Dolores Ludeña

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.

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.

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.

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.

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.

402 Effect of Antenatal Corticosteroids in Activity and Expression of Secretory Phospholipase A2 and TNF Alfa in Lung of Newborn Rats

Introduction The sPLA2 plays an important role in the development of acute respiratory distress syndrome. It is regulated by many factors including steroids and TNFa. Antenatal corticosteroids are recommended for preventing respiratory distress syndrome in preterm infants. Recent studies suggest that betamethasone might be a better choice than dexamethasone. The aim of this study is evaluate differences between both antenatal corticosteroids in the regulation of sPLA2 and TNFa. Methods Dexamethasone, betamethasone or saline were administered intravenously to pregnant Wistar rats on the 20th and 21st days of gestation. We evaluated pulmonary sPLA2 and TNFa mRNA in newborn rats at birth by RT-PCR. We also evaluated sPLA2 activity by an ultrasensitive non-radioactive method on microplate and the TNFa protein expression by ELISA. Differences between the groups were determined by one way ANOVA (p<0.05). Results We observed a statistically significant decrease in the sPLA2 mRNA in the betamethasone (0.61) and dexamethasone (0.26) groups respect the control (1.05) group and a decrease in the sPLA2 activity in the betamethasone group (33.78) respect the control group (50.74). We observed a statistically significant decrease in the TNFa protein in the betamethasone group (472.61) respect the dexamethasone group (768.65). Conclusions Antenatal glucocorticoids inhibits the expression of sPLA2 through the reduction of TNFa in the lung of newborn rats. These potential beneficial effects are more evident in the group treated with antenatal betamethasone. Our studies also support the notion that betamethasone could be the drug of choice for treating pregnant women at risk of preterm delivery.

Effects of Oxidative Stress and Antenatal Corticosteroids on the Pulmonary Expression of Vascular Endothelial Growth Factor (VEGF) and Alveolarization

The lung is the human organ most susceptible to oxidative damage. The transition from fetal, a low-oxygen environment, to FiO2: 0.21, induces a relative degree of oxidative stress for all newborns, but especially in the case of preterm neonates whose lung development has been interrupted. A supplementary supply of oxygen (hyperoxia) is often used to treat preterm newborns and these infants may not yet be prepared to protect their lungs from oxidative injury.