A multiscale optimisation method for bone growth scaffolds based on triply periodic minimal surfaces.

Tissue engineered bone scaffolds are potential alternatives to bone allografts and autografts. Porous scaffolds based on triply periodic minimal surfaces (TPMS) are good candidates for tissue growth because they offer high surface-to-volume ratio, have tailorable stiffness, and can be easily fabricated by additive manufacturing. However, the range of TPMS scaffold types is extensive, and it is not yet clear which type provides the fastest cell or tissue growth while being sufficiently stiff to act as a bone graft. Nor is there currently an established methodology for TPMS bone scaffold design which can be quickly adopted by medical designers or biologists designing implants. In this study, we examine six TPMS scaffold types for use as tissue growth scaffolds and propose a general methodology to optimise their geometry. At the macro-scale, the optimisation routine ensures a scaffold stiffness within suitable limits for bone, while at the micro-scale it maximises the cell growth rate. The optimisation procedure also ensures the scaffold pores are of sufficient diameter to allow oxygen and nutrient delivery via capillaries. Of the examined TPMS structures, the Lidinoid and Split P cell types provide the greatest cell growth rates and are therefore the best candidates for bone scaffolds.

Polymeric micelles in drug delivery: An insight of the techniques for their characterization and assessment in biorelevant conditions.

Polymeric micelles, i.e. aggregation colloids formed in solution by self-assembling of amphiphilic polymers, represent an innovative tool to overcome several issues related to drug administration, from the low water-solubility to the poor drug permeability across biological barriers. With respect to other nanocarriers, polymeric micelles generally display smaller size, easier preparation and sterilization processes, and good solubilization properties, unfortunately associated with a lower stability in biological fluids and a more complicated characterization. Particularly challenging is the study of their interaction with the biological environment, essential to predict the real in vivo behavior after administration. In this review, after a general presentation on micelles features and properties, different characterization techniques are discussed, from the ones used for the determination of micelles basic characteristics (critical micellar concentration, size, surface charge, morphology) to the more complex approaches used to figure out micelles kinetic stability, drug release and behavior in the presence of biological substrates (fluids, cells and tissues). The techniques presented (such as dynamic light scattering, AFM, cryo-TEM, X-ray scattering, FRET, symmetrical flow field-flow fractionation (AF4) and density ultracentrifugation), each one with their own advantages and limitations, can be combined to achieve a deeper comprehension of polymeric micelles in vivo behavior. The set-up and validation of adequate methods for micelles description represent the essential starting point for their development and clinical success.

inPentasomes: An innovative nose-to-brain pentamidine delivery blunts MPTP parkinsonism in mice.

Preclinical and clinical evidences have demonstrated that astroglial-derived S100B protein is a key element in neuroinflammation underlying the pathogenesis of Parkinson's disease (PD), so much as that S100B inhibitors have been proposed as promising candidates for PD targeted therapy. Pentamidine, an old-developed antiprotozoal drug, currently used for pneumocystis carinii is one of the most potent inhibitors of S100B activity, but despite this effect, is limited by its low capability to cross blood brain barrier (BBB). To overcome this problem, we developed a non-invasive intranasal delivery system, chitosan coated niosomes with entrapped pentamidine (inPentasomes), in the attempt to provide a novel pharmacological approach to ameliorate parkinsonism induced by subchronic MPTP administration in C57BL-6 J mice. inPentasomes, prepared by evaporation method was administered daily by intranasal route in subchronic MPTP-intoxicated rodents and resulted in a dose-dependent manner (0.001-0.004 mg/kg) capable for a significant Tyrosine Hydroxylase (TH) positive neuronal density rescue in both striatum and substantia nigra of parkinsonian mice. In parallel, inPentasomes significantly decreased the extent of glial-related neuroinflammation through the reduction of specific gliotic markers (Iba-1, GFAP, COX-2, iNOS) with consequent PGE2 and NO2- release reduction, in nigrostriatal system. inPentasomes-mediated S100B inhibition resulted in a RAGE/NF-κB pathway downstream inhibition in the nigrostriatal circuit, causing a marked amelioration of motor performances in intoxicated mice. On the basis of our results, chitosan coated niosomes loaded with pentamidine, the inPentasome system, self-candidates as a promising new intranasal approach to mitigate parkinsonism in humans and possibly paves the way for a possible clinical repositioning of pentamidine as anti-PD drug.

Directional K+ channel insertion in a single phospholipid bilayer: Neutron reflectometry and electrophysiology in the joint exploration of a model membrane functional platform.

We investigated the insertion of small potassium (K+) channel proteins (KcvMA-1D and KcvNTS) into model membranes and the lipid-protein structural interference, combining neutron reflectometry and electrophysiology. Neutron reflectometry experiments showed how the transverse structure and mechanical properties of the bilayer were modified, upon insertion of the proteins in single model-membranes, either supported on solid substrate or floating. Parallel electrophysiology experiments were performed on the same channels reconstituted in free-standing planar lipid bilayers, of both typical composition and matched to the neutron reflectometry experiment, assessing their electrical features. Functional and structural results converge in detecting that the proteins, conical in shape, insert with a directionality, cytosolic side first. Our work addresses the powerful combination of the two experimental approaches. We show here that membrane structure spectroscopy and ion channel electrophysiology can become synergistic tools in the analysis of structural-functional properties of biomimetic complex environment.

Water response to ganglioside GM1 surface remodelling.

BACKGROUND: Gangliosides are biological glycolipids participating in rafts, structural and functional domains of cell membranes. Their headgroups are able to assume different conformations when packed on the surface of an aggregate, more lying or standing. Switching between different conformations is possible, and is a collective event. Switching can be induced, in model systems, by concentration or temperature increase, then possibly involving ganglioside-water interaction. In the present paper, the effect of GM1 ganglioside headgroup conformation on the water structuring and interactions is addressed. METHODS: Depolarized Rayleigh Scattering, Raman Scattering, Quasielastic Neutron Scattering and NMR measurements were performed on GM1 ganglioside solutions, focusing on solvent properties. RESULTS: All used techniques agree in evidencing differences in the structure and dynamics of solvent water on different time-and-length scales in the presence of either GM1 headgroup conformations. CONCLUSIONS: In general, all results indicate that both the structural properties of solvent water and its interactions with the sugar headgroups of GM1 respond to surface remodelling. The extent of this modification is much higher than expected and, interestingly, ganglioside headgroups seem to turn from cosmotropes to chaotropes upon collective rearrangement from the standing- to the lying-conformation. SIGNIFICANCE: In a biological perspective, water structure modulation could be one of the physico-chemical elements contributing to the raft strategy, both for rafts formation and persistence and for their functional aspects. In particular, the interaction with approaching bodies could be favoured or inhibited or triggered by complex-sugar-sequence conformational switch. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.

Mechanistic understanding of gene delivery mediated by highly efficient multicomponent envelope-type nanoparticle systems.

We packaged condensed DNA/protamine particles in multicomponent envelope-type nanoparticle systems (MENS) combining different molar fractions of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3ß-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipids dioleoylphosphocholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE). Dynamic light scattering (DLS) and microelectrophoresis allowed us to identify the cationic lipid/DNA charge ratio at which MENS are small sized and positively charged, while synchrotron small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) revealed that MENS are well-shaped DNA/protamine particles covered by a lipid monobilayer. Transfection efficiency (TE) experiments indicate that a nanoparticle formulation, termed MENS-3, was not cytotoxic and highly efficient to transfect Chinese hamster ovary (CHO) cells. To rationalize TE, we performed a quantitative investigation of cell uptake, intracellular trafficking, endosomal escape, and final fate by laser scanning confocal microscopy (LSCM). We found that fluid-phase macropinocytosis is the only endocytosis pathway used by MENS-3. Once taken up by the cell, complexes that are actively transported by microtubules frequently fuse with lysosomes, while purely diffusing systems do not. Indeed, spatiotemporal image correlation spectroscopy (STICS) clarified that MENS-3 mostly exploit diffusion to move in the cytosol of CHO cells, thus explaining the high TE levels observed. Also, MENS-3 exhibited a marked endosomal rupture ability resulting in extraordinary DNA release. The lipid-dependent and structure-dependent TE boost suggests that efficient transfection requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.

Neutrons for rafts, rafts for neutrons.

The determination of the structure of membrane rafts is a challenging issue in biology. The selection of membrane components both in the longitudinal and transverse directions plays a major role as it determines the creation of stable or tunable platforms that host interactions with components of the outer environment. We focus here on the possibility to apply neutron scattering to the study of raft mimics. With this aim, we realized two extreme experimental models for the same complex membrane system (phospholipid : cholesterol : ganglioside GM1), involving two of the characteristic components of glycolipid-enriched rafts. One consists of a thick stack of tightly packed membranes, mixed and symmetric in composition, deposited on a silicon wafer and analyzed by neutron diffraction. The other consists of a free floating individual membrane, mixed and asymmetric in composition in the two layers, studied by neutron reflection. We present here results on the ganglioside-cholesterol coupling. Ganglioside GM1 is found to force the redistribution of cholesterol between the two layers of the model membranes. This causes cholesterol exclusion from compositionally symmetric ganglioside-containing membranes, or, alternatively, asymmetric cholesterol enrichment in raft-mimics, where gangliosides reside into the opposite layer.

Diagnóstico de infertilidad masculina: necesidad de valoraciones funcionales y cromatínicas / Diagnosis of Male Infertility: A Need of Functional and Chromatin Evaluation

Objetivos: Estudiar la incidencia de alteraciones funcionales y cromatínicas en espermatozoides de pacientes, agrupados según normalidad de los parámetros seminales estándar en función de la quinta edición del manual de la Organización Mundial de la Salud (OMS). Identificar y correlacionar las características alteradas con mayor frecuencia en la subpoblación normal para los parámetros estándar. Materiales y métodos: Estudio prospectivo en el que se evaluaron en muestras de semen de 110 pacientes y 6 donantes fértiles (control), los parámetros seminales estándar (volumen, concentración, motilidad, morfología, células redondas, células peroxidasa-positivas) según las directrices de la OMS, así como test complementarios funcionales (sobrevida 24 horas, test hipo-osmótico, test de estrés modificado) y adicionales (azul de anilina, nitroblue-tetrazolium, TUNEL). Según el resultado de los análisis estándar los pacientes se dividieron en dos grupos (A: todos los parámetros estándar normales; B: al menos uno de los parámetros estándar alterado). Resultados: El 96,61% de las muestras presentó alterada al menos una de las variables analizadas. Los grupos A y B mostraron diferencia estadísticamente significativa en todos los test complementarios. En el grupo A el 93,68% de las muestras presentó al menos un test complementario alterado, y la variable afectada con mayor frecuencia fue la fragmentación del ADN espermático (16,95%). Conclusiones: La profundización en el estudio seminal, introduciendo en la rutina ensayos funcionales y cromatínicos, redunda en un diagnóstico más afinado de la infertilidad del varón. Los parámetros de la OMS deben considerarse como un abordaje primario (AU)

Objectives: To evaluate the incidence of functional and chromatin alterations on spermatozoids in patients grouped according to normality of standard semen parameters based on the 5th edition of the World Health Organization (WHO) guidelines. To identify and correlate the most frequently altered characteristics in the normal standard semen parameters sub-population. Materials and Methods: A prospective study was performed. It evaluated standard semen parameters (volume, sperm concentration, motility and morphology, round cells, peroxidase-positive cells) according to WHO guidelines, as well as functional tests (24hours survival, hypoosmotic swelling test, modified stress test), and additional assays (aniline blue, nitroblue-tetrazolium, TUNEL) in 110 semen samples from patients and 6 from fertile donors (control). Based on standard semen parameters values, patients were divided into two groups (A: all standard parameters normal; B: one altered standard parameter at least). Results: At least one of the variables analyzed was altered in 96.61% of the samples. Groups A and B showed statistically significant differences in all the complementary tests. At least one of the complementary tests were altered in 93.68% of the samples in group A, and the most frequently affected variable was sperm DNA fragmentation (16.95%). Conclusions: Performing a more in-depth seminal study within the routine functional and chromatin assays provides a more precise diagnosis of male infertility. The WHO standards should be considered as a primary approach (AU)

[Diagnosis of male infertility: a need of functional and chromatin evaluation]. / Diagnóstico de infertilidad masculina: necesidad de valoraciones funcionales y cromatínicas.

OBJECTIVES: To evaluate the incidence of functional and chromatin alterations on spermatozoids in patients grouped according to normality of standard semen parameters based on the 5th edition of the World Health Organization (WHO) guidelines. To identify and correlate the most frequently altered characteristics in the normal standard semen parameters sub-population. MATERIALS AND METHODS: A prospective study was performed. It evaluated standard semen parameters (volume, sperm concentration, motility and morphology, round cells, peroxidase-positive cells) according to WHO guidelines, as well as functional tests (24 hours survival, hypoosmotic swelling test, modified stress test), and additional assays (aniline blue, nitroblue-tetrazolium, TUNEL) in 110 semen samples from patients and 6 from fertile donors (control). Based on standard semen parameters values, patients were divided into two groups (A: all standard parameters normal; B: one altered standard parameter at least). RESULTS: At least one of the variables analyzed was altered in 96.61% of the samples. Groups A and B showed statistically significant differences in all the complementary tests. At least one of the complementary tests were altered in 93.68% of the samples in group A, and the most frequently affected variable was sperm DNA fragmentation (16.95%). CONCLUSIONS: Performing a more in-depth seminal study within the routine functional and chromatin assays provides a more precise diagnosis of male infertility. The WHO standards should be considered as a primary approach.

Ganglioside GM1 forces the redistribution of cholesterol in a biomimetic membrane.

Neutron reflectivity has been applied to investigate different mixed asymmetric lipid systems, in the form of single "supported+floating" bilayers, made of phospholipids, cholesterol and GM1 ganglioside (Neu5Acα2-3(Galß1-3GalNAcß1-4)Galß1-4Glcß1Cer)) in bio-similar mole ratios. Bilayer preparation was carried out layer-by-layer with the Langmuir-Blodgett Langmuir-Schaefer techniques, allowing for compositional asymmetry in the system buildup. It is the first time that such a complex model membrane system is reported. Two important conclusions are drawn. First, it is experimentally shown that the presence of GM1 enforces an asymmetry in cholesterol distribution, opposite to what happens for a GM1-free membrane that, submitted to a similar procedure, results in a full symmetrization of cholesterol distribution. We underline that natural cholesterol has been used. Second, and most interesting, our results suggest that a preferential asymmetric distribution of GM1 and cholesterol is attained in a model membrane with biomimetic composition, revealing that a true coupling between the two molecular species occurs.

DNA sperm damage correlates with nuclear ultrastructural sperm defects in teratozoospermic men.

Sperm morphology has consistently been the best indicator of male fertility. Transmission electron microscopy currently provides the most information on the subcellular details of sperm structure. Recently, assessment of sperm DNA damage has been employed to assess fertility potential. The purpose of this work was to link sperm DNA damage, evaluated by an intercalated fluorescent dye, with the structural characteristics of sperm. Conventional semen analysis was performed on samples from men undergoing fertility evaluation. Thirty men were evaluated and assigned to three subgroups based on strict criteria for sperm morphology: normal morphology (>14% normal forms), intermediate morphology (5-14% normal forms), and poor morphology (<5% normal forms). By quantifying acridine orange-positive cells and ultrastructural sperm defects, we found that the poor morphology pattern group showed a positive association between sperm carrying damaged DNA and the percentage of sperm nucleus with vacuoles (P = 0.01). No statistically significant correlations were established in other ultrastructural characteristics of sperm, including immature chromatin, lytic changes, or abnormal sperm tails. These results suggest that zones without chromatin in the sperm nucleus reflect underlying chromosomal or DNA defects in severe teratozoospermic men. This association should be considered in the evaluation of male fertility.

Lamellar stacking split by in-membrane clustering of bulky glycolipids.

We developed a simple model to investigate the effect of lipid clustering on the local interlayer distance in a cluster of interacting lamellae. The model, based on nonequilibrium thermodynamics and linear stability theories, explores the early stages of the lamella-lamella phase separation process where the lateral diffusion is much faster than the interlamellar lipid exchange. Results indicate, in the early stages, the presence of locally distorted regions with a higher concentration of one lipid component and an anomalous repeat distance. Experimental cases are presented, consisting of multilamellar-oriented depositions of phospholipids containing minority amounts of ganglioside or sphingomyelin under a low-hydration condition. The minority components are known to form domains within the phospholipid bilayer matrix. The low water content inhibits the lipid exchange among nearby lamellae and strengthens lamella-lamella interaction, allowing for a straightforward comparison with the model. Small-angle and wide-angle neutron diffraction experiments were performed in order to detect interlayer distances and local chain order, respectively. Lamellar stacking splitting has been observed for the ganglioside-containing lamellae, induced by in-phase lipid clustering. In excess water and after long equilibration times, these local structures may further evolve, leading to coexisting lamellar phases with different lipid compositions and interlayer distances.

Structure of self-organized multilayer nanoparticles for drug delivery.

The combined use of cryo-TEM, dynamic light scattering, and small-angle X-ray and neutron scattering techniques allows a detailed structural model of complex pharmaceutical preparations of soybean lecithin/chitosan nanoparticles used as drug vectors to be worked out. Charge-driven self-organization of the lipid(-)/polysaccharide(+) vesicles occurs during rapid injection, under mechanical stirring, of an ethanol solution of soybean lecithin into a chitosan aqueous solution. We conclude that beyond the charge inversion region of the phase diagram, i.e., entering the redissolution region, the initial stages of particle formation are likely to be affected by a re-entrant condensation effect at the nanoscale. This behavior resembles that at the mesoscale which is well-known for polyion/amphiphile systems. Close to the boundary of the charge inversion region, nanoparticle formation occurs under a maximum condensation condition at the nanoscale and the complexation-aggregation process is driven toward a maximum multilamellarity. Interestingly, the formulation that maximizes vesicle multilamellarity corresponds to that displaying the highest drug loading efficiency.

Intermicellar interactions may induce anomalous size behavior in micelles carrying out bulky heads with multiple spatial arrangements.

We report experimental and theoretical results on the concentration dependence of the micellar size of GM1 and GM1acetyl gangliosides, five-sugar-headed anionic glycolipids. Contrary to one of the mainstays of colloid science, that the aggregation number of amphiphile aggregates grows with concentration, an anomalous region is found at intermediate concentrations, where a sharp decrease of the aggregation number occurs. Experiments were performed by small-angle X-ray and neutron scattering (SAXS and SANS). Two models are discussed, reproducing the observed behavior of either GM1acetyl or GM1. The first one is a conventional picture of interacting micelles where a reduction in the molecular surface area, leading to an increase of the aggregate dimension, is paid to reduce intermicellar interactions: it foresees a monotonous increase of the aggregation number with concentration. The second one accounts for a conformational bistability of the bulky headgroups of GM1, modifying the amphiphilic molecular surface area and protrusion from the aggregate surface, and contributing to the inter- and intramicellar interaction balance. Energy minimization leads to a complex behavior of the aggregation number, which is consistent with the anomalous behavior of GM1.

DC(13)PC bilayers from anomalous swelling to main transition: an X-ray scattering investigation.

We have performed small-angle (SAXS) and wide-angle X-ray scattering (WAXS) measurements on the lamellar phase and on large unilamellar vesicles (LUVs) of DC(13)PC in the temperature range corresponding to the anomalous swelling regime of multibilayer systems, adjacent to the chain melting transition, and across the transition. Our SAXS measurements indicate that on cooling from the L(alpha) phase, a uniform progressive swelling of the lamellar system to anomalous distances, starting approximately 2 degrees C above the main transition, is followed by a region of coexistence, covering the width of the transition ( approximately 0.6 degrees C). Across the transition region, a progressively increasing volume fraction of gel phase with a constant P (beta') interlamellar distance coexists with a decreasing amount of nongel phase that keeps on swelling to longer distances. Along both the swelling and the transition regions, anomalies in the specific heat are observed revealing a two-step process. Simultaneous WAXS experiments show a progressive "density" increase along the swelling region, constituting a direct spectroscopic evidence of an "evolving membrane" approaching the transition in a bulk real system. Calorimetric and densitometric measurements on LUVs are also presented, together with WAXS results, that show the existence of a double step main transition in a single component nanosized closed bilayer.

Glomerular filtration is required for transfection of proximal tubular cells in the rat kidney following injection of DNA complexes into the renal artery.

Gene transfer to the kidney can be achieved with various DNA vectors, resulting in transgene expression in glomerular or tubular districts. Controlling transgene destination is desirable for targeting defined renal cells for specific therapeutic purposes. We previously showed that injection of polyplexes into the rat renal artery resulted in transfection of proximal tubular cells. To investigate whether this process involves glomerular filtration of the DNA-containing particles, fluorescent polyethylenimine polyplexes were prepared, containing fluoresceinated poly-L-lysine. This allowed visualization of the route of the particles into the kidney. Our polyplexes were filtered through the glomerulus, since fluorescent proximal tubuli were observed. Conversely, fluorescent lipopolyplexes containing the cationic lipid DOTAP were never observed in tubular cells. Size measurements by laser light scattering showed that the mean diameter of polyplexes (93 nm) was smaller than that of lipopolyplexes (160 nm). The size of the transfecting particles is therefore a key parameter in this process, as expected by the constraints imposed by the glomerular filtration barrier. This information is relevant, in view of modulating the physico-chemical properties of DNA complexes for optimal transgene expression in tubular cells. Gene Therapy (2000) 7, 279-285.

Effect of continuously warmed i.v. fluids on intraoperative hypothermia.

The investigators examined the effect of infusing continuously warmed (ie, 37.0 degrees C [98.6 degrees F]) i.v. fluids in two groups of middle-aged female patients undergoing laparoscopic cholecystectomy procedures. They hypothesized that increasing i.v. fluid temperature during surgery would decrease patients' risk for hypothermia. One group of patients received prewarmed i.v. fluids that cooled to room temperature during surgery. The second group received i.v. fluids that were warmed continuously by a fluid warmer during the surgical procedures. Analyses of covariance, with the first intraoperative temperature measurement treated as the covariate, revealed nonsignificant results at the P < .05 level. The results suggest that administering continuously warmed i.v. fluids intraoperatively has no significant effect on maintaining patients' body temperatures during short laparoscopic surgical procedures.

Experimental evidence of a temperature-related conformational change of the hydrophilic portion of gangliosides.

The present paper reports the experimental observation of an interesting thermodynamic process which could be biologically important: such behaviour, shown by some gangliosides, is likely to be peculiar of these glycosidic compounds as it has never been observed for other membrane-type amphiphilic molecules. In water solution, gangliosides have been found to present a bistable behaviour between two stable states (called A and B) which does not involve any change in the primary structure of the molecule. The interconversion between state A and state B, and vice versa, does not occur spontaneously, but has to be triggered by some external agent, which makes this system a potentially regulated process with important biological correlations. In the present experiments, state B is reached from state A with a temperature rise in the range 30-55 degrees C. The new state is stable regardless of any possible temperature cycle. The initial state A is then regained when the ganglioside solution is dried and the solute is redissolved. The two states are believed to correspond to two different conformations of the hydrophilic portion of the molecule. The bistable behaviour is shown by the gangliosides GM2, GM1, GD1a, GD1b and Fuc-GD1b, GT1b, however, does not show such an effect.

Physicochemical behavior of the ganglioside GM3 in dilute aqueous solution.

The aggregative properties of the ganglioside GM3 have been studied with small-angle X-ray scattering and dynamic light scattering in dilute aqueous solutions. Dynamic light scattering experiments show that GM3 solutions are very polydisperse containing a large amount of small aggregates (hydrodynamic radius of 7-9 nm) in addition to a quite broad distribution of aggregates with an average hydrodynamic radius of 50-60 nm and a small fraction of very large aggregates (> 200 nm). This together with small-angle X-ray scattering scattering experiments and model calculations suggests the coexistence of a lamellar phase (vesicles or extended lamellae) with small aggregates (modelled as lamellar platelets). The latter can also be viewed as lamellar fragments coming from GM3 vesicles which constantly break and reform.