Developing scaffolds for tissue engineering using the Ca2+-induced cold gelation by an experimental design approach

The Ca2+-induced cold gelation technique was found suitable to prepare highly porous biodegradable scaffolds based on bovine serum albumin (BSA) and alpha-casein from bovine milk for tissue engineering. A 23 full factorial design was used to study the influence and impact of each factor on the several responses of the scaffolds. In vitro degradation (ID), swelling ratio (SR), porosity (PO), and pore size (PS) as well cytotoxicity (CT) were evaluated and shown to be dependent on the pH of sample preparation and on the amount of BSA and casein present, making these scaffolds tunable structures. Under optimized working conditions (4.19% of BSA, 0.69% of Casein, pH 7.07), the ID attained was 37.97%, the SR observed was 11.87, the PO was 82.11%, the PS measured was 180.63 μm at surface, and 175.91 μm at fracture, whereas maximum cell viability was 84% in comparison to controls. Moreover, the scaffold supported cell adhesion and proliferation. These results, consistent with the prediction by the experimental design approach, support the use of this methodology to develop tunable scaffolds for tissue engineering using the Ca2+-induced cold gelation.Contract grant sponsor: FEDER through POFC-COMPETE and by national funds from FCT; contract grant number: PEst-C/BIA/UI4050/201

Medical Termination of Delayed Miscarriage: Four-Year Experience with an Outpatient Protocol

Purpose: To evaluate the efficacy of an outpatient protocol with vaginal misoprostol to treat delayed miscarriage. Methods: Retrospective analysis of prospectively collected data on women medically treated for missed abortion with an outpatient protocol. The inclusion criteria were: ultrasound-based diagnosis of missed abortion with less than 10 weeks; no heavy bleeding, infection, inflammatory bowel disease or misoprostol allergy; no more than 2 previous spontaneous abortions; the preference of the patient regarding the medical management. The protocol consisted of: 1) a single dose of 800 µg of misoprostol administered intravaginally at the emergency department, after which the patients were discharged home; 2) clinical and ultrasonographic evaluation 48 hours later – if the intrauterine gestational sac was still present, the application of 800 µg of vaginal misoprostol was repeated, and the patients were discharged home; 3) clinical and ultrasonography evaluation 7 days after the initiation of the protocol – if the intrauterine gestational sac was still present, surgical management was proposed. The protocol was introduced in January 2012. Every woman received oral analgesia and written general recommendations. We also gave them a paper form to be presented and filled out at each evaluation. Results: Complete miscarriage with misoprostol occurred in 340 women (90.2%). Surgery was performed in 37 (9.8%) patients, representing the global failure rate of the protocol. Miscarriage was completed after the first misoprostol administration in 208 (55.2%) women, with a success rate after the second administration of 78.1% (132/169). The average age of the women with complete resolution using misoprostol was superior to the average age of those who required surgery (33.99 years versus 31.74 years; p = 0.031). Based on the ultrasonographic findings in the first evaluation, the women diagnosed with fetal loss achieved greater success rates compared with those diagnosed with empty sac (p = 0.049). Conclusions: We conclude this is an effective and safe option in the majority of delayed miscarriage cases during the first trimester, reducing surgical procedures and their consequences

Improved Poly (D,L-lactide) nanoparticles-based formulation for hair follicle targeting

Objective Hair follicles are widely recognized as the preferential target and site of accumulation for nanoparticles after topical application. This feature is of particular importance for hair cosmetics, having the potential to refine the treatment of several hair follicle-related disorders. The aim of this work was to improve the preparation of Poly (D,L-lactide) (PLA) nanoparticles for in vivo follicular target and drug delivery. Methods Envisaging a future industrial scale-up of the process, nanoprecipitation method was used to prepare PLA nanoparticles: the effect of several processing parameters on their properties was examined and the yield of nanoparticles formation determined. Encapsulation efficiencies and in vitro release profiles of lipophilic and hydrophilic model compounds were also assessed. In vitro cytotoxicity and ex vivo penetration studies were performed on a reference skin cell line (NCTC2455, human skin keratinocytes) and porcine skin, respectively. Results Using acetone : ethanol (50 : 50, v/v) as the solvent phase, 0.6% (w/w) of Pluronic® F68 as a surfactant agent and agitation to mix the solvent and non-solvent phases, a monodispersed population of non-cytotoxic spherical nanoparticles of approximately 150 nm was obtained. The yield of nanoparticles for this formulation was roughly 90%. After encapsulation of model compounds, no significant changes were found in the properties of particles and the entrapment efficiencies were above 80%. The release kinetics of dyes from PLA nanoparticles indicate an anomalous transport mechanism (diffusion and polymer degradation) for Nile Red (lipophilic) and a Fickian diffusion of first order for fluorescein 5(6)-isothiocyanate (hydrophilic). Ex vivo skin penetration studies confirmed the presence of nanoparticles along the entire follicular ducts. Conclusions The optimized method allows the preparation of ideal PLA nanoparticles-based formulations for hair follicle targeting. PLA nanoparticles can effectively transport and release lipophilic and hydrophilic compounds into the hair follicles, and the yields obtained are acceptable for industrial purposes.Objectif Les follicules pileux sont largement reconnus comme la cible préférentielle et le site de l'accumulation des nanoparticules après application topique. Cette caractéristique est particulièrement importante pour les produits cosmétiques pour les cheveux, ayant la possibilité d'affiner le traitement de plusieurs troubles des follicules de cheveux. Le but de ce travail était d'améliorer la préparation de nanoparticules poly (D,L-lactide) (PLA) pour une administration folliculaire in vivo ciblée de drogues. Méthodes En envisageant un avenir à l’échelle industrielle du procédé, une méthode de nanoprécipitation a été utilisé pour préparer des nanoparticules de PLA: l'effet de plusieurs paramètres de traitement sur leurs propriétés a été examiné et le rendement de la formation des nanoparticules a été déterminé. Les efficacités d'encapsulation et de profils de libération in vitro de composés modèles lipophiles et hydrophiles ont également été évaluées. La cytotoxicité in vitro et ex vivo des études de pénétration a été effectuée sur une lignée de cellules de peau de référence (NCTC2455, des kératinocytes de peau humaine) et la peau de porc, respectivement. Resultats En utilisant l'acétone : éthanol (50 : 50, v/v) comme phase solvant, 0,6% (p/p) de Pluronic® F68 à titre d'agent tensioactif et l'agitation pour mélanger les phases de solvant et de non-solvant, une population monodispersée des nanoparticules sphériques non cytotoxiques d'environ 150 nm a été obtenue. Le rendement de nanoparticules pour cette formulation était d'environ 90%. Après encapsulation de composés modèles, aucune modification significative n'a été observée dans les propriétés des particules et les efficacités de piégeage ont été supérieures à 80%. La cinétique de libération de colorants de nanoparticules de PLA indique un mécanisme de transport anormal (diffusion et dégradation de polymère) pour le rouge Nil (lipophile) et une diffusion selon Fick de premier ordre pour FITC (hydrophile). Les études de pénétration ex vivo de la peau ont confirmé la présence de nanoparticules sur tous les conduits folliculaires. Conclusions La méthode optimisée permet la préparation de formulations à base de nanoparticules de PLA, idéales pour ciblage du follicule pileux. Les nanoparticules de PLA peuvent effectivement transporter et libérer des composés lipophiles et hydrophiles dans les follicules pileux; les rendements obtenus sont acceptables à des fins industrielles.The authors thank to the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and to the Project “BioHealth – Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE- 07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER. This work was partly supported by FEDER through POFC–582 COMPETE and by Portuguese funds from FCT– Fundacão Para a Ciência e a Tecnologia through the project PEst-OE/BIA/UI4050/2014. The authors thank C. Botelho for technical assistance

Folic acid-tagged protein nanoemulsions loaded with CORM-2 enhance the survival of mice bearing subcutaneous A20 lymphoma tumors

Folic Acid (FA)-tagged protein nanoemulsions were found to be preferentially internalized on B-cell lymphoma cell line (A20 cell line), which, for the first time, are reported to express folate receptor (FR)-alpha. Carbon monoxide releasing molecule-2 (CORM-2) was incorporated in the oil phase of the initial formulation. FA-functionalized nanoemulsions loaded with CORM-2 exhibited a considerable antitumor effect and an increased survival of BALB/c mice bearing subcutaneous A20 lymphoma tumors. The developed nanoemulsions also demonstrated to be well tolerated by these immunocompetent mice. Thus, the results obtained in this study demonstrate that FA-tagged protein nanoemulsions can be successfully used in cancer therapy, with the important ability to delivery drugs intracellularly.SFRH/BD/81479/2011 and SFRH/BD/81269/2011 scholarships from Fundação para a Ciência e a Tecnologia (FCT). This work has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. This work was supported by FEDER through POFC-COMPETE and by Portuguese funds from FCT through the project PEst-OE/BIA/UI4050/2014

Study of sardine oil antioxidant properties for the development of topical therapeutic formulations

Sardine is one of the most common fish of the Portuguese coast with important nutritional features. Sardine oil is also a natural source of nutrients with proven benefits for human health, being rich in omega-3 fatty acids, particularly EPA and DHA (polyunsaturated fatty acids-PUFAs) [1]. Several studies show that there is a direct link between a diet enriched in omega-3 and the prevention of diseases such as cardiovascular disease, inflammatory conditions, mental disorders and prevention of various types of cancer [2]. The aim of this work was to characterize the antioxidant role of sardine oil for the development of topical applications. To evaluate the antioxidant effect of sardine oil on skin, human fibroblasts (BJ-5ta), human melanocytes (A375) and human keratinocytes (NCTC2544) were used. Concentrations of oil higher than 8 mg/ml affected significantly the cell viability while for lower concentrations the effect was reduced. The lowest concentrations, 0.5 and 4 mg/ml, were tested to evaluate the protective role of sardine oil in situation of induced oxidative stress. These two concentrations were able to protect cells from damage with a higher effect measured for the fibroblasts. Moreover the incubation of cells with the sardine oil was able to activate a transcription factor Nuclear factor-erythroid-2-related factor 2 (Nrf2) which plays a crucial role in the coordinated induction of genes encoding many stress-responsive and cytoprotective enzymes and related proteins [3]. These results open the opportunity to develop new therapeutic and cosmetic applications based on sardine-derived compounds. Their incorporation in topical creams may contribute to a better treatment of inflammation and in the prevention of skin aging

Functionalized protein nanoemulsions by incorporation of chemically modified BSA

The incorporation of bioactive compounds in stealth nanoparticles or nanoemulsions enhances their half-life in systemic circulation and can overcome the problems associated with the free drug. Bovine serum albumin (BSA)-drug conjugates were produced with either methotrexate (MTX), a potent anticancer agent, or vancomycin (VCM), a potent antibiotic. Those conjugates were used to produce functionalized BSA nanoemulsions in a formulation composed by aqueous phase and organic phase. BSA-Folic acid (FA) conjugates were also produced allowing specific folate receptor (FR) mediated targeting of cancer cells (KB cell line). All conjugates had similar effects either in solution or in the form of nanoemulsions: BSA-MTX as anti-proliferative over Caco-2 cell line and BSA-VCM as lower minimum inhibitory concentration (MIC) comparatively to VCM solution on Staphylococcus aureus strain Newman. The production of nanoemulsions using BSA-drug conjugates for obtaining vectors loaded with stabilized drugs offers a good, flexible template for a wide range of medical applications.Ana Loureiro (SFRH/BD/81479/2011) holds a scholarship from Fundacao para a Ciencia e a Tecnologia (FCT). Goncalo J. L. Bernardes is a Royal Society University Research Fellow at the Department of Chemistry, University of Cambridge and an Investigador FCT at the Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa. This work has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. This work was supported by FEDER through POFC - COMPETE and by Portuguese funds from FCT through the project PEst-OE/BIA/UI4050/2014

Enzymatic synthesis of poly(catechin)-antibiotic conjugates : an antimicrobial approach for indwelling catheters

Biofilm formation in urinary indwelling catheters is one of the most critical issues that patients face. Catheters were coated with poly(catechin)-antibiotic conjugates with enhanced antimicrobial properties. Catechin was conjugated with two antibiotics, namely trimethoprim (TMP) and sulfamethoxazole (SMZ) via activation with N,N′-disuccinimidyl carbonate (DSC) and subsequent coupling to molecules containing α-amine moieties. Silicone and polyurethane catheters were functionalized in situ through laccase oxidation of catechin-antibiotic conjugates. Four antimicrobial coatings were produced, namely with poly(catechin), poly(catechin)- TMP, poly(catechin)-SMZ and poly(catechin)-TMP-SMZ. The bacterial adhesion reduction was tested on the functionalized devices using gram-negative and gram-positive strains. The most significant reduction in adhesion was observed with poly(catechin)-TMP (gram-negative −85 % and grampositive −87 %) and with poly(catechin)-TMP-SMZ (gramnegative −85 % and gram-positive −91 %). The cytotoxicity to mammalian cells was tested by indirect contact for 5 days and revealed that all the tested coatings supported more than 90 % of viable cells. A promising approach for the increase of the indwelling catheter lifespan was developed aiming to reduce catheter-associated chronic infections.The authors would like to acknowledge Pronefro (Portugal) and Degania (Israel) for supplying polyurethane and silicone catheters, respectively. The author Idalina Goncalves would like to acknowledge the NOVO project (FP7-HEALTH-2011.2.3.1-5) for funding. This work was partly supported by FEDER through POFC-COMPETE and by Portuguese funds from Fundacao para a Ciencia e a Tecnologia (FCT) through the project PEst-OE/BIA/UI4050/2014. The authors Carla Silva and Teresa Matama would like to acknowledge FCT for their scholarships SFRH/BPD/46515/2008 and SFRH/BPD/47555/2008

Development of therapeutic and cosmetic formulations based on sardine-based products

Sardine is one of the most common fish of the Portuguese coast and has important nutritional features. Sardine oil is also a source of nutrients with proven benefits for human health, being rich in polyunsaturated fatty acids (PUFAs) [1]. Several studies show that there is a direct link between a diet enriched in omega-3 and the prevention of diseases such as cardiovascular disease, inflammatory conditions such as rheumatoid arthritis or asthma, mental disorders and prevention of various types of cancer [2]. The aim of this work was to characterize in a systematic way the potential protective role of sardine oil and derived PUFAs. To evaluate the antioxidant and antiinflammatory effect of sardine oil and PUFAs, human fibroblasts (BJ-5ta), human melanocytes (A375) and human keratinocytes (NCTC2544) were used. Cell viability was affected for concentrations higher than 8mg/ml for sardine oil and higher than 0.1mg/ml for PUFAs. However and regarding PUFAs, melanocytes revealed a higher susceptibility. With the lowest tested concentrations, sardine-based compounds promoted cell proliferation and protected cells from induced oxidative stress, with higher protection conferred by PUFAs. These results open the opportunity to develop new therapeutic and cosmetic applications based on sardine-derived compounds. Their incorporation in topical creams may contribute to a better treatment of inflammation and in the prevention of skin aging

Albumin/asparaginase capsules prepared by ultrasound to retain ammonia

"Published online: 17 June 2016"Asparaginase reduces the levels of asparagine in blood, which is an essential amino acid for the proliferation of lymphoblastic malign cells. Asparaginase converts asparagine into aspartic acid and ammonia. The accumulation of ammonia in the bloodstream leads to hyperammonemia, described as one of the most significant side effects of asparaginase therapy. Therefore, there is a need for asparaginase formulations with the potential to reduce hyperammonemia. We incorporated 2 % of therapeutic enzyme in albumin-based capsules. The presence of asparaginase in the interface of bovine serum albumin (BSA) capsules showed the ability to hydrolyze the asparagine and retain the forming ammonia at the surface of the capsules. The incorporation of Poloxamer 407 in the capsule formulation further increased the ratio aspartic acid/ammonia from 1.92 to 2.46 (and 1.10 from the free enzyme), decreasing the levels of free ammonia. This capacity to retain ammonia can be due to electrostatic interactions and retention of ammonia at the surface of the capsules. The developed BSA/asparaginase capsules did not cause significant cytotoxic effect on mouse leukemic macrophage cell line RAW 264.7. The new BSA/asparaginase capsules could potentially be used in the treatment of acute lymphoblastic leukemia preventing hyperammonemia associated with acute lymphoblastic leukemia (ALL) treatment with asparaginase.Fundação para a Ciência e a Tecnologia - SFRH\BPD\98388\2013 ; UID/BIO/04469/2013 ; UID/BIA/04050/2013COMPETE 2020 (POCI-01-0145-FEDER-006684

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat