Phenotypic analysis of cell surface markers and gene expression of human mesenchymal stem cells and chondrocytes during monolayer expansion.

Both chondrocytes and mensenchymal stem cells (MSCs) are the most used cell sources for cartilage tissue engineering. However, monolayer expansion to obtain sufficient cells leads to a rapid chondrocyte dedifferentiation and a subsequent ancillary reduced ability of MSCs to differentiate into chondrocytes, thus limiting their application in cartilage repair. The aim of this study was to investigate the influence of the monolayer expansion on the immunophenotype and the gene expression profile of both cell types, and to find the appropriate compromise between monolayer expansion and the remaining chondrogenic characteristics. To this end, human chondrocytes, isolated enzymatically from femoral head slice, and human MSCs, derived from bone marrow, were maintained in monolayer culture up to passage 5. The respective expressions of cell surface markers (CD34, CD45, CD73, CD90, CD105, CD166) and several chondrogenic-related genes for each passage (P0-P5) of those cells were then analyzed using flow cytometry and quantitative real-time PCR, respectively. Flow cytometry analyses showed that, during the monolayer expansion, some qualitative and quantitative regulations occur for the expression of cell surface markers. A rapid increase in mRNA expression of type 1 collagen occurs whereas a significant decrease of type 2 collagen and Sox 9 was observed in chondrocytes through the successive passages. On the other hand, the expansion did not induced obvious change in MSCs gene expression. In conclusion, our results suggest that passage 1 might be the up-limit for chondrocytes in order to achieve their subsequent redifferentiation in 3D scaffold. Nevertheless, MSCs could be expanded in monolayer until passage 5 without loosing their undifferentiated phenotypes.

Effect of alginate culture and mechanical stimulation on cartilaginous matrix synthesis of rat dedifferentiated chondrocytes.

To investigate whether the application of alginate culture and mechanical stimulation will improve the synthesis of cartilaginous matrix in dedifferentiated chondrocytes, rat chondrocytes underwent dedifferentiation upon serial monolayer culture up to passage 6, and then were encapsulated in 2% alginate gel and subject to static culture. After 28 days culture in static, the beads were exposed to 48 h of mechanical stimulation with continuous agitation. The sGAG content in alginate bead was measured by alcian blue staining. The expression of collagen protein was detected using immunofluorescence. After 28 days culture in alginate bead, the dedifferentiated chondrocytes remained round in shape and re-synthesized the chondrocyte-specific matrix. Compared with static culture, mechanical stimulation induced statistically increases in the production of glycosaminoglycan (p< or =0.01), as well as in the synthesis of collagen type II protein (p< or =0.05). On the contrary, no positive expression of collagen type I protein was observed at the end of culture. Our results demonstrated that both of alginate culture and mechanical stimulation help to restore chondrocyte phenotype and promotes the synthesis of cartilaginous matrix.

Influence of polyelectrolyte multilayer films on the ICAM-1 expression of endothelial cells.

Recently, the use of polyelectrolyte films has been suggested as a new versatile technique of surface modification aimed at tissue engineering. In the present study, we evaluated the expression of intercellular adhesion molecule (ICAM)-1 of endothelial cells (ECs) seeded on two types of polyelectrolyte multilayer films either terminated by poly(D-lysine) (PDL) or poly(allylamine hydrochloride) (PAH). This work showed that chemical stimulations with tumor necrosis factor (TNF)-alpha induced the ICAM-1 expression of ECs differently depending largely on the film architecture employed. Compared with PAH-ending films, the PDL-ending ones upregulated the ICAM-1 expression of the ECs after a prolonged exposition to TNF-alpha, rendering this film type less favorable in tissue engineering. Cytochalasin D (an F-actin disrupting agent) showed the involvement of the cytoskeleton in the upregulation of ICAM-1 for cells deposited on films terminated by PDL. The PAH-ending films did not perturb the ICAM-1 expression of ECs and might thus enhance the seeding of ECs in vascular engineering.

Effect of cyclic stretching and foetal bovine serum (FBS) on proliferation and extra cellular matrix synthesis of fibroblast.

It is well known today that mechanical forces are one of the important factors that induce a variety of cellular responses including morphological changes, protein synthesis, and gene expression and which are involve in tissue remodelling. We studied the effect of uniaxial cyclic stretching on the proliferation, collagens, and tenascin C mRNA expression of fibroblasts under different concentrations of foetal bovine serum. Proliferation was studied by cell cycle analysis, mRNA expression of collagen and tenascin C was studied by RT-PCR. Human fibroblasts were grown in silicon sheet coated with 1% gelatin. Cyclic stretching (5% elongation) was applied at 0.5 Hz (30 cycle/min), for 24 h with two concentrations of the serum (0.5%, 10% FBS). We showed that stretching enhances the synthesis of collagen and tenascin C, but do not act on the proliferation. In contrast, higher concentration of serum enhances the proliferation. These findings suggest that both mechanical stretching and serum concentration can modulate proliferation and extra cellular matrix synthesis in human fibroblasts.

Endothelial cell--interactions with polyelectrolyte multilayer films.

The seeding of endothelial cells (ECs) on biomaterial surfaces became a major challenge, allowing to improve the non-thrombogenic properties of these surfaces. Recently, the use of polyelectrolyte films has been suggested as a new versatile technique of surface modification aimed at tissue engineering. In this study, we evaluate the adhesion properties of ECs on two types of polyelectrolyte films ending either by poly(D-lysine) (PDL), or poly(allylamine hydrochloride) (PAH), and compared them to data obtained on PDL or PAH monolayers, glass and fibronectin (Fn)-coated glass. ECs seeded on polyelectrolyte films showed a good morphology, allowing ECs to resist physiological shear stress better compared to ECs seeded on glass or Fn. The expression of beta1 integrins was slightly lower on polyelectrolyte films than on control surfaces. However, the phosphorylation of focal adhesion kinase, involved in the transduction of adhesion signal, was not modified on PAH ending films compared to control surfaces; whereas it became lower on PDL ending films. Finally, PAH ending films improve strongly ECs adhesion without disturbing the adhesion mechanism, necessary for the development of a new endothelium. These types of films or similar build-ups could thus be used in the future as a way to modify surfaces for vascular tissue engineering.

[Chondrocyte mecanobiology. Application in cartilage tissue engineering]. / Mécanobiologie du chondrocyte. Application à l'ingénierie du cartilage.

Cartilage is a hydrated connective tissue that withstands and distributes mechanical forces within joints. Chondrocytes utilize mechanical signals to maintain cartilaginous tissue homeostasis. They regulate their metabolic activity through complex biological and biophysical interactions with the extracellular matrix (ECM). Some mechanotransduction mechanisms are known, while many others no doubt remain to be discovered. Various aspects of chondrocyte mechanobiology have been applied to tissue engineering, with the creation of replacement tissue in vitro from bioresorbable or non-bioresorbable scaffolds and harvested cells. The tissues are maintained in a near-physiologic mechanical and biochemical environment. This paper is an overview of both chondrocyte mechanobiology and cartilage tissue engineering

Interactions of mechanotransduction pathways.

Integrins may serve as mechanosensors in endothelial cells (ECs): shear stress causes integrin-Shc association, assembly of the signaling complex and then leads to JNK activation. Flow also mediates selective and cell-specific alterations in vascular cell G-protein expression that correlate with changes in cell-signalling, G-protein functionality and modulate Ca2+ concentration. In this study, we explored the cross-talks between EC membrane mechanosensors, such as integrins, ion channels, and G-proteins in shear stress-induced signal transduction by their specific inhibition. Confluent monolayer of bovine aortic endothelial cells (BAECs) were incubated with or without specific inhibitors prior to shearing experiments. Our results showed an attenuation of integrin-Shc association under shear stress with RGD, and with PTX, but not with BAPTA/AM. The inhibitions of shear-activated JNK are similar for RGD and PTX. However, unlike for integrin association, the chelation of calcium reduced JNK activation. These results provide several lines of evidence of the interactions between different mechanosensors in ECs. First, integrin-Shc association required cell attachment and G-protein activity, but not intracellular calcium. Second, shear-induced JNK activation is regulated by multiple mechano-sensing mechanisms such as integrin, G-protein and calcium concentration.

Changes of vasodilator-stimulated phosphoprotein (VASP) and its phosphorylation in endothelial cells exposed to laminar flow.

Vasodilator-stimulated phosphoprotein (VASP), an actin filaments-associated protein expressed mainly in focal adhesions and dynamic membrane regions of endothelial cells (ECs), serves as a substrate for cAMP and cGMP-dependent protein kinases. In this work, we studied the effect of laminar shear stress in vitro on the location and expression of the VASP as well as its phosphorylation associated with actin reorganisation in human umbilical endothelial cells (HUVECs). The distributions of VASP and microfilaments were observed by a fluorescent double staining. The level of VASP expression in cells was quantified by western blot. Experiments showed that, after exposure to a shear stress of 10 dyn/cm(2) for 24 h, besides the elongation and orientation of the cells, and spots of VASP were found along thick stress fibres, particularly at their two extremities. Western blot data showed conversions up to 11.4% and 8.9% of the 46 kD non-phosphorylated form VASP to its 50 kD phosphorylated form after exposures of 1 h and 4 h respectively to the same shear stress. The shear stress of 15 dyn/cm(2) had qualitatively the same effect as that of 10 dyn/cm(2), but with a lower magnitude. However, the shear stress of 2 dyn/cm(2) had much slower and weaker effects on the same tendency. These results suggest that a laminar shear stress can induce VASP translocation and phosphorylation that proceed actin filaments rearrangement along with the flow direction in HUVECs.

[Changes in the VASP expression feature of endothelial cells under steady laminar flow].

To investigate the effects of physiological shear stress on the vasodilator-stimulated phosphoprotein (VASP) location and expression changes associated with actin remodeling, we isolated and cultured human umbilical endothelial cells(HUVECs) with trypsin digestion. A parallel-plated flow chamber device was used to create laminar shear stress in vitro. The distributions of VASP and microfilaments in cells were observed by double staining with Alexa488 and rhodamine-phalloidin. Changes of VASP expression and phosphorylation were analyzed quantitatively with Western blot before and after exposure to shear flow for different times. We found that, under a shear stress of 10 dyn/cm2, HUVECs were elongated and oriented gradually to the flow direction. Microfilaments were recruited and oriented also to the flow direction with thicker VASP, specially targeted to their extremities. Western blotting data showed a rapid phosphorylation of VASP, and an increase of total VASP expression which peaked at 2 h (2 folds), then recovered until 8 h, followed by a slow increase again. These results suggest that VASP is a potential component which participates in the regulation of cell actin remodelling induced by shear flow.

Optimisation of biochemical condition and substrates in vitro for tissue engineering of ligament.

In this work, we analysed the effect of growth factors on in vitro cell proliferation and collagens synthesis by fibroblasts cultured for 72 h on different substrates (silicon sheet with or without 1% gelatin, and glass as control surface) for ligament tissue engineering. A human fibroblast cell line (CRL-2703) was used. The synthesis of type I and type III collagens were evaluated qualitatively and quantitatively by RT-PCR and confocal microscopy, respectively. Cell proliferation was evaluated by two methods: (1) MTT assay (2) cell cycle analysis. It was found that PDGF-AB stimulate the proliferation of fibroblast cultured on gelatin coated silicon sheet in dose dependant manner with a maximum effect at 10 ng ml(-1). The exogenous TGF-beta1 induced the expression of type I and type III collagens in a dose and substrate-dependant manner. We deduce from this work that biochemical conditions and substrates have an important impact for optimisation of the tissue neo synthesis.

Polyelectrolyte multilayers and degradable polymer layers as multicompartment films.

Polyelectrolyte multilayers are now a well established concept with numerous potential applications in particular as biomaterial coatings. To timely control the biological activity of cells in contact with a substrate, multicompartment films made of different polyelectrolyte multilayers deposited sequentially on the solid substrate constitute a promising new approach. In a first paper (Langmuir 2004, 20, 7298) we showed that such multicompartment films can be designed by alternating exponentially growing polyelectrolyte multilayers acting as reservoirs and linearly growing ones acting as barriers. In the present study, we first demonstrate however that these barriers composed of synthetic polyelectrolytes are not degraded despite the presence of phagocytic cells. We propose an alternative approach where exponentially growing poly(L-lysine)/hyaluronic acid (PLL/HA) multilayers, used as reservoirs, are alternated with biodegradable polymer layers consisting in poly(lactic-co-glycolic acid) (PLGA) and acting as barriers for PLL chains that diffuse within the PLL/HA reservoirs. We first show that these PLGA layers can be deposited alternatively with PLL/HA multilayers leading to polyelectrolyte multilayer/hydrolyzable polymeric layer films and acting as a reservoirs/barriers system. Bone marrow cells seeded on these films ending by a PLL/HA reservoir rapidly degrade it and internalize the PLL chains confined in this reservoir. Then the cells degraded locally the PLGA barrier and internalize the PLL localized in a lower (PLL/HA) compartment after 5 days of seeding. By changing the thickness of the PLGA layer, we hope to be able to tune the time delay of degradation. Such mixed architectures made of polyelectrolyte multilayers and hydrolyzable polymeric layers could act as coatings allowing us to induce a time scheduled cascade of biological activities. We are currently working on the use of comparable films with compartments filled by proteins or peptides and in which the degradation of the barriers results from a hydrolysis over tunable time scales.

[Changes of VASP in shear stress induced cytoskeleton reorganization in endothelial cells].

AIM: To investigate the effects of different level of laminar shear stresses on the vasodilator-stimulated phosphoprotein (VASP) location and expression changes associated with actin reorganization and it's mechanism. METHODS: A parallel-plate flow chamber device was used to create laminar shear stress in vitro on cultured human umbilical endothelial cells (HUVECs). The distribution of VASP and microfilament were observed by double immunofluorescence staining. RT-PCR was used to test VASP mRNA level, while VASP parameters were analyzed quantitatively with Western blot. RESULTS: After exposure to a flow of 10 dyn/cm2 flow for 24 h, HUVECs were elongated and oriented gradually to the direction of the flow. The microfilaments were recruited and oriented to the direction of flow with thicker VASP, specially targeted to the ends of stress fibres. RT-PCR result indicated shear could induce VASP mRNA increase. Western blotting data showed a dynamic reversible phosphorylation of VASP during 24 h, and total VASP expression increased rapidly, peaked at 2 h, then recovered at 8h followed by a slow increase again. H89, a cAMP inhibitor could inhibit shear induced VASP expression increase and phosphorylation. CONCLUSION: VASP is an potential important component which participates in the regulation of cell cytoskeleton reorganization and morphology modification induced by shear flow via a cAMP/cAK pathway.

Multicompartment films made of alternate polyelectrolyte multilayers of exponential and linear growth.

The layer by layer deposition process of polyelectrolytes is used to construct films equipped with several compartments containing "free polyelectrolytes". Each compartment corresponds to a stratum of an exponentially growing polyelectrolyte multilayer film, and two consecutive compartments are separated by a stratum composed of a linearly growing multilayer that acts as a barrier preventing polyelectrolyte diffusion from one compartment to another. We use hyaluronic acid/poly(L-lysine) as the system to build the compartments and the poly(styrene sulfonate)/poly(allylamine) system for the barrier. Using confocal microscopy, it is shown that poly(L-lysine) diffuses only within the compartment in which it was initially introduced during the film construction and is thus unable to cross the barriers. Using fluorescein isothiocyanate as a pH indicator, it is also shown that although poly(styrene sulfonate)/poly(allylamine) multilayers act as a barrier for polyelectrolytes, they do not prevent proton diffusion through the film. Such films open the route for multiple functionalization of biomaterial coatings.