Adhesion, intracellular signalling and osteogenic differentiation of mesenchymal progenitor cells and preosteoblasts on poly(epsilon)caprolactone films functionalized by peptides derived from fibronectin and/or BMP-9.

Biomaterials that can control the behaviour of stem cells play a major role in regenerative medicine and tissue engineering. We previously showed that poly(epsilon)caprolactone (PCL) films functionalized with adhesive peptides containing sequences of both cell binding domain (RGD) and synergistic site (PHSRN) of the fibronectin (pFibro) enhanced the osteoblastic commitment of C3H10T1/2 mesenchymal progenitor cells (C3H10T1/2 cells) induced by soluble BMP-9 or its derived peptide SpBMP-9. Here, the effect of PCL films functionalized with pFibro and/or SpBMP-9 or its negative peptide NSpBMP-9 on adhesion and intracellular signalling of C3H10T1/2 cells was determined. The differentiation of adherent C3H10T1/2 cells and MC3T3-E1 preosteoblasts into osteoblasts was also analysed with or without IGF-2, since this growth factor can favour the osteoblastic differentiation induced by BMP-9. We found that pFibro and SpBMP-9 co-functionalization on PCL films promoted the adhesion of C3H10T1/2 cells with well-organized focal adhesion points and FAK activation. In these mesenchymal progenitor cells, PCL-SpBMP-9 and PCL-pFibro/SpBMP-9 induced the activation and nuclear translocation of Smad 1/5 after 4 h, and enhanced the protein expression of RUNX2 (3 d) and alkaline phosphatase (ALP) activity (7 d), two markers of osteoblast differentiation. No PPARγ, a marker of adipogenic differentiation, was detected. C3H10T1/2 cells attached to PCL-SpBMP-9 also contained more SOX9, a marker of chondroblastic lineage, compared with other experimental conditions. The use of inactive peptides NSpBMP-9 confirmed the specificity and effectiveness of SpBMP-9 on cell adhesion, intracellular signalling and osteoblastic differentiation. Adding IGF-2 only significantly improved the differentiation of MC3T3-E1 preosteoblasts into osteoblasts as shown by the increase in gene expression encoding Osterix (mRNA Sp7) and ALP (mRNA Alpl), probably because of the lack of serum in the medium. Therefore, material surface co-functionalized with pFibro and SpBMP-9 could be most useful for developing scaffolds with both osseointegrative and osteoinductive properties for bone application and tissue engineering strategy when combined with IGF-2 in serum free medium.

The effect of substrate bulk stiffness on focal and fibrillar adhesion formation in human abdominal aortic endothelial cells.

Endothelial cell (EC) dysfunction contributes to atherosclerosis, which is associated with arterial stiffening and fibronectin (FN) deposition, by ECs and smooth muscle cells (SMCs). The effect of stiffness on the EC/FN interaction and fibrillar adhesion formation has been poorly studied. An in vitro model was prepared that included FN-coated polydimethylsiloxane (PDMS) films with similar hydrophobicity and roughness but distinct Young's modulus values, mimicking healthy (1.0 MPa) and atherosclerotic (2.8 MPa) arteries. Human aortic abdominal endothelial cells (HAAECs) seeded on 1.0 MPa PDMS films spread over time and reached their maximum surface area faster than on 2.8 MPa PDMS films. In addition, HAAECs appeared to organize focal adhesion more rapidly on 1.0 MPa PDMS films, despite the similar cell binding domain accessibility to adsorbed FN. Interestingly, we also observed up to a ~5-fold increase in the percentage of HAAECs that had a well-developed fibrillar adhesion on 1.0 MPa compared to 2.8 MPa PDMS films as verified by integrin α5 subunits, tensin, and FN staining. This variation did not affect EC migration. These results suggest that there are favourable conditions for FN matrix assembly by ECs in early atherosclerosis rather than at advanced stages. Our in vitro model will therefore be helpful to understand the influence of bulk stiffness on cells involved in atherosclerosis.

High-purity biodiesel production from microalgae and added-value lipid extraction: a new process.

A new process was tested in order to produce and purify biodiesel from microalgae lipids and to recover unsaponifiable (added-value) lipids. This process is a two-step biodiesel production including a saponification reaction step followed by an esterification reaction step. The process includes a recovery of the unsaponified lipids between both reaction steps. Among the conditions tested, the following conditions were found to be the best: temperature for both steps (90 °C), saponification time (30 min), esterification time (30 min), sulfuric acid/potassium hydroxide (1.21, w/w), and methanol-lipid ratio (13.3 mL/g). Under these conditions, the fatty acid methyl ester (FAME) yield and the biodiesel purity were, respectively, 32% (g FAME/g lipid) and 77% (g FAME/g biodiesel). This study also showed that the two-step biodiesel process allows a FAME mass composition rich in palmitate (27.9-29.4 wt%), palmitoleate (24.9-26.0 wt%), elaidate (14.8-15.2 wt%), and myristate (12.1-13.0 wt%).

Effect of BMP-9 and its derived peptide on the differentiation of human white preadipocytes.

Several studies have shown that bone morphogenetic proteins (BMPs) can influence adipogenic and osteogenic cell lineages. We have shown that a peptide derived from BMP-9 (pBMP-9) at 400 ng/ml inhibits the proliferation of preosteoblasts and induces differentiation. We have now determined the effects of pBMP-9 (400 ng/ml) and equimolar concentrations of BMP-2 (100 ng/ml), BMP-9 (84.6 ng/ml) and pBMP-9 (9.04 ng/ml) on human white preadipocytes (HWP). pBMP-9 dose dependently reduced the proliferation of HWP without affecting the number of apoptotic cells. Incubation of the cells for 1 h with BMP-2, BMP-9 or pBMP-9 activated the Smad1/5/8 pathway, while incubation for 7 days in adipocyte differentiation (AD) serum-free medium containing ciglitazone and equimolar concentrations of BMP-2, BMP-9 or pBMP-9 enhanced the levels of mRNA of the adipogenic markers aP2 and adipoQ and increased the number of lipid vesicles. Thus, pBMP-9, like BMP-9, can increase the AD of HWP in AD serum-free medium.

Cell responses to bone morphogenetic proteins and peptides derived from them: biomedical applications and limitations.

The bone morphogenetic proteins (BMPs) are cytokines of the transforming growth factor beta family. Some BMPs such as BMP-2 and BMP-7 play a major role in the development of the skeleton and the maintenance of homeostasis during bone remodelling. To date, only BMP-2 and BMP-7 have been approved by the Food and Drug Administration for specific orthopaedic applications. However, due to BMP cost, peptides derived from their knuckle epitope with osteogenic properties have been developed. BMPs are involved in many other biological events, including embryogenesis, angiogenesis and cancer. BMPs therefore have great biomedical potential as osteogenic factors and as anti-cancer agents. This review focuses on the use of BMPs and their derived peptides in biomedical delivery systems and gene therapy.

Wettability of substrata controls cell-substrate and cell-cell adhesions.

The maintenance of endothelial cell (EC) monolayer architecture requires stable adhesions not only between neighboring cells but also between cells and the extracellular matrix. While the influence of biomaterials surface wettability on cell-substratum adhesion is rather well studied, its impact on cell-cell cohesion has not been extensively investigated. In the present study a model system consisting of hydrophilic and hydrophobic glass pre-coated with fibronectin and fibrinogen was used to study the influence of surface wettability on both types of cell adhesions. It was demonstrated that the substrate wettability controls the adhesion and cytoskeletal organization of endothelial cells, which has an impact on the subsequent ability of cells to establish stable cell-cell cohesions. These effects were related to the accessibility of specific domains of the adsorbed proteins. While the hydrophobic substratum promoted cell-cell cohesion, on hydrophilic substrata cell-substrate adhesion was dominant. In addition, evidence for an influence of surface wettability on the cross talk between integrins and cadherins was found.

Differentiation of preosteoblasts using a delivery system with BMPs and bioactive glass microspheres.

Bone morphogenetic proteins (BMPs) and 45S5 Bioglass microspheres (bioactive GM) can increase the differentiation of osteoblasts. Recombinant human BMP-2 (rhBMP-2) is presently the BMP most frequently used in delivery systems and it has already been used in clinical bone healing studies. We have developed a delivery system that combines a collagen Type I gel, BMP and bioactive GM. Since BMP-9 seems to be more osteogenic than BMP-2, we compared the differentiation of MC3T3-E1 preosteoblasts induced by our delivery system containing either a peptide derived from BMP-9 (pBMP-9), or rhBMP-2, both at 100 ng/mL. After 5 days, alkaline phosphatase staining showed that pBMP-9 induced more differentiation than rhBMP-2 in all experimental conditions. Also, bioactive GM increased this BMP effect. Since preosteoblasts secreted matrix metalloproteinases (MMPs) that can degrade collagen, we then studied the influence of the delivery system on MMPs production. We observed that MMP-2 was the major MMP involved in all experimental conditions. In addition, pBMP-9 with bioactive GM generated less MMP-2 than did rhBMP-2 on days 3 and 5. Thus, a delivery system using collagen Type I gel with pBMP-9 and bioactive GM seems to be a promising system for bone regeneration.

Self-assembled monolayers with different terminating groups as model substrates for cell adhesion studies.

Cell shapes induced by cell-substratum interactions are linked with proliferation, differentiation or apoptosis of cells. To clarify the relevance of specific surface characteristics, we applied self-assembled monolayers (SAM) of alkyl silanes exhibiting a variety of terminating functional groups. We first characterised the SAMs on glass or silicon wafers by measuring wettability, layer thickness and roughness. Water contact angle data revealed that methyl (CH(3)), bromine (Br), and vinyl (CH=CH(2)) groups lead to hydrophobic surfaces, while amine (NH(2)) and carboxyl (COOH) functions lead to moderately wettable surfaces, and polyethylene glycol (PEG) and hydroxyl (OH) groups created wettable substrata. The surfaces were found to be molecular smooth except for one type of NH(2) surface. The SDS-PAGE analysis of proteins adsorbed from bovine serum to the SAMs showed less protein adsorption to PEG and OH than to CH(3), NH(2) and COOH. Immunoblotting revealed that a key component of adsorbed proteins is vitronectin while fibronectin was not detectable. The interaction of human fibroblasts with CH(3), PEG and OH terminated SAMs was similarly weak while strong attachment, spreading, fibronectin matrix formation and growth were observed on COOH and NH(2). The strong interaction of fibroblasts with the latter SAMs was linked to an enhanced activity of integrins as observed after antibody-tagging of living cells.

Gap junction communication between cells aggregated on a cellulose-coated polystyrene: influence of connexin 43 phosphorylation.

The appropriate functioning of tissues and organ systems depends on intercellular communication such as gap junctions formed by connexin (Cx) protein channels between adjacent cells. We have previously shown that Swiss 3T3 cells aggregated on hydrophilic cellulose substratum Cuprophan (CU) establish short linear gap junctions composed of Cx 43 in cell surface plaques. This phenomenon seems to depend on the high intracellular cyclic AMP (cAMP) concentration triggered by attachment of the cells to CU. We have now used a cellulose-coated polystyrene inducing the same cell behaviour to analyse the gap junction communication between aggregated cells. The transfer of the dye Lucifer Yellow (LY) between cells showed that cells aggregated on cellulose substratum rapidly (within 90 min) establish functional gap junctions. Inhibitors of cAMP protein kinase (PKI) or protein kinase C (GF109203X) both inhibited the diffusion of LY between neighbouring cells. Western blot analysis showed that this change in permeability was correlated with a decrease in Cx 43 phosphorylation. Thus, cellulose substrata seem to induce cell-cell communication through Cx 43 phosphorylation modulated by PKA and PKC. To understand the mechanisms by which a substratum regulates gap junctional communication is critically important for the emerging fields of tissue engineering and biohybrid devices.

Cyclic AMP-dependent cell shape changes induced by mechanical forces.

Biomaterials used in some biomedical devices are exposed to flow of physiological fluids. The flow-induced forces may influence the morphological and the biochemical responses of adhering cells. The objective of this work is to examine the capacity of a mechanical stress to cause changes in cell/substratum and cell/cell interactions via the second messenger cAMP pathway (cyclic Adenosine Monophosphate). Cyclic AMP is known to modulate cell shape, cell adhesion and intercellular communication in static conditions. A specially designed flow chamber was used to analyze the responses of mouse 3T3 fibroblasts spread on biocompatible substrata and submitted to controlled shear stresses. A 1.1-Pa shear stress induced: cell rounding, disruption of vitronectin receptors clusters and clustering of connexins 43 at cell-cell apposition points. These cell responses were cAMP-dependent. These investigations should help provide a better understanding of the early biochemical events triggered by mechanical forces.

Cyclic AMP--dependent aggregation of Swiss 3T3 cells on a cellulose substratum (Cuprophan) and decreased cell membrane Rho A.

Cell surface integrin receptors and Rho family GTPases function together to mediate adhesion-dependent events in cells. We have shown that the attachment of Swiss 3T3 cells to a cellulose substratum (Cuprophan, CU) activates adenylyl cyclase, which catalyses cyclic AMP (cAMP) production. CU adsorbs vitronectin poorly, prevents cell spreading and causes cells to aggregate. By contrast, spread cells on polystyrene (PS) contain low cAMP concentrations. We have now investigated the shift between integrin signalling-Rho A and the cAMP pathway. CU did not support the formation of focal contacts and stress fibres. The plasma membranes of cells on CU had less Rho A than those of cells on PS. Also, blocking vitronectin (VN) or fibronectin (FN)-integrin receptors with echistatin, which activates cAMP production, decreased Rho A in the plasma membrane of cells attached to PS. But adsorption of VN or FN onto CU, which limits the production of the cAMP, increased the cell membrane Rho A. Adding an inhibitor of cAMP-dependent protein kinase PKA to the medium also increased the plasma membrane Rho A in aggregated cells attached to CU. These results highlight the importance of cAMP, generated by cell attachment to substratum, as a gating element in integrin-Rho A signalling.

Organization of cyclic AMP-dependent connexin 43 in Swiss 3T3 cells attached to a cellulose substratum.

We have previously shown that the adenylyl cyclase, which produces cyclic AMP (cAMP) in Swiss 3T3 cells, is activated by their attachment to a cellulose substratum (Cuprophan, CU). This substratum adsorbs vitronectin poorly, prevents cell spreading and causes them to aggregate. By contrast, cells spread out on polystyrene and contain low concentrations of cAMP. We have found that Connexin 43 (Cx 43) gap junction plaques are involved in this cell aggregation. MDL 12330 A, a specific inhibitor of adenylyl cyclase, prevented cell aggregation on CU and abolished Cx 43 channel clustering. But forskolin, a direct activator of adenylyl cyclase, and SBr cAMP, a cell-permeable analogue of cAMP, caused Cx 43 channel clustering in cells attached to polystyrene. Hence, Cx 43 channel clustering is regulated by cAMP in Swiss 3T3 cells. In addition, neither brefeldin A nor monensin (inhibitors of transit through the endoplasmic reticulum and Golgi apparatus), abolished Cx 43 channel clustering in cells aggregated on CU. Thus, the Cx 43 that form clusters in cells attached to CU are not dependent upon the trafficking of Cx 43 from intracellular storage sites, but are probably reorganised from the plasma membrane.

Accumulation of cyclic AMP in Swiss 3T3 cells adhering to a cellulose biomaterial substratum through interaction with adenylyl cyclase.

Controlling cell shape induced by cell-substrata interaction appears of prime importance to influence subsequent biological processes such as cell migration, proliferation, differentiation or apoptosis. Studies on Swiss 3T3 fibroblasts have recently provided evidence that cell spreading is mediated by integrins and RhoA. Our previous studies showed that on Cuprophan, a cellulose membrane (CU) to which vitronectin adhesive protein is poorly adsorbed, Swiss 3T3 cells are rounded and undergo cAMP-dependent aggregation. In contrast, on a polyacrylonitrile membrane (AN69) that favours the adsorption of vitronectin and fibronectin, cells spread out and contain low concentrations of cAM P. We have now examined the parts played by the three components in the cAMP pathway (receptors, G-proteins and adenylyl cyclase itself) in cAM P-dependent cell aggregation on CU. Experiments with intact cells showed no interaction between the CU and receptors, or between the CU and G-proteins. Assays on membrane preparations plus the Mn-ATP substrate, which uncouples G-proteins and adenylyl cyclase, demonstrated that activation of the cAMP pathway by CU depends primarily on the catalytic activity of the adenylyl cyclase. These investigations provide essential data for the development of biomaterials that favour cell functionality.

Activation of the cyclic AMP pathway in cells adhering to biomaterials: regulation by vitronectin- and fibronectin-integrin binding.

Our previous studies have shown that cells adhering to biomaterials in serum-free conditions increase their content of cyclic AMP (cAMP) and become aggregated. In cells on an acrylonitrile membrane (AN69), these biochemical and morphological changes are prevented by adding 10% foetal calf serum (FCS) to the medium; cells on the cellulose membrane Cuprophan (CU) remain unaffected. The present study examines the roles of vitronectin (VN)- and/or fibronectin (FN)-integrin binding in this inhibition. Competitively blocking VN- and FN-receptors with echistatin increased intracellular cAMP significantly and caused cells on AN69 to aggregate, but did not modify cAMP-dependent cell aggregation on CU. VN or FN adsorbed onto CU also inhibited cAMP production by attached cells and prevented their aggregation, whereas adsorbed BSA had no effect. Therefore, the binding of VN or FN to cell-surface integrins seems to limit the activation of the cAMP pathway initiated by the substratum itself.

cAMP levels in cells attached to AN69 and Cuprophan: cAMP dependence of cell aggregation and the influence of serum.

We have examined the link between the aggregation or spreading of cells adhering to substrata of differing biocompatibility and activation of the cyclic AMP (cAMP) pathway. We compared the rate at which the Mouse Swiss 3T3 fibroblasts attached to Cuprophan (CU), AN69 and a control plastic in the presence and absence of foetal calf serum (FCS). Serum had no effect on the kinetics of cell attachment to CU or AN69. Cells incubated in culture medium containing 10% FCS aggregated on CU, whereas they spread on AN69 and plastic. Aggregated cells contained significantly higher concentrations of cAMP than cells spreading, and aggregation was prevented by treatment with miconazole, an inhibitor of adenylyl cyclase. cAMP-dependent cell aggregation occurred on all three substrata in serum-free medium, suggesting that proteins adsorbed onto AN69 and plastic in the presence of serum helped protect the cells. Far less serum protein was adsorbed onto CU than onto AN69 or plastic, consistent with the similar increases in cAMP in cells attached to CU with or without serum.

Cyclic AMP in cells adhering to bioincompatible (Cuprophan) and biocompatible (AN69) substrates.

The processing of signals produced when cells contact biomaterials was examined. Of the several possible pathways, this study focuses on the amount of cAMP that accumulated in NIH 3T3 cells during the first 45 min after the cells contacted the bioincompatible membrane Cuprophan (CU) and the biocompatible membrane AN69. The cells that adhered to CU contained more cAMP than those that attached to AN69. This might be because the cells did not spread but remained rounded up under scanning electron microscopy. There was no increase in cAMP in the cells that did not adhere to CU. The cAMP-modulating agents, forskolin and isoproterenol, were used to assess the cAMP-generating capacity of adenylylcyclase in cells adhering to CU and AN69. This capacity was not affected by a high concentration (100 microM) of forskolin. Isoproterenol had no effect on the cAMP content of the cells, demonstrating that beta adrenergic receptors are not implicated in the activation of cAMP production by membranes. The bioincompatibility of CU seems to be responsible for the greater amount of cAMP in adherent cells, and this parameter could provide an index for assessing biocompatibility.