Biodegradable poly (lactic acid-co-glycolic acid) scaffolds as carriers for geneticallymodified fibroblasts

Recent advances in gene delivery into cells allow improved therapeutic effects in gene therapy trials. To increase the bioavailability of applied cells, it is of great interest that transfected cells remain at the application site and systemic spread is minimized. In this study, we tested clinically used biodegradable poly(lactic acid-co-glycolic acid) (PLGA) scaffolds (Vicryl & Ethisorb) as transient carriers for genetically modified cells. To this aim, we used human fibroblasts and examined attachment and proliferation of untransfected cells on the scaffolds in vitro, as well as the mechanical properties of the scaffolds at four time points (1, 3, 6 and 9 days) of cultivation. Furthermore, the adherence of cells transfected with green fluorescent protein (GFP) and vascular endothelial growth factor (VEGF165) and also VEGF165 protein secretion were investigated. Our results show that human fibroblasts adhere on both types of PLGA scaffolds. However, proliferation and transgene expression capacity were higher on Ethisorb scaffolds most probably due to a different architecture of the scaffold. Additionally, cultivation of the cells on the scaffolds did not alter their biomechanical properties. The results of this investigation could be potentially exploited in therapeutic regiments with areal delivery of transiently transfected cells and may open the way for a variety of applications of cell-based gene therapy, tissue engineering and regenerative medicine

Fetus Sound Stimulation: Cilia Memristor Effect of Signal Transduction

Background. This experimental study evaluates fetal middle cerebral artery (MCA) circulation after the defined prenatal acoustical stimulation (PAS) and the role of cilia in hearing and memory and could explain signal transduction and memory according to cilia optical-acoustical properties. Methods. PAS was performed twice on 119 no-risk term pregnancies. We analyzed fetal MCA circulation before, after first and second PAS. Results. Analysis of the Pulsatility index basic (PIB) and before PAS and Pulsatility index reactive after the first PAS (PIR 1) shows high statistical difference, representing high influence on the brain circulation. Analysis of PIB and Pulsatility index reactive after the second PAS (PIR 2) shows no statistical difference. Cilia as nanoscale structure possess magnetic flux linkage that depends on the amount of charge that has passed between two-terminal variable resistors of cilia. Microtubule resistance, as a function of the current through and voltage across the structure, leads to appearance of cilia memory with the “memristor” property. Conclusion. Acoustical and optical cilia properties play crucial role in hearing and memory processes. We suggest that fetuses are getting used to sound, developing a kind of memory patterns, considering acoustical and electromagnetically waves and involving cilia and microtubules and try to explain signal transduction

Lymphocyte glucocorticoid receptor expression level and hormone-binding properties differ between war trauma-exposed men with and without PTSD

<p>Objective: Posttraumatic stress disorder (PTSD) has been shown to be associated with altered glucocorticoid receptor (GR) activity. We studied the expression and functional properties of the receptor in peripheral blood mononuclear cells (PBMCs) from non-traumatized healthy individuals (healthy controls; n = 85), and war trauma-exposed individuals with current PTSD (n = 113), with life-time PTSD (n = 61) and without PTSD (trauma controls; n = 88). The aim of the study was to distinguish the receptor alterations related to PTSD from those related to trauma itself or to resilience to PTSD.</p><p>Methods: Functional status of the receptor was assessed by radioligand binding and lysozyme synthesis inhibition assays. The level of GR gene expression was measured by quantitative PCR and immunoblotting.</p><p>Results: Current PTSD patients had the lowest, while trauma controls had the highest number of glucocorticoid binding sites (B-max) in PBMCs. Hormone-binding potential (B-max/K-D ratio) of the receptor was diminished in the current PTSD group in comparison to all other study groups. Correlation between B-max and K-D that normally exists in healthy individuals was decreased in the current PTSD group. Contrasting B-max data, GR protein level was lower in trauma controls than in participants with current or life-time PTSD.</p><p>Conclusions: Current PTSD is characterized by reduced lymphocyte GR hormone-binding potential and by disturbed compensation between B-max and hormone-binding affinity. Resilience to PTSD is associated with enlarged fraction of the receptor molecules capable of hormone binding, within the total receptor molecule population in PBMCs. (C) 2013 Elsevier Inc. All rights reserved.</p>

Mineralocorticoid receptor and heat shock protein expression levels in peripheral lymphocytes from war trauma-exposed men with and without PTSD

Alterations in the number and functional status of mineralocorticoid (MR) and glucocorticoid receptors (GR) may contribute to vulnerability to posttraumatic stress disorder (PTSD). Corticosteroid receptors are chaperoned by heat shock proteins Hsp90 and Hsp70. We examined relations between corticosteroid receptor and heat shock protein expression levels, and related them with war trauma exposure, PTSD and resilience to PTSD. Relative levels of MR. Hsp90 and Hsp70 were determined by immunoblotting in lymphocytes from war trauma-exposed men with current PTSD (current PTSD group, n=113), with lifetime PTSD (life-time PTSD group, n=61) and without PTSD (trauma control group, n=88), and from non-traumatized healthy controls (healthy control group, n=85). Between-group differences in MR, Hsp90 and Hsp70 levels and in MR/GR ratio were not observed. The level of MR was correlated with both Hsp90 and Hsp70 levels in trauma control and healthy control groups. On the other hand, GR level was correlated only with Hsp90 level, and this correlation was evident in current PTSD and trauma control groups. In conclusion, PTSD and exposure to trauma are not related to changes in lymphocyte MR, Hsp90 or Hsp70 levels, but may be associated with disturbances in corticosteroid receptors interaction with heat shock proteins. (C) 2013 Elsevier Ireland Ltd. All rights reserved.European Commision {[}INCO-CT-2004-509213]; Ministry of Education, Science and Technological Development of the Republic of Serbia {[}41009, 179018

Determination of mechanical properties for Vicryl and Ethisorb scaffolds with and without seeded cells.

<p>The mechanical properties of Vicryl (A) and Ethisorb (B) scaffolds were measured by using a uniaxial test system. Meshes were clamped along their long side with an initial length L₀ of 20 mm. Maximum force values for the two types of scaffolds (both with and without Hs27 cells) were measured in a single experiment at day (d) 1, 3, 6 and 9 and presented graphically (C). All error bars attached to the mean values represent the SEM of five technical replicates (n = 5; two-way ANOVA comparing maximum force values of Vicryl and Ethisorb scaffolds for each time point: ****p<0.00005).</p

Visualisation of Hs27 cell growth on Vicryl and Ethisorb scaffolds.

<p>During the co-cultivation period of 9 days, Hs27 fibroblast cells on Vicryl (A) and Ethisorb (B) scaffolds were investigated at four different time points by means of light microscopy with 100-fold magnification. In the upper row of each panel, the scaffolds containing no cells (control) are presented. The fibroblasts were visualized with Giemsa staining and can be seen in the images of the bottom row of each panel as small blue dots.</p

Analysis of scaffold fibers and Hs27 cell morphology by scanning electron microscopy.

<p>Hs27 fibroblasts are grown on Vicryl and Ethisorb scaffolds and visualized at day 1, day 3 day6 and day 9 in culture by means of scanning electron microscopy (Scale bars in the upon two rows in A and B are 100μm, in the lowest row are 25μm)</p

Visualisation of GFP-expressing Hs27 cells adhered on Vicryl and Ethisorb scaffolds.

<p>5 x 10⁶ Hs27 cells were electroporated with 20 μg GFP plasmid by using Nucleofector transfection apparatus. After a short period in culture, transfected cells were transferred on scaffolds. The expression of GPF protein in Hs27 fibroblast grown on Vicryl (A) and Ethisorb (B) scaffolds was recorded at day 1, 3, 6 and 9 by a fluorescent microscope with 100-fold magnification. In the upper row of each panel, the scaffolds containing no cells are depicted.</p