuo, discreto movimento de gaveta e compressão tibial negativa 15 dias após a cirurgia. Aos 30 dias, 26 casos tinham suporte firme (FS); aos 45 dias, 24 casos tinham FS e oito casos sem claudicação (WC). Durante avaliações radiográficas subsequentes, observou-se a incorporação progressiva da união do enxerto e da osteotomia. Neste estudo, a maior parte do CCLD diagnosticado ocorreu em machos, divergindo dos resultados obtidos por outros autores que encontraram maior frequência em fêmeas. Suporte sem claudicação foi observado na maioria dos casos de implantes aos 60 dias. Foi concluído que o enxerto ósseo cortical alogênico conservado foi capaz de promover a união óssea na TTA de cães com CCLD. Nenhum dos animais apresentou sinais de contaminação, infecção da ferida cirúrgica ou rejeição relacionada à presença do enxerto, demonstrada pela incorporação completa do enxerto ósseo observada precocemente aos 45 dias em alguns animais. A glicerina foi um bom meio de conservação para os fragmentos destinados à enxertia porque, além do menor custo, manteve os fragmentos ósseos livres de contaminação, reduzindo a antigenicidade e preservando as funções de osteoindução e osteocondução. A possibilidade de moldagem do enxerto à necessidade do animal é uma característica favorável à execução da técnica modificada que pode ser moldada de acordo com o tamanho do animal, possibilitando perfeita adaptação ao local osteotomizado em diferentes raças. Intercorrências comumente observadas na TTA com luxação patelar, lesões meniscais, fratura da crista tibial e deslocamento não foram encontradas nos animais deste estudo, provavelmente devido à melhor distribuição de forças entre a passagem do parafuso no TT e a placa do TTA, confirmando que tem boa adaptação à técnica conferindo às vantagens da TTA modificada em relação à TTA convencional.(AU)

Cryopreservation of banana's cv Grand Naine in vitro rhizomes

ABSTRACT The preservation of banana genetic material is usually performed through seedlings. However, most banana cultivars do not produce seed and are propagated vegetatively. Therefore, cryopreservation is a feasible technique that allows the preservation of banana genotypes indefinitely. For the success of cryopreservation protocols, the selection of cryoprotectants and pre-freezing techniques are important factor. Therefore, the objective of this study was to verify the effects of different cryoprotectants with and without 1% phloroglucinol and pre-cooling periods on the development of a protocol for cryopreservation of in vitro rhizomes ofMusa accuminata(AAA) cv Grand Naine banana. The addition of 1% phloroglucinol to the cryoprotective solutions, such as PVS2 enhanced recovery of cryopreserved banana rhizomes. In addition, pre-cooling of explants in ice for 3 hours in PVS2 + 1% of phloroglucinol allowed efficient cryopreservation of banana rhizomes, followed by successful recovery and regeneration of in vitro shoots of banana cv Grand Naine.

Blocking or enhancing effects of some basic emollients in UVA penetration

Abstract: Background: Topical agents used in combination with phototherapy or photochemotherapy may have both blocking or enhancing effects in ultraviolet rays. Objective: In this in vivo study, the effects of topical petrolatum, basis cream, glycerine, and olive oil on the transmission of ultraviolet A radiation were investigated. Methods: A test was performed to determine the minimal phototoxic dose on 29 volunteers with only psoralen plus ultraviolet A (PUVA) and then the same test was repeated with white petrolatum, basis cream, glycerine, olive oil, and sunscreen (0.3cc/25cm2). The effects of each agent on the minimal phototoxic dose were determined after 72 h. Results: When compared to pure PUVA, there was a statistically significant increase in the mean minimal phototoxic dose values by the application of white petrolatum (P = 0.011), but there was no significant increase or decrease in the mean minimal phototoxic dose values after the application of basis cream (P = 0.326), glycerine (P = 0.611) or olive oil (P = 0.799). Study limitations: Low number of patients Conclusion: The application of white petrolatum, which has a blocking effect, and also of basis cream immediately before PUVA therapy should not be recommended. Although we specify that glycerine and maybe olive oil can be used before photochemotherapy, there is a need for further research in larger series.

Antitumor compounds from Streptomyces sp. KML-2, isolated from Khewra salt mines, Pakistan

BACKGROUND: Actinomycetes are gram positive bacteria with high G + C content in their DNA and are capable of producing variety of secondary metabolites. Many of these metabolites possess different biological activities and have the potential to be developed as therapeutic agents. The aim of the present study was to screen actinomycetes inhabiting halophilic environment such as Khewra salt mines present in Pakistan for cytotoxic and antitumor compounds. RESULTS: An actiomycetes strain designated as Streptomyces sp. KML-2 was isolated from a saline soil of Khewra salt mines, Pakistan. The strain Streptomyces sp. KML-2 showed 84 % cytotoxic activity against larvae of Artemiasalina. In the screening phase, the strain exhibited significant antitumor activity with IC50 values of 12, 48 and 56 µg/ml against Hela, MDBK and Vero cell lines, respectively. After that extract from 20 l fermentation was used to purify secondary metabolites by several chromatographic techniques. Structure elucidation of isolated compounds revealed that it is highly stable producer of Chromomycin SA (1) and 1-(1H-indol-3-yl)-propane-1,2,3-triol (2). Both of the isolated compounds showed significant antitumor activity against Hela and MCF-7 cancer cell lines (IC50 values 8.9 and 7.8 µg/ml against Hela; 12.6 and 0.97 µg/ml against MCF-7, respectively). The 16S rRNA gene sequence (1437 bp) of the strain confirm its identity (99 %) with Streptomyces griseus. CONCLUSIONS: From this research work we were successful in isolating two potent antitumor compounds, Chromomycin SA and 1-(1H-indol-3-yl)-propane-1,2,3-triol from Streptomyces KML-2 strain, isolated from Khewra salt mine. As such this is the second report which confirms that S. griseus can produce Chromomycin SA without introducing any mutagenesis in its biosynthesizing gene cluster and isolated indole derivative is being reported first time from any member of actinomycetes group with having novel antitumor activity against Hela and MCF-7 cells Nucleotide sequences: Nucleotide sequence data reported are available in the GenBank database under the accession number: GenBank KJ009562.

Optimization of biomass production of a mutant of Yarrowia lipolytica with an increased lipase activity using raw glycerol

The yeast Yarrowia lipolytica accumulates oils and is able to produce extracellular lipases when growing in different carbon sources including glycerol, the principal by-product of the biodiesel industry. In this study, biomass production of a novel mutant strain of Y. lipolytica was statistically optimized by Response Surface Methodology in media containing biodiesel-derived glycerol as main carbon source. This strain exhibited distinctive morphological and fatty acid profile characteristics, and showed an increased extracellular lipase activity. An organic source of nitrogen and the addition of 1.0 g/l olive oil were necessary for significant lipase production. Plackett-Burman and Central Composite Statistical Designs were employed for screening and optimization of fermentation in shaken flasks cultures, and the maximum values obtained were 16.1 g/l for biomass and 12.2 Units/ml for lipase, respectively. Optimized batch bioprocess was thereafter scaled in aerated bioreactors and the values reached for lipase specific activity after 95 % of the glycerol had been consumed, were three-fold higher than those obtained in shaken flasks cultures. A sustainable bioprocess to obtain biomass and extracellular lipase activity was attained by maximizing the use of the by-products of biodiesel industry.

Optimización de la producción de biomasa usando glicerol crudo, de una cepa mutante de Yarrowia lipolytica con actividad incrementada de lipasa. La levadura Yarrowia lipolytica acumula aceites y produce una lipasa extracelular al crecer en diferentes fuentes de carbono, entre ellas el glicerol, principal subproducto de la creciente industria del biodiésel. En el presente trabajo, se optimizó mediante la metodología de superficies de respuesta la producción de biomasa de una nueva cepa mutante de Y. lipolytica, empleando medios con glicerol derivado de la industria del biodiésel como principal fuente de carbono. Esta cepa presentó características morfológicas y perfil de ácidos grasos distintivos, y una mayor actividad de lipasa extracelular. Para obtener una producción significativa de lipasa extracelular, fue necesario el agregado de una fuente orgánica de nitrógeno y de 1 g/l de aceite de oliva. Se utilizaron los diseños estadísticos de Plackett-Burman y central compuesto para la selección y la optimización de las fermentaciones en frascos agitados; los máximos valores de biomasa y de lipasa obtenidos fueron de 16,1 g/l y 12,2 unidades/ml, respectivamente. Luego, el bioproceso en lote optimizado se escaló a biorreactores aireados, y los valores de actividad específica de lipasa alcanzados después de haberse consumido el 95 % del glicerol fueron tres veces más altos que los obtenidos en los cultivos en frascos agitados. En suma, se desarrolló un bioproceso sostenible para la obtención de biomasa y de una actividad de lipasa extracelular, que a la vez maximiza el uso de subproductos de la industria del biodiésel.

Osteoblast response to commercially available demineralized bone matrices - An in-vitro study.

Objective: Reconstruction of lost attachment apparatus is a major goal of periodontal therapy. Although various osteoinductive bone replacement grafts (BRGs) have been used with apparent clinical success, unequivocal evidence of osteoinductivity may be obtained only through the demonstration of increased osteoblastic/osteoclastic differentiation following exposure to these materials. Materials and Methods: Bone marrow stem cells (BMSCs) obtained from rat femur were cultured in Dulbecco's Modified Eagles Medium (DMEM) and 10% fetal bovine serum (FBS). They were then exposed to two demineralized bone matrices (DBM's) - Grafton and Osseograft, and divided into three groups, comprising of a negative control (BMSC + DMEM + 10% FBS), Grafton, Osseograft. An osteogenic medium (OM) (10 hm dexamethasone, 10 hm b-glycerophosphate, and 50 μg/ml ascorbic acid) was added to create three subgroups comprising of a positive control (OM), Grafton with OM, Osseograft with OM. Results: After an initial phase (up to day 5), both Grafton and Osseograft induced an increased proliferative activity in the BMSCs, which reached a plateau after day 10. These grafts also induced increased alkaline phosphatase activity when compared to the control groups and to BMSCs with an OM. Conclusion: Both Osseograft and Grafton are capable of inducing osteoblastic proliferation and differentiation.

Influence of serum and necrotic soft tissue on the antimicrobial effects of intracanal medicaments

The purpose of this study was to investigate the influence of serum and necrotic soft tissue on the antimicrobial activity of intracanal medicaments. The medicaments tested were: calcium hydroxyde/glycerin paste, calcium hydroxide/chlorhexidine paste, calcium hydroxide/camphorated paramonochlorophenol/glycerin paste, and chlorhexidine/zinc oxide paste. Survival of Enterococcus faecalis and Candida albicans exposed to the medicaments tested in the presence or absence of serum or necrotic tissue was monitored in three in vitro experiments where samples for culturing were taken at different time periods. The overall results demonstrated that the antimicrobial activity of all intracanal medicaments tested was slowed down in the presence of necrotic tissue. Calcium hydroxide pastes in glycerin or chlorhexidine were significantly affected by serum. Of the medicaments tested in this study, the least affected was the calcium hydroxide/camphorated paramonochlorophenol/glycerin paste.

O objetivo deste estudo foi avaliar a influência do soro e de tecido mole necrosado na atividade antimicrobiana de medicamentos intra-canais. Os medicamentos testados foram pastas de hidróxido de cálcio/glicerina, hidróxido de cálcio/clorexidina, hidróxido de cálcio/paramonoclorofenol canforado/glicerina e clorexidina/óxido de zinco. A sobrevivência de Enterococcus faecalis e Candida albicans expostos aos medicamentos na presença ou ausência de soro ou tecido necrosado foi monitorada em três experimentos in vitro nos quais amostras para cultura foram avaliadas em diferentes períodos de tempo. No geral, os resultados demonstraram que a atividade antimicrobiana de todos os medicamentos testados foi retardada na presença de soro ou de tecido necrosado. As pastas de hidróxido de cálcio em glicerina ou clorexidina foram significativamente afetadas pelo soro. Dos medicamentos testados, o menos afetado foi a pasta de hidróxido de cálcio/paramonoclorofenol canforado/glicerina.

Concentration of hydrogen ions in several calcium hydroxide pastes over different periods of time

The purpose of this in vitro study was to measure the concentration of hydrogen ions (pH) of calcium hydroxide [(Ca(OH)2] pastes combined with different vehicles over 7 periods of time. The Ca(OH)2 was manipulated with the following vehicles: i: sterile water; ii: iodoform plus sterile water; iii: local anesthetics (Lydocaine 2 percent with 1: 100,000 epinephrine); iv: polyethyleneglycol; v: glycerin; vi: 2.0 percent chlorhexidine gel; vii: camphorated paramonochlorophenol (CMCP); viii: (CMCP) + glycerin; and ix: polyethyleneglycol plus CMCP. The pastes were made on a glass plate to toothpaste consistency and the pH was measured at the following times: 5 min, 1, 24, 48 h; 7, 14 and 28 days. The data were statistically analyzed (Kruskal-Wallis at p<0.05). At 5 min, 1 and 24 h, the pH of all tested pastes ranged from 13.05 to 11.16. At 48 h and 7 days the pH of all tested pastes ranged from 11.66 to 8.92. At 14 and 28 days almost all pastes had pH means lower than 10. In conclusion, the mean pH of all tested calcium hydroxide pastes decreased with the time. Pastes made with aqueous vehicles (especially with sterile water), followed by oily vehicles (especially with CMCP + glycerin), held the highest pH means over the periods of time tested.

O objetivo deste estudo in vitro foi mensurar a concentração de ions hidroxila (pH) de pastas de hidróxido de cálcio manipuladas com diversos veículos em 7 intervalos de tempo. As pastas foram manipuladas com os seguintes veículos: (i) água destilada estéril; (ii) idodofórmio + água destilada estéril; (iii) anestésico local (Lidocaína a 2 por cento com 1:100.000 epinefrina); (iv) polietilenoglicol (Calen); (v) glicerina; (vi) clorexidina gel 2 por cento; (vii) paramonoclorofenol canforado (PMCC); (viii) PMCC + glicerina; e (ix) PMCC + polietilenoglicol (Calen PMCC). As pastas foram manipuladas em consistência de pasta de dente e os pH mensurados 5 min; 1, 24, 48 h; 7, 14 e 28 dias após manipulação. Os resultados foram analisados estatisticamente através do teste Kruskal-Wallis (p<0,05). Aos 5 min, 1 e 2 h após manipulação o pH de todas as pastas ficou entre 13.05 e 11.16. Aos 48 h e 7 dias após a manipulação, o pH de todas as pastas testadas variou de 11.66 a 8.92. Aos 14 e 28 dias, quase todas as pastas mostraram pH menor que 10. Concluiu-se que o pH de todas as pastas hidróxido de cálcio decresceram de acordo com o tempo. Pastas feitas com veículos aquosos (especialmente com água destilada), seguida de veículos oleosos (especialmente com CMCP + glicerina) mantiveram as maiores médias de pH durante os períodos testados.

Effects of intraperitoneal injection of phenol, glycerin and acetic acid on neoplastic ascitis in guinea pigs / Efeitos da injeção intraperitoneal de fenol, glicerina e ácido acético na ascite neoplásica em cobaias

PURPOSE: To investigate the histolytic action of a solution composed by phenol, glycerin and acetic acid on neoplastic ascitis in guinea pigs. METHODS: Thirty-two guinea pigs were used. The animals were randomly distributed in experimental and control groups, and the effects of the peritoneal injection of the testing solution were studied. Saline solution was used for the control groups. Biochemical and anatomopathological (heart, lungs, kidneys, spleen and peritoneal serous membrane) were evaluated at 24 hours and 4 weeks of development. RESULTS: It was observed that solution E, when infused into the peritoneal cavity, caused no clinical, histological or laboratory alterations in these animals when compared to those in the control group. CONCLUSION: Given our results, it would be interesting to study the effects of the proposed solution on cases with experimental neoplastic ascites with a later view to treating it in humans.

OBJETIVO: Investigar a ação hìstolítica de uma solução composta de fenol, glicerina e ácido acético na ascite neoplásica em cobaias. MÉTODOS: Foram utilizadas 32 cobaias, distribuídas por sorteio, em grupos experimentais e controles e estudados os efeitos da injeção peritonial da solução teste. Nos grupos controles empregou-se solução fisiológica. Foram estudadas alterações bioquímicas, anatomopatológicas (coração, pulmões, rins, baço e serosa peritonial), com 24 horas e 4 semanas de evolução. RESULTADOS: Verificou-se que a solução E quando instilada na cavidade peritonial não provocou nenhuma alteração clinica, histologica ou laboratorial nestes animais, quando comparados com o grupo controle. CONCLUSÃO: Frente aos resultados obtidos, consideramos interessante estudar os efeitos da solução proposta em casos de ascite neoplásica experimental em animais, com posterior estudo em seres humanos.

Biomechanical and histological evaluation of glycerol-preserved human sclerae

PURPOSE: To determine the histological and biomechanical characteristics of glycerol-preserved human sclera. METHODS: A total of 114 paired human sclerae were cleaned and preserved with 98 percent glycerol under refrigeration at 4 to 8ºC. The samples were divided into a control group with no preservation and 5 groups of 19 sclerae in 7, 15, 30, 90 and 180 days of preservation. Each specimen was submitted to histological examination and tested for traction distensibility functions. RESULTS: Preservation in glycerol did not cause alterations in the histological architecture of the scleral tissue. The mean load required to break the scleral tissue increased according to preservation time as a sigmoid function. A significant increase in mechanical resistance and decrease in distension of scleral tissue occurred after 90 days of preservation. CONCLUSIONS: Scleral preservation in glycerol keeps tissue integrity. The preserved material is less distensible after 90 days. Surgeons who use sclera in ophthalmic procedures should be aware of the mechanical characteristics of glycerol-preserved sclera and take into account tissue preservation time.

OBJETIVO: Determinar as características histológicas e biomecânicas de escleras humanas preservadas em glicerol. MÉTODOS: Escleras de ambos os olhos de 55 doadores foram limpas e preservadas com glicerol a 98 por cento sob refrigeração (4 a 8ºC). A amostra foi dividida em grupo controle sem preservação e 5 grupos de 19 escleras com 7, 15, 30, 90 e 180 dias de preservação. Todas as amostras foram submetidas à avaliação histológica e aos testes de tração e distensão. RESULTADOS: A preservação em glicerol não provocou alterações na arquitetura histológica do tecido escleral. A carga média necessária para romper o tecido escleral aumentou com o tempo de preservação segundo uma função sigmóide. Um incremento significativo na resistência mecânica e diminuição da elasticidade do tecido ocorreram após 90 dias de preservação. CONCLUSÕES: A preservação escleral com glicerol mantém a integridade tecidual. O material preservado torna-se menos distensível após 90 dias de preservação. Cirurgiões que usam esclera preservada em procedimentos oftalmológicos devem estar conscientes das propriedades mecânicas do material e levar em conta o tempo de preservação do material.

Análise ultraestrutural e de fatores de crescimento de diferentes métodos de preservação da membrana amniótica utilizada em cirurgia ocular / Ultrastructural and growth factor analysis of amniotic membrane preserved by different methods for ocular surger

OBJETIVO: Comparar, por microscopia eletrônica, a integridade anatômica e a presença de fatores de crescimento e citocinas da membrana amniótica preservada com glicerol/MEM (1:1) e dimetilsulfóxido puro. MÉTODOS: As membranas amnióticas preservadas em glicerol/MEM (1:1) ou dimetilsulfóxido puro foram processadas para microscopia eletrônica de transmissão e varredura. Como controle, membrana amniótica fresca foi imediatamente fixada após coleta e processada para microscopia eletrônica. As citocinas e os fatores de crescimento avaliados foram: TGF-beta- fator transformador de crescimento beta; TGF-beta ativ- fator transformador de crescimento beta ativado; EGF- fator recombinante de crescimento epitelial humano; FGF-4- fator de crescimento fibroblástico 4; FGF-beta- fator de crescimento fibroblástico básico; IL-4- interleucina 4; PGE2- prostaglandina E2; IL-10- interleucina 10; KGF- fator de crescimento de queratinócito; HGF- fator de crescimento de hepatócito. RESULTADOS: As membranas amnióticas do grupo controle apresentavam epitélio íntegro, com microvilos na superfície e complexos juncionais entre as células e a membrana basal. As membranas amnióticas preservadas em glicerol/MEM tinham aspecto semelhante às do controle, com maior altura das células epiteliais. Já as membranas amnióticas preservadas em dimetilsulfóxido mostraram redução das junções intercelulares e destacamento do epitélio da membrana basal. As citocinas e fatores de crescimento não apresentaram diferenças entre os grupos, exceto FGF-4, FGF-beta, PGE2 e KGF. CONCLUSÕES: A membrana amniótica preservada em meio glicerol/MEM apresentou melhor integridade tecidual, com menor desprendimento do epitélio da membrana basal, em comparação com a preservada no dimetilsulfóxido puro. Os fatores de crescimento e citocinas estavam, em sua maior parte, preservados com as duas técnicas de preservação.

PURPOSE: To compare the anatomical structure and the presence of growth factors and cytokines of amniotic membrane preserved in glycerol/MEM (1:1) or undiluted dimethyl sulfoxide through electron microscopy. METHODS: Amniotic membrane preserved in glycerol/MEM (1:1) or undiluted dimethyl sulfoxide were processed for transmission and scaning electron microscopy. As control, freshly collected amniotic membrane was fixed and processed for electron microscopy. The cytokines and growth factors assessed were: TGF-beta (transforming growth factor beta); TGF-b activ (activated transforming growth factor beta); EGF (epidermal growth factor); FGF-4 (fibroblast growth factor 4); bFGF (basic fibroblast growth factor); IL-4 (interleukin 4); PGE2 (prostaglandin E2); IL-10 (interleukin 10); KGF (keratinocyte growth factor); HGF (hepatocyte growth factor). RESULTS: Amniotic membrane from the control group showed intact epithelium, with surface microvilli and junctional complexes between the cells and the basal membrane. Glycerol/MEM preserved amniotic membrane had similar aspect to the control, with higher epithelial cells. Those amniotic membranes preserved in dimethyl sulfoxide disclosed less intercellular junction and detachment of the epithelium from the basal membrane. The cytokines and growth factors did not disclose significant differences, except for FGF-4, bFGF, PGE2 and KGF. CONCLUSIONS: Amniotic membrane preserved in glycerol/MEM showed a better tissue structure, with less detachment of the epithelium from the basal membrane, in comparison to undiluted dimethyl sulfoxide. The majority of the growth factors and cytokines were kept with both techniques of preservation.

Comparative study of cryopreserved bone tissue and tissue preserved in a 98 percent glycerol solution / Estudo comparativo entre o tecido ósseo criopreservado e o conservado em glicerol a 98 por cento

OBJECTIVE: To compare the bone graft cryopreservation method (at -80°C) with a preservation method using a 98 percent glycerol solution at room temperature (10°C-35°C), by testing the antibacterial and fungal effects of 98 percent glycerol and comparatively analyzing the observed histological changes resulting from the use of both methods. METHOD: This study was of 30 samples of trabecular bone tissue from 10 patients undergoing total hip arthroplasty. Each femoral head provided 3 samples that were randomized into 3 groups, namely, the control group, the cryopreserved group, and the group preserved in a 98 percent glycerol at room temperature for 1 year. The samples were submitted to histomorphologic, cell feasibility, and microbiologic analyses. The results were statistically analyzed using the McNemar test, with a statistical significance index of 0.05. RESULTS: Values obtained using the McNemar test to compare probability distributions of histomorphologic variables (mature or lamellar bone, immature bone, and necrosis) and cell feasibility (osteoblasts and osteoclasts) indicated that there is no difference between the distributions of variables under the 3 experimental conditions. Microbiological analysis of the 98 percent glycerol solution and bone fragments from samples stored for 1 year at room temperature did not show bacterial or fungal growth. The histological and microbiological investigation were performed at 2 different time points: immediately after the sample processing and after 1 year. CONCLUSION: The method used to preserve bone grafts kept in 98 percent glycerol at room temperature (10°C-35°C) was similar to cryopreservation in terms of bone matrix preservation; no bacteria or fungi were found in the samples.

OBJETIVO: Comparar o método da criopreservação de enxertos ósseos (- 80° C) com o da conservação em glicerol a 98 por cento em temperatura ambiente (10° C a 35° C), testando os efeitos antibacterianos e antifúngicos do glicerol a 98 por cento e analisando comparativamente as alterações histológicas verificadas e decorrentes do emprego dos dois métodos. MÉTODO: Este estudo foi constituído de 30 amostras de tecido ósseo trabecular provenientes de 10 pacientes, submetidos a Artroplastia Total do Quadril. Cada cabeça femoral forneceu 3 amostras e estas foram divididas aleatoriamente em 3 grupos, a saber: controle, criopreservado e conservado em glicerol a 98 por cento à temperatura ambiente durante um ano. As amostras foram encaminhadas à Anatomia Patológica para estudo histomorfologico, de viabilidade celular, e microbiológico. Os resultados foram analisados estatisticamente pelo método de McNemar, com índice de significância de 0,05. RESULTADOS: A análise dos valores obtidos no teste de McNemar na comparação das distribuições de probabilidades das variáveis da histomorfologia (osso maduro ou lamelar, osso imaturo e necrose) e da viabilidade celular (osteoblastos e osteoclastos) indica não haver diferença entre as distribuições das variáveis nas três condições experimentais. A análise microbiológica da solução de glicerol a 98 por cento e dos fragmentos ósseos das amostras armazenadas durante um ano em temperatura ambiente não apresentou crescimento bacteriano ou de fungos. As espécimens do grupo controle foram analisadas histológica e microbiologicamente logo após a coleta das mesmas. CONCLUSÃO: O método de conservação de enxertos ósseos mantidos no glicerol a 98 por cento em temperatura ambiente (10°C a 35°C) foi similar ao da criopreservação quanto à preservação da matriz óssea e à ausência de crescimento de bactérias ou fungos.

Standardization of serum free media for cryopreservation of RK13 cells.

Foetal calf serum present in the media used for cryopreservation was replaced by various synthetic polymer such as gelatin, glycerol, carboxymethyl cellulose and dimethyl sulphoxide at various concentration. Growth pattern of cells, % survival and karyological studies have been done in the present study. It was found that optimum concentration of carboxymethyl cellulose was 0.1% in combination with 10% glycerol and 10% DMSO. At this concentration percentage survival of cells was found maximum and karyotype was found normal without any abnormality in the chromosomes. It was concluded from the study that serum free media can be employed for the cryopreservation of these cells which are further used for production of tissue culture vaccines without causing any adverse affects.

Rat liver responsiveness to gluconeogenic substrates during insulin-induced hypoglycemia

Hepatic responsiveness to gluconeogenic substrates during insulin-induced hypoglycemia was investigated. For this purpose, livers were perfused with a saturating concentration of 2 mM glycerol, 5 mM L-alanine or 5 mM L-glutamine as gluconeogenic substrates. All experiments were performed 1 h after an ip injection of saline (CN group) or 1 IU/kg of insulin (IN group). The IN group showed higher (P<0.05) hepatic glucose production from glycerol, L-alanine and L-glutamine and higher (P<0.05) production of L-lactate, pyruvate and urea from L-alanine and L-glutamine. In addition, ip injection of 100 mg/kg glycerol, L-alanine and L-glutamine promoted glucose recovery. The results indicate that the hepatic capacity to produce glucose from gluconeogenic precursors was increased during insulin-induced hypoglycemia.

Esquema terapéutico en las úlceras de miembros inferiores: experiencia con la bota de Unna modificada / Therapeutic schedule in the lower limb ulcers: experience with modified Unna boot

Las úlceras de miembros inferiores por insuficiencia venosa son una patología frecuente en pacientes por sobre la cuarta década de la vida, incapacitante, refractaria a la terapia y causal de hospitalización prolongada. La Pasta de Unna ha sido uno de los tratamientos más utilizados desde los últimos 100 años. Mostramos en este trabajo los beneficios del uso de un esquema secuencial de la cicatrización completa o preparación para un ulterior injerto de dichas úlceras; esta experiencia se fundamenta en otras investigaciones publicadas con anterioridad. Realizamos un estudio prospectivo en 23 pacientes, de ambos sexos, adultos, con úlceras de miembros inferiores, de diferentes etiologías, durante el periodo comprendido entre enero de 1986 hasta octubre de 1998. Se utilizaron diversos productos cicatrizantes y antibacterianos previos a la aplicación de la Pasta de Unna Modificada, además de un método práctico de utilización de ésta. Del total de 23 pacientes estudiados, la úlcera más frecuente fue por insuficiencia venosa en 21 pacientes (91,30 por ciento). Se excluyeron 6 pacientes por presentar: vasculopatía periférica no venosa, reinfección, evolución tórpida del injerto o retiro de la consulta. Se incluyen 17 pacientes susceptibles de evaluar. La respuesta con cicatrización fue buena en 14 pacientes (82,35 por ciento). Nuestra experiencia demuestra que el reposo, limpieza y antibioticoterapia tópica y/o sistémica previa y el recambio semanal de la bota hasta lograr su epitelización o apta para colocar injerto dermoepidérmico son necesarios. Es un método efectivo para el tratamiento de las úlceras por insuficiencia venosa

Atividade antibacteriana da pasta de hidróxido de cálcio/paramonoclorofenol canforado/glicerina contendo diferentes proporçöes de iodofórmio sobre bactérias anaeróbias estritas e facultativas / The antibacterial activity of calcium hydroxide camphorated paramonochlorophenol glycerin past containing differet amounts of iodorform against obligate and facultative anaerobic bacteria

A atividade antibacteriana de pasta à base de hidróxido de cálcio/paramonoclorofenol cnforado/glicerina (H/P/G) contendo diferentes proporções de iodofórmio foi testada contra 3 bactérias anaeróbias estritas (Porphyromonas endodontalis, Prophyromonas gingivalis e Prevotella intermedia) e 3 anaeróbias facultativas (enterococcus faecallis, Staphylococcus aureuse e Streptococcus sanguis). Para fins comparativos, testou-se também os efeitos antibacterianos de pastas à base de iodofórmio ou hidróxido de cálcio em glicerina. Os resultados demonstraram que a adição de iodofórmio à pasta H/P/G näo interferiu em sua propriedades antibacterianas. A pasta de hidróxido de cálcio e glicerina não paresentou qualquer efeito inibitório sobre as espécies bacterianas testadas.

Aggregation of banana pyrophosphate fructose 6-phosphate 1-phosphotransferase by glycerol.

Addition of glycerol during purification of banana (Musaceae, Musa cavendishii) pyrophosphate fructose 6-phosphate 1-phosphotransferase [(PFP), EC 2.7.1.90] initiated molecular aggregation of the enzyme. The aggregation process was dependent on the glycerol concentration. The native enzyme (66 kDa molecular mass) showed enhanced activity at 3% (V/V) or less of glycerol concentration. Glycerol concentration between 4 and 5% (V/V) affected a gradual and sequential aggregation of native form of the enzyme. These aggregated forms had molecular masses of 135, 200 and 270 kDa. The 135 and 200 kDa forms were stable for about 72 hrs and prolonged storage over 2 weeks resulted in the formation of the 270 kDa form. Concentration over 5% could reduce the time required for aggregation. Fru2.6 bis P activated the enzyme over ten fold, but did not help in the aggregation process. Studies on the role of glycerol on PFP specific activity suggested a difference in the activation process compared to that by Fru2.6bis P. Replacement of Hepes buffer by Tris increased the Fru2.6 bis P requirement for maximum activation by around 10 fold. Removal of glycerol from the buffer media resulted in almost complete inactivation of the enzyme.

Estudo histomorfológico sobre reimplante mediato de incisivo superior de rato. Efeitos da conservaçäo do dente em glicerina à 98 por cento / Histomorphologic study on mediate replant of superior incisor in rats. Effect of tooth preservation in glycerol

Tendo em vista as boas propriedades da glicerina como agente de preservaçäo de tecidos, foi avaliado neste trabalho, os efeitos desta substância sobre reimplantes dentais mediatos. Foram empregados 24 ratos divididos em 2 grupos de 12. No grupo I, o incisivo superior direito, após extraçäo foi mantido durante 48 horas em soro fisiológico. A seguir, foi removida a polpa, o canal obturado com hidróxido de cálcio e reimplantado em seu respectivo alvéolo. No grupo II, o dente foi conservado em glicerina à 98 por cento durante o mesmo período e após mesmo procedimento do grupo anterior, foi reimplantado em seu respectivo alvéolo. Em número de 6 para cada grupo, os ratos foram sacrificados após 20 e 60 dias. As peças obtidas após processamento laboratorial de rotina foram incluídas em parafina. Os cortes foram corados pela hematoxilina e eosina e pelo tricrômico de Masson. Os dentes conservados em glicerina, quando comparados ao soro fisiológico, mostraram menor migraçäo da aderência epitelial e reabsorçäo cemento-dentária mais discreta após 60 dias. Além disso, o ligamento periodontal encontrava-se mais organizado