Cell culture in autologous fibrin scaffolds for applications in tissue engineering.

In tissue engineering techniques, three-dimensional scaffolds are needed to adjust and guide cell growth and to allow tissue regeneration. The scaffold must be biocompatible, biodegradable and must benefit the interactions between cells and biomaterial. Some natural biomaterials such as fibrin provide a structure similar to the native extracellular matrix containing the cells. Fibrin was first used as a sealant based on pools of commercial fibrinogen. However, the high risk of viral transmission of these pools led to the development of techniques of viral inactivation and elimination and the use of autologous fibrins. In recent decades, fibrin has been used as a release system and three-dimensional scaffold for cell culture. Fibrin scaffolds have been widely used for the culture of different types of cells, and have found several applications in tissue engineering. The structure and development of scaffolds is a key point for cell culture because scaffolds of autologous fibrin offer an important alternative due to their low fibrinogen concentrations, which are more suitable for cell growth. With this review our aim is to follow methods of development, analyze the commercial and autologous fibrins available and assess the possible applications of cell culture in tissue engineering in these three-dimensional structures.

Differentiation within autologous fibrin scaffolds of porcine dermal cells with the mesenchymal stem cell phenotype.

Porcine mesenchymal stem cells (pMSCs) are an attractive source of cells for tissue engineering because their properties are similar to those of human stem cells. pMSCs can be found in different tissues but their dermal origin has not been studied in depth. Additionally, MSCs differentiation in monolayer cultures requires subcultured cells, and these cells are at risk of dedifferentiation when implanting them into living tissue. Following this, we attempted to characterize the MSCs phenotype of porcine dermal cells and to evaluate their cellular proliferation and differentiation in autologous fibrin scaffolds (AFSs). Dermal biopsies and blood samples were obtained from 12 pigs. Dermal cells were characterized by flow cytometry. Frozen autologous plasma was used to prepare AFSs. pMSC differentiation was studied in standard structures (monolayers and pellets) and in AFSs. The pMSCs expressed the CD90 and CD29 markers of the mesenchymal lineage. AFSs afforded adipogenic, osteogenic and chondrogenic differentiation. The porcine dermis can be proposed to be a good source of MSCs with adequate proliferative capacity and a suitable expression of markers. The pMSCs also showed optimal proliferation and differentiation in AFSs, such that these might serve as a promising autologous and implantable material for use in tissue engineering.

Lymphoma outbreak in a GASH:Sal hamster colony.

We have detected a high incidence of lymphomas in a colony of GASH:Sal Syrian golden hamsters (Mesocricetus auratus). This strain is characterised by its ability to present convulsive crises of audiogenic origin. Almost 16 % (90 males and 60 females) of the 975 animals were affected during a 5-year period by the development of a progressing lymphoid tumour and exhibited similar clinical profiles characterised by lethargy, anorexia, evident abdominal distension, and a rapid disease progression resulting in mortality within 1 to 2 weeks. A TaqMan® probe-based real-time PCR analysis of genomic DNA from different tissue samples of the affected animals revealed the presence of a DNA sequence encoding the hamster polyomavirus (HaPyV) VP1 capsid protein. Additionally, immunohistochemical analysis using HaPyV-VP1-specific monoclonal antibodies confirmed the presence of viral proteins in all hamster tumour tissues analysed within the colony. An indirect ELISA and western blot analysis confirmed the presence of antibodies against the VP1 capsid protein in sera, not only from affected and non-affected GASH:Sal hamsters but also from control hamsters from the same breeding area. The HaPyV genome that accumulated in tumour tissues typically contained deletions affecting the noncoding regulatory region and adjacent sequences coding for the N-terminal part of the capsid protein VP2.

Clinical, genetic and pharmacological data support targeting the MEK5/ERK5 module in lung cancer.

Despite advances in its treatment, lung cancer still represents the most common and lethal tumor. Because of that, efforts to decipher the pathophysiological actors that may promote lung tumor generation/progression are being made, with the final aim of establishing new therapeutic options. Using a transgenic mouse model, we formerly demonstrated that the sole activation of the MEK5/ERK5 MAPK route had a pathophysiological role in the onset of lung adenocarcinomas. Given the prevalence of that disease and its frequent dismal prognosis, our findings opened the possibility of targeting the MEK5/ERK5 route with therapeutic purposes. Here we have explored such possibility. We found that increased levels of MEK5/ERK5 correlated with poor patient prognosis in lung cancer. Moreover, using genetic as well as pharmacological tools, we show that targeting the MEK5/ERK5 route is therapeutically effective in lung cancer. Not only genetic disruption of ERK5 by CRISPR/Cas9 caused a relevant inhibition of tumor growth in vitro and in vivo; such ERK5 deficit augmented the antitumoral effect of agents normally used in the lung cancer clinic. The clinical correlation studies together with the pharmacological and genetic results establish the basis for considering the targeting of the MEK5/ERK5 route in the therapy for lung cancer.

Factors influencing malignant evolution and long-term survival in solitary fibrous tumours of the pleura.

Solitary pleuro-pulmonary fibrous tumours are relatively uncommon neoplasms that are difficult to manage therapeutically and which, cytogenetically, have been poorly studied. The aim of the present work was to analyse the characteristics of a series of consecutive operated solitary pleural fibrous tumours in an attempt to discover a malignant pattern of evolution. This was a retrospective observational study of 19 cases. Samples were studied for clinical, histological, immunohistochemical and cytogenetic characteristics (aCGH, FISH). Descriptive statistics were used: the Kapplan-Meyer log-rank test and the Cox-regression model for survival analysis. Analysis of malignant evolution was achieved using 2x2 tables; significant factors were included in a binary logistic regression model. Parietal pleural implantation of the primary tumour, high mib1 expression, and low p53 expression were seen to be statistically significant factors for survival. We recommend a close follow-up for patients with a malignant primary tumour and low p53 expression and a regular long-term follow-up for benign primary tumours with a high mib1 index, high positive p53, and deletions. These findings need confirmation in more extensive series.

Dexamethasone and betamethasone for prenatal lung maturation: differences in vascular endothelial growth factor expression and alveolarization in rats.

BACKGROUND: Fetal and postnatal lung development is regulated by glucocorticoids. The use of antenatal corticosteroids is reported to produce effects on vascular endothelial growth factor (VEGF), which plays a crucial role in pulmonary development. OBJECTIVES: The purpose of this study was to compare pulmonary VEGF expression in newborn rats that were exposed to antenatal betamethasone versus dexamethasone and to evaluate its impact on the alveolarization period of rats (0-14 days of life). METHODS: Betamethasone, dexamethasone or equivalent saline solution (control group) was administered to pregnant rats on 20th and 21st days of gestation. Pulmonary VEGF mRNA, VEGF protein expression, and alveolarization changes were evaluated at birth and at 14 days of life. RESULTS: Betamethasone and dexamethasone were observed to have different actions on VEGF expression with a correlation with alveolarization on both days of study. Antenatal dexamethasone decreased VEGF expression, betamethasone tended to produce the induction of the expression of VEGF, and moreover, betamethasone did not produce a decrease in alveolarization as seen in the animals that received dexamethasone. CONCLUSIONS: Our results support the notion that betamethasone could be a better choice than dexamethasone for antenatal lung maturation.

Autologous fibrin scaffolds cultured dermal fibroblasts and enriched with encapsulated bFGF for tissue engineering.

Autologous fibrin scaffolds (AFSs) enriched with cells and specific growth factors represent a promising biocompatible scaffold for tissue engineering. Here, we analyzed the in vitro behavior of dermal fibroblasts (DFs) (cellular attachment, distribution, viability and proliferation, histological and immunohistochemical changes), comparing AFS with and without alginate microcapsules loaded with basic fibroblast growth factor (bFGF), to validate our scaffold in a future animal model in vivo. In all cases, DFs showed good adhesion and normal distribution, while in scaffolds with bFGF at 14 days, the cell counts detected in proliferation and viability assays were greatly improved, as was the proliferative state, and there was a decrease in muscle specific actin expression and collagen synthesis in comparison with the scaffolds without bFGF. In addition, the use of plasma without fibrinogen concentration methods, together with the maximum controlled release of bFGF at 14 days, favored cell proliferation. To conclude, we have been able to create an AFS enriched with fully functional DFs and release-controlled bFGF that could be used in multiple applications for tissue engineering.

Effects of antenatal betamethasone and dexamethasone on the lung expression of vascular endothelial growth factor and alveolarization in newborn rats exposed to acute hypoxia and recovered in normoxia or hyperoxia.

The use of antenatal corticosteroids is widespread and it is important to know their effect(s) on vascular endothelial growth factor (VEGF), which plays a crucial role in pulmonary development. The purpose of this study was to compare pulmonary VEGF expression in newborn rats that were exposed to antenatal betamethasone versus dexamethasone under normoxia, hypoxia and oxidative stress, and to evaluate its impact on alveolarization. Betamethasone, dexamethasone or equivalent saline solution (control group) was administered to pregnant rats. Newborn rats were randomized to room air, hypoxia followed by hyperoxia, or hypoxia followed by air. Pulmonary VEGF protein, VEGF mRNA, and alveolarization were evaluated at 4 days of life. Betamethasone and dexamethasone were observed to have different actions on VEGF expression with a correlation with alveolarization. Antenatal dexamethasone decreased VEGF expression, and dexamethasone and hyperoxia had an additive effect on the inhibition of VEGF with a reduction in alveolar development. Betamethasone appeared to have an effect on the induction of the expression of VEGF, and it seemed to inhibit the negative action of hyperoxia on VEGF. Moreover, betamethasone did not produce a decrease in alveolarization. Our results support the notion that betamethasone could be better than dexamethasone for antenatal lung maturation.

Pulmonary expression of vascular endothelial growth factor (VEGF) and alveolar septation in a newborn rat model exposed to acute hypoxia and recovered under conditions of air or hyperoxia.

Vascular endothelial growth factor (VEGF) is an endothelial cell growth factor expressed in normal lung tissue. The aim of the study was to investigate the expression of VEGF and its repercussions as regards alveolarization in the developing rat lung. We studied pulmonary VEGF expression at 0 and 14 days of life in Wistar rats. Rat pups were exposed to hypoxia for two hours during the first hours of life and recovered under conditions of hyperoxia or normoxia for a further two hours, or not recovered. The animals of the control group were only exposed to conditions of normoxia. Our results showed that VEGF was increased in the lungs of the animals that were exposed to hypoxia but we did not find any correlation with the septation. The VEGF was decreased in the lungs of animals exposed to hyperoxia after neonatal hypoxia. We observed this at 0 and 14 days of life, and it was correlated with a lower degree of alveolarization at 14 days of life. Our data suggest that hyperoxia after neonatal hypoxia at birth may give rise to a decrease in the expression of VEGF, possibly permanently, together with a reduction in alveolar development.

Nodulation of Lupinus albus by strains of Ochrobactrum lupini sp. nov.

The nodulation of legumes has for more than a century been considered an exclusive capacity of a group of microorganisms commonly known as rhizobia and belonging to the alpha-Proteobacteria. However, in the last 3 years four nonrhizobial species, belonging to alpha and beta subclasses of the Proteobacteria, have been described as legume-nodulating bacteria. In the present study, two fast-growing strains, LUP21 and LUP23, were isolated from nodules of Lupinus honoratus. The phylogenetic analysis based on the 16S and 23S rRNA gene sequences showed that the isolates belong to the genus Ochrobactrum. The strains were able to reinfect Lupinus plants. A plasmid profile analysis showed the presence of three plasmids. The nodD and nifH genes were located on these plasmids, and their sequences were obtained. These sequences showed a close resemblance to the nodD and nifH genes of rhizobial species, suggesting that the nodD and nifH genes carried by strain LUP21T were acquired by horizontal gene transfer. A polyphasic study including phenotypic, chemotaxonomic, and molecular features of the strains isolated in this study showed that they belong to a new species of the genus Ochrobactrum for which we propose the name Ochrobactrum lupini sp. nov. Strain LUP21T (LMG 20667T) is the type strain.