New injectable self-assembled hydrogels that promote angiogenesis through a bioactive degradation product.

Hydrogels used in regenerative medicine are often designed to allow cellular infiltration, degradation, and neovascularization. Low molecular weight hydrogels (LMWHs), formed by self-assembly via non-covalent interactions, are gaining significant interest because they are soft, easy to use and injectable. We propose LMWHs as suitable body implant materials that can stimulate tissue regeneration. We produced four new LMWHs with molecular entities containing nucleic acid and lipid building blocks and analyzed the foreign body response upon subcutaneous implantation into mice. Despite being infiltrated with macrophages, none of the hydrogels triggered detrimental inflammatory responses. Most macrophages present in the hydrogel-surrounding tissue acquired an immuno-modulatory rather than inflammatory phenotype. Concomitantly, no fibrotic capsule was formed after three weeks. Our glyconucleolipid LMWHs exhibited different degradation kinetics in vivo and in vitro. LMWHs with high angiogenic properties in vivo, were found to release glyconucleoside (glucose covalently linked to thymidine via a triazole moiety) as a common by-product of in vitro LMWH degradation. Chemically synthesized glyconucleoside exhibited angiogenic properties in vitro in scratch assays with monolayers of human endothelial cells and in vivo using the chick chorioallantoic membrane assay. Collectively, LMWHs hold promise as efficient scaffolds for various regenerative applications by displaying good biointegration without causing fibrosis, and by promoting angiogenesis through the release of a pro-angiogenic degradation product. STATEMENT OF SIGNIFICANCE: The main limitations of biomaterials developed in the field of tissue engineering remains their biocompatibility and vascularisation properties. In this context, we developed injectable Low Molecular Weight Hydrogels (LMWH) exhibiting thixotropic (reversible gelation) and thermal reversible properties. LMWH having injectability is of great advantage since it allows for their delivery without wounding the surrounding tissues. The resulting gels aim at forming scaffolds that the host cells colonize without major inflammation, and that won't be insulated by a strong fibrosis reaction. Importantly, their molecular degradation releases a product (a glycosyl-nucleoside conjugate) promoting angiogenesis. In this sense, these LMWH represent an important advance in the development of biomaterials promoting tissue regeneration.

Strontium-doped hydroxyapatite polysaccharide materials effect on ectopic bone formation.

Previous studies performed using polysaccharide-based matrices supplemented with hydroxyapatite (HA) particles showed their ability to form in subcutaneous and intramuscular sites a mineralized and osteoid tissue. Our objectives are to optimize the HA content in the matrix and to test the combination of HA with strontium (Sr-HA) to increase the matrix bioactivity. First, non-doped Sr-HA powders were combined to the matrix at three different ratios and were implanted subcutaneously for 2 and 4 weeks. Interestingly, matrices showed radiolucent properties before implantation. Quantitative analysis of micro-CT data evidenced a significant increase of mineralized tissue formed ectopically with time of implantation and allowed us to select the best ratio of HA to polysaccharides of 30% (w/w). Then, two Sr-substitution of 8% and 50% were incorporated in the HA powders (8Sr-HA and 50Sr-HA). Both Sr-HA were chemically characterized and dispersed in matrices. In vitro studies performed with human mesenchymal stem cells (MSCs) demonstrated the absence of cytotoxicity of the Sr-doped matrices whatever the amount of incorporated Sr. They also supported osteoblastic differentiation and activated the expression of one late osteoblastic marker involved in the mineralization process i.e. osteopontin. In vivo, subcutaneous implantation of these Sr-doped matrices induced osteoid tissue and blood vessels formation.

Chitosan-based hydrogels for developing a small-diameter vascular graft: in vitro and in vivo evaluation.

AIMS: Vascular grafts made of synthetic polymers perform poorly in small-diameter applications (cardiac and peripheral bypass). Chitosan is a biocompatible natural polymer that can provide a novel biological scaffold for tissue engineering development. The goal of this study was to demonstrate the biocompatibility of a novel chitosan preparation in vitro and in vivo, and to assess its potential as a scaffold for vascular applications. METHODS AND RESULTS: A series of experiments of increasing complexity, ranging from in vitro biocompatibility and hemocompatibility tests to in vivo studies in small and large animals (rats and sheep), was performed to provide a comprehensive analysis of chitosan hydrogels' biological properties. In vitro studies established that: (i) chitosan supported human endothelial progenitor cells adhesion, proliferation and resistance to physiological shear stress; (ii) chitosan did not activate platelets, the complement system, or the intrinsic coagulation pathway. In vivo results showed: (iii) no resorption of chitosan and no chronic inflammation at 60 days in a rat heterotopic implantation model (magnetic resonance imaging and histology); (iv) no flow obstruction (Doppler ultrasound) and no thrombus formation (histology and scanning electron microscopy) at 2 h after a carotid arteriotomy repair with chitosan patches in sheep. Finally, two chitosan tubes were implanted as carotid interposition grafts for 3 days in sheep showing that chitosan was strong enough to be sutured, to withstand arterial pressure, and no flow obstruction was observed through this short period. CONCLUSION: Chitosan-based hydrogels displayed promising in vitro biocompatibility and hemocompatibility properties as well as in vivo short-term performance.

In vitro assessment of a collagen/alginate composite scaffold for regenerative endodontics.

AIM: To develop a biological scaffold that could be moulded to reproduce the geometry of a gutta-percha point with precision and allow the differentiation of mesenchymal stem cells into osteoblasts to be used as a regenerative endodontic material. METHODOLOGY: A collagen/alginate composite scaffold was cast into a sodium alginate mould to produce a gutta-percha point-like cone. Prior to gelation, the cone was seeded with human stem cells from the apical papilla (SCAPs) to evaluate cell/scaffold interactions. The reconstructed tissue was characterized after 8 days in culture. Elastic modulus, tissue compaction and cell differentiation were assessed. Student t-tests and the Mann-Whitney U test were performed. RESULTS: The fabrication method developed enabled the shape of a gutta-percha point to be mimicked with great accuracy and reproducibility (P = 0.31). Stem cells seeded into this composite scaffold were able to spread, survive and proliferate (P < 0.001). Moreover, they were able to differentiate into osteoblasts and produce calcified osseous extracellular matrix (P < 0.001). The construct showed no significant contraction after 8 days, preserving its shape and tip diameter (P = 0.58). CONCLUSIONS: The composite scaffold could present substantial benefits compared to synthetic materials. It could provide a favourable healing environment in the root canal conducive for regenerative endodontics and is therefore appropriate to be evaluated in vivo in further studies.

Attachment of human endothelial cells to polyester vascular grafts: pre-coating with adhesive protein assemblies and resistance to short-term shear stress.

Cardiovascular prosthetic bypass grafts do not endothelialize spontaneously in humans, and so they pose a thrombotic risk. Seeding with cells improves their performance, particularly in small-caliber applications. Knitted tubular polyethylene-terephthalate (PET) vascular prostheses (6 mm) with commercial type I collagen (PET/Co) were modified in the lumen by the adsorption of laminin (LM), by coating with a fibrin network (Fb) or a combination of Fb and fibronectin (Fb/FN). Primary human saphenous vein endothelial cells were seeded (1.50 × 10(5)/cm2), cultured for 72 h and exposed to laminar shear stress 15 dyn/cm(2) for 40 and 120 min. The control static grafts were excluded from shearing. The cell adherence after 4 h on PET/Co, PET/Co +LM, PET/Co +Fb and PET/Co +Fb/FN was 22%, 30%, 19% and 27% of seeding, respectively. Compared to the static grafts, the cell density on PET/Co and PET/Co +LM dropped to 61% and 50%, respectively, after 120 min of flow. The cells on PET/Co +Fb and PET/Co +Fb/FN did not show any detachment during 2 h of shear stress. Pre-coating the clinically-used PET/Co vascular prosthesis with LM or Fb/FN adhesive protein assemblies promotes the adherence of endothelium. Cell retention under flow is improved particularly on fibrin-containing (Fb and Fb/FN) surfaces.

Cell interactions between human progenitor-derived endothelial cells and human mesenchymal stem cells in a three-dimensional macroporous polysaccharide-based scaffold promote osteogenesis.

Several studies have reported the benefits of mesenchymal stem cells (MSCs) for bone tissue engineering. However, vascularization remains one of the main obstacles that must be overcome to reconstruct large bone defects. In vitro prevascularization of the three-dimensional (3-D) constructs using co-cultures of human progenitor-derived endothelial cells (PDECs) with human bone marrow mesenchymal stem cells (HBMSCs) appeared as a potential strategy. However, the crosstalk between the two lineages has been studied in two-dimensional (2-D), but remains unknown in 3-D. The aim of this study is to investigate the cell interactions between PDECs and HBMSCs in a porous matrix composed of polysaccharides. This biodegradable scaffold promotes cell interactions by inducing multicellular aggregates composed of HBMSCs surrounded by PDECs. Cell aggregation contributes to the formation of junctional proteins composed of Connexin43 (Cx43) and VE-cadherin, and an activation of osteoblastic differentiation of HBMSCs stimulated by the presence of PDECs. Inhibition of Cx43 by mimetic peptide 43GAP27 induced a decrease in mRNA levels of Cx43 and all the bone-specific markers. Finally, subcutaneous implantations for 3 and 8 weeks in NOG mice revealed an increase in osteoid formation with the tissue-engineered constructs seeded with HBMSCs/PDECs compared with those loaded with HBMSCs alone. Taking together, these results demonstrate that this 3-D microenvironment favored cell communication, osteogenesis and bone formation.

Interaction between a bisphosphonate, tiludronate and nanocrystalline apatite: in vitro viability and proliferation of HOP and HBMSC cells.

Nanocrystalline apatites (NCA) are the inorganic components of mineralized tissues and they have been recently proposed as biomaterials for drug delivery systems. Bisphosphonates (BPs) are currently the reference drugs used to treat diseases involving bone disorders such as osteoporosis. Nevertheless, the interaction phenomena between BP molecules and apatite nanocrystals of bone are not well understood. Therefore, the adsorption characteristics have been examined and cellular activity of tiludronate molecules on NCA as models of bone mineral has been investigated. Adsorption experiments of tiludronate onto NCA were carried out and revealed a Langmuir-type adsorption isotherm. The uptake of tiludronate molecules is associated with a release of phosphate ions, indicating that the main reaction is an ion exchange process involving surface anions. The results evidence the strong affinity of BP molecules for the apatitic surface. The interactions of NCA-tiludronate associations with human osteoprogenitor cells and human bone marrow stromal cells do not reveal any cytotoxicity and evidence the activity of adsorbed tiludronate molecules. Moreover, an evolution of the physico-chemical characteristics of the apatitic substrate during biological study was observed, highlighting the existence of dynamic interactions. This work contributes to clarifying the reaction mechanisms between BPs and biomimetic apatites.

Whatever their differentiation status, human progenitor derived - or mature - endothelial cells induce osteoblastic differentiation of bone marrow stromal cells.

Association of the bone-forming osteoblasts (OBs) and vascular endothelial cells (ECs) into a biomaterial composite provides a live bone graft substitute that can repair the bone defect when implanted. An intimate functional relationship exists between these cell types. This communication is crucial to the coordinated cell behaviour necessary for bone development and remodelling. Previous studies have shown that direct co-culture of primary human osteoprogenitors (HOPs) with primary human umbilical vein endothelial cells (HUVECs) stimulates HOPs differentiation and induces tubular-like networks. The present work aims to test the use of human bone marrow stromal cells (HBMSCs) co-cultured with human endothelial progenitor cells in order to assess whether progenitor-derived ECs (PDECs) could support osteoblastic differentiation as mature ECs do. Indeed, data generated from the literature by different laboratories considering these co-culture systems appear difficult to compare. Monocultures of HUVECs, HOPs, HBMSCs (in a non-orientated lineage), PDECs (from cord blood) were used as controls and four combinations of co-cultures were undertaken: HBMSCs-PDECs, HBMSCs-HUVECs, HOPs-PDECs, HOPs-HUVECs with ECs (mature or progenitor) for 6 h to 7 days. At the end of the chosen co-culture time, intracellular alkaline phosphatase (ALP) activity was detected in HOPs and HBMSCs and quantified in cell extracts. Quantitative real-time polymerase chain reaction (qPCR) of ALP was performed over time and vascular endothelial growth factor (VEGF) was measured. After 21 days, calcium deposition was observed, comparing mono- and co-cultures. We confirm that ECs induce osteoblastic differentiation of mesenchymal stem cells in vitro. Moreover, HUVECs can be replaced by PDECs, the latter being of great interest in tissue engineering.

Carbon nanotubes have a deleterious effect on the nose: the first in vitro data.

BACKGROUND: The information currently available concerning carbon nanotubes toxicity is disturbing and conflicting. Moreover, little is known about their effect on the nasal cavities, which are the first target for nanoparticles. MATERIAL AND METHOD: We investigated the cytotoxicity (50 to 0.5 microg/mL) of double-walled carbon nanotube with two independent tests (MTT, Wst-1) on normal human nasal epithelial cells after 12-day exposure (control untreated nasal cells and A549). Nasal cell differentiation function, oxidative stress, the morphological features of cells in contact with DWCNTs and the localizations of the latter were also investigated. RESULTS: Exposure revealed a dose-dependent decrease in cell metabolic activity and cell growth. In nearly all conditions, normal human nasal epithelial cells were more sensitive than malignant ones. Even with both tests, the cytotoxic threshold dose could not be accurately determined because of dye adsorption by DWCNTs. Nasal cells showed stronger cytokeratin 7 and persistent UEA-I immunostaining. Cytokeratin 19 production was increased at 25 microg/mL and mucus production was stimulated from 0.5 microg/mL. A significant increase in Reactive Oxygen Species was observed from 25 microg/mL. The cell plasma membrane showed several holes and DWCNTs were present in the cytoplasm. CONCLUSION: DWCNTs seem to have a deleterious effect on nasal cells after 12-day exposure.

Magnetic resonance imaging tracking of human adipose derived stromal cells within three-dimensional scaffolds for bone tissue engineering.

For bone tissue engineering, human Adipose Derived Stem Cells (hADSCs) are proposed to be associated with a scaffold for promoting bone regeneration. After implantation, cellularised scaffolds require a non-invasive method for monitoring their fate in vivo. The purpose of this study was to use Magnetic Resonance Imaging (MRI)-based tracking of these cells, labelled with magnetic agents for in vivo longitudinal assessment. hADSCs were isolated from adipose tissue and labelled with USPIO-rhodamine (Ultrasmall SuperParamagnetic Iron Oxide). USPIO internalisation, absence of toxicity towards hADSCs, and osteogenic differentiation of the labelled cells were evaluated in standard culture conditions. Labelled cells were then seeded within a 3D porous polysaccharide-based scaffold and imaged in vitro using fluorescence microscopy and MRI. Cellularised scaffolds were implanted subcutaneously in nude mice and MRI analyses were performed from 1 to 28 d after implantation. In vitro, no effect of USPIO labelling on cell viability and osteogenic differentiation was found. USPIO were efficiently internalised by hADSCs and generated a high T2* contrast. In vivo MRI revealed that hADSCs remain detectable until 28 d after implantation and could migrate from the scaffold and colonise the area around it. These data suggested that this scaffold might behave as a cell carrier capable of both holding a cell fraction and delivering cells to the site of implantation. In addition, the present findings evidenced that MRI is a reliable technique to validate cell-seeding procedures in 3D porous scaffolds, and to assess the fate of hADSCs transplanted in vivo.

High-throughput laser printing of cells and biomaterials for tissue engineering.

In parallel with ink-jet printing and bioplotting, biological laser printing (BioLP) using laser-induced forward transfer has emerged as an alternative method in the assembly and micropatterning of biomaterials and cells. This paper presents results of high-throughput laser printing of a biopolymer (sodium alginate), biomaterials (nano-sized hydroxyapatite (HA) synthesized by wet precipitation) and human endothelial cells (EA.hy926), thus demonstrating the interest in this technique for three-dimensional tissue construction. A rapid prototyping workstation equipped with an IR pulsed laser (tau=30 ns, lambda=1064 nm, f=1-100 kHz), galvanometric mirrors (scanning speed up to 2000 mm s(-1)) and micrometric translation stages (x, y, z) was set up. The droplet generation process was controlled by monitoring laser fluence, focalization conditions and writing speed, to take into account its mechanism, which is driven mainly by bubble dynamics. Droplets 70 microm in diameter and containing around five to seven living cells per droplet were obtained, thereby minimizing the dead volume of the hydrogel that surrounds the cells. In addition to cell transfer, the potential of using high-throughput BioLP for creating well-defined nano-sized HA patterns is demonstrated. Finally, bioprinting efficiency criteria (speed, volume, resolution, integrability) for the purpose of tissue engineering are discussed.

Polyelectrolyte multilayer films allow seeded human progenitor-derived endothelial cells to remain functional under shear stress in vitro.

There is considerable interest in making multilayer films for various applications, among which are cell contacting biomaterials, allowing new opportunities to prepare functionalized biomaterials. In this study we have explored the capability of poly(sodium-4-styrene sulfonate)/poly(allylamine hydrochloride) polyelectrolyte multilayer films (PMFs) as functional coatings for human progenitor-derived endothelial cells (PDECs), since the latter are a potential source of endothelial-type cells to be used in bioartificial vascular substitutes. We performed investigations with PDECs derived from peripheral blood and characterized as endothelial cells. After forming a confluent monolayer on PMFs they were exposed to laminar pulsatile physiological shear stress. We investigated whether PDECs were able to withstand shear stress and to respond at the mRNA (microarray analysis) and protein levels (thrombomodulin and tissue factor functional activity), in comparison with collagen I and fibrin glue used as controls. After shear stress the PDECs remained spread on the substrates, with a resulting increase in the number of expressed genes. Considering the functional significance of our findings for the regulation of coagulation and fibrinolytic factors, mRNA tissue plasminogen activator and thrombomodulin, profibrinolytic and thrombin inhibiting respectively, were overexpressed in PDECs after 6h shear stress. von Willebrand factor showed down-regulation, while tissue factor was up-regulated. We can speculate that PMFs could favour anti-thrombogenic activity by PDECs because activated protein C generation, measuring thrombomodulin activity, was particularly high on PMFs, but unchanged after 6h shear stress. Thus, PMFs could represent suitable coatings able to provide functional surfaces for endothelialization with PDECs.

Cyclo-DfKRG peptide modulates in vitro and in vivo behavior of human osteoprogenitor cells on titanium alloys.

The first aim of the present study was to investigate the capacity of a cyclo-DfKRG-coated hydroxyapatite-titanium alloy (Ti-HA-RGD) to activate in vitro human osteoprogenitor cells adhesion and differentiation. The second purpose was to examine in vivo the role of a autologous cell seeding on cyclo-DfKRG-functionalized materials to provide bone repair after implantation in femoral condyle of rabbits. Our in vitro results have demonstrated that both titanium alloy functionalized with hydroxyapatite (Ti-HA-RGD and Ti-HA) contributed to higher cell adhesion than titanium alloy alone respectively 85 and 55% vs 15% compared to tissue culture polystyrene after one hour of cell seeding. As for differentiation, after 3 days of culture, Ti-HA presented the highest increase of ALP mRNA of all surfaces studied. Ti-HA-RGD showed an intermediate value about half as high as Ti-HA. Moreover after 3 days, both Ti-HA and Ti-HA-RGD surfaces showed the highest increase of cbfa1 mRNA expression. Two weeks following implantation, in vivo findings revealed that percentage of lacunae contact observed with pre-cellularized Ti-HA-RGD samples remains significantly lower than with Ti-HA group (10.5+/-9.6 % vs 33.7+/-11.5 %, P<0.03). Meanwhile, RGD peptide coating had no significant additional effect on the bone implant contact and area. Moreover, histomorphometry analysis revealed that implantation of pre-cellularized RGD coated materials with ROP cells increased significantly peri-implant fibrous area (24+/-11.6% vs 3+/-1.7% for Ti-HA-RGD, P<0.02). RGD coatings demonstrated osteoblastic adhesion, differentiation and in vivo bone regeneration at most equivalent to HA coatings.

Interaction between human umbilical vein endothelial cells and human osteoprogenitors triggers pleiotropic effect that may support osteoblastic function.

Osteogenesis occurs in striking interaction with angiogenesis. There is growing evidence that endothelial cells are involved in the modulation of osteoblast differentiation. We hypothesized that primary human umbilical vein endothelial cells (HUVEC) should be able to modulate primary human osteoprogenitors (HOP) function in an in vitro co-culture model. In a previous study we demonstrated that a 3 day to 3 week co-culture stimulates HOP differentiation markers such as Alkaline Phosphatase (ALP) activity and mineralization. In the present study we addressed the effects induced by the co-culture on HOP within the first 48 hours. As a prerequisite, we validated a method based on immuno-magnetic beads to separate HOP from HUVEC after co-culture. Reverse transcription-real time quantitative PCR studies demonstrated up-regulation of the ALP expression in the co-cultured HOP, confirming previous results. Surprisingly, down-regulation of runx2 and osteocalcin was also shown. Western blot analysis revealed co-culture induced down-regulation of Connexin43 expression in both cell types. Connexin43 function may be altered in co-cultured HOP as well. Stimulation of the cAMP pathway was able to counterbalance the effect of the co-culture on the ALP activity, but was not able to rescue runx2 mRNA level. Co-culture effect on HOP transcriptome was analyzed with GEArray cDNA microarray showing endothelial cells may also modulate HOP extracellular matrix production. In accordance with previous work, we propose endothelial cells may support initial osteoblastic proliferation but do not alter the ability of the osteoblasts to produce extracellular mineralizing matrix.

Calcium carbonate-calcium phosphate mixed cement compositions for bone reconstruction.

The feasibility of making calcium carbonate-calcium phosphate (CaCO(3)-CaP) mixed cements, comprising at least 40% (w/w) CaCO(3) in the dry powder ingredients, has been demonstrated. Several original cement compositions were obtained by mixing metastable crystalline CaCO(3) phases with metastable amorphous or crystalline CaP powders in aqueous medium. The cements set within at most 1 h at 37 degrees C in atmosphere saturated with water. The hardened cement is microporous and exhibits weak compressive strength. The setting reaction appeared to be essentially related to the formation of a highly carbonated nanocrystalline apatite phase by reaction of the metastable CaP phase with part or almost all of the metastable CaCO(3) phase. The recrystallization of metastable CaP varieties led to a final cement consisting of a highly carbonated poorly crystalline apatite analogous to bone mineral associated with various amounts of vaterite and/or aragonite. The presence of controlled amounts of CaCO(3) with a higher solubility than that of the apatite formed in the well-developed CaP cements might be of interest to increase resorption rates in biomedical cement and favors its replacement by bone tissue. Cytotoxicity testing revealed excellent cytocompatibility of CaCO(3)-CaP mixed cement compositions.

Functionalization of biomaterials and cell to cell communication.

In the field of osseous substitution, the possibilities being offered to the surgeons prove sometimes difficult to apply in particular in the case of great losses of osseous substance. For these reasons, it is necessary to develop innovative techniques to satisfy the request increasing for substitutes and to see appearing on the market solutions combining availability, perenniality and biosecurity of the implants. The implantation of stem cells in a biomaterial opens a way of development of therapeutic substitute. Moreover, in order to optimize the rehabitation of the biomaterials by the cells and the host tissues, the second approach consists in modifying the surface of materials by the coating or the grafting of adhesive factors in order to stimulate their colonization. At least, one cannot consider a tissue mechanism of repair without a better knowledge of the respective role of the various cell populations implied in the rebuilding of this tissue and their cell to cell communication processes.

Mineralization of regenerated cellulose hydrogels induced by human bone marrow stromal cells.

The proliferation of cultured human bone marrow stromal cells (HBMSC) on regenerated cellulose hydrogels was assessed. Regenerated cellulose hydrogels showed good rates of HBMSC proliferation, the cells exhibiting a flattened morphology, and after 22 days in culture, the cells had homogeneously colonized the surface of the materials. Moreover, since the early days in culture, between the surface of the materials and attached cells a continuous granulated hydroxyapatite layer was formed. It has been previously demonstrated in vitro, but without cells, that these materials did not mineralize. Hence, it seems that HBMSC promoted the mineralization of the surface.

In vitro endothelial cell susceptibility to xenobiotics: comparison of three cell types.

In three different endothelial cell (EC) cultures (primary human umbilical cord vein, so-called HUVEC; and immortalized cell lines HBMEC and EA-hy-926), the effects of different xenobiotics were studied in order to standardize vascular EC models for in vitro pharmacotoxicological studies. Cell characteristics were first investigated by the production and the mRNA levels of known endothelial markers in the three EC culture models. EC secretory products, tissue plasminogen activator (tPA) and von Willebrand factor (vWF), were present in the supernatant of the immortalized cell lines. The mRNA levels of vWF, tPA, platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), and beta -integrin subunit, which are involved in the control of platelet function, coagulation, and fibrinolysis as well as in cell-matrix interactions, were investigated in all EC types. For at least three parameters, cultured cells provided marked characteristics of EC phenotype, in HUVEC and in immortalized cell lines, regardless of their origin from the macro- or microcirculation. Toxicity experiments were assessed after 24 h exposure to cadmium, cyclosporin A and cisplatin by MTT assay. These experiments show nonsignificant difference in susceptibility to cyclosporin A and cadmium on HUVEC, HBMEC, and EA-hy-926. However, HBMEC, seems to be highly susceptible to cisplatin compared to HUVEC, the latter being more sensitive than EA-hy-926. For experiments conducted with cyclosporin and cadmium, cell lines could constitute an alternative material for routine cytotoxicity studies.

Effect of several sterilisation techniques on homogeneous self assembled monolayers.

Understanding how cells sense their environment and are able to regulate their metabolism is of great importance for the success of biomaterials implantation. Self assembled monolayers (SAMs) are in use nowadays to model the surface of such materials. They permit the control of different surface parameters (like chemistry, surface energy and topography) enabling to get a greater insight in cells behaviour when interacting with surfaces and thus, in the future, to enhance surface properties of biomaterials. As sterilisation is the compulsory step for in vitro and in vivo assays with living biological materials, it is important to know how SAMs react under sterilisation techniques in use on biomaterials. In this work, the effect of three types of sterilisation techniques: gamma-irradiation, mostly used on biomaterials, dry heat and steam autoclaving, have been investigated on NH2 and CH3 terminated SAMs. Gamma-irradiation destructs drastically the NH2 and partially the CH3 monolayers by producing oxidative compounds (COOH, C=O, C-OH). The main product induced by gamma-irradiation on NH2 monolayers is carboxylic acid, whereas CH3 shows an important increase in the amount of alcoholic groups. This difference in deterioration is assumed to be due to the higher stability of the CH3 monolayer. Steam autoclaving to a lesser extent gives the same results on NH2 monolayers. Dry heat seems to be the most reliable technique, which can be used on such surfaces as it removes physically adsorbed organic contaminants without affecting the integrity of the surface.