Striatin knock out induces a gain of function of INa and impaired Ca2+ handling in mESC-derived cardiomyocytes.

AIM: Striatin (Strn) is a scaffold protein expressed in cardiomyocytes (CMs) and alteration of its expression are described in various cardiac diseases. However, the alteration underlying its pathogenicity have been poorly investigated. METHODS: We studied the role(s) of cardiac Strn gene (STRN) by comparing the functional properties of CMs, generated from Strn-KO and isogenic WT mouse embryonic stem cell lines. RESULTS: The spontaneous beating rate of Strn-KO CMs was faster than WT cells, and this correlated with a larger fast INa conductance and no changes in If. Paced (2-8 Hz) Strn-KO CMs showed prolonged action potential (AP) duration in comparison with WT CMs and this was not associated with changes in ICaL and IKr. Motion video tracking analysis highlighted an altered contraction in Strn-KO CMs; this was associated with a global increase in intracellular Ca2+, caused by an enhanced late Na+ current density (INaL) and a reduced Na+/Ca2+ exchanger (NCX) activity and expression. Immunofluorescence analysis confirmed the higher Na+ channel expression and a more dynamic microtubule network in Strn-KO CMs than in WT. Indeed, incubation of Strn-KO CMs with the microtubule stabilizer taxol, induced a rescue (downregulation) of INa conductance toward WT levels. CONCLUSION: Loss of STRN alters CMs electrical and contractile profiles and affects cell functionality by a disarrangement of Strn-related multi-protein complexes. This leads to impaired microtubules dynamics and Na+ channels trafficking to the plasma membrane, causing a global Na+ and Ca2+ enhancement.

Cardiovascular magnetic resonance-derived left ventricular mechanics-strain, cardiac power and end-systolic elastance under various inotropic states in swine.

BACKGROUND: Cardiovascular magnetic resonance (CMR) strain imaging is an established technique to quantify myocardial deformation. However, to what extent left ventricular (LV) systolic strain, and therefore LV mechanics, reflects classical hemodynamic parameters under various inotropic states is still not completely clear. Therefore, the aim of this study was to investigate the correlation of LV global strain parameters measured via CMR feature tracking (CMR-FT, based on conventional cine balanced steady state free precession (bSSFP) images) with hemodynamic parameters such as cardiac index (CI), cardiac power output (CPO) and end-systolic elastance (Ees) under various inotropic states. METHODS: Ten anaesthetized, healthy Landrace swine were acutely instrumented closed-chest and transported to the CMR facility for measurements. After baseline measurements, two steps were performed: (1) dobutamine-stress (Dobutamine) and (2) verapamil-induced cardiovascular depression (Verapamil). During each protocol, CMR images were acquired in the short axisand apical 2Ch, 3Ch and 4Ch views. MEDIS software was utilized to analyze global longitudinal (GLS), global circumferential (GCS), and global radial strain (GRS). RESULTS: Dobutamine significantly increased heart rate, CI, CPO and Ees, while Verapamil decreased them. Absolute values of GLS, GCS and GRS accordingly increased during Dobutamine infusion, while GLS and GCS decreased during Verapamil. Linear regression analysis showed a moderate correlation between GLS, GCS and LV hemodynamic parameters, while GRS correlated poorly. Indexing global strain parameters for indirect measures of afterload, such as mean aortic pressure or wall stress, significantly improved these correlations, with GLS indexed for wall stress reflecting LV contractility as the clinically widespread LV ejection fraction. CONCLUSION: GLS and GCS correlate accordingly with LV hemodynamics under various inotropic states in swine. Indexing strain parameters for indirect measures of afterload substantially improves this correlation, with GLS being as good as LV ejection fraction in reflecting LV contractility. CMR-FT-strain imaging may be a quick and promising tool to characterize LV hemodynamics in patients with varying degrees of LV dysfunction.

Biomaterials and biophysical stimuli for bone regeneration.

First, we review basic concepts of Tissue Engineering, that is, how the tensegrity is able to modulate the cell behavior. Then, we review our experimental results regarding the bone tissue engineering via biomaterials and bioreactors.

Wool fibril sponges with perspective biomedical applications.

Sheep's wool was used as a natural source to prepare keratin microfibril sponges for scaffolding, by disruption of the histological structure of the fibres through mild alkali treatment, followed by ultrasonication, casting and salt-leaching. The wool sponges showed highly interconnected porosity (93%) and contain intrinsic sites of cellular recognition that mimic the extracellular matrix (ECM). They displayed good thermal and water stability due to the conversion of disulphide cystine bonds into shorter monosulphide lanthionine intermolecular bonds, but significantly swelled in water, because of the high hydrophilicity and porosity, with a volume increasing up to 38%. Nevertheless, sponges were stable in water without structural changes, with a neutral pH in aqueous media, and showed excellent resilience to repeated compression stresses. According to in vitro biocompatibility assays, wool fibril sponges showed a good cell adhesion and proliferation as proved by MTT, FDA assays and SEM observations. The unique structure of the cortical cell network made by wool keratin proteins with controlled-size macro-porosity suitable for cell guesting, and nutrient feeding, provides an excellent scaffold for future tissue engineering applications.

Electromagnetic stimulation to optimize the bone regeneration capacity of gelatin-based cryogels.

One of the key challenges in reconstructive bone surgery is to provide living constructs that possess the ability to integrate in the surrounding host tissue. Bone graft substitutes and biomaterials have already been widely used to heal critical-size bone defects due to trauma, tumor resection and tissue degeneration. In the present study, gelatin-based cryogels have been seeded with human SAOS-2 osteoblasts followed by the in vitro culture of the cells. In order to overcome the drawbacks associated with static culture systems, including limited diffusion and in homogeneous cell-matrix distribution, the present work describes the application of a bioreactor to physically enhance the cell culture in vitro using an electromagnetic stimulus. The results indicate that the physical stimulation of cell-seeded gelatin-based cryogels upregulates the bone matrix production. We anticipate that the scaffolds developed consisting of human bone proteins and cells could be applied for clinical purposes related to bone repair.

AUTOCOUNTER, an ImageJ JavaScript to analyze LC3B-GFP expression dynamics in autophagy-induced astrocytoma cells.

An ImageJ JavaScript, AUTOCOUNTER, was specifically developed to monitor and measure LC3B-GFP expression in living human astrocytoma cells, namely T98G and U373-MG. Discrete intracellular GFP fluorescent spots derived from transduction of a Baculovirus replication-defective vector (BacMam LC3B-GFP), followed by microscope examinations at different times. After viral transgene expression, autophagy was induced by Rapamycin administration and assayed in ph-p70S6K/p70S6K and LC3B immunoblotting expression as well as by electron microscopy examinations. A mutated transgene, defective in LC3B lipidation, was employed as a negative control to further exclude fluorescent dots derived from protein intracellular aggregation. The ImageJ JavaScript was then employed to evaluate and score the dynamics changes of the number and area of LC3B-GFP puncta per cell in time course assays and in complex microscope examinations. In conclusion, AUTOCOUNTER enabled to quantify LC3B-GFP expression and to monitor dynamics changes in number and shapes of autophagosomal-like vesicles: it might therefore represent a suitable algorithmic tool for in vitro autophagy modulation studies.

Alzheimer's disease, autoimmunity and inflammation. The good, the bad and the ugly.

Alzheimer's disease (AD) has been recognized as the most common cause of sporadic dementia. It represents both a medical and social problem, as it affects 10% of over-65 population. Even if the elderly are the most involved population, aging alone cannot be considered as the only cause of this disease. In this review we wanted to focus on the last hypotheses on the possible causes of this neuronal affection. We focused in particular on the role of inflammation and alteration of the inflammatory status that is typical of the elderly and may lead to chronic inflammation. The inflammation seems to be a cause of neuronal impairment and loss. Some studies have proposed a protective role of antiinflammatory drugs. Then we analyzed the role of genetic polymorphisms of some pro-inflammatory substances that seem to be linked to some cases of dementia. The complement system seems to have a role too, as some factors have been found in senile plaques, representing a possible involvement of classical complement pathway. One of the latest hypotheses is about the role of blood-brain barrier (BBB), as its loss of integrity may lead to a passage of proteins in cerebro spinal fluid (CSF), causing a compromised role of BBB in preserving the brain as an "immune sanctuary".

Effects of electromagnetic stimulation on osteogenic differentiation of human mesenchymal stromal cells seeded onto gelatin cryogel.

Bone tissue engineering typically uses biomaterial scaffolds, osteoblasts or cells that can become osteoblasts, and biophysical stimulations to promote cell attachment and differentiation. In this study, we investigated the effects of an electromagnetic wave on mesenchymal stromal cells isolated from the bone marrow and seeded upon gelatin cryogel disks. In comparison with control conditions without electromagnetic stimulus, the electromagnetic treatment (magnetic field, 2 mT; frequency, 75 Hz) increased the cell proliferation and differentiation and enhanced the biomaterial surface coating with bone extracellular matrix proteins. Using this tissue-engineering approach, the gelatin biomaterial, coated with differentiated cells and their extracellular matrix proteins, may be used in clinical applications as an implant for bone defect repair.

Surface modification of a porous polyurethane through a culture of human osteoblasts and an electromagnetic bioreactor.

There is increasing interest in new biomaterials and new culture methods for bone tissue engineering, in order to produce, in vitro, living constructs able to integrate in the surrounding tissue. Using an electromagnetic bioreactor (magnetic field intensity, 2 mT; frequency, 75 Hz), we investigated the effects of electromagnetic stimulation on SAOS-2 human osteoblasts seeded onto a porous polyurethane. In comparison with control conditions, the electromagnetic stimulation caused higher cell proliferation, increased surface coating with decorin and type-I collagen, and higher calcium deposition. The immunolocalization of decorin and type-I collagen showed their colocalization in the cell-rich areas. The use of an electromagnetic bioreactor aimed at obtaining the surface modification of the porous polyurethane in terms of cell colonization and coating with calcified matrix. The superficially modified biomaterial could be used, in clinical applications, as an implant for bone repair.

Stimulation of osteoblast growth by an electromagnetic field in a model of bone-like construct.

The histogenesis of bone tissue is strongly influenced by physical forces, including magnetic fields. Recent advances in tissue engineering has permitted the generation of three dimensional bone-like constructs. We have investigated the effects of electromagnetic stimulation on human osteoblast cells grown in a hydrophobic polyurethane scaffold. Bone-like constructs were stimulated by pulsed electromagnetic fields in a bioreactor. Proliferation, bone protein expression and calcified matrix production by osteoblasts were measured using histochemical methods. In stimulated cultures, the number of cells was significantly higher compared to static (control) cultures. In both stimulated and control cultures, cells were immunoreactive to osteoblast markers, including type-I collagen, osteocalcin and osteopontin, thus suggesting that the expression of bone-related markers was maintained throughout the in vitro experiments. Morphometric analysis of von Kossa-stained sections revealed that stimulation with electromagnetic field significantly increased matrix calcification. The data lend support to the view that the application of a magnetic field can be used to stimulate cell growth in bone-like constructs in vitro. This finding may be of interest for the production of biomaterials designed for clinical applications.

Calcified matrix production by SAOS-2 cells inside a polyurethane porous scaffold, using a perfusion bioreactor.

The repair and regeneration of damaged or resected bone are problematic. Bone autografts show optimal skeletal incorporation, but often bring about complications. Hence, there is increasing interest in designing new biomaterials that could potentially be used in the form of scaffolds as bone substitutes. In this study we used a hydrophobic cross-linked polyurethane in a typical tissue-engineering approach, that is, the seeding and in vitro culturing of cells within a porous scaffold. The polyurethane porous scaffold had an average pore diameter of 624 microm. Using a perfusion bioreactor, we investigated the effect of shear stress on SAOS-2 human osteoblast proliferation and calcified matrix production. The physical, morphological, and compressive properties of the polyurethane foam were characterized. At a scaffold perfusion rate of 3 mL/min, in comparison with static conditions without perfusion, we observed 33% higher cell proliferation; higher secretion of osteopontin, osteocalcin, decorin, and type I collagen (9.16-fold, 71.9-fold, 30.6-fold, and 18.12-fold, respectively); and 10-fold increased calcium deposition. The design of the bioreactor and the design of the polyurethane foam aimed at obtaining cell colonization and calcified matrix deposition. This cultured biomaterial could be used, in clinical applications, as an osteoinductive implant for bone repair.