Mathilde Hindié

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 fibroblastsin vitro and analyzed cell death. J774.2 macrophages and L929 fibroblasts were incubated with 0.43 and 2.81 μm alumina particles or 0.45 and 3.53 μm 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 μm alumina particles (which aggregate at high concentrations) and they internalized 0.45 μm 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 μm PS beads, and apoptosis, mainly due to 0.45 μm 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 cytotoxicityin vitro.

Nanofilm Biomaterials: Localized Cross-Linking To Optimize Mechanical Rigidity and Bioactivity

Nanofilm biomaterials, formed by the layer-by-layer assembly of charged macromolecules, are important systems for a variety of cell-contacting biomedical and biotechnological applications. Mechanical rigidity and bioactivity are two key film properties influencing the behavior of contacting cells. Increased rigidity tends to improve cells attachment, and films may be rendered bioactive through the incorporation of proteins, peptides, or drugs. A key challenge is to realize films that are simultaneously rigid and bioactive. Chemical cross-linking of the polymer framework--the standard means of increasing a films rigidity--can diminish bioactivity through deactivation or isolation of embedded biomolecules or inhibition of film biodegradation. We present here a strategy to decouple mechanical rigidity and bioactivity, potentially enabling nanofilm biomaterials that are both mechanically rigid and bioactive. Our idea is to selectively cross-link the outer region of the film, resulting in a rigid outer skin to promote cell attachment, while leaving the film interior (with any embedded bioactive species) unaffected. We propose an approach whereby an N-hydroxysulfosuccinimide (sulfo-NHS) activated poly(L-glutamic acid) is added as the terminal layer of a multilayer film and forms (covalent) amide bonds with amino groups of poly(L-lysine) placed previously within the film. We characterize film assembly and cross-linking extent via quartz crystal microbalance with dissipation monitoring (QCMD), Fourier transform infrared spectroscopy in attenuated total reflection mode (FTIR-ATR), and laser scanning confocal microscopy (LSCM) and measure the attachment and metabolic activity of preosteoblastic MC3T3-E1 cells. We show cross-linking to occur primarily at the film surface and the subsequent cell attachment and metabolic activity to be enhanced compared to native films. Our method appears promising as a means to realize films that are simultaneously mechanically rigid and bioactive.

Medical-grade silicone induces release of proinflammatory cytokines in peripheral blood mononuclear cells without activating T cells

For more than 40 years, silicone implants had been employed in aesthetic, cosmetic medicine, and plastic surgery. Although adverse reactions produced by these products are rare, cases of immuno-mediated reactions have been reported. To evaluate the aspects of immuno-reactivity to medical-grade silicone dermal filler, peripheral blood mononuclear cells (PBMC) of 39 individuals were studied. PBMC used include individuals with silicone injection-related delayed adverse reactions, with silicone injections, and healthy control. Silicone induced production of TNF-alpha and IL-6 in all three groups. Notably, elevated production of IL-6 was observed in nonstimulated PBMC and also the percentage of CD4(+)CD69(+) T cells was higher in PHA-stimulated PBMC from individuals with silicone injection-related adverse reactions when compared with other two groups. However, IFN-gamma was not released in silicone-stimulated or silicone+LPS-stimulated PBMC from any group and no production of IL-2 was measured indicating no proliferative response of PBMC. Subsequently, no CD4(+)CD69(+) T cells were observed in these conditions. Finally, the inflammatory response in silicone-stimulated cultures of monocyte-derived macrophages with autologous lymphocytes is lesser than that observed in PBMC. In conclusion, silicone induces a release of proinflammatory cytokines but does not act as a polyclonal activator of CD4(+) T cells. Thus, silicone is mounting an immune response in individuals with silicone-related adverse effects but is not silicone antigen-dependent.

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 β‐catenin transcripts in cells cultured on CEL and tPS were similar, but those on CEL contained less β‐catenin protein. Immunoprecipitation and immunostaining showed that both N‐cadherins and β‐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.

Pre-osteoblasts on poly(l-lactic acid) and silicon oxide: Influence of fibronectin and albumin adsorption

Cell adhesion and subsequent viability are critical initial steps in biomaterial-tissue integration and are strongly dependent on the material properties and the presence of matrix proteins. In the present study MC3T3-E1 osteoblast-like cell behavior on silicon oxide (SO) and poly(L-lactic acid) (PLLA) substrates has been examined, with a focus on the influence of the adhesive protein fibronectin and the non-adhesive protein albumin adsorbed on the substrates. Quartz crystal microgravimetry showed adsorption of fibronectin and albumin to be nearly identical on SO and PLLA. Subsequent exposure a previously adsorbed fibronectin layer to albumin decreased the rigidity of the adsorbed layer without any measurable increase in adsorbed mass. Cell adhesion and spreading were significantly enhanced on both SO and PLLA substrates coated with fibronectin or with fibronectin and albumin, compared with uncoated or albumin-coated substrates. The only statistically significant difference between the two substrates in these assays was increased spreading on PLLA compared with SO in the presence of fibronectin and albumin. Cell proliferation was significantly higher on SO compared with PLLA after 7 days culture, but depended on the presence of fibronectin only in the PLLA system. In contrast, mitochondrial activity was higher on PLLA than on SO, and was enhanced by fibronectin on both substrates. PLLA substrates coated with fibronectin and subsequently exposed to albumin exhibited the highest level of cell differentiation, as assayed via alkaline phosphatase activity. These results demonstrate the importance of adsorbed proteins on osteoblast-like cell-surface interactions.

Bioengineered hyaluronic acid elicited a nonantigenic T cell activation: Implications from cosmetic medicine and surgery to nanomedicine

Hyaluronan is known to act as a filling material of extracellular matrices and as an adhesive substrate for cellular migration. Consequently, it is widely used in aesthetic medicine and surgery, and it would be expected to be used in nanomedicine. Previous clinical case reports associated hyaluronic acid implants to delayed immune-mediated adverse effects. A series of experiments to evaluate immune cell activation supported by this dermal filler and nanomedical biomaterial were performed. The study comprised a total of 12 individuals. Four healthy individuals, none with cosmetically injected dermal filler, were considered as control. Five individuals carried injections of hyaluronic acid dermal filler. Three individuals carried injections of hyaluronic acid dermal filler and presented delayed adverse effects related to the dermal filler. Hyaluronic acid-stimulated peripheral blood mononuclear cells (PBMC) produced low levels of pro-inflammatory cytokines. Phytohemagglutinine (PHA)-stimulated PBMC from patients with hyaluronic implants presenting adverse effects showed a slight increase in the production of interferon (IFN)-gamma and higher expression of CD25, CD69, or CD71. In conclusion, hyaluronic acid administration elicited a laboratory evidence of immune cell activation. Production of low levels of proinflammatory cytokines in vitro could be an observation for low-grade inflammation in vivo resulting in T cell activation.

The inhibition of cell spreading on a cellulose substrate (cuprophan) induces an apoptotic process via a mitochondria-dependent pathway

Cell shape was found to be a strong indicator of whether individual cells grow or die, and may play an important role in controlling apoptosis as well as cell growth. We compared here the behaviour of rounded Swiss 3T3 cells aggregated on a cellulose cuprophan membrane to those cultured on dish polystyrene. We demonstrated that cells aggregated on cellulose substrates for up to 48 h underwent programmed cell death that was associated with phosphatidylserine flipping and caspase 9 and caspase 3 activation, suggesting a mitochondria‐dependent apoptotic process. In addition, we found that this phenomenon cannot be entirely explained by disengagement of α5β1 integrin ligation.

Nanotemplated polyelectrolyte films as porous biomolecular delivery systems: Application to the growth factor BMP-2

Biomaterials capable of delivering controlled quantities of bioactive agents, while maintaining mechanical integrity, are needed for a variety of cell contacting applications. We describe here a nanotemplating strategy toward porous, polyelectrolyte-based thin films capable of controlled biomolecular loading and release. Films are formed via the layer-by-layer assembly of charged polymers and nanoparticles (NP), then chemically cross-linked to increase mechanical rigidity and stability, and finally exposed to tetrahydrofuran to dissolve the NP and create an intra-film porous network. We report here on the loading and release of the growth factor bone morphogenetic protein 2 (BMP-2), and the influence of BMP-2 loaded films on contacting murine C2C12 myoblasts. We observe nanotemplating to enable stable BMP-2 loading throughout the thickness of the film, and find the nanotemplated film to exhibit comparable cell adhesion, and enhanced cell differentiation, compared with a non-porous cross-linked film (where BMP-2 loading is mainly confined to the film surface).

Effects of human fibronectin and human serum albumin sequential adsorption on preosteoblastic cell adhesion.

Fibronectin (Fn) is widely reported to promote cell adhesion and spreading, and recent reports attest to the synergistic effect of coadsorbed albumin (unexpected due to the passivating character of the latter protein). In this study, the sequential adsorption of fibronectin and albumin, and the morphology of cultured MC3T3-E1 preosteoblastic cells are investigated on three important biomaterial surfaces: silicon oxide, poly(styrene) (PS), and hydroxyapatite (HA). Using quartz crystal microgravimetry with dissipation analysis, the adsorbed protein composition and mechanics are determined. Interestingly, cell morphological changes correlate neither with the amount of Fn nor the rigidity of the protein layer. On the PS surface, Alb is seen to significantly diminish cell spreading, possibly due to Alb aggregation with a partially denatured initially placed Fn layer. HA appears to be a particularly favorable substrate for osteoblast adhesion, despite having low Fn adsorption and protein layer rigidity.

Fabrication of functional fibronectin patterns by nanosecond excimer laser direct write for tissue engineering applications

Laser direct write techniques represent a prospective alternative for engineering a new generation of hybrid biomaterials via the creation of patterns consisting of biological proteins onto practically any type of substrate. In this paper we report on the characterization of fibronectin features obtained onto titanium substrates by UV nanosecond laser transfer. Fourier-transform infrared spectroscopy measurements evidenced no modification in the secondary structure of the post-transferred protein. The molecular weight of the transferred protein was identical to the initial fibronectin, no fragment bands being found in the transferred protein’s Western blot migration profile. The presence of the cell-binding domain sequence and the mannose groups within the transferred molecules was revealed by anti-fibronectin monoclonal antibody immunolabelling and FITC-Concanavalin-A staining, respectively. The in vitro tests performed with MC3T3-E1 osteoblast-like cells and Swiss-3T3 fibroblasts showed that the cells’ morphology and spreading were strongly influenced by the presence of the fibronectin spots.