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Almost all cells in the human body are subjected to mechanical stresses. These forces can vary from a few Pascals (shear stress) to some mega Pascals (on hip cartilage). It is now well known that mechanical forces have a decisive effect on cellular physiology. In 1880, W. Roux introduced the concept of functional adaptation; which can be defined as a quantitative autoregulation controlled by stimuli like mechanical forces. These stresses influence functionality and cellular metabolism and can lead to appropriate tissue remodelling by triggering a cascade of reactions (mechanotransduction), being the signal for the adaptation of cells and tissues. However, although the main biological effects of mechanical forces are well documented, the relation between mechanical forces and physiological phenomena is largely unknown. In this paper, some effects of mechanical stresses on different cells (mesenchymal stem cells, bone cells, chondrocyte, endothelial cells, vascular or muscular cells, etc.) are summarized.

International audience; Background & AimMesenchymal stromal/stem cells (MSCs) are a heterogeneous population characterized by self-renewing and ability to differentiate into mesodermal cell origin. Wharton Jelly MSC (WJ-MSC) are a promising candidate for cell therapy due to their ease of isolation and their tissue regeneration capabilities. Moreover, they can regulate the immune response by secreting a set of soluble factors and by establishing direct interactions with the cells of immune system. These immunomodulation properties are used in the treatment of different pathologies such as graft versus host disease. In order to use WJ-MSCs for clinical applications, an in vitro expansion step is critical to obtain the sufficient dose. This step imposes several physicochemical and hydrodynamic constraints that can influence the quality and functional properties of cells.The objective of this work was to identify and validate predictive markers related to immunomodulatory properties of WJ-MSCs. For this purpose, CD119, CD200 and Notch1 were chosen and studied.Methods, Results & ConclusionAt first, markers distribution was studied by flow cytometry. Secondly, immunomodulatory potential of WJ-MSC was evaluated by a coculture of these cells with CD4, CD8 lymphocytes, followed by the study of lymphocytes proliferation by flow cytometry. These experimentss were performed under different conditions: WJ-MSCs cultivated in normoxia (N) and hypoxia (H), early passage and senescent cells, and under stimulation with gamma interferon (IFN-γ).Our results showed a heterogeneity of distribution of the markers studied. In addition, our study revealed that WJ-MSC cultivated in H was more immunosuppressive than those cultivated in N. Moreover, we showed that the cellular senescence induced a reduction of the immunosuppressive properties, and that IFN-γ stimulation increased WJ-MSC immunosuppression potential. Finally, our study highlighted a trend towards a correlation between the expression of CD119 and Notch1 by WJ-CSM and their immunomodulatory properties.To conclude, the culture conditions and cell senescence affect the immunomodulatory properties of WJ-CSM hence the interest of verifying these properties after the cell expansion procedures. Furthermore CD119 and Notch1 receptors may be predictive markers of the immunomodulatory properties of WJ -CSM to be checked during in vitro amplification.

International audience; Background & AimMesenchymal stromal/stem cells (MSCs) are fibroblast-like multipotent cells capable of proliferation, self-renewal and differentiation into different mesodermal cell-lineages. They are known to be implicated in many key biological processes like tissue regeneration, secretion of bioactive factors which give them immunomodulatory and trophy properties. MSCs isolated from umbilical cord Wharton's Jelly (WJ-MSC) aroused much attention due to several advantages: multipotency capacity, painless, safety for allogenic therapeutic purposes and efficiency for treating certain diseases. In order to use MSCs for therapeutic purpose, these cells must be expanded in long-term, which inevitably triggers cell senescence, a process of cell proliferation arrest that occurs in vivo and in vitro as a response of cells to excessive stresses. This phenomenon is characterized by several markers and features and it can affect cell behavior.Because of senescent cells influence the outcome of a variety of physiological and pathological processes, and because of WJ-MSCs are used in cell therapy, the aim of the study was to characterize senescence WJ-MSCs after in vitro expansion, to control the quality of these cells in terms of function and phenotype.Methods, Results & ConclusionTo induce senescence process, we used in vitro model called “replicative senescence”. Cells were cultured in normoxia (N) and hypoxia (H) conditions and analyzed in early and late passages. Proliferation analysis of MSCs demonstrated a high rate of proliferation in H condition. In addition, senescence analysis using a Beta-Galactosidase test indicated that MSCs in H condition senesce slower than those in N. On the other hand, cell-surface biomarker analysis revealed that all MSCs derived from WJ expressed the common surface markers among which CD44, CD105 without great changes during expansion. However other markers presented changes during expansion (like a decrease in CD146). Senescent cells presented variable immunosuppressive potential compared to MSCs in early passage. Known that senescent cells are characterized by changes in chromatin structure, we studied proteins implicated in chromatin remodeling like HMGB1. Immunofluorescence study showed a translocation of HMGB1 from MSC nucleus in senescent cells. All these results will improve the characterization of MSCs senescence and may provide new parameters for establishing the quality of MSCs prior to their therapeutic application.

International audience; Background & AimMesenchymal Stromal/Stem Cells (MSC) are a heterogeneous population of self-renewal multipotent cells which can be obtained from adult sources (Bone Marrow MSC (BMMSC)) and fetal sources (Wharton Jelly MSC (WJMSC)). MSC are a very interesting tool for tissue engineering because of their differentiation properties and for cell therapy because of their immunomodulation properties. Indeed, MSC secrete soluble factors which can modulate the immune response.Before their clinical use, an in vitro expansion step must be performed in order to obtain the sufficient dose. However, this expansion step could affect MSC quality and need to be controlled in order to increase the therapeutic efficacy.The first challenge was to optimize the culture conditions and to choose the most appropriate media for the MSC culture to ensure that they retain their functional properties and thus their therapeutic potential. The second challenge was to know the impact of in vitro expansion on MSC functional properties.Methods, Results & ConclusionWe firstly studied the impact of culture conditions (normoxia (N), hypoxia (H), fetal calf serum (FCS) supplement, human platelet lysate (hPL) supplement on the behavior of MSC. Our results showed that better proliferation properties were obtained for MSC expanded with human platelet lysate (hPL) in hypoxia for WJMSC and in normoxia for BMMSC. Moreover, we observed that WJMSC and BMMSC expanded with hPL have better clonogenicity and are less senescent. Using neutralizing antibodies, we confirmed the involvement of high concentration of growth factors (PDGF, EGF,…) in hPL in WJMSC proliferation properties. The main surface markers and differentiation capacities were found to be equivalent for WJMSC and BMMSC for the different culture conditions.We secondly studied the impact of in vitro expansion on MSC immunomodulation properties. We showed that WJMSC and BMMSC expanded in hypoxia have better immunosuppressive properties when they are co-cultivated with CD4+ T cells and that senescence and interferon-γ (IFN-γ) stimulation could affect the MSC immunomodulatory properties.To conclude MSC expansion with hPL gives better clonogenicity properties and better proliferation properties in normoxia for BMMSC and in hypoxia for WJMSC without affecting surface markers and differentiation properties. Moreover, WJMSC expanded in hypoxia with hPL with low senescence are more immunosuppressive. This property can be improved by IFN-γ stimulation.

International audience; End-stage liver disease can be the termination of acute or chronic liver diseases, with manifestations of liver failure; transplantation is currently an effective treatment for these. However, transplantation is severely limited due to the serious lack of donors, expense, graft rejection and requirement of long-term immunosuppression. Mesenchymal stem cells (MSCs) have attracted considerable attention as therapeutic tools as they can be obtained with relative ease and expanded in culture, along with features of self-renewal and multidirectional differentiation. Many scientific groups have sought to use MSCs differentiating into functional hepatocytes to be used in cell transplantation with liver tissue engineering to repair diseased organs. In most of the literature, hepatocyte differentiation refers to use of various additional growth factors and cytokines, such as hepatocyte growth factor (HGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), oncostatin M (OSM) and more, and most are involved in signalling pathway regulation and cell-cell/cell-matrix interactions. Signalling pathways have been shown to play critical roles in embryonic development, tumourigenesis, tumour progression, apoptosis and cell-fate determination. However, mechanisms of MSCs differentiating into hepatocytes, particularly signalling pathways involved, have not as yet been completely illustrated. In this review, we have focused on progress of signalling pathways associated with mesenchymal stem cells differentiating into hepatocytes along with the stepwise differentiation procedure.

International audience; 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.

International audience; Most human tissues do not regenerate spontaneously, which is why "cell therapy" are promising alternative treatments. The Principe is simple: patients' or donors' cells are collected and introduced into the injured tissues or organs directly or in a porous 3D material, with or without modification of their properties. This concept of regenerative medicine is an emerging field which can be defined as "the way to improve health and quality of life by restoring, maintaining, or enhancing tissue and organ functions".There is an extraordinarily wide range of opportunities for clinical applications: artheropathies, diabetes, cartilage defects, bone repair, burns, livers or bladder regeneration, organs reconstruction (lung, heart, liver...) neurodegenerative disorders, sepsis...Different stem cells (SC) with different potential can be used and characterised (totipotent, mesenchymal of different origins, especially those present in tissues...). Today it is undeniable that cells like bone marrow, adipose tissue or Wharton Jelly stem cells, are of potential interest for clinical applications because they are easily separated and prepared and no ethical problems are involved in their use.In this paper some potential clinical applications in the vascular field are considered: peripheral arteriopathy in diabetic patients, cardiac insufficiency, traitment of erectile dysfunction, or organ regeneration with liver as example. But the regeneration of tissue or organ is and will remain a challenge for the future development of cell therapy. Many problems remain to be solved that could lead to the development of innovative strategies to facilitate cell differentiation, increase the yield of cells and ensure a standardised product, overcome the risks of teratogenic effects and/or immune reactions, enable grafting via direct cell or biotissue transplantation and avoid legal issues involved in national regulations.

International audience; 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.

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.

For this study, we have considered a new large field of view imaging dedicated to matrix collagen (no stained samples). To integrate a multidimensional scale (non-sliced samples), a femtosecond oscillator (two photon excitation laser) has been coupled with a large field optical setup to collect SHG signal. We introduced an index (F-SHG) based on decay time response measured by TCSPC for, respectively, Fluorescence (F) and Second Harmonic Generation (SHG) values. For samples where protein collagen is the major component of extracellular matrix (skin) or not (nacre), we compared the index distribution (from 2 to 12) obtained with large field optical setup. In this work, we showed for the first time that multiscale large field imaging combined to multimodality approaches (SHG-TCSPC) could be an innovative and non invasive technique to detect and identify some biological interest molecules (collagen) in biomedical topics.

Human tissues don't regenerate spontaneously, explaining why regenerative medicine and cell therapy represent a promising alternative treatment (autologous cells or stem cells of different origins). The principle is simple: cells are collected, expanded and introduced with or without modification into injured tissues or organs. Among middle-term therapeutic applications, cartilage defects, bone repair, cardiac insufficiency, burns, liver or bladder, neurodegenerative disorders could be considered.

Tissue engineering is a multidisciplinary field that applies the principles of engineering, life sciences, cell and molecular biology toward the development of biological substitutes that restore, maintain, and improve tissue function. In Western Countries, tissues or cells management for clinical uses is a medical activity governed by different laws. Three general components are involved in tissue engineering: (1) reparative cells that can form a functional matrix; (2) an appropriate scaffold for transplantation and support; and (3) bioreactive molecules, such as cytokines and growth factors that will support and choreograph formation of the desired tissue. These three components may be used individually or in combination to regenerate organs or tissues. Thus the growing development of tissue engineering needs to solve four main problems: cells, engineering development, grafting and safety studies.

Cartilage tissue engineering gives the ability to product adaptable neocartilage to lesion with autologous cells. Our work aimed to develop a stratified scaffold with a simple and progressive spraying build-up to mimic articular cartilage environment. An Alginate/Hyaluronic Acid (Alg/HA) hydrogel seeded with human Mesenchymal Stem Cells (hMSC) was construct by spray. First, cells repartition and actin organization were study with confocal microscopy. Then, we analyzed cells viability and finally, metabolic activity. Our results indicated a homogenous cells repartition in the hydrogel and a pericellular actin repartition. After 3 days of culture, we observed about 52% of viable cells in the scaffold. Then, from day 7 until the end of culture (D28), the proportion of living cells and their metabolic activity increased, what indicates that culture conditions are not harmful for the cells. We report here that sprayed method allowed to product a scaffold with hMSCs that confer a favorable environment for neocartilage construction: 3D conformation and ability of cells to increase their metabolic activity, therefore with few impact on hMSCs.

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.

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.

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.

Some of vascular endothelial dysfunctions are currently caused by impairment in nitric oxide biodisponibility, involving resistance arteries and leading to increase the systemic blood pressure and thus leading to hypertension. Hence, antihypertensive agents, that reverse endothelial dysfunction and lower blood pressure, might improve the prognosis of patients with hypertension. Many studies investigated the underlying mechanism of Nebivolol (NEB)-inducing vasodilation and reported the highly role of selective β1 adrenoceptor blocker with its additional vasodilating mechanisms. However, the way that mediates these actions remains controversial. A lot of hypothesizes suggested a NO endotheliumdependent release induced by NEB in static conditions. This NO production was due to stimulation of β3 adrenoreceptors and oestrogen receptors and was dependent on e-NOS activation, then implicating PI3-K and Akt signaling pathway. Other findings reported an endothelium independent mechanism, then independent from Ca+2/Calmodulin, e-NOS and β-adrenoreceptor pathway (Fig. 1). Other ways are also described, so this paper presents an overview of the NEB signaling pathways in terms of endothelium dependence as well as the involved receptors.

The quality of an antibody is related to its affinity expressed by the equilibrium constant. The biological activity of an antibody can be estimated by the strength of antigen–antibody bridges. An optical method is proposed to estimate the specific binding energy using the dissociation behavior of suspended RBC agglutinates in a shear flow and measuring laser backscattering. A constant increase of intensity was observed when the shear stress raises, pointing to a progressive dissociation of RBC agglutinates into smaller ones. The final break-up of two-cell rouleaux is produced at critical shear stress ( τ c ), which reflects the mechanical action required to dissociate the molecular bridges between cells. The critical shear stress permitted to define the specific surface adhesive energy ( Γ ) by using the Derjaguin relation. The determination of τ c provides a good way to assess the functional characterization of specific immunoglobulins, which could be very useful for antibody quality control.

Tissue engineering is an emerging multidisciplinary field involving surgery medicine, biology, chemistry, mechanics and engineering to improve the health and quality of life by restoring, maintaining or enhancing tissue and organ function [6;11]. The principle is simple: cells are collected and introduced -with or without modification of their biological properties- into an environment in which physico-chemical and mechanical parameters are kept stable. When they reach maturity, the biotissue or cells can be grafted. The main parameters are: cells, scaffold, biological molecules and mechanical forces. A combination of these parameters can promote cellular differentiation and proliferation. But it should be emphasized that at this time, signalling pathways and the rationale behind the physiological design remain to be elucidated. Among the main medium-term clinical applications are cardiac insufficiency, atherosclerosis, osteoarthritis, diabetes, liver diseases, bladder, and skin. In this paper the examples of cartilage and vascular engineering will be examined.

5th China-France International Symposium, Kunming, PEOPLES R CHINA, DEC 12-14, 2013; International audience; Since the 1960s and the therapeutic use of hematopoietic stem cells of bone marrow origin, there has been increasing interest in the study of undifferentiated progenitors that have ability to proliferate and differentiate in different tissues. Different stem cells (SC) with different potential can be isolated and characterised. Despite the promise of embryonic stem cells, in many cases, adult stem cells provide a more interesting approach to clinical applications. It is undeniable that mesenchymal stem cells (MSC) from bone marrow, adipose tissue or MSC of Wharton Jelly, which have limited potential, are of interest for clinical applications in regenerative medicine because they are easily separated and prepared and no ethical problems are involved in their use. During the last 10 years, these multipotent cells have generated considerable interest and in particular have been shown to escape allogeneic immune response and be capable of immunomodulatory activity. These properties may be of a great interest for regenerative medicine. Different clinical applications are under study (cardiac insufficiency, atherosclerosis, stroke, bone, cartilage, diabetes, ophthalmology, urology, liver, organ's reconstruction ...).

Since the 1960s and the therapeutic use of hematopoietic stem cells of bone marrow origin, there has been an increasing interest in the study of undifferentiated progenitors that have the ability to proliferate and differentiate into various tissues. Stem cells (SC) with different potency can be isolated and characterised. Despite the promise of embryonic stem cells, in many cases, adult or even fetal stem cells provide a more interesting approach for clinical applications. It is undeniable that mesenchymal stem cells (MSC) from bone marrow, adipose tissue, or Wharton's Jelly are of potential interest for clinical applications in regenerative medicine because they are easily available without ethical problems for their uses. During the last 10 years, these multipotent cells have generated considerable interest and have particularly been shown to escape to allogeneic immune response and be capable of immunomodulatory activity. These properties may be of a great interest for regenerative medicine. Different clinical applications are under study (cardiac insufficiency, atherosclerosis, stroke, bone and cartilage deterioration, diabetes, urology, liver, ophthalmology, and organ's reconstruction). This review focuses mainly on tissue and organ regeneration using SC and in particular MSC.

International audience

International audience; Nitric oxide is implicated in the target action of Nebivolol, a selective beta 1 adrenoceptor blocker used in hypertension treatment. As the Nitric Oxide (NO) production and the actin cytoskeleton are linked, the aim of this work was to study the involvement of actin cytoskeleton on mechanism of action of Nebivolol in cultured endothelial cells. We studied the effect of Nebivolol (200 mu M) on actin filaments remodeling and its impact on NO production and eNOS activation. Results showed that Nebivolol perturbs actin filaments polymerization, increases NO production and eNOS activity between 30 minutes and 1 h. Stabilization of actin filaments with phalloidine (50 mu M) abolishes Nebivolol effects on eNOS activation and NO production. Furthermore, Rho-kinase activity decreased during the first hour of Nebivolol treatment, then increased after 3 h, while actin filaments repolymerized, eNOS activation and NO production decreased. In SMCs, Nebivolol induced a decrease in the Rhokinase activity from 1 h until 24 h of incubation. In conclusion, we suggest that Nebivolol induced NO production in Endothelial Cells (ECs) via complementary actions between actin cytoskeleton remodeling inducing eNOS activation and Rho-kinase implication. The effect of Nebivolol on ECs occurs during the first hour, this effect on SMCs seems to be maintained until 24 h, explaining persisted action of Nebivolol observed in vivo.

supplementing culture medium with LV and polybrene. Transduction efficiency was measured as % eGFP+ MSCs via flow cytometry. IL-1Ra secretion into culture media was quantified by ELISA. The chondrogenic potential of MSCs following transduction with constitutive IL-1Ra or eGFP LV was assessed in pellet culture. After 21 days in chondrogenic medium (+10 ng/mL rhTGF-b3) or a basal medium, pellets were processed for histology and stained to evaluate sulfated glycosaminoglycan (s-GAG) and collagen production as measures of chondrogenesis. Results: Transduced MSCs constitutively produced IL-1Ra in monolayer culture for 27 days at concentrations of 3-10 ng per 10K cells every 3 days (Fig 1, A). Doxycycline-induced IL-1Ra production exceeded constitutive expression for the first 21 days. Non-transduced cells did not produce detectable levels of IL-1Ra. The magnitude of inducible IL1Ra secretionwas effectively tuned from 2-200 ng/mL with doxycycline doses ranging from 1-1000 ng/mL (Fig 1, B). IL-1Ra-transduced, eGFPtransduced (90% eGFP+), or control MSCs were differentiated in pellet culture for 21 days. Pellets in chondrogenic culture conditions produced s-GAGs far exceeding the production in basal medium, with similar levels observed in transduced and control cell populations (Fig 2, A). IL1Ra secretion in transduced pellets ranged from approximately 100-250 ng/mL throughout the culture period (a magnitude shown to inhibit pathophysiologic IL-1 levels) (Fig 2, B). These findings show that IL-1Raproducing MSCs can undergo chondrogenesis following transduction while simultaneously producing therapeutic levels of IL-1Ra. Conclusions: We have developed MSCs capable of tunable IL-1Ra production at therapeutically relevant concentrations. We have shown that these MSCs retain their chondrogenic potential in pellet culture. Future studies will examine the dynamic response of MSCs in response to IL-1 challenge during chondrogenesis in pellet culture and in a tissueengineered construct. This system shows potential to enable growth and differentiation of tissue-engineered cartilage constructs within the inflammatory environment of an OA joint. In addition to a protective effect on the engineered cartilage, release of IL-1Ra into the surrounding tissue may have therapeutic effects throughout the joint. Figure 2. A. Safranin-O/fast green/haematoxylin staining for s-GAGs, collagen, and cell nuclei in MSC pellets following 21 days in culture in chondrogenic (top row) or basal medium (bottom row). 6 mm sections, scale bar 1⁄4 500 mm. B. IL-1Ra secretion into media at 3 day intervals (mean SEM, n1⁄43).

International audience; Collagen gels could be used as carriers in tissue engineering to improve cell retention and distribution in the defect. In other respect hydroxyapatite could be added to gels to improve mechanical properties and regulate gel contraction. The aim of this work was to analyze the feasibility to incorporate hydroxyapatite into collagen gels and culture mesenchymal stem cells inside it. Human bone marrow mesenchymal stem cells (hMSC-BM) were used in this study. Gels were prepared by mixing rat tail type I collagen, hydroxyapatite microparticles and MSCs. After polymerization gels were kept in culture while gel contraction and mechanical properties were studied. In parallel, cell viability and morphology were analyzed. Gels became free-floating gels contracted from day 3, only in the presence of cells. A linear rapid contraction phase was observed until day 7, then a very slow contraction phase took place. The incorporation of hydroxyapatite improved gel stability and mechanical properties. Cells were randomly distributed on the gel and a few dead cells were observed all over the experiment. This study shows the feasibility and biocompatibility of hydroxyapatite supplemented collagen gels for the culture of mesenchymal stem cells that could be used as scaffolds for cell delivery in osteoarticular regenerative medicine.

Injuries to articular cartilage are one of the most challenging issues of musculoskeletal medicine due to the poor intrinsic ability of this tissue for repair. Despite progress in orthopaedic surgery, cell-based surgical therapies such as autologous chondrocyte transplantation (ACT) have been in clinical use for cartilage repair for over a decade but this approach has shown mixed results. Moreover, the lack of efficient modalities of treatment for large chondral defects has prompted research on cartilage tissue engineering combining cells, scaffold materials and environmental factors. This paper focuses on the main parameters in tissue engineering and in particular, on the potential of mesenchymal stem cells (MSCs) as an alternative to cells derived from patient tissues in autologous transplantation and tissue engineering. We discussed the prospects of using autologous chondrocytes or MSCs in regenerative medicine and summarized the advantages and disadvantages of these cells in articular cartilage engineering.

The FoxO family of Forkhead transcription factors functions at the interface of tumor suppression, energy metabolism and organismal longevity. FoxO factors are key downstream targets of insulin, growth factor, nutrient and oxidative stress stimuli that coordinate a wide-range of cellular outputs. These transcription factors could participate in the regulation of different phenomena found in the osteoarthritis pathology, like apoptosis, chondrocyte proliferation, cell dedifferentiation or resistance to oxidative stress. Moreover, we found recently that FoxO transcription factors could be involved on Diacerhein mode of action, a drug that reduces the IL-1β deleterious effects on osteoarthritis cartilage through inhibition of the expression of degrading enzymes. It could explain the downregulated proliferation and the increased p27 expression observed on human osteoarthritic chondrocytes in the presence of Diacerhein.

In the last years, there were many studies based on the use of human bone marrow mesenchymal stem cells (hMSCs) in cell therapy and tissue engineering. Although hMSCs can be easily obtained and expanded in culture, a large number of cells are often needed. The expansion of hMSCs depends on the culture conditions, such as media, cell density or culture flasks. Moreover, growth factors are often added to improve cell proliferation. In this study, we compared the effect of two culture media (DMEM and alpha-MEM), two culture flasks (75 or 25 cm2) and two different mononuclear cell seeding densities (1 x 10(4) or 5 x 10(4) MNC/cm2) on the isolation of hMSCs from bone marrow samples and analyzed if the isolation conditions affected the expansion of these cells in the first two passages. Experiments were performed without the addition of exogenous growth factors. Our results showed that alpha-MEM is the optimal culture medium for both, isolation and expansion of mesenchymal stem cells. Moreover, the cell seeding density of 50,000 MNC/cm2 in 25 cm2 culture flasks seems to be the best condition for the isolation step.

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.

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.

International audience; 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.

Several works have recently shown that Mesenchymal Stem Cells (MSC) collected from bone marrow can differentiate in vitro into cartilage cells under the effects of transforming growth factors or critical transcription factors [1]. The transforming growth factor TGF-β is a family of multifunctional cytokines controlling cell growth, differentiation, and extra cellular matrix deposition. The biological effects of TGF-β are mediated by type I (TBR-I) and II (TBR-II) receptors. In this work we presented a preliminary study to characterize the localization and mobility of TBR-II using mesenchymal stem cells derived from bone marrow by Fluorescence Correlation Spectroscopy (FCS). This sensitive technique is mainly used for biological applications for measuring dynamic processes (number density, interaction fractions and molecular dynamics) in a fluorescent signal on the nanosecond to second time scales [2]. To apply this analytical method to living cells, some steps of standardization have to be optimized in the case of indirect immunolabeling.

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.

A problem in immunohematology is to define the antibody quality which is related to its affinity expressed by the equilibrium constant. The activity of an antibody can be measured by the strength of its interaction, related to the adhesive energy exchanged during RBC agglutination which depends on the antigen-antibody liaison strength. To estimate this adhesive energy, two methods are used in this paper. Firstly, the dissociation behaviour of suspended RBC agglutinates was analysed by laser backscattering intensity (r) in a Couette flow. Backscattered intensity issued from shear-induced mechanical dissociation is recorded and submitted to a numerical process to obtain the energy parameter (ED). Secondly, a modification of this technique is proposed for measuring specific binding energy. Samples were exposed to increasing shear stress, and backscattered intensity was recorded. A constant increase of this intensity with raising shear stress was observed, pointed to a progressive dissociation of RBC agglutinates into smaller ones. Considering that complete dissociation of agglutinates is only approached asymptotically it is assumed that the final break-up of doublets (two-cell agglutinates) is produced at a critical shear stress (tauC) reflecting the work done to breaking-up the molecular bridges between both adjacent cells. This shear stress is defined by the extrapolation of the linear part of the curves [r-log tau] to the backscattered signal (r0) corresponding to the complete dispersion of RBCs. These approaches permit to define the specific surface adhesive energy (Gamma) by using the Derjaguin relation and to assess the functional characterization of specific immunoglobulins. In conclusion, two parameters characterizing monoclonal antibody agglutination properties, ED and Gamma, were estimated by laser backscattering methods, which could be very useful for antibodies quality control.

progenitor cells. The aim of this study is to determinate the useful of the hAMSC and the hAEC on regenerating human joint cartilage in an in vitro model. Methods: HAM was used as support for the culture of hAMSCs or hAECs. Focal injuries in human joint cartilage biopsies were done. Later, a pellet of cells (hAMSCs or hAECs, depending on the repair model developed) was implanted into the focal defects of cartilage. HAM, with the cells grown on it, was placed in direct contact with the cartilage surface to be repaired. These implants were cultured in DMEM+10% FBS for 8 weeks. The repair tissues were analyzed by histological and histochemistry analysis considering the ICRS macroscopic evaluation of cartilage repair. Results: hAMSCs and hAECs cultured on HAM and transplanted onto focal injuries of cartilage penetrated into the nearby surface of the chondral defect. The Hematoxylin and Eosin staining showed that hAMSC or hAECs pellet filled the chondral defect. There was a good integration between the repair tissue and native cartilage. Type II collagen and aggrecan stainings of repair tissue were slightly positive on the extracellular matrix, and positive inside the cytoplasm of the cells. The safranin O staining expressed the presence of proteoglycans. Finally, type I collagen stainings were weak or totally negative (Figure 1A). We realized an ICRS macroscopic evaluation of cartilage repair to compare both kinds of progenitor cells in the in vitro model. hAMSCs displayed better degree of defect repair, greatest integration to border zone and, in general, higher repair assessment (Figure 1B). Conclusions: We get reduce the area of the defect with quality integration. The morphology of the repair tissue showed a fibrocartilaginous appearance and a high cellularity. hAMSCs showed better results considering the ICRS macroscopic evaluation of cartilage repair.