[Treatment by stem cell therapy of erectile dysfunction of diabetic origin: State of the art]. / Traitement par les cellules souches de la dysfonction érectile d'origine diabétique : état des lieux.

PURPOSE: Review of various publications on stem cell therapy to treat erectile dysfunction of diabetic origin. MATERIAL AND METHODS: Bibliographic search in PUBMED performed using the keywords cell therapy strain/erectile dysfunction associated with diabetes. Among the 51 articles obtained from the PUBMED research, we selected 16 articles for their specificity of studying erectile dysfunction (DE) related to diabetes. RESULTS: Different types of stem cells have been studied: adipose derived mesenchymal stem cells/bone marrow derived mesenchymal stem cells as well as progenitor endothelial cells. The experimental protocols are quite similar from one study to the next with nevertheless some specifications concerning the studied cells and the monitoring of the latter. Intracavernous pressure (ICP) measured after the injection of stem cells into the corpus cavernosum was always significantly higher than the control populations. The addition of certain growth factors to stem cells by gene transfection improve the efficacy of the cells. No ideal tracking markers of the cells have been identified. CONCLUSION: The positive effect of the injection of stem cells on the ICP belongs to the cellular trans-differentiation effect but especially to the paracrine effects which have not yet been completely elucidated.

In vitro inhibition of IL-1beta catabolic effects on cartilage: a mechanism involved on diacerein anti-OA properties.

Osteoarthritis (OA) is a progressive joint disease which represents a combination of several disorders leading to cartilage degradation. The main characteristic of OA is an imbalance between chondrocyte anabolic and catabolic activities. Cytokines produced by the synovium and chondrocytes, especially interleukin 1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha), play a significant role in the degradation of cartilage. They stimulate the production of nitric oxide (NO), which is involved in cartilage catabolism and also may induce the apoptosis of chondrocytes. The IL-1beta produced in activated chondrocytes or synovium may modulate disease progression in OA and should therefore be considered a potential target for therapeutic interventions. Drug and non-drug treatments are used to relieve pain and/or swelling in OA. Diacerein is a slow-acting drug that may slow down the breakdown of cartilage and relieve pain and swelling. It is not clear whether diacerein works but it has been proposed that diacerein acts as a symptom-modifying and perhaps disease-structure modifying drug.

Interest of second harmonic generation imaging for diagnosis in thick and opaque tissue.

In articular hyaline cartilage, chondrocytes are surrounded by an extracellular matrix which is mainly composed by collagen and proteoglycanes. Pathological specimens show a partial or complete degradation of this matrix. Therefore, it could be interesting to know how mechanical or biochemical constraints applied to cartilage specimens induce modifications of the cartilage network. Multiphoton technology combined to Second Harmonic Generation (SHG) enables to image cartilage specimens in a non-invasive mode with high resolution at deep penetration. By placing a band pass filter in front of the transmitted light detector, SHG signal with frequency doubled can be isolated for a new contrast imaging. SHG (second harmonic generation) is a diffusion process generated from organized structures and does not need any fluorescent staining. Due to their non-centrosymetric structure, collagen fibrilles present a high second-order non-linear susceptibility and thus give rise to a strong SHG signal when exposed to high enough electric fields produced by a focal point of a femtosecond pulsed laser (multiphoton microscopy). As the extracellular matrix of cartilage is in part constituted by collagen fibers, it can be imaged with this contrast tool. The intensity of SHG signals strongly depends on the organization of collagen fibers. Thus a modification of the extracellular matrix in terms of 3D-organization of collagen induced by mechanical stress can be shown with this contrast tool.

Influences of construct properties on the proliferation and matrix synthesis of dedifferentiated chondrocytes cultured in alginate gel.

To investigate whether the chondrocytes-alginate construct properties, such as cell seeding density and alginate concentration might affect the redifferentiation, dedifferentiated rat articular chondrocytes were encapsulated at low density (LD: 3 x 10(6) cells/ml) or high density (HD: 10 x 10(6) cells/ml) in two different concentrations of alginate gel (1.2% or 2%, w/v) to induce redifferentiation. Cell viability and cell proliferation of LD culture was higher than those of HD culture. The increase in alginate gel concentration did not make an obvious difference in cell viability, but reduced cell proliferation rate accompanied with the decrease of cell population in S phase and G2/M phase. Scan electron microscopy observation revealed that chondrocytes maintained round in shape and several direct cell-cell contacts were noted in HD culture. In addition, more extracellular matrix was observed in the pericellular region of chondrocytes in 2% alginate culture than those in 1.2% alginate culture. The same tendency was found for the synthesis of collagen type II. No noticeable expression of collagen type I was detected in all constructs at the end of 28-day cultures. These results suggested that construct properties play an important role in the process of chondrocytes' redifferentiation and should be considered for creating of an appropriate engineered articular cartilage.

Polyelectrolyte multilayer films: effect of the initial anchoring layer on the cell growth.

In tissue engineering, surface characteristics of a biomaterial are one of most important factors determining the compatibility with the environment. They influence attachment and growth of cells onto the material. In many cases, the surface should to be modified and engineered in the desired direction. The modification of non-adhesive surfaces with polyelectrolyte multilayer films (PMF) was recently depicted as a powerful technique to promote the growth of different cell lines. In this study, we evaluated the possible use of two different PMF as surface modification for the culture of mesenchymal stem cells (MSC). We used two types of PMF which differed by the nature of the initial anchoring layer which was poly(ethylenimine) (PEI) or poly(allylamine hydrochloride) (PAH). This initial polyelectrolytes adsorption was followed by the alternated deposition of poly(sodium 4-styrenesulfonate) (PSS) and (PAH) in order to obtain a PEI-(PSS-PAH)(3) film or a PAH-(PSS-PAH)(3) film. In order to control the behaviour of MSC, the cell viability was evaluated by Alamar Blue assay and the actin cytoskeleton was labelled and visualised in a confocal microscope. The behaviour of cells on the two PMF was compared to cells cultivated on surfaces treated with fibronectin. The results showed that PAH-(PSS-PAH)(3) PMF improve the growth of cells, inducing a higher cell viability compared to PEI-(PSS-PAH)(3) PMF and fibronectin at 2, 3 and 7 days of culture. Moreover, those cells showed a well-organized actin cytoskeleton. In conclusion, PAH-(PSS-PAH)(3) polyelectrolyte multilayer film seems to constitute an excellent material for MSC seeding.

Review: behaviour of endothelial cells faced with hypoxia.

Hypoxia is a diminution of oxygen quantity delivered to tissue for cellular need to product energy. Hypoxia derives from two major conditions in health diseases: anemia and ischemia. Anemic hypoxia comes from damage to O(2) transport like red blood cells diminution or disease. Ischemic hypoxia is a diminution of blood flow following a diminution of blood volume after a hemorrhagic shock. After hypoxia, vessels dilate to increase blood flow allowing a better oxygenation of peripheral tissues. This vasodilation appears immediately after the beginning of hypoxia and can be maintained during several hours. Today, the molecular mechanisms of this vasodilation stay unclear. But it seems that potassic channels, ATP concentration and medium acidification in addition to vasodilator/vasoconstrictor balance play a great role to facilitate the oxygenation of the ischemic areas.As endothelial cells (EC) are lining the vasculature, they are always in contact with blood, which carries, amongst other compounds, oxygen. In this way, they are the first target for an oxygen partial pressure (PO(2)) diminution. EC, through different mechanosensors, can sense a variation in PO(2) and adapt their metabolism to maintain ATP production. Under hypoxia, EC switch into hypoxic metabolism, leading to the production of reactive oxygen species (ROS). Indeed, when PO(2) is low, the respiratory chain in the mitochondria runs slower. Furthermore, cytochrome C capacity to trap O(2) is reduced; this phenomenon alters the cellular redox potential and leads to the accumulation of electrons that induce the formation of ROS.This review presents an overview of the behaviour of endothelial cells face to hypoxia. We propose to focus on nitric oxide, hypoxia inducible factor (HIF), lactate and ROS productions. Then we present the different mode of culture of EC under hypoxia. Finally, we conclude on the difficulty to study hypoxia because of the various types of system developed to reproduce this phenomenon and the different signalling ways that can be activated.

Effect of cyclic stretching and TGF-beta on the SMAD pathway in fibroblasts.

Tissue engineering requires the response of the cells to different stimuli inducing the synthesis of the extracellular matrix (ECM). It was been shown that mechanical and biochemical stimuli acted on the synthesis of ECM, particularly type I and III collagens. Growth factors implied in transduction pathways are multiple, but the main is TGF-beta. Member of the transforming growth factor-beta (TGF-beta) family bind to type II and type I serine/threonine kinase receptors, which initiate intracellular signals through activation of SMADs proteins. Nevertheless, the effects of mechanical stress of this pathway remain unknown. The aim of this work was to study the pathway of TGF-beta via the SMADs proteins under mechanical (stretching) and biochemical (TGF-beta) stimulations. Endogenous SMADs expression and its modulation by biochemical and mechanical stimulations were evaluated by both flow cytometry and confocal microscopy. Our results demonstrate that 10 ng of TGF-beta and stretching (5%, 1 Hz) applied during 15 min induced a negative feed back loop which blocks the signalling pathway to control TGF-beta activity. This inhibition effect was raised after 1 h of stimulation. Nevertheless, these preliminary studies should be continued by study of expression and localization of inhibitory SMADs (SMAD7).

In vitro effects of diacerein on NO production by chondrocytes in response to proinflammatory mediators.

Osteoarthritis (OA) is a progressive joint disease which represents a combination of several disorders leading to cartilage degradation. The production of nitric oxide (NO) by iNOS, which is stimulated by proinflammatory mediators, is involved in cartilage catabolism and should therefore be considered a potential target for therapeutic interventions. Diacerein is a slow-acting drug that may slow down the breakdown of cartilage and relieve pain and swelling. Diacerhein, in contrast to an NSAID, is a potent inhibitor of IL-1beta induced NO production by chondrocytes and cartilage. This effect appeared to result from iNOS transcriptional and/or post-transcriptional events, maybe by the inhibition of the NF-kappaB transcription factor. This paper presents results on the influence of Diacerein on NO production.

Three-dimensional sprayed active biological gels and cells for tissue engineering application.

Complex three-dimensional structures can "a priori" be built layer-by-layer with a large number of different components, including various cell types, polyelectrolytes, drugs, proteins, peptides or DNA. Our approach is based on the spraying of such elements in order to form a highly functionalized and structured biomaterial. The proposed route will allow the control at the surface and in depth the distribution of the different included elements (matrix and cells).The main objective of this work concerns the buildup of biomaterials aimed to reconstruct biological tissue. The proposed ways are highly innovative and consist in a simple and progressive spraying of all the elements constituting finally the biomaterial.We report here that it is possible (i) to build an alginate gel by alternate spraying of alginate and Ca(2+); (ii) to spray active alginate gel and cells; (iii) to build layer-by-layer an active reservoir under and on the top of this sprayed gel and cells; (iv) to follow the activity of these sprayed cells with time; (v) to propose a three-dimensional sprayed structure for tissue engineering application.

Chimerism analysis following nonmyeloablative stem cell transplantation using a new cell subset separation method: Robosep.

Chimerism analysis has become an important tool to manage patients in the peri-transplant period of allogenic stem cell transplantation. During this period, cells of donor and host origin can coexist and increasing proportion of cells of host origin is considered as a recurrence of the underlying disease. We currently performed chimerism analysis on separate peripheral blood cell subsets, lymphocytes and granulocytes. To improve our isolation method, a new automated device from Stem Cell Technology Roboseptrade mark was tested and compared to our manual separation technique. The results obtained on T cell purification showed an improvement of the purity (98.42% with Robosep vs. 92.42% with the manual technique Rosettesep) and of the recovery (63.43% with Robosep and 38% with Rosettesep). The results were significantly improved on patient samples with less than 10% CD3 positive cells (purity: 90% vs. 44.44%; recovery: 73.79% vs. 43.98%). Granulocytes separation was based on CD15 expression. The results showed an improvement of the purity with Robosep (96.90% vs. 86.20% with the manual technique Polymorphprep) but the recovery was impaired (35.2% vs. 52.30%). Using a myeloid (CD66/CD33) cocktail, recovery was improved with the Robosep device (64.04% with the myeloid cocktail vs. 22.4% with the CD15 cocktail). Our data demonstrated that Robosep allowed a performant cell purification in the early period post-transplantation even for populations representing less than 10% of the peripheral blood cells.

Multiphoton microscopy for blood vessel imaging: new non-invasive tools (Spectral, SHG, FLIM).

Imaging thick and opaque tissue, like blood vessel, in a noninvasive mode with high resolution, is nowadays possible with multiphoton technology. A near-infrared excitation presents the advantage to be compatible with living specimens and allows a deep penetration into tissues. The nonlinear excitation process is followed by several deactivation ways, among which fluorescence emission can be represented with Spectral or Lifetime imaging. Applied to ex vivo blood vessel imaging, these techniques enabled us to discriminate cell structures (nucleus, cytoskeleton) by fluorescent labelling (Hoechst, QDots). Another method, based on 2-photon excitation and which doesn't need any exogenous dye has also been experimented on arteries: SHG (Second Harmonic Generation) is a diffusion process generated from organized structures. Collagen molecules give rise to a strong SHG signal, enabling us to image the arterial wall (3-dimensional extracellular matrix).

Potential relation between cytoskeleton reorganization and e-NOS activity in sheared endothelial cells (Effect of rate and time of exposure).

Endothelial cells (ECs) which participate the interface between the blood and the vessel wall undergo morphologic changes in response to shear stress induced by blood flow, liable for the important regulation on physiologic and pathophysiologic function of blood vessels. Shear stress induced changes in cell morphology, begin with elongation in the direction of shearing and end by a reorientation and assembly of F-actin stress fibers. Shear stress is also implicated in many important ECs functions such as: decrease of platelet aggregation, anti-thrombogenic and anti-adhesive effects, inhibition of vascular smooth muscle cell (SMC) proliferation and regulation of their contraction and arterial tonicity, via a regulation of vasodilator and vasoconstrictor secretion molecules such as nitric oxide (NO), endothelin I, prostacyclin and angiotensin II. Besides, many of human diseases such as hypercholesterolemia, diabetes and hypertension, are strongly linked to a disturbance of the production of several vasodilator or vasoconstrictor molecules. The aim of this in-vitro study was to evaluate the potential balance between time and rate effects of shearing in cell shape changes and e-NOS activity. Two unidirectional steady laminar flow rates (1.2 Pa and 2.0 Pa) were applied on EC monolayers, each one for a short and a long period, (6 h and 24 h). Cytoskeleton reorganization was evaluated by actin filaments labelling and observed by confocal microscopy. NO production was evaluated by a colorimetric method using the Griess reagent kit for nitrite determination. Results showed that laminar flow affected cell rearrangement by inducing cytoskeleton reorientation and increased production of NO. Laminar shear rate at 2.0 Pa for 24 h did not upregulate NO release. Whereas at 1.2 Pa for 24 h, NO release increased by 33% compared with the static conditions. Both 1.2 Pa and 2.0 Pa for 6 h increased NO release by 17% and 24% respectively as compared with the static conditions. These observations suggested that stress fiber assembly, which controls EC reorientation and NO production, are dependent on rate and time of shearing. In addition, there appear to be a relation between the cytoskeleton reorganization stage and NO production. These results could promote the parameters to evaluate the more appropriate pattern of shearing, to evaluate a potential pharmacological effect on hypertension disorder decrease.

Introduction to endothelial cell biology.

Vascular endothelial cells form a monocellular layer on blood vessel walls with an estimated mass of 1.5 kg. One of the roles of endothelial cells is to control the hemodynamics through various metabolic activities affecting homeostasis, vascular tonus, blood fluidity, coagulating properties and blood cell adhesion. In other respects thousands of studies have underlined the crucial role of local blood flow conditions on their properties. However, the hemodynamic forces are different according to the anatomical site and to the type of blood vessels (arteries, veins, venules, ...). In microcirculation, the endothelial cells in the venules are particularly active and constitute the physiological site of liquid exchange (permeability) and above all cellular transit. During critical ischemia, the post-capillary venules are deeply involved. In other respects the properties of endothelial cells may be impaired in many diseases as atherosclerosis, hypertension, inflammation and metabolic diseases.

In vitro impact of physiological shear stress on endothelial cells gene expression profile.

In the vascular system, the shear applied to the vascular wall activates mechano-sensors located on endothelial cells (ECs) leading to a modification in the gene expression profile. We applied laminar shear stress at 1 Pa on ECs for 6 h and measured by quantitative real time PCR the expression modulation of genes implied in inflammation (ICAM-1 and E-selectin), oxidative stress sensing (HO-1) and vascular tone modulation (eNOS). We showed that all these genes are shear stress inducible. ICAM-1 is more up-regulated than E-selectin suggesting different levels of implication in inflammatory responses and different modes of induction (SSRE, cytokine). Laminar shear stress induces an oxidative stress translated into HO-1 up-regulation, and a possible vasodilatation through the induction of eNOS. Our laminar shear stress system opens a novel and interesting frame in the evaluation of the impact on ECs and blood cells of new pharmacological substances injected in the bloodstream.

Organ reconstruction: Dream or reality for the future.

The relevance of research on reconstructed organs is justified by the lack of organs available for transplant and the growing needs for the ageing population. The development of a reconstructed organ involves two parallel complementary steps: de-cellularization of the organ with the need to maintain the structural integrity of the extracellular matrix and vascular network and re-cellularization of the scaffold with stem cells or resident cells.Whole organ engineering for liver, heart, lung or kidneys, is particularly difficult because of the structural complexity of organs and heterogeneity of cells. Rodent, porcine and rhesus monkey organs have been de-cellularized to obtain a scaffold with preserved extracellular matrix and vascular network. As concern the cells for re-cellularization, embryonic, foetal, adult, progenitor stem cells and also iPS have been proposed.Heart construction could be an alternative option for the treatment of cardiac insufficiency. It is based on the use of an extra-cellular matrix coming from an animal's heart and seeded with cells likely to reconstruct a normal cardiac function. Though de-cellularization techniques now seem controlled, the issues posed by the selection of cells capable of generating the various components of cardiac tissue are not settled yet. In addition, the recolonisation of the matrix does not only depend on the phenotype of cells that are used, but it is also impacted by the nature of biochemical signals emitted.Recent researches have shown that it is possible to use decellularized whole liver treated by detergents as scaffold, which keeps the entire network of blood vessels and the integrated extracellular matrix (ECM). Beside of decellularized whole organ scaffold seeding cells selected to repopulate a decellularized liver scaffold are critical for the function of the bioengineered liver. At present, potential cell sources are hepatocyte, and mesenchymal stem cells.Pulmonary regeneration using engineering approaches is complex. In fact, several types of local progenitor cells that contribute to cell repair have been described at different levels of the respiratory tract. Moving towards the alveoles, one finds bronchioalveolar stem cells as well as epithelial cells and pneumocytes. A promising option to increase the donor organ pool is to use allogeneic or xenogeneic decellularized lungs as a scaffold to engineer functional lung tissue ex vivo.The kidney is certainly one of the most difficult organs to reconstruct due to its complex nature and the heterogeneous nature of the cells. There is relatively little research on auto-construction, and experiments have been performed on rats, pigs and monkeys.Nevertheless, before these therapeutic approaches can be applied in clinical practice, many researches are necessary to understand and in particular the behaviour of cells on the decellularized organs as well as the mechanisms of their interaction with the microenvironment. Current knowledges allow optimism for the future but definitive answers can only be given after long term animal studies and controlled clinical studies.

Mechanobiology of mesenchymal stem cells: Which interest for cell-based treatment?

Thanks to their immune properties, the mesenchymal stem cells (MSC) are a promising source for cell therapy. Current clinical trials show that MSC administrated to patients can treat different diseases (graft-versus-host disease (GVHD), liver cirrhosis, systemic lupus, erythematosus, rheumatoid arthritis, type I diabetes…). In this case, the most common mode of cell administration is the intravenous injection, and the hemodynamic environment of cells induced by blood circulation could interfere on their behavior during the migration and homing towards the injured site. After a brief review of the mechanobiology concept, this paper will help in understanding how the mechanical environment could interact with MSC behavior once they are injected to patient in cell-based treatment.

Aging of bone marrow mesenchymal stromal/stem cells: Implications on autologous regenerative medicine.

With their proliferation, differentiation into specific cell types, and secretion properties, mesenchymal stromal/stem cells (MSC) are very interesting tools to be used in regenerative medicine. Bone marrow (BM) was the first MSC source characterized. In the frame of autologous MSC therapy, it is important to detect donor's parameters affecting MSC potency. Age of the donors appears as one parameter that could greatly affect MSC properties. Moreover, in vitro cell expansion is needed to obtain the number of cells necessary for clinical developments. It will lead to in vitro cell aging that could modify cell properties. This review recapitulates several studies evaluating the effect of in vitro and in vivo MSC aging on cell properties.

Influence of mechanical stress on cell viability.

The cartilage is a hydrated connective tissue in joints that withstands and distributes mechanical forces. The chondrocytes utilize mechanical signals to regulate their metabolic activity through complex biological and biophysical interactions with the extracellular matrix (ECM). The aim of this work was to study the influence of mechanical stress on cells behavior cultured in 3D biosystems (alginate and alginate supplemented with hyaluronate). After mechanical stimulation, cell viability and cell death process were the main studied parameters. Our results indicated that viability and cell cycle progression were inhibited under mechanical stimulation, as far as the extracellular matrix was not yet synthesized. In contrast, on day 21, the mechanical stimulation had positive effect on these parameters.

IL-1beta synthesis by chondrocyte analyzed by 3D microscopy and flow cytometry: effect of Rhein.

Several factors are known to be involved in the destruction of the articular cartilage. Interleukin-1 (IL-1) plays an important role in the pathogenesis of osteoarthritis (OA) either directly or through the stimulation of catabolic factors. The action of IL-1 on articular cartilage is multifaceted and it most likely plays an important role in the mechanism of cartilage destruction. IL-1 suppresses the synthesis of the cartilage matrix components and promotes the degradation of cartilage matrix macromolecules. Diacerein is an anthraquinone molecule that has been shown to reduce the severity of OA, both in man and in animal models. The present study was designed to evaluate in vitro effects of diacerein on IL-1beta expression in LPS or IL-1alpha stimulated chondrocytes. Intracellular IL-1beta production was analysed in articular chondrocytes cultured in monolayer or in alginate 3D-biosystems in the presence of lipopolysaccharide (LPS) or IL-1alpha, with or without diacerein. The results show that LPS and IL-1alpha increase intracellular IL-1beta and Diacerein inhibited LPS-induced and IL-1alpha induced IL-1beta production by articular chondrocytes. Moreover, the effect of mechanical stimulation was analysed. An inhibitory effect of DAR at therapeutic concentrations on IL-1beta production in articular chondrocytes is suggested.

Cell and tissue engineering and clinical applications: an overview.

Most human tissues do not regenerate spontaneously; this is why cell therapies and tissue engineering are promising alternatives. The principle is simple: cells are collected in a patient and introduced in the damaged tissue or in a tridimentional porous support and harvested in a bioreactor in which the physico-chemical and mechanical parameters are controlled. Once the tissues (or the cells) are mature they may be implanted. In parallel, the development of biotherapies with stem cells is a field of research in turmoil given the hopes for clinical applications that it brings up. Embryonic stem cells are potentially more interesting since they are totipotent, but they can only be obtained at the very early stages of the embryo. The potential of adult stem cells is limited but isolating them induces no ethical problem and it has been known for more than 40 years that bone marrow does possess the regenerating functions of blood cells. Finally, the properties of foetal stem cells (blood cells from the umbilical cord) are forerunners of the haematopoietic system but the ability of these cells to participate to the formation of other tissues is more problematic. Another field for therapeutic research is that of dendritic cells, antigen presenting cells. Their efficiency in cell therapy relies on the initiation of specific immune responses. They represent a promising tool in the development of a protective immune response against antigens which the host is usually unable to generate an efficient response (melanomas, breast against cancer, prostate cancer, ..). Finally, gene therapy, has been nourishing high hopes but few clinical applications can be envisaged in the short term, although potential applications are multiple (haemophilia, myopathies, ..). A large number of clinical areas stand as candidates for clinical applications: leukaemia and cancers, cardiac insufficiency and vascular diseases, cartilage and bone repair, ligaments and tendons, liver diseases, ophthalmology, diabetes, neurological diseases (Parkinson, Huntington disease, ..), .. Various aspects of this new regenerative therapeutic medicine are developed in this work.