Effect of Lyophilization on Stability of PEG-Protein Conjugate: A Case Study with Peginterferon alfa-2b

Abstract The purpose of the present study was to develop stable lyophilized formulation of peginterferon alfa-2b which is acquiescent to the short lyophilization process. The present study evaluates the effect of buffering components and cryoprotectant(s) on depegylation of the peginterferon alfa-2b in combination with lyophilization process. Finally, a short lyophilization process was identified which can produce a stable pharmaceutical form of peginterferon alfa-2b without any depegylation during long-term storage. Formulations were analyzed mainly for depegylation by HP-size exclusion chromatography and in-vitro antiviral activity. Residual moisture content in the lyophilized product was also used as a key indicating parameter to check its role with respect to depegylation upon storage under various temperature conditions. It was observed that the peginterferon alfa-2b when formulated in presence of cryoprotectant like sucrose requires longer lyophilization process of about 5 days, irrespective of the buffering components used, to reduce the level of residual moisture content and thereby to produce the stable formulation without depegylation. A stable formulation in presence of high concentration of lactose as a cryoprotectant was developed which can withstand stresses exerted to protein-polymer conjugate during lyophilization phases without any significant depegylation. A short lyophilization process of about 48 hours can be utilized for peginterferon alfa-2b when formulated in presence of lactose as a cryoprotectant through which a stable lyophilized formulation can be produced as against longer process required when sucrose is used a cryoprotectant, which is essential from commercial point of view as lyophilization is a costly process.

Processamento e conservação de enxertos de pele: uma abordagem com foco nos métodos de criopreservação, liofilização e glicerolização / Skin grafts processing and conservation: an approach focusing on cryopreservation, freeze-drying and glycerol conservation methods

Introdução: o uso de substitutos cutâneos para o tratamento de diversas feridas graves é uma forma eficiente de prevenir infecções e favorecer o processo de reepitelização. No entanto, tecidos biológicos estão suscetíveis a degradação e contaminação. Por isso, devem ser submetidos a rigorosos protocolos de processamento e testes que comprovem suas contribuições benéficas e segurança de aplicação. Objetivo: trazer uma abordagem sobre as principais características dos métodos de criopreservação, glicerolização e liofilização e sua consequencia nos aspectos imunológicos, microbiológicos e de viabilidade tecidual de enxertos de pele humana. Metodologia: foi realizada uma busca online utilizando as palavras chaves "criopreservação", "liofilização", "glicerolização", "enxertos", "processamento tecidual" e "engenharia dos tecidos" em múltiplas combinações nos bancos de dados PubMed, LILACS e ScienceDirect. Resultados: 200 artigos científicos foram obtidos, 26 excluídos por duplicidade, 92 selecionados para leitura integral a partir da leitura de seus resumos e 27 utilizados na construção desta revisão. A liofilização e a glicerolização são métodos semelhantes considerando a viabilidade tecidual. O uso de glicerol traz como principal desvantagem sua citotoxicidade quando comparado aos outros métodos. A criopreservação mantém os tecidos viáveis. Contudo, pode ser mais cara e trazer riscos de transmissão de microorganismos patogênicos. De modo geral, não é bem estabelecido quais os melhores métodos de conservação para uma adequada conservação da viabilidade dos enxertos de pele. Considerações Finais: os 3 métodos, liofilização, glicerolização e criopreservação, possuem aplicabilidade na conservação de enxertos. A falta de padronização na aplicação de enxertos apesar de sua frequente aplicação e a escassez de estudos recentes sobre o tema justificam o presente estudo.

Introduction: the use of skin substitutes for treatment of several wounds is an efficient way to prevent infections and allow the re-epithelialization process. However, biological tissues are susceptible to degradation and contamination. Therefore, they must undergo rigorous processing and testing protocols that prove their beneficial contributions and application security. Objective:to bring an approach on the main characteristics of cryopreservation, freeze-drying and glycerol conservation methods and their implications on immunological, microbiological and tissue viability aspects when applied to human skin grafts. Methodology:a mostly online search was performed using the keywords "cryopreservation", "freeze-drying", "glycerol conservation", "grafts", "tissue processing" and "tissue engineering" in multiple combinations in PubMed, LILACS and ScienceDirect databases. Results: 200 scientific articles were rescued, 26 excluded by duplicity, 92 selected for full reading from the reading of their abstracts and 27 used in the construction of this review. Freeze-drying and glycerol conservation are similar methods, with glycerol conservation having greater economic advantage. The use of glycerol presents cytotoxicity when compared to the other methods. Cryopreservation keeps tissues viable, however, is more expensive and carry risks of transmission of pathogenic microorganisms. Overall, there is a lack of clarity about the importance of viability in the performance of skin grafts. Final considerations: the 3 methods have applicability in graft conservation. The lack of standardization in graft application despite its frequent application and the scarcity of recent studies on the subject justify the present study.

Development of a new protocol for freeze-drying preservation of Pseudoalteromonas nigrifaciens and its protective effect on other marine bacteria

Background: Freeze-drying is known as one of the best methods to preserve bacterial strains. Protectant is the key factor affecting the survival rate of freeze-dried strains. In addition, salinity, bacterial suspension concentration, drying time, and other factors can also affect the survival rate of strains to varying degrees. At present, there are relatively few studies on freeze-drying preservation of marine bacteria. In the present study, we performed the freeze-drying protectant screening and optimized the preservation conditions for Pseudoalteromonas nigrifaciens, which is widely distributed in marine environment. The protective effects of the screened protectants were verified by 18 other marine bacterial strains. Results: The results indicated that the combination of 5.0% (w/v) lactose, 5.0% (w/v) mannitol, 5.0% (w/v) trehalose, 10.0% (w/v) skim milk powder, 0.5% (w/v) ascorbic acid and 0.5% (w/v) gelatin was the best choice for the preservation of P. nigrifaciens. The suggested salinity and concentration of initial cell suspension were 10 g/L NaCl and 1.0 × 109 CFU/mL, respectively. Furthermore, stationary-phase cells were the best choice for the freeze-drying process. The highest survival rate of P. nigrifaciens reached 52.8% when using 5­10% (w/v) skim milk as rehydration medium. Moreover, the other 18 marine strains belonging to Pseudoalteromonas, Vibrio, Photobacterium, Planomicrobium, Edwardsiella, Enterococcus, Bacillus, and Saccharomyces were freezedried under the abovementioned conditions. Their survival rates were 2.3­95.1%. Conclusion: Collectively, our results supported that the protectant mixture and parameters were beneficial for lyophilization of marine bacteria

Effects of drying and storage conditions on the stability of TSH in blood spots

ABSTRACT Objective To evaluate the influence of sample drying and storage temperature on TSH stability in neonatal screening. Subjects and methods Blood samples from 29 adult volunteers as a surrogate for neonatal blood (10 with normal TSH, 9 with overt hypothyroid and 10 with subclinical hypothyroidism) were spotted on filter paper and dried at 22°C or 35°C for 3 hours. The samples were then stored at 22°C, -4°C, or -20°C, and TSH measurements were performed at day 0 (D0), D7, D30, D60, D180, and D360 of storage. Results The drying temperature did not interfere with TSH measurement on D0. TSH values remained stable up to D30 when stored at 22°C and were stable up to D60 when stored in a refrigerator or freezer. Samples stored at 22°C had a greater decrease in TSH values than samples stored in a refrigerator or a freezer. Conclusions Freezer storage is not advantageous compared to storage in the refrigerator. At the end of one year, if confirmation of the initial result is required, a reduction of TSH concentrations should be taken into account.

Chitosan and carboxymethylated derivative nanoparticles as delivery systems for biological products: preparation, characterization, stability and in vitro/in vivo evaluation / Nanopartículas de quitosana e derivado carboximetilado como sistemas de fornecimento (delivery) de produtos biológicos: preparo, caracterização, estabilidade e avaliação in vitro/in vivo

Chitosan is a biocompatible and biodegradable mucoadhesive polymer with unique advantages, such as the distinct trait of opening the junctions to allow paracellular transport of antigen and good tolerability. However, the poor solubility of chitosan in neutral or alkalinized media has restricted its applications in the pharmaceutical field. Chitosan can be easily carboxymethylated to improve its solubility in aqueous media, while its biodegradability and biocompatibility are preserved. Apart from this, carboxymethyl chitosan (CMCS) can be easily processed into nanoparticles which highlight its suitability and extensive usage for preparing different drug delivery formulations. The present study deals with the development and characterization of a delivery system based on CMCS nanoparticles using ovalbumin as model protein. We demonstrated that ovalbumin loaded nanoparticles were successfully synthetized using calcium chloride as a cross-linker by ionic gelation. The nanoparticles exhibited an average size of approximately 169 nm and presented a pseudo-spherical shape. The nanoparticles size increased according to the addition of CaCl2 due to the strong electrostatic attraction. During storage the nanoparticles size increased was attributed to swelling and aggregation. The loading efficiency of ovalbumin was found to be 17%. Confocal microscopy clearly showed the association between ovalbumin and CMCS chains into nanoparticles. Therefore, we suggest these nanoparticles can be considered as an attractive and promising carrier candidate for proteins and antigens. The major challenge that limits the use of such carriers is their instability in an aqueous medium. Thus, the next step of this work was to determine the robustness of several formulations using distinct freeze-drying protocols. This study demonstrated that mannitol in concentration of 10% (w/v) is well suited to preserve ovalbumin loaded CMCS nanocapsules from aggregation during lyophilization and subsequent reconstitution. Importantly, the results showed that an annealing step has a huge impact on porosity of freeze-dried cake by nearly complete crystallization of mannitol, once the crystalline matrix prevents the partial collapse and the formation of larger pores observed without annealing. Therefore, the usual observation that annealing increases the pore size due to growth of ice crystal size does not always apply, at least when crystallization of solute is involved. Since all characterizations and stability studies had been performed, the main purpose of this study was to develop a stable antigen delivery system for oral immunization using CMCS and inactivated rabies virus (RV) as the antigen. RV loaded nanoparticles was found to enhance both systemic (IgG) and local (IgA) immune responses against RV after oral delivery in mice. The effective doses 50% were 50-times higher than the negative controls, indicating that the immune response started only after the third boosting dose. Furthermore, enough neutralizing antibodies was produced to be protected against the harmful effects of the rabies virus. It is therefore concluded, that the CMCS nanoparticles formulated in this study, are suitable for oral vaccine delivery, and can be suggested as a promising delivery system for a diverse range of antigens as well as a gene/protein delivery system, especially for those positively charged. Since several approaches show that effective intervention in airway allergic inflammation can be achieved with allergen-activated interleukin-10-secreting cells, the final part of this work was dedicated to assessing whether IL-10 loaded chitosan nanoparticles (IL10-CSNPs) could be used as a possible inhalable therapeutic tool for preventing exacerbations in asthmatic patients. As positive controls, we also assess whether interleukin 17A and interleukin 9 have the ability to stimulate human airway smooth muscle (HASM) cell contractility using magnetic twisting cytometry (MTC). Significant decreased baseline cell stiffness was observed in HASM cells pre-treated with IL-10, but not with IL10-CSNPs, whereas treatment with IL-17A significantly enhanced baseline cell stiffening. Our findings reveal a previously unknown mechanism underlying immunotherapy for prevention and treatment of asthma

A quitosana é um polímero mucoadesivo biocompatível e biodegradável, com vantagens únicas, tais como a característica distinta de abrir as junções que permitim o transporte paracelular de antígenos e boa tolerabilidade. No entanto, sua baixa solubilidade em meios neutros ou alcalinizados tem restringido suas aplicações no campo farmacêutico. A quitosana pode ser facilmente carboximetilada para melhorar de sua solubilidade em meios aquosos, enquanto sua biodegradabilidade e biocompatibilidade são preservadas. Além disso, a carboximetilquitosana (CMCS) pode ser facilmente processada na forma de nanopartículas, o que destaca sua adequabilidade para uso extensivo no preparo de sistemas de delivery de medicamentos. O presente estudo trata do desenvolvimento e caracterização de um sistema de delivery baseado em nanopartículas de CMCS utilizando ovalbumina como proteína modelo. Nós demonstramos que as nanopartículas carregadas com ovalbumina foram sintetizadas com sucesso utilizando cloreto de cálcio como agente de reticulação por gelificação iônica. As nanopartículas exibiram um tamanho médio de aproximadamente 169 nm e apresentaram uma forma pseudo-esférica. O tamanho das nanopartículas aumentou de acordo com a adição de CaCl2 devido à forte atração eletrostática. Durante o armazenamento, o tamanho aumentado das nanopartículas foi atribuído a incorporação de água e agregação. A eficiência de encapsulamento da ovalbumina foi de aproximadamente 17%. A microscopia confocal mostrou claramente a associação entre ovalbumina e a cadeias de CMCS nas nanopartículas. Sugerimos, portanto, que tal sistema pode ser considerado como candidato atraente e promissor para o carreamento de proteínas e antígenos. O principal desafio que limita o uso desses carreadores consiste na instabilidade em meio aquoso. Assim, o próximo passo deste trabalho foi determinar a robustez de várias formulações utilizandose diferentes protocolos de liofilização. Este estudo demonstrou que o manitol em uma concentração de 10% (p/v) é adequado para preservar da agregação as nanocápsulas de CMCS carregadas com ovalbumina durante a liofilização e subsequente reconstituição. Mais importante, os resultados mostraram que uma etapa de annealing tem um enorme impacto sobre a porosidade da amostra liofilizada devido a quase completa cristalização do manitol, uma vez que a matriz cristalina evita o colapso parcial e a formação de poros maiores observados na ausência do annealing. Portanto, a observação comum de que o annealing aumenta o tamanho doporos devido ao crescimento dos cristais de gelo nem sempre se aplica, pelo menos quando a cristalização de um soluto está envolvida. Uma vez que todas as caracterizações e estudos de estabilidade foram realizados, o principal objetivo deste estudo foi desenvolver um sistema estável de delivery de antígeno para imunização oral utilizando CMCS e vírus rábico inativado (RV) como antígeno. Verificou-se que as nanopartículas carregadas com RV aumentam as respostas imune sistêmica (IgG) e local (IgA) contra o RV após administração oral em camundongos. As doses efetivas 50% foram 50 vezes maiores que os controles negativos, indicando que a resposta imune foi iniciada apenas após a terceira dose da vacina. Além disso, foram produzidos anticorpos neutralizantes suficientes para proteção contra os efeitos nocivos do vírus rábico. Conclui-se, portanto, que as nanopartículas de CMCS formuladas neste estudo, são adequadas para o delivery oral de vacinas, e podem ser sugeridas como um sistema promissor de delivery para uma gama diversa de antígenos, bem como para o delivery de genes/proteínas, especialmente para aqueles carregados positivamente. Uma vez que diversas abordagens mostram que uma intervenção efetiva em casos de inflamação alérgica de vias aéreas pode ser conseguida por meio de células secretoras de interleucina 10 (IL-10) mediante ativação por alergenos, a parte final deste trabalho esteve dedicada a avaliação de nanopartículas de quitosana carregadas com IL-10 (IL10-CSNPs) como possível ferramenta terapêutica inalável para prevenção de exacerbações em pacientes asmáticos. Como controles positivos, avaliou-se adicionalmente se as interleucinas 17A (IL-17A) e 9 (IL-9) possuem a capacidade de estimular a contratilidade de células humanas de músculo liso de vias aéreas humanas (HASM) por meio de citometria de torção magnética (MTC). Uma diminuição significativa da rigidez celular basal foi observada em células HASM pré-tratadas com IL-10, mas não com IL10-CSNPs, enquanto que o tratamento com IL-17A aumentou significativamente a magnitude rigidez celular basal. Nossos resultados revelam um mecanismo previamente desconhecido subjacente à imunoterapia para prevenção e tratamento da asma

Desenvolvimento, caracterização e avaliação da formação de complexos de inclusão de rutina, avobenzona e ρ-metoxicinamato de octila na presença de ciclodextrinas / Development, characterization and evaluation of inclusion complexes between rutin, avobenzone and octyl ρ-methoxycinnamate with cyclodextrins

A radiação UV pode causar danos à pele humana, e, evitar estes danos, é uma preocupação crescente para a população e um desafio à comunidade científica. Para uma ação efetiva de fotoproteção, a associação de filtros, como avobenzona (BMBM) e ρ-metoxicinamato de octila (EHMC), são empregados. Devido à semelhança estrutural com os filtros solares químicos, a rutina (RUT), tal como outros flavonoides, apresenta atividade fotoprotetora. Apesar da disponibilidade de diferentes classes de filtros solares, o desenvolvimento de fotoprotetores contendo filtros químicos é um desafio, devido à instabilidade inerente a certos filtros orgânicos. As ciclodextrinas (CDs) são oligossacarídeos cíclicos, de formato tronco-cônico, cuja estrutura externa é hidrófilica e sua cavidade interna central hidrofóbica, com a capacidade de acomodar substâncias lipofílicas, formando complexos de inclusão. A formação dos complexos de inclusão pode levar à alterações de propriedades físico-químicas da molécula hóspede, tais como, solubilidade, fotoestabilidade e biodisponibilidade. O objetivo deste trabalho foi desenvolver, caracterizar e avaliar a formação de complexos de inclusão entre RUT, BMBM e EHMC e as CDs (HPßCD e SBEßCD). Os complexos de inclusão (RUT:HPßCD, RUT:SBEßCD, BMBM:HPßCD, BMBM:SBEßCD, EHMC:HPßCD e EHMC:SBEßCD) foram obtidos pelo método de liofilização e quantificados por cromatografia líquida de alta eficiência (CLAE). Os sistemas binários foram caracterizados em solução, pelo método de equilíbrio de solubilidade, e, no estado sólido, empregando calorimetria exploratória diferencial (DSC), termogravimetria (TG/DTG) e difração de raios-X de pó (PDRX). As substâncias isoladas e os complexos binários foram avaliados quanto à fotoestabilidade em estado sólido, e, em solução. Incremento na solubilidade (X mcg mL-1) foi observado para os complexo RUT:HPßCD (4,13x); RUT:SBEßCD (4,38x); BMBM:HPßCD (43,3x); BMBM:SBEßCD (53,3x); EHMC:HPßCD (12,7x); e EHMC:SBEßCD (70,0x). Os ensaios de DSC, TG/DTG, e P-DRX indicaram a formação de complexos de inclusão para os todos os sistemas, onde a supressão dos eventos endotérmicos característicos das substâncias isoladas foram observados; porém, nos complexos de BMBM, a presença de avobenzona livre no meio foi detectada, sugerindo, que a complexação não foi completa. A formação dos complexos de inclusão promoveu o aumento da fotoestabilidade em todos os sistemas avaliados, tanto no estado sólido, como em solução. Os resultados reportados neste estudo, indicaram que a complexação de substâncias fotoprotetoras com HPßCD e SBEßCD, pode representar, uma estratégia promissora quanto ao aumento da solubilidade e da fotoestabilidade

UV radiation may cause demage on human skin, and preventing it, is an increasing worry for the population and a challenge to the scientific community. For an effective action of photoprotection, the association of filters, like avobention (BMBM) and octyl ρ-methoxycinnamate (EHMC), are used. Due to the structural similarity with the chemical solar filters, the rutin (RUT), like other flavonoids, shows photoprotective activity. Despite the availability of different classes of sunscreens, the development of photoprotectors containing chemical filters is a challenge, due to the inherent instability of certain organic filters. The cyclodextrins (CD) are cyclic oligosaccharides of truncated conical structure, which external structure is hydrophilic and its internal central hydrophobic cavity, with capacity to accommodate lipophilic substances, forming inclusion complexes. The formation of the inclusion complexes can lead to changes in physicalchemical properties of the host molecule, such as, solubility, photostability and bioavailability. The objective of this work was to develop, characterize and evaluate the formation of the inclusion complexes between RUT, BMBM and EHMC and the CDs (HPßCD and SBEßCD). The inclusion complexes (RUT:HPßCD, RUT:SBEßCD, BMBM:HPßCD, BMBM:SBEßCD, EHMC:HPßCD and EHMC:SBEßCD) were obtained by the lyophilization method and quantified by high performance liquid chromatography (HPLC). The binary systems were characterized in solution, by solubility equilibrium method and in solid state, using differential scanning calorimetry (DSC), thermogravimetry (TG/DTG) and powder X-ray diffraction (P-XRD). The isolated substances and binary complexes were evaluated the photostability in solid state, and in solution. The increase in solubility (X mcg mL-1) was observed for the complexes RUT:HPßCD (4.13x); RUT:SBEßCD (4.38x); BMBM:HPßCD (43.3x); BMBM:SBEßCD (53.3x); EHMC:HPßCD (12.7x); and EHMC:SBEßCD (70.0x). The analysis of DSC, TG/DTG, and P-DRX indicated the formation of inclusion complexes for all systems, where the suppression of the endothermic events characteristic of the isolated substances were observed; however, in the BMBM complexes, the presence of free avobenzone was detected, suggesting that the complexation was not complete. The formation of inclusion complexes promoted the increase of photostability in all evaluated systems, as in solid state as in solution. The results reported in this study indicated that the complexation of photoprotective substances with (HPßCD e SBEßCD). may represent a promising strategy for increasing solubility and photostability

Tratamiento de defectos periodontales con matriz derivada del esmalte: serie de casos / Treatment of periodontal defects with enamel matrix derivative: case series

Objetivo: Presentar los resultados clínicos y radiográficos obtenidos a 6 meses del tratamiento de defectos intraóseos con la técnica quirúrgica mínimamente invasiva y el uso de matriz derivada del esmalte sola o en combinación con hueso bovino desmineralizado. Casos clínicos: se seleccionaron 13 sitios con profundidad de sondaje mayor a 5 mm y pérdida ósea vertical. Se evaluaron los siguientes párámetros clínicos: profundidad de sondaje, nivel de inserción clinica y recesión gingival. Los defectos se trataron con matriz derivada del esmalte sola o en combinación con hueso bovino desmineralizado. La profundidad de sondaje inicial promedio era de 6,31 (rango: 5-9) y a los 6 meses, de 3,15 (rango 1-4), lo que significa que la reducción en el promedio de la profundidad de sondaje fue de 3,16. La ganancia en el nivel de inserción clínica fue de 2,9 mm. La evaluación radiográfica evidenció llenado óseo en los sitios tratados. Conclusiones: en la serie de casos presentados, los resultados fueron exitosos y coinciden con los informados en la literatura. La estabilidad del colgajo y el cierre primario son requisitos fundamentales para el éxito y la predictibilidad de cualquier procedimiento regenerativo. Las técnicas quirúrgicas mínimamente invasivas -en comparación con el abordaje tradicional- mejoran notablemente estos parámetros, al reducir los tiempos quirúrgicos y la morbilidad, disminuyendo el malestar del paciente.

Ascorbic acid from lyophilized camu-camu fruit: stability and quality control of hard capsules

Camu-camu, a fruit found in the Amazon Basin river banks and lake shores, is known for its high ascorbic acid content together with, other antioxidants. This feature shows high potential for being exploited in agribusiness industry and pharmaceutical processes. However, its high acidity, as well as peel bitterness associated with the phenolic substances content, has discouraged its consumption while still fresh. The development of alternative forms for consuming this fruit, while still preserving its ascorbic acid and polyphenol content, in addition to its great potential for maintaining human health, has become a major economic activity in coastal communities. The present study evaluated the ascorbic acid stability found in camu-camu capsules. Lyophilization was performed with the fruit pulp and peel. Both freeze-dried fruit and powder-flled capsules were stored at 5° C. Ascorbic acid stability was monitored for 90 days using HPLC assay technique. The encapsulation process of freezedried pulp was considered satisfactory in the ascorbic acid conservation, since there was only a loss of 10% of its initial concentration throughout the study period for 60 days.(AU)

Lyophilized allografts without pre-treatment with glutaraldehyde are more suitable than cryopreserved allografts for pulmonary artery reconstruction

Various methods are available for preservation of vascular grafts for pulmonary artery (PA) replacement. Lyophilization and cryopreservation reduce antigenicity and prevent thrombosis and calcification in vascular grafts, so both methods can be used to obtain vascular bioprostheses. We evaluated the hemodynamic, gasometric, imaging, and macroscopic and microscopic findings produced by PA reconstruction with lyophilized (LyoPA) grafts and cryopreserved (CryoPA) grafts in dogs. Eighteen healthy crossbred adult dogs of both sexes weighing between 18 and 20 kg were used and divided into three groups of six: group I, PA section and reanastomosis; group II, PA resection and reconstruction with LyoPA allograft; group III, PA resection and reconstruction with CryoPA allograft. Dogs were evaluated 4 weeks after surgery, and the status of the graft and vascular anastomosis were examined macroscopically and microscopically. No clinical, radiologic, or blood-gas abnormalities were observed during the study. The mean pulmonary artery pressure (MPAP) in group III increased significantly at the end of the study compared with baseline (P=0.02) and final [P=0.007, two-way repeat-measures analysis of variance (RM ANOVA)] values. Pulmonary vascular resistance of groups II and III increased immediately after reperfusion and also at the end of the study compared to baseline. The increase shown by group III vs group I was significant only if compared with after surgery and study end (P=0.016 and P=0.005, respectively, two-way RM ANOVA). Microscopically, permeability was reduced by ≤75% in group III. In conclusion, substitution of PAs with LyoPA grafts is technically feasible and clinically promising.

Estudio clínico e histológico de aloinjerto de hueso liofilizado en alvéolos pos extracción: presentación de caso clínico / Clinical and histological study of bone freeze drying allograft in postextraction sockets: report of a clinical case

Objetivos: evaluar las respuestas clínicas e histológicas y determinar la calidad del hueso obtenido por medio del aloinjerto utilizado en un alvéolo posextracción en el que se realizó una técnica de preservación del volumen alveolar, a fin de colocar implantes. Caso clínico: una paciente de 47 años recibió un tratamiento de preservación del volumen alveolar posextracción mediante aloinjerto de hueso liofilizado (matriz ósea UNC en polvo) contenido por una lámina ósea cortical (matriz ósea UNC en membrana). A los 120 días, se tomó biopsia y se colocaron implantes. La muestra se observó con microscopía óptica. Conclusión: histológicamente, se identificaron restos de partículas y de lámina ósea, e intensos fenómenos de angiogénesis y neoformación ósea. La observación clínica permitió visualizar los márgenes nítidos del reborde y verificar la conservación del volumen ósea en el lugar en el que se realizó la fijación primaria de los implantes. La técnica de preservación con los biomateriales citados permite la colocación del implante en una posición adecuada, con resultados funcionales y estéticos.

Early onset intellectual disability in chromosome 22q11.2 deletion syndrome / Inicio temprano de discapacidad intelectual en el Síndrome de deleción del cromosoma 22q11.2

Chromosome 22q11.2 deletion syndrome, or DiGeorge syndrome, or velocardiofacial syndrome, is one of the most common multiple anomaly syndromes in humans. This syndrome is commonly caused by a microdelection from chromosome 22 at band q11.2. Although this genetic disorder may reflect several clinical abnormalities and different degrees of organ commitment, the clinical features that have driven the greatest amount of attention are behavioral and developmental features, because individuals with 22q11.2 deletion syndrome have a 30-fold risk of developing schizophrenia. There are differing opinions about the cognitive development, and commonly a cognitive decline rather than an early onset intellectual disability has been observed. We report a case of 22q11.2 deletion syndrome with both early assessment of mild intellectual disabilities and tetralogy of Fallot as the only physic manifestation.

El síndrome del cromosoma 22q11.2, también conocido como supresión o síndrome de DiGeorge o síndrome velocardiofacial, es uno de los síndromes más comunes de anomalías múltiples en los seres humanos. Este síndrome es comúnmente causado por una microdeleción del cromosoma 22 en q11.2 banda. Aunque este trastorno genético muestra varias anomalías clínicas y diferentes grados de compromiso orgánico, las características clínicas que han atraído la mayor atención son el comportamiento y el desarrollo, porque las personas con síndrome de deleción 22q11.2 tienen un riesgo 30 veces mayor de desarrollar esquizofrenia. Hay diferentes opiniones sobre el desarrollo cognitivo, y comúnmente se se ha observado un deterioro cognitivo en lugar de un inicio temprano de discapacidad intelectual. Presentamos un caso de síndrome de deleción 22q11.2 tanto con la evaluación temprana de discapacidades intelectuales leves como con la tetralogía de Fallot como única manifestación física.

Estabilidade de micro-organismos potencialmente probióticos em barra de cereais - efeito da técnica de incorporação / Stability of microorganisms potentially probiotics in cereal bar - Effect of technique of incorporation

Este trabalho objetivou desenvolver formulações de barras de cereais contendo Lactobacillus acidophilus e Saccharomyces boulardii e avaliar a estabilidade destes micro-organismos potencialmente probióticos no produto elaborado durante o tempo de armazenamento (shelf life). Foram formuladas barras de cereais contendo separadamente os agentes probióticos S. boulardii e L. acidophilus, ambos nas formas liofilizada, granulados com lactose e encapsulados em alginato de cálcio. Foi avaliada a estabilidade dos micro-organismos durante o tempo de armazenamento. Tanto a levedura S. boulardii quanto a bactéria L. acidophilus apresentaram maior sobrevida na forma liofilizada, quando comparado com as outras formas de incorporação, mantendo contagem na faixa de 7 log UFC/g, durante oito semanas e na faixa de 7 log UFC/g, durante seis semanas, respectivamente. Comparando os resultados da incorporação dos dois micro-organismos na forma livre liofilizada observouse que a população de S. boulardii apresentou melhores resultados de viabilidade quando comparada com a população de L. acidophilus. Este estudo mostrou que é possível incorporar micro-organismos potencialmente probióticos em barras de cereais, preferencialmente na forma liofilizada, por proporcionar maior sobrevida dos micro-organismos.(AU)

This study aims to develop formulations of cereal bars that contain Lactobacillus acidophilus and Saccharomyces boulardii and evaluate the stability of these potentially probiotic microorganisms in the elaborated product during the time of storage (shelf life). Cereal bars were designed containing separately the probiotic agents S. boulardii and L. acidophilus, both in the lyophilized form, granulated with lactose form and encapsulated in calcium alginate form. The stability of microorganisms during storage time was evaluated. Both, the yeast S. boulardii as well as the bacterium L. acidophilus showed a higher survival in the lyophilized form when compared with the other forms of incorporation, keeping the count in the range of 7 log UFC/g for eight weeks and in the range 7 log UFC/g for six weeks, respectively. Comparing the results of the incorporation of the two microorganisms in the free lyophilized form, one could observe that the population of S. boulardii showed better results of viability when compared with the population of L. acidophilus. This study showed that it is possible to incorporate potentially probiotic microorganisms in cereal bars, preferably in the lyophilized form, in order to provide a higher survival of the microorganisms.(AU)

Evaluación del cultivo liofilizado de Candida albicans utilizado en esquemas de certificaciones de calidad / Evaluation of the lyophilized culture of Candida albicans used in quality certification schemes

Introducción: el laboratorio de control microbiológico de la UEB Laboratorios Liorad dispone de una colección de cultivos microbianos para la conservación de microorganismos, donde se encuentra depositada la levadura Candida albicans que se emplea en esquemas de certificaciones de calidad establecidos para la evaluación de ensayos como: promoción de crecimiento de los medios de cultivos, validación de técnicas microbiológicas, entre otros. Objetivo: evaluar los resultados de la conservación de esta cepa por el método de liofilización durante un periodo de ocho años. Métodos: para el crecimiento de la cepa se utilizó el medio de cultivo Caldo Saboraud y variantes de sustancias lioprotectoras puras como: (leche descremada al 20 por ciento, glicerol 20 por ciento, sacarosa al 10 por ciento y peptona 5 por ciento) así como la mezcla de lioprotectores (leche 10 por ciento, sacarosa 5 por ciento, glicerol 5 por ciento). Se evaluó viabilidad, pureza y estabilidad genética de esta cepa durante el tiempo objeto de estudio. Resultados: las características propias de la especie estudiada se mantuvieron inalterables con un elevado grado de pureza en todas las variantes estudiadas. En cuanto a la supervivencia, cuando se emplearon las sustancias lioprotectoras puras se evidenció una marcada disminución de la viabilidad. No así al emplear la mezcla de lioprotectores que mantuvo niveles de viabilidad por encima del límite establecido durante todo el tiempo objeto de estudio. Conclusiones: los valores obtenidos en cuanto a la supervivencia de este microorganismo permiten inferir que para la conservación por largos periodos de tiempo la variante donde se empleó mezclas de lioprotectores resultó una buena opción para la conservación de C. albicans(AU)

Introduction: the microbiological control laboratory at the Basic Enterprise Unit Liorad Laboratories stores a collection of microbial cultures for the preservation of microorganisms, including the Candida albicans yeast used in the quality certification schemes established for the evaluation of assays such as fostering of culture medium growth and validation of microbiological techniques, among others. Objective: evaluate the results obtained in the preservation of this strain by the lyophilization method during a period of eight years. Methods: for strain growth, use was made of Saboraud broth culture medium as well as variants of pure lyoprotective substances such as 20 percent skimmed milk, 20 percent glycerol, 10 percent saccharose and 5 percent peptone, and the mixture of lyoprotectors (10 percent milk, 5 percent saccharose, 5 percent glycerol). An evaluation was conducted of the viability, purity and genetic stability of the strain during the study period. Results: characteristics typical of the study species remained unchanged with a high degree of purity in all the variants examined. As to survival, a marked reduction in viability was observed when pure lyoprotective substances were used, but not with the mixture of lyoprotectors, in which case viability levels exceeded the established limit during the entire study period. Conclusions: the survival values obtained for this microorganism indicate that preservation for long periods with mixtures of lyoprotectors was a good option for the preservation of C. albicans(AU)

Validation of analytical methodology for quantification of cefazolin sodium pharmaceutical dosage form by high performance liquid chromatography to be applied for quality control in pharmaceutical industry

A reversed-phase high performance liquid chromatography method was validated for the determination of cefazolin sodium in lyophilized powder for solution for injection to be applied for quality control in pharmaceutical industry. The liquid chromatography method was conducted on a Zorbax Eclipse Plus C18 column (250 x 4.6 mm, 5 μm), maintained at room temperature. The mobile phase consisted of purified water: acetonitrile (60: 40 v/v), adjusted to pH 8 with triethylamine. The flow rate was of 0.5 mL min-1 and effluents were monitored at 270 nm. The retention time for cefazolin sodium was 3.6 min. The method proved to be linear (r2=0.9999) over the concentration range of 30-80 µg mL-1. The selectivity of the method was proven through degradation studies. The method demonstrated satisfactory results for precision, accuracy, limits of detection and quantitation. The robustness of this method was evaluated using the Plackett–Burman fractional factorial experimental design with a matrix of 15 experiments and the statistical treatment proposed by Youden and Steiner. Finally, the proposed method could be also an advantageous option for the analysis of cefazolin sodium, contributing to improve the quality control and to assure the therapeutic efficacy.

Um método cromatográfico em fase reversa foi validado para a determinação de cefazolina sódica em pó liofilizado, a ser aplicado no controle de qualidade em indústrias farmacêuticas. O método por cromatografia líquida foi conduzido em coluna Zorbax Eclipse Plus C18 (250 × 4,6 mm, 5 µm) mantida à temperatura ambiente. A fase móvel consistiu de água purificada: acetonitrila (60 : 40 v/v), com o pH ajustado para 8 com trietilamina. A vazão usada foi de 0,5 mL min-1 e os analitos de interesse foram monitorizados a 270 nm. O tempo de retenção da cefazolina sódica foi de 3,6 min. As áreas dos picos de cefazolina sódica foram lineares na faixa de concentração de 30-80 µg mL-1 (r2 = 0,9999). A seletividade do método foi demonstrada através de estudos de degradação. O método demonstrou resultados satisfatórios para precisão, exatidão, limites de detecção e de quantificação. A robustez do método foi avaliada utilizando o esquema fatorial de Plackett-Burman com uma matriz de 15 experimentos simultâneos, e analisados por tratamento estatístico proposto por Youden e Steiner. Finalmente, o método proposto pode ser também uma opção de êxito para a análise de cefazolina sódica, contribuindo para o controle de qualidade e para garantir a eficácia terapêutica.

Aumento horizontal de rebordo maxilar anterior com enxerto de osso de origem bovina / Horizontal augmentation of anterior maxillary alveolar ridge using bovine bone graft

A reabsorção do rebordo alveolar muitas vezes deixa volume ósseo insuficiente para a instalação de implantes osseointegráveis. Poucos estudos têm investigado a quantidade e a qualidade da formação óssea em humanos após o aumento do rebordo alveolar horizontal. A técnica de posição utilizando um biomaterial de origem bovina (Bonefill – Bionnovation – São Paulo) em combinação com osso autógeno para o aumento horizontal do rebordo alveolar antes da colocação do implante, está  em crescente uso devido preocupações referentes … morbidade associada com  áreas doadoras intra e extraorais, pois há  a necessidade de intervir cirurgicamente em mais de uma região. Este caso cl¡nico relata o aumento de volume anterior em maxila utilizando enxerto bovino em grânulos em mistura com 30% de osso autógeno colhido da paciente durante o ato cirúrgico. Após 180 dias, cinco implantes osseointegráveis foram instalados e estes receberam uma prótese de carga imediata em resina 48 horas após o procedimento cirúrgico. A paciente optou por trocar a prótese em resina por uma prótese fixa em cerâmica após 180 dias da instalação dos implantes. Não houve registro de complicações protéticas ou perda de implantes em 5 anos de acompanhamento

The alveolar ridge resorption often leaves a contour bone insufficient for the installation of osseointegrated implants. Few studies have investigated the quantity and quality of human bone formation after increasing alveolar ridge height. The utilization of technique combining animal origin biomaterial and autogenous bone to augment the alveolar ridge before implant installation has become more frequent due to the concerns regarding morbity of intra and extra-oral donor sites since surgical intervention is required in more than one region. The present paper presents a clinical case of horizontal augmentation of maxillary alveolar ridge, using bovine graft in grains mixed with 30% of autogenous bone removed from the patient during surgery. After 180 days, five osseointegrated implants were installed and received immediate load resin prosthesis 48 hours after the surgical procedure. The patient has chosen to substitute the resin prosthesis for a metal-ceramic prosthesis after 6 months. In a 5 years follow-up, no complication or loss of prosthetic implant was observed

Técnica de preservación del volumen alveolar post-extracción utilizando hueso liofilizado y lámina ósea cortical / Preservation techniques of alveolar post-extraction volume using liophilisate bone and cortical bone sheets

Se evaluó la eficacia del hueso liofilizado humano (Matriz Ósea UNC en Polvo) injertado en cavidades alveolares post-extracción, recubierto por una lámina ósea cortical (Matriz Ósea UNC en membrana), en el tratamiento de preservación del perfil volumétrico del reborde alveolar.La metodología de trabajo se fundamentó en: 1) El estudio de una casuística de 27 casos clínicos en pacientes de ambos sexos que poseían elementos dentarios unirradiculares con indicación de extracción. Se injertó en las cavidades óseas resultantes hueso liofilizado, contenido in situ mediante una lámina ósea cortical parcialmente desmineralizada. Los pacientes fueron evaluados clínica y radiográficamente, mediante modelos de estudio, Rx convencional y radiovisiografía que permitieron mensurar las modificaciones producidas por resorción durante un año. Se realizaron controles pre y post-operatorios, a los 7, 15, 30, 60, 90, 120, 180 y 360 días. A los modelos de yeso preliminares y a los obtenidos a los 120 y 360 días se les efectuaron cortes transversales en las zonas de estudio y se los escaneó. Las imágenes obtenidas se procesaron mediante un analizador de imágenes (Image Pro-Plus). Los datos se analizaron estadísticamente con software específico (SPSS). El estudio demostró que las mayores modificaciones dimensionales del reborde se observaron sobre el área superficial del alvéolo y en los primeros 4 meses post-extracción. La lámina cortical presentó características físicas, estructurales y biológicas que le permitieron actuar como barrera física oclusiva, minimizando los fenómenos de inhibición celular heterotípica y favoreciendo los procesos osteogénicos por el mecanismo de osteopromoción...

Human efficacy lyophilized bone (UNC Bone Matrix Powder) grafted post-extraction alveolar sacs, covered by a cortical bone plate (UNC Bone Matrix membrane), in the treatment volume preservation ridge profile was evaluated. The working methodology was based on: 1) The study of a case series of 27 clinical cases in patients of both sexes who had single-rooted tooth elements indicating extraction. It was grafted bone cavities in the resulting lyophilized bone content in situ by a partially demineralized cortical bone plate. Patients were evaluated clinically and radiographically, using study models, and conventional Rx radiovisiography that allowed mensurar resorption induced changes for a year. Pre and post-operative controls at 7, 15, 30, 60, 90, 120, 180 and 360 days were performed. A preliminary plaster models and those obtained at 120 and 360 days transects were conducted in the study areas and were scanned. The images obtained were processed by an image analyzer (Image Pro-Plus). The data were statistically analyzed with software (SPSS) .The study showed that older flange dimensional changes were observed on the surface area of the alveoli and in the first 4 months post-extraction. The cortical sheet submitted physical, structural, and biological characteristics that allowed him to act as occlusive physical barrier, minimizing heterotypic cellular inhibition phenomena and processes favoring osteogenic mechanism osteopromoción...

Evaluation of three methods for preservation of Azotobacter chroococcum and Azotobacter vinelandii

La preservación de bacterias es asunto de granimportancia debido a que muchas de ellas son usadas enprocesos biotecnológicos que requieren mantener su viabilidad ypropiedades genéticas. En este estudio, se evaluaron tres métodospara la preservación de A. chroococcum C26 y A. vinelandii C27;criopreservación, liofilización, e inmovilización en polímerossecos, durante 60, 30 y 60 días, respectivamente. A su vez, seestudió el efecto de tres agentes protectivos para la liofilizacióny para la criopreservación y cuatro polímeros. La eficiencia delos métodos fue evaluada contando células viables y midiendoactividad como fijación de nitrógeno. Los resultados mostraronque la mejor técnica, la cual mantuvo la viabilidad y la actividad,fue la liofilización, seguida por inmovilización y criopreservación.La liofilización mantuvo estable la habilidad bacteriana para fijarnitrógeno, la tasa de sobrevivencia bacteriana (TSB) fue superioral 80%; y el mejor resultado se evidenció cuando se usó S/BSAcomo agente protectivo. La inmovilización mantuvo la BSRsuperior al 80%, y la fijación de nitrógeno fue disminuida en 20%.La criopreservación tuvo pérdida sustancial de viabilidad para C26(TSB aprox. 70%); mientras que C27 se preservó bien. La fijaciónde nitrógeno fue significativamente disminuida para ambas cepasindependientemente del agente crioprotectivo usado (P < 0.05).Los resultados sugieren que el éxito de los métodos de preservaciónpara Azotobacter depende de la técnica, el agente protectivo y la cepausada; siendo la liofilización con S/BSA la técnica con mejoresresultados para preservar las bacterias de este género...

Because the use of bacteria for biotechnological processes requires maintaining their viability and geneticstability, preserving them becomes essential. Here, we evaluated three preservation methods for A.chroococcum C26 and A. vinelandii C27; preservation methods: cryopreservation and immobilization in drypolymers for 60 days, and freeze-drying for 30. We evaluated their efficiency by counting viable cells andmeasuring nitrogen fixation activity. Additionally, we assessed the effect of three protective agents forfreeze-drying, three for cryopreservation, and four polymers. Freeze-drying proved the best technique tomaintain viability and activity, followed by immobilization and cryopreservation. Bacterial nitrogen fixingability remained unchanged using the freeze-drying method, and bacterial survival exceeded 80%; S/BSAwas the best protective agent. Immobilization maintained bacterial survival over 80%, but nitrogen fixationwas decreased by 20%. Lastly, cryopreservation resulted in a dramatic loss of viability for C26 (BSRapprox. 70%), whereas C27 was well preserved. Nitrogen fixation for both strains decreased regardless ofthe cryoprotective agent used (P < 0.05). In conclusion, the success of Azotobacter preservation methodsdepend on the technique, the protective agent, and the strain used. Our results also indicated that freezedryingusing S/BSA is the best technique to preserve bacteria of this genus...

Porque o uso de bactérias para processos biotecnológicos,requer a manutenção da sua viabilidade e estabilidade genética,preserva-las é essencial Avaliaram-se três métodos de preservação deA. chroococcum C26 e A. vinelandii C27; criopreservação, liofilização, eimobilização de polímeros secos. Examinamos também o efeito deagentes protetores para liofilizar, para a criopreservação, e polímeros.A eficiência foi avaliada contando as células viáveis e medindoa atividade como a fixação do azoto. Os resultados mostraramque a melhor técnica foi a liofilização seguida de imobilização ecriopreservação. A liofilização manteve inalterada a capacidadeda bactéria para fixar o azoto, e o melhor resultado foi observadoquando se usou S/BSA como agente protetor. A criopreservaçãoresultou em uma perda dramática de viabilidade para C26 (TSBaprox. 70%.), enquanto que C27 foi bem preservada. A fixaçãode azoto foi significativamente diminuída para ambas as estirpes,independentemente do agente crioprotector utilizado (P < 0.05).Em conclusão, os resultados sugerem que o êxito dos métodosde conservação de Azotobacter dependem da técnica, do agente deproteção, e da estirpe utilizada, sendo a liofilizacao com S/BSA amelhor técnica para preservar as bactérias deste género...

Comparative study and histomorphometric analysis of bone allografts lyophilized and sterilized by autoclaving, gamma irradiation and ethylene oxide in rats

PURPOSE: To compare three sterilization methods (autoclave, gamma irradiation and ethylene oxide) over non demineralized lyophilized bone allografts. METHODS: Bone allografts were implanted on paravertebral muscles of 21 rats. After 30 days animals were sacrificed and grafts underwent comparative analysis regarding histomorphometric and macroscopic parameters. RESULTS: Allografts that underwent the three sterilization methods presents similar weight gain, cortical thickness similar to control group, and less fibrosis than the control group. Grafts that underwent sterilization in autoclave presented less presence of multinucleated giant cells, although not statistically significant. There was also no statistically significant difference regarding mineralization on the three groups. CONCLUSION: The three sterilization methods cause similar effects on bone allografts regarding macroscopic and histomorphometric parameters.

Characterization and optimization of lyophilization and storage conditions of Leech saliva extract from the tropical leech Hirudinaria manillensis

The medicinal Malaysian leeches have been used in traditional medicine to treat many different ailments. In this study, leech saliva extract [LSE] was collected from the medicinal Malaysian leech Hirudinaria manillensis. Gel electrophoresis of LSE was carried out to estimate the peptide and protein molecular weights of its content. Results showed that LSE contains more than 60 peptides and proteins with molecular masses ranging from 1.9-250kDa. Thrombin time assay in vitro was employed to assess the collected LSE antithrombin activity. First, to study its stability, LSE was lyophilized under the following different conditions: pre-freezing temperature, type of container and lyophilization cycle. Pre-freezed LSE sample at -20°C and lyophilized for 24 hours retained about 100-95% of its original biological activities. Second, the LSE antithrombin activity was monitored for a period of six months. Storage temperature, type of the container and photosensitivity effects on antithrombin activity of the lyophilized [solid state] and non-lyophilized [liquid state] were investigated. Results showed that storage temperature drastically affected the biological activity of LSE with -20°C as the optimum temperature. Samples stored at ambient temperature and +4°C were light photosensitive and adversely affected when stored in polypropylene tubes. Lyophilized samples were more stable than non-lyophilized ones over the period of study. To sum up, in order to have a biologically active stock of LSE, it has to be lyophilized for no more than 24 hours following freezing at -20°C and has to be stored at -20°C in glass tubes protected from light

Preparation and evaluation of [99m]Tc-DMSA lyophilized kit for renal imaging

Dimercaptosuccinic acid [DMSA] has been evaluated and used with technetium 99m [[99m]Tc] in imaging of kidneys. DMSA lyophilized kits were prepared and radiolabelled with [99m]Tc. Paper and thin-layer chromatography have been employed using various eluent systems for the radiochemical analysis, percentage labeling and binding capacity of [99m]Tc-DMSA. Female albino rabbits were used for this study. Biological data obtained after intravenous injection of radiolabelled DMSA to female albino rabbits revealed 32.42% uptake and long retention time in the kidneys. On the basis of animal biodistribution data, it is suggested that DMSA when labeled with [99m]Tc is useful complex for renal imaging and can be successfully applied as a diagnostic tool in nuclear medicine. Clinical biodistribution and radiation dosimetry studies are planned in future