Decellularization of porcine esophageal tissue at three diameters and the bioscaffold modification with EETs-ECM gel.

Damaged complex modular organs repair is a current clinical challenge in which one of the primary goals is to keep their biological response. An interesting case of study it is the porcine esophagus since it is a tubular muscular tissue selected as raw material for tissue engineering. The design of esophageal constructs can draw on properties of the processed homologous extracellular matrix (ECM). In this work, we report the decellularization of multilayered esophagus tissue from 1-, 21- and 45-days old piglets through the combination of reversible alkaline swelling and detergent perfusion. The bioscaffolds were characterized in terms of their residual composition and tensile mechanical properties. The biological response to esophageal submucosal derived bioscaffolds modified with ECM gel containing epoxyeicosatrienoic acids (EETs) was then evaluated. Results suggest that the composition (laminin, fibronectin, and sulphated glycosaminoglycans/sGAG) depends on the donor age: a better efficiency of the decellularization process combined with a higher retention of sGAG and fibronectin is observed in piglet esophageal scaffolds. The heterogeneity of this esophageal ECM is maintained, which implied the preservation of anisotropic tensile properties. Coating of bioscaffolds with ECM gel is suitable for carrying esophageal epithelial cells and EETs. Bioactivity of EETs-ECM gel modified esophageal submucosal bioscaffolds is observed to promote neovascularization and antiinflammatory after rabbit full-thickness esophageal defect replacement.

Bioreduction of precious and heavy metals by Candida species under oxidative stress conditions.

The aim of the present work was to evaluate whether Candida species can reduce both precious and toxic pure metals from the respective molecular ions. From these results, the nanoparticles formed were studied using scanning electron microscopy with energy-dispersive spectroscopy, Raman spectroscopy, X-ray fluorescence spectroscopy and synchrotron radiation. Our results showed that the metal ions were reduced to their corresponding metallic nanoconglomerate or nanoparticles by Candida species. This is the first report on how yeasts of this genus are capable of achieving homeostasis (resilience) in the presence of metal ions of both precious and toxic metals by reducing them to a metallic state.

Effect of varying the crosslinking degree of a pericardial implant with oligourethane on the repair of a rat full-thickness abdominal wall defect.

The stability and bioactivity of biologic implants rely mainly on the control of the crosslinking process of collagen. However, the most common methods have no control on the crosslinking degree producing it excessively. This study outlines the role of crosslinking of collagen-based implants with oligourethane on the host response following reconstruction of a rat full-thickness abdominal wall defect. We decellularized and crosslinked bovine pericardial tissue to achieve two crosslinking degrees. For the decellularized implants, named as non-crosslinked (N-CL), the collagen-amines were 0.42 ± 0.02 mmol/mg. Crosslinking by the oligourethane reduced the primary amine concentration to 0.28 ± 0.01 and 0.19 ± 0.01 mmol/mg; these values were classified as low (∼30%, L-CL) and medium crosslinking (∼50%, M-CL), respectively. By imaging the implants using second harmonic generation microscopy, we observed undulated bundles of collagen fibers organized in multi-directed layers localized in N-CL and L-CL samples. Post-implantation, a negligible change in the organization of collagen fibers in the crosslinked implants was observed, suggesting that the in vivo biodegradation was delayed. An enlargement of the implant area was also observed, without rupture, in all three (N-CL, L-CL, M-CL) materials, whereas adhesion to the omentum, but not to the bowel, was observed. The number of blood vessels after 90-day implantation in N-CL and L-CL was 13 ± 1 and 12 ± 1 per field, respectively, while the number significantly decreased to 2 ± 1 in M-CL. The results suggest that the controlled degree of crosslinking in oligourethane-modified biologic implants can be used as a strategy to balance biodegradation and remodeling in surgical repair of soft tissues.

Moonlighting proteins induce protection in a mouse model against Candida species.

In recent years, C. albicans and C. glabrata have been identified as the main cause of candidemia and invasive candidiasis in hospitalized and immunocompromised patients. In order to colonize the human host, these fungi express several virulence factors such as the response to oxidative stress and the formation of biofilms. In the expression of these virulence factors, the cell wall of C. albicans and C. glabrata is of fundamental importance. As the outermost structure of the yeast, the cell wall is the first to come in contact with the reactive oxygen species (ROS) generated during the respiratory outbreak, and in the formation of biofilms, it is the first to adhere to organs or medical devices implanted in the human host. In both processes, several cell wall proteins (CWP) are required, since they promote attachment to human cells or abiotic surfaces, as well as to detoxify ROS. In our working group we have identified moonlighting CWP in response to oxidative stress as well as in the formation of biofilms. Having identified moonlighting CWP in Candida species in response to two virulence factors indicates that these proteins may possibly be immunodominant. The aim of the present work was to evaluate whether proteins of this type such as fructose-bisphosphate aldolase (Fba1), phosphoglycerate kinase (Pgk) and pyruvate kinase (Pk), could confer protection in a mouse model against C. albicans and C. glabrata. For this, recombinant proteins His6-Fba1, His6-Pgk and His6-Pk were constructed and used to immunize several groups of mice. The immunized mice were infected with C. albicans or C. glabrata, and subsequently the liver, spleen and kidney were extracted and the number of CFU was determined. Our results showed that Pk confers immunity to mice against C. albicans, while Fba1 to C. glabrata. This data allows us to conclude that the moonlighting CWP, Fba1 and Pk confer in vivo protection in a specific way against each species of Candida. This makes them promising candidates for developing specific vaccines against these pathogens.

The component leaching from decellularized pericardial bioscaffolds and its implication in the macrophage response.

The extracellular matrix molecules remaining in bioscaffolds derived from decellularized xenogeneic tissues appear to be important for inducing cell functions conducting tissue regeneration. Here, we studied whether decellularization methods, that is, detergent Triton X-100 (TX) alone and TX combined with reversible alkaline swelling (STX), applied to bovine pericardial tissue, could affect the bioscaffold components. The in vitro macrophage response, subdermal biodegradation, and cell infiltration were also studied. The results indicate a lower leaching of fibronectin, but a higher leaching of laminin and sulfated glycosaminoglycans from tissues decellularized with STX and TX, respectively. The in vitro secretion of interleukin-6 and monocyte chemoattractant protein by RAW264.7 macrophages is promoted by decellularized bioscaffold leachates. A lower polymorphonuclear cell density is observed around decellularized bioscaffolds at 1-day implantation; concurrently showing a higher cell infiltration in STX- than in TX-implant. Cells infiltrated into TX-implant show a fibroblastic morphology at 7-day implantation, concurrently the capillary formation is observed at 14-day. Pericardial bioscaffolds suffer biodegradation more pronounced in STX- than in TX-implant. Both TX and STX decellularization methods favor a high leaching of basal lamina components, which presumably promotes a faster macrophage stimulation compared to nondecellularized tissue, and appear to be associated with an increased host cell infiltration in a rat subdermal implantation. Meanwhile, the connective tissue components leaching from TX decellularized bioscaffolds, unlike the STX ones, appear to be associated with an enhanced angiogenesis accompanied by an early-promoted fibroblastic cell transition. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2810-2822, 2016.

Changes in GDPase/UDPase enzymatic activity in response to oxidative stress in four Candida species.

The terminal processing of proteins and lipids occurs in the Golgi apparatus and involves the transport of sugar nucleotides into the Golgi lumen by specific carriers and the accumulation of nucleoside diphosphates (NDPs) as a result of oligosaccharide-protein glycosyltransferase activity. NDPs are converted into the corresponding nucleoside monophosphates (NMPs) by nucleoside diphosphatases (NDPases), thus relieving inhibition of sugar transferases. In addition, NMPs are then exchanged for equimolecular amounts of cytosolic sugar nucleotides by antiport transport systems. NDPases, commonly GDPase and UDPase, thus play a critical role in glycoprotein maturation and may influence fungal pathogenesis, morphogenesis, and cell wall properties. Interest of this laboratory has recently focused on the effect of reactive oxygen species (ROS) on enzymes involved in detoxification of these oxidants and on the metabolism of biomolecules such as lipids, nucleic acids, and proteins in human pathogenic Candida species. We therefore consider it important to extend these studies to determine how GDPase and UDPase are affected after exposure of cells to oxidants such as menadione, a superoxide (O2 (•-))-generator, and H2O2. Results indicate that activity of both enzymes decrease in response to these agents suggesting that ROS may also affect other critical cell functions such as protein glycosylation.

Influence of culture media on biofilm formation by Candida species and response of sessile cells to antifungals and oxidative stress.

The aims of the study were to evaluate the influence of culture media on biofilm formation by C. albicans, C. glabrata, C. krusei, and C. parapsilosis and to investigate the responses of sessile cells to antifungals and reactive oxygen species (ROS) as compared to planktonic cells. For biofilm formation, the Candida species were grown at different periods of time in YP or YNB media supplemented or not with 0.2 or 2% glucose. Sessile and planktonic cells were exposed to increasing concentrations of antifungals, H2O2, menadione or silver nanoparticles (AgNPs). Biofilms were observed by scanning electron microscopy (SEM) and quantified by the XTT assay. C. albicans formed biofilms preferentially in YPD containing 2% glucose (YPD/2%), C. glabrata in glucose-free YNB or supplemented with 0.2% glucose (YNB/0.2%), while C. krusei and C. parapsilosis preferred YP, YPD/0.2%, and YPD/2%. Interestingly, only C. albicans produced an exopolymeric matrix. This is the first report dealing with the in vitro effect of the culture medium and glucose on the formation of biofilms in four Candida species as well as the resistance of sessile cells to antifungals, AgNPs, and ROS. Our results suggest that candidiasis in vivo is a multifactorial and complex process where the nutritional conditions, the human immune system, and the adaptability of the pathogen should be considered altogether to provide an effective treatment of the patient.

Synthesis and characterization of protected oligourethanes as crosslinkers of collagen-based scaffolds.

This paper describes the preparation and characterization of water-soluble urethane oligomers bearing protected isocyanate groups. It also points out its ability to crosslink decellularized pericardium, as a model collagen scaffold, and to adjust their structural characteristics. A library of oligourethanes was synthesized by varying the molecular weight (Mw 400, 600, 1000 or 2000 g mol-1) of the poly(ethylene glycol) and the type of aliphatic diisocyanate (isophorone diisocyanate/IPDI or hexamethylene diisocyanate/HDI). 1H and 13C NMR, FTIR and mass spectrometry demonstrated that the crosslinkers are composed of chains with carbamoylsulfonate end groups that have trimeric and pentameric oligourethanes, and monomeric diisocyanate. The degree of crosslinking and hence the in vitro degradation susceptibility of the decellularized pericardium were inversely related to the Mw of the oligourethanes. The toxicity of the extractable products from oligourethane-collagen materials toward fibroblasts and macrophages was found to be lower for the crosslinker derived from IPDI than for those derived from HDI. On the other hand, the resistance to collagenase or oxidative degradation of the bovine pericardium crosslinked with HDI/oligourethane was higher than the one prepared with IPDI/oligourethane. As the Mw of the oligomers regulates the degree of crosslinking while the chemical composition influences the cytocompatibility and biodegradation of decellularized pericardium, these urethane oligomers can be used as safer crosslinkers for other protein-based biomaterials.

Litter size determines circadian periodicity of nursing in rabbits.

Nursing in rabbits occurs inside the nest with circadian periodicity. To determine the contribution of suckling stimulation in regulating such periodicity, we varied the size of the litters provided (1, 2, 4, or 6-8 pups). Nursing does, kept under a 14:10 (L:D) photoperiod, were continuously videotaped from parturition into lactation day 15. Although parturitions occurred throughout the day, a significant negative linear correlation (p < 0.0001; r = -0.68) was evident between time of delivery and time of nursing on lactation day 1, regardless of newborn number: longer intervals between these two events were seen in does delivering in the early morning than in those that gave birth late in the day. In rabbits suckling 6-8 pups, a Rayleigh analysis revealed that the population vector best describing their nursing pattern (across lactation days 1-15) had a phase angle = 58° (corresponding to solar time 0352 h and rho = 0.78; p < 0.001). In contrast, the nursing pattern of does nursing litters smaller than 6 pups did not show circadian periodicity; rather, mothers showed multiple entrances into the nest box throughout the day. Cluster analysis revealed that the main equilibrium point of intervals between suckling bouts shifted from 24 h (6-8 pups) to 6 h (4 and 2 pups) and to as low as 4 h with 1 pup. In the groups nursing 2, 4, or 6-8 pups, most nursing episodes were followed by food and water intake. Those mothers also showed self-grooming of the ventrum and nipples after nursing. The incidence of these behaviors was lower in does nursing 1 pup. In conclusion, nursing in rabbits spontaneously occurs with circadian periodicity, but it is largely modulated by a threshold of suckling stimulation.

Stability and mechanical evaluation of bovine pericardium cross-linked with polyurethane prepolymer in aqueous medium.

The present study investigates the potential use of non-catalyzed water-soluble blocked polyurethane prepolymer (PUP) as a bifunctional cross-linker for collagenous scaffolds. The effect of concentration (5, 10, 15 and 20%), time (4, 6, 12 and 24 h), medium volume (50, 100, 200 and 300%) and pH (7.4, 8.2, 9 and 10) over stability, microstructure and tensile mechanical behavior of acellular pericardial matrix was studied. The cross-linking index increased up to 81% while the denaturation temperature increased up to 12 °C after PUP crosslinking. PUP-treated scaffold resisted the collagenase degradation (0.167±0.14 mmol/g of liberated amine groups vs. 598±60 mmol/g for non-cross-linked matrix). The collagen fiber network was coated with PUP while viscoelastic properties were altered after cross-linking. The treatment of the pericardial scaffold with PUP allows (i) different densities of cross-linking depending of the process parameters and (ii) tensile properties similar to glutaraldehyde method.

Astrogliosis is temporally correlated with enhanced neurogenesis in adult rat hippocampus following a glucoprivic insult.

2-Deoxy-d-glucose (2-DG) administration causes transient depletion of glucose derivates and ATP. Hence, it can be used in a model system to study the effects of a mild glycoprivic brain insult mimicking transient hypoglycemia, which often occurs when insulin or oral hypoglycemic agents are administered for diabetes control. In the present study, the effect of a single 2-DG application (500mg/kg, a clinically applicable dose) on glial reactivity and neurogenesis in adult rat hippocampus was examined, as well as a possible temporal correlation between these two phenomena. Post-insult (PI) glial reactivity time course was assessed by immunoreaction against glial-fibrillary acidic protein (GFAP) during the following 5 consecutive days. A clear increase of GFAP immunoreactivity in hilus was observed from 48 to 96h PI. Moreover, enhanced labeling of long radial processes in the granule cell layer adjacent to hilus was evidenced. On the other hand, a transient increase of progenitor cell proliferation was detected in the subgranular zone, prominently at 48h PI, coinciding with the temporal peak of glial activation. This increase resulted in an augment of neuroblasts double labeled with 5-bromo-deoxyuridine (BrdU) and with double cortin (DCX) at day 7 PI. Around half of these cells survived 28 days showing matured neuronal phenotype double labeled by BrdU and a neuronal specific nuclear protein marker (NeuN). These findings suggest that a transient neuroglycoprivic state exerts a short-term effect on glial activation that possibly triggers a long-term effect on neurogenesis in hippocampus.

Dietary tryptophan restriction in rats triggers astrocyte cytoskeletal hypertrophy in hippocampus and amygdala.

We have previously reported that dietary tryptophan (TRP) restriction in a rat crucial postnatal developmental stage induces depression-like behavior and alters dendritic spine density in CA1 pyramidal neurons and granule cells of the hippocampus. Due to astrocyte involvement in critical brain mechanisms, it seems worth to investigate possible adaptive changes in the glial population with TRP restriction. Experimental rats were fed with low TRP diet (20% of TRP level of the laboratory rat chow) from postnatal days 30-60. Antibody against glial fibrillary acidic protein (GFAP), a principal intermediate filament in astrocytes, was used to evaluate cytoskeletal hypertrophy and glial proliferation. Our results showed an increase in size and branching of GFAP-immunoreactive (IR) cells in the dorsal hippocampus and amygdala, characteristics of an astrocytic activation. No significant differences were found regarding the number of GFAP-IR cells in both regions. These results indicate that dietary TRP restriction can induce astrocytic activation, hence, provide further evidences supporting the hypothesis that serotonin may also modulate glial morphology.

Maternal hyperthyroidism in rats impairs stress coping of adult offspring.

Given the evidence that maternal hyperthyroidism (MH) compromises expression of neuronal cytoskeletal proteins in the late fetal brain by accelerated neuronal differentiation, we investigated possible consequences of MH for the emotional and cognitive functions of adult offspring during acute and subchronic stress coping. Experimental groups consisted of male rat offspring from mothers implanted with osmotic minipumps infusing either thyroxine (MH) or vehicle (Ctrl) during pregnancy. Body weight and T4 level were monitored during the first 3 postnatal months, and no differences were found with the controls. We analyzed hippocampal CA3 pyramidal neurons and dentate granular cell morphology during several postnatal stages and found increased dendritic arborization. On postnatal day 90 a modified subchronic mild stress (SCMS) protocol was applied to experimental subjects for 10 days. The Morris water maze was used before, during, and after application of the SCMS protocol to measure spatial learning. The tail suspension test (TST) and forced-swimming test (FST) were used to evaluate behavioral despair. The MH rats displayed normal locomotor activity and spatial memory prior to SCMS, but impaired spatial learning after acute and chronic stress. In both the FST and TST we found that MH rats spent significantly more time immobile than did controls. Serum corticosterone level was found to increase after 30 min of restraint stress, and corticotropin-releasing factor immunoreactivity was found to be increased in the central nucleus of the amygdala. Our results suggest that MH in rats leads to the offspring being more vulnerable to stress in adulthood.

Rats subjected to extended L-tryptophan restriction during early postnatal stage exhibit anxious-depressive features and structural changes.

Serotonin transmission dysfunction has been suggested to play an important role in depression and anxiety. This study reports the results of a series of experiments in which rats were subjected to extended maize-based tortilla diets during early postnatal stages. This diet contains only approximately 20% of the L-tryptophan in normal diets of laboratory rodents. Compared with controls, experimental rats displayed a significant increase of immobility counts in the forced swimming test and exhibited anxiety-like behavior in the elevated plus maze test after 1 month of diet treatment. Low levels of serotonin contents were found in prefrontal cortex, striatum, hippocampus, and brainstem using high-performance liquid chromatography. Immunocytochemical reactions against 5-Bromo-2'-deoxyuridine revealed a significant decrease in the proliferation rate for the subgranular zone of dentate gyrus. c-Fos expression after the forced swimming test was found reduced in prefrontal cortex, dentate gyrus, CA1, and hilus of hippocampus and amygdala. Moreover, dendrite arbor atrophy and decreased spine density were evident in Golgi-Cox-impregnated CA1 pyramidal neurons. Abnormal dendrite swelling in dentate gyrus granule cells was also observed. These findings indicate an involvement of hyposerotoninergia in emotional disturbance produced by L-tryptophan restriction during critical developmental stages and suggest that neuroplasticity changes might underlie these changes.

Continuous monitoring of upper-limb activity in a free-living environment.

OBJECTIVE: To develop and evaluate a system for the objective measurement of upper-limb activity during a person's activities of daily living. DESIGN: Construct validation study. SETTING: Home and work environment. PARTICIPANTS: Ten able-bodied participants and 10 stroke patients. INTERVENTIONS: A novel activity monitor was developed for monitoring upper-limb activity. The Strathclyde Upper-Limb Activity Monitor (SULAM) gave a signal proportional to the vertical displacement of the wrist with respect to the shoulder. Participants wore the SULAM on both upper limbs for 8 hours while performing their normal daily activities. MAIN OUTCOME MEASURES: SULAM-derived variables quantified the total upper-limb activity and the range in which movement of the upper limb occurred. RESULTS: Data showed that the dominant arm of the able-bodied participants was 19% more active than the nondominant arm, whereas the unaffected arms of the group of stroke patients was used 3 to 6 times more than their affected arm. For all the variables, in both groups, there were significant differences between their upper limbs except for movement below midtrunk. There were also significant differences between groups for bimanual movement time ( P =.000) and composite movement time ( P =.000). CONCLUSIONS: The results suggest that the quantification of vertical position of the wrist with respect to the shoulder might reflect upper-limb activity. By using the SULAM, it is possible to monitor upper-limb activity over the course of a person's normal day, with minimal interference. This study provides preliminary evidence of the SULAM as a useful tool for objectively evaluating interventions aimed at improving upper-limb activity and function.