Inhibition of LPS-induced proinflammatory responses of J774.2 macrophages by immobilized enzymatically tailored pectins.

The surface of an implant device can be modified by immobilizing biological molecules on it to improve its integration into the host tissue. We have previously demonstrated that enzymatically tailored plant pectins are promising nanocoatings for biomaterials. This study investigates whether a coating of modified hairy region (rhamnogalacturonan-I) from apple pectin (MHR-alpha) which has anti-adhesive properties can inhibit the generation of inflammatory mediators by lipopolysaccharide (LPS)-activated macrophages. For that purpose, J774.2 murine macrophages were cultured for 24h on MHR-alpha-coated Petri dishes and tissue culture polystyrene controls, with and without LPS. Cell morphology, cell growth, nitrite and TNF-alpha secretion were studied. The results indicate that MHR-alpha coating inhibits the LPS-induced activation of macrophages.

Fibronectin modulates osteoblast behavior on Nitinol.

We have previously demonstrated that primary rat osteoclasts behave differently when cultured on austenite and martensite Nitinol. In this study, we coated the two phases of Nitinol with plasma fibronectin and studied if this modifies the proliferation and cell cycle of MC3T3-E1 osteoblasts. The influence of the crystalline structure of Nitinol on the remodeling and conformation of fibronectin was also studied. The results on austenite demonstrated that fibronectin was more strongly remodeled and the cells spread better compared with the martensite phase. Interestingly, the conformation of the protein showed no differences between austenite and martensite. In addition, fibronectin improved cell proliferation in both phases, but the effect of fibronectin coating was stronger on the austenite surface. In addition, in both Nitinol phases, the proportion of cells in the G(1) phase was observed to grow in the presence of fibronectin. This could indicate cell differentiation on Nitinol.

Microstructure, mechanical properties and cytocompatibility of stable beta Ti-Mo-Ta sintered alloys.

We have synthesized titanium-based alloys containing molybdenum and tantalum elements by powder metallurgy. The microstructure, the residual porosity and the mechanical properties of the sintered Ti-Mo and Ti-Ta-Mo alloys were investigated by using optical and electronic microscopy, X-ray diffraction, microhardness and compression tests. The cytocompatibility of the different alloys was evaluated by the assessment of bone cell density, migration and adhesion after 14 days incubation. All the alloys present a high ductility and an excellent cytocompatibility, which make these materials useful for medical implants.

Adaptation of rat soleus muscle spindles after 21 days of hindlimb unloading.

Spindle discharges are affected by muscle unloading, and changes in passive stiffness of the muscle-tendon unit may contribute to the changes in spindle solicitation. To test this hypothesis, we determined the spindle sensitivity from electroneurograms of the soleus nerve, and, concomitantly, we measured the incremental passive muscle tension. Both measurements were done from ramp and hold stretches imposed to the soleus muscle after the Achilles tendon was severed. The ratio between the spindle sensitivity and the passive stiffness gave a "spindle efficacy index" (SEI). The experiments were conducted on control rats (C, n = 12) and on rats that had undergone hindlimb unloading (HU, n = 12) for 21 days. The muscle threshold lengths for electroneurogram to discharge (neurogram length, Ln) and for detecting passive tension (slack length, Ls) were determined, and, when these lengths differed, the stretches were imposed at these two initial lengths. The contralateral muscles were used to count muscle spindles and spindle fibers (ATPase staining) and to identify MyHC isoforms by immunostaining. Ln and Ls values were identical for the C muscles, while after HU, Ln was significantly shorter than Ls, which indicated that spindle afferents were more sensitive since they discharged before any passive tension was developed by the soleus muscle. At Ln, spindle sensitivity and passive stiffness did not differ for C and HU muscles. Consequently, when calculated at this relatively short initial muscle length, the SEI was maintained (or even slightly increased) after HU. This held under dynamic conditions (ramp phase of the stretch) and under static conditions (hold phase of the stretch). At Ls, the dynamic and static incremental stiffness values increased significantly after HU. Under dynamic conditions, the spindle sensitivity also increased after HU but to a less degree than incremental stiffness, which led to a significant decrease in SEI. Under static conditions, the spindle sensitivity presented a high increase, and, consequently, SEI was not modified. These functional changes were associated with structural adaptations: HU did not alter the total number of muscle spindles, but the number of spindles containing three nuclear chain fibers increased significantly. The main change in intrafusal MyHC content concerned the slow type I MyHC isoform. In conclusion, after a period of muscle unloading, the spindle discharges were maintained or even enhanced in several experimental conditions. This may be due to a better transmission of the external stretch to muscle spindles through stiffer elastic structures but also to own muscle spindle adaptations which reinforce the spindle sensitivity, notably under static conditions.

Interactions of B16F10 melanoma cells aggregated on a cellulose substrate.

There is evidence that the shape of cells and their contact with a matrix direct the growth and the differentiation of both normal and cancer cells. Cells in 3D culture resemble the in vivo situation more closely than do those in conventional 2D cultures. We have studied the interactions and functions of B16F10 mouse melanoma cells, which spread and grow well on tissue culture polystyrene (tPS), when they were made to aggregate on cellulose-coated Petri dishes (CEL). This aggregation of melanoma cells on CEL was Ca2+ dependent and mediated by N-cadherins. The levels of N-cadherin and beta-catenin transcripts in cells cultured on CEL and tPS were similar, but those on CEL contained less beta-catenin protein. Immunoprecipitation and immunostaining showed that both N-cadherins and beta-catenins were present at the membranes of cells on CEL. Cells proliferated significantly more slowly after 48 h on CEL and the cellulose coating caused most of them to arrest in G1. We also compared the melanin contents and tyrosinase activity of cells on CEL and controls grown on tPS. Melanogenesis was induced in cells aggregated on CEL. A cellulose substrate thus appears to be an outstanding tool for studying cell-cell interactions and cell functions in 3D cultures.

Culture of melanoma cells as aggregates on cellulose substratum.

Cells aggregate on an original cellulose substratum (CEL). This influences the signaling programs of adhering cells. CEL thus appears to be a suitable tool for studying the regulation of cell-substratum and cell-cell interactions.

Development of a beta-type Ti-12Mo-5Ta alloy for biomedical applications: cytocompatibility and metallurgical aspects.

Ti-based biocompatible alloys are especially used for replacing failed hard tissue. Some of the most actively investigated materials for medical implants are the beta-Ti alloys, as they have a low elastic modulus (to inhibit bone resorption). They are alloyed with elements such as Nb, Ta, Zr, Mo, and Fe. We have prepared a new beta-Ti alloy that combines Ti with the non-toxic elements Ta and Mo using a vacuum arc-melting furnace and then annealed at 950 degrees C for one hour. The alloy was finally quenched in water at room temperature. The Ti-12Mo-5Ta alloy was characterised by X-ray diffraction, optical microscopy, SEM and EDS and found to have a body-centred-cubic structure (beta-type). It had a lower Young's modulus (about 74 GPa) than the classical alpha/beta Ti-6Al-4V alloy (120 GPa), while its Vickers hardness remained very high (about 303 HV). This makes it a good compromise for a use as a bone substitute. The cytocompatibility of samples of Ti-12Mo-5Ta and Ti-6Al-4V titanium alloys with various surface roughnesses was assessed in vitro using organotypic cultures of bone tissue and quantitative analyses of cell migration, proliferation and adhesion. Mechanically polished surfaces were prepared to produce unorientated residual polished grooves and cells grew to a particularly high density on the smoother Ti-12Mo-5Ta surface tested.

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.

Comparative particle-induced cytotoxicity toward macrophages and fibroblasts.

The cytotoxicity caused by the debris resulting from wear of prostheses can produce major damage to tissues around the implant. We have compared particle internalization by macrophages and fibroblasts in vitro and analyzed cell death. J774.2 macrophages and L929 fibroblasts were incubated with 0.43 and 2.81 microm alumina particles or 0.45 and 3.53 microm polystyrene (PS) beads. Incubation of J774.2 cells with alumina particles of both sizes and 0.5 and 1.0 mg/ml PS beads significantly decreased cell numbers in a particle concentration-dependent manner. L929 cells were not affected by lower concentrations of 0.43 microm alumina particles (which aggregate at high concentrations) and they internalized 0.45 microm PS beads without any decrease in cell numbers. Particles were more cytotoxic for macrophages than for fibroblasts. Particles caused the size of both types of cells to increase in correlation with cytotoxicity. Trypan blue exclusion and lactate dehydrogenase release showed cell membrane leakage for both types of cells incubated with PS beads for 24 h. Apoptosis was assessed by annexin V-FITC, propidium iodide staining and assay of caspase 3 activity. Macrophage death appeared to depend on both necrosis, caused mainly by 3.53 microm PS beads, and apoptosis, mainly due to 0.45 microm PS beads. The release of the inflammatory cytokine IL-6 appears to be nonlinearly correlated with cytotoxicity. Thus, the size of the internalized particles affects macrophages and fibroblasts differently, and the increase in cell size can be used as a preliminary criterion of particle cytotoxicity in vitro.

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.

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.

Embolic risk of the different stages of carotid bifurcation balloon angioplasty: an experimental study.

PURPOSE: Embolic events during carotid angioplasty are a challenging problem. This experimental study was undertaken to determine the embolic risk after each stage of carotid angioplasty procedure. METHODS: Five ex vivo carotid artery balloon angioplasties were performed on fresh carotid specimens. The carotid specimens were obtained from five patients who underwent an internal carotid artery bypass for stenosis >75%. Before the endovascular maneuvers and after each stage of the procedures, the specimens were flushed with 20 mL of saline solution. Small particulate emboli (diameter, <60 microm) were searched in all the effluents according to the Coulter technique. After this procedure, each effluent was also submitted to scanning electron microscopy. RESULTS: When the stenosis was crossed with the guidewire or the balloon catheter, the number and the mean diameter of embolic particles did not change with three plaques (CP1, CP2, and CP3) and were increased with two plaques (CP4 and CP5). The maximal size of particles was 220 microm (CP5). After balloon angioplasty, the number and the mean diameter of particles increased with CP1, CP2, and CP3. With CP4 and CP5, the number of particles decreased, but their size increased. The maximal size of particles was 1100 microm (CP4). CONCLUSION: Carotid balloon angioplasty generates embolic particles after each stage of the procedure. Techniques of prevention should then be effective from the initial step of the angioplasty procedure, and the selection of patients for carotid angioplasty remains crucial.

Neutral red assay of the cytotoxicity of fluorocarbon-coated polymethylmethacrylate intraocular lenses in vitro.

Polymethylmethacrylate (PMMA) intraocular lenses (IOLs) were coated with Teflon AF, an amorphous, transparent Teflon, to render them highly hydrophobic. Teflon-coated PMMA IOLs were immersed in culture medium for 30 days at 37 degrees C. Four concentrations of the IOL leachables, 2 concentrations of a toxic control (phenol), and complete liquid culture medium (nontoxic control) were incubated for 24 h in a 96-well plate containing confluent L-929 fibroblasts. The cytotoxic effect of each solution on the fibroblasts was quantitatively assessed by measuring the uptake of neutral red by the viable cells. After the extraction of the neutral red using 1% acetic acid-50% ethanol, the optical densities were measured with a microplate reader at 550 nm. Scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) were used to analyze the surfaces of the IOLs. Only the optical densities in the wells containing fibroblasts that had been in contact with the phenol solutions were significantly lower than those in the wells incubated with the nontoxic control solution (p < 0.01). There were no signs of surface alteration by SEM, apart from some crystals on the IOLs. The crystals were composed of Na and Cl, as demonstrated by XPS. Aqueous extractables from the Teflon-coated IOLs produced no cytotoxic effects in the neutral red assay used.

Evaluation of teflon-coated intraocular lenses in an organ culture method.

An amorphous and transparent form of Teflon is proposed as a coating of polymethylmethacrylate (PMMA) intraocular lenses (IOLs), rendering them highly hydrophobic. We used an organ culture method to evaluate cell adhesion, proliferation, and migration on Teflon-coated IOLs. Corneal explants from 14-day-old chicken embryos were placed on a semisolid culture medium and covered with uncoated PMMA (n = 36) and Teflon-coated PMMA (n = 36) IOLs and two controls, Thermanox (n = 84) and latex (n = 36). After incubation (7 days at 37 degrees C), a digital imaging system was used to measure the areas of the cell migration layers on the materials. The cells were then removed with tripsin-ethylenediaminetetraacetic acid and the cells detached at times up to 75 min were counted (Coulter(R) Multisizer System). The values were used to construct a cell disconnecting curve for each material. The areas of cell migration layers on uncoated and Teflon-coated IOLs were significantly different (p <.05). Cell disconnecting curves demonstrated that cells adhered less strongly to Teflon-coated IOLs than to the other materials. This organ culture method demonstrated that the coating of PMMA IOLs with Teflon AF(R) is correlated with antiadhesive and antiproliferative properties.

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.

Effect of the interaction between transforming growth factor beta and erythropoietin on the proliferation of normal erythroid progenitors and leukemic UT-7 cells: action of transforming growth factor beta on the erythropoietin receptor.

The actions of transforming growth factor beta (TGF beta) and erythropoietin (Epo) were studied using normal erythroid progenitors from fetal rat liver and spleen at 18, 19 and 20 days. rhTGF beta 1 inhibited the growth of late BFUe colonies significantly at each age and in both organs in methylcellulose cultures containing 2 U/ml rhEpo. There was no significant inhibition of CFUe proliferation, except for spleen CFUe at 18 days, suggesting different CFUe sensitivities to growth factors at a given fetal age, 18 days, in liver and spleen. The colorimetric MTT assay was used to examine the inhibition of the growth of human leukemic UT-7 cells by TGF beta 1. TGF beta 1 inhibited the proliferation of UT-7 cells in cultures without Epo at 24 h and in cultures with Epo at 24 and 72 h. The specific binding of [125I]Epo to UT-7 surface was decreased by TGF beta 1 without any change in non-specific binding. TGF beta 1 also inhibited the expression of Epo-receptors on UT-7 cells, without changing receptor affinity. The inhibition of hematopoietic progenitor cell growth by TGF beta could involve altering the cell surface expression of growth factor receptors.