PolyNaSS grafting on titanium surfaces enhances osteoblast differentiation and inhibits Staphylococcus aureus adhesion.

Titanium surface modifications to simultaneously prevent bacterial adhesion but promote bone-cell functions could be highly beneficial for improving implant osseointegration. In the present in vitro study, the effect of sulfonate groups on titanium surfaces was investigated with respect to both S. aureus adhesion and osteoblast functions pertinent to new bone formation. Commercial pure titanium (cpTi) squares were oxydized (Tiox), grafted with poly(sodium styrene sulfonate) groups (Tigraft) by covalent bonding using radical polymerization, and were characterized by infrared spectroscopy (HATR-FTIR) and colorimetry. Bacterial adhesion study showed that Tigraft exhibited high inhibition of S. aureus adhesion S at levels >90 %, when compared to cpTi (P < 0.05). In contrast osteoblasts adhesion was similar on all three titanium surfaces. While the kinetics of cell proliferation were similar on the three titanium surfaces, Alkaline phosphatase-specific activity of osteoblasts cultured on Tigraft surfaces was twofold higher than that observed on either on Tiox or cpTi surfaces (P < 0.01). More importantly, the amount and the distribution of calcium-containing nodules was different. The total area covered by calcium-containing nodules was 2.2-fold higher on the Tigraft as compared to either Tiox or cpTi surfaces (P < 0.01). These results provide evidence that poly(sodium styrene sulfonate) groups grafting on cpTi simultaneously inhibits bacteria adhesion but promote osteoblast function pertinent to new bone formation. Such modified titanium surfaces offer a promising strategy for preventing biofilm-related infections and enhancing osteointegration of implants in orthopaedic and dental applications.

Development of proteomic tools to study protein adsorption on a biomaterial, titanium grafted with poly(sodium styrene sulfonate).

It is known that protein adsorption is the initial interaction between implanted biomaterials and biological environment. Generally, a complex protein layer will be formed on material surfaces within a few minutes and the composition of this layer at the interface determines the biological response to the implanted material, and therefore the long-term compatibility of the biomaterial. Despite different techniques exist to observe protein adsorption on biomaterials, none of them led to the identification of adsorbed proteins. In this paper, we report a chromatographic technique coupled to proteomics to analyse and identify proteins from complex biological samples adsorbed on biomaterial surfaces. This approach is based on (1) elaboration of the chromatographic support containing the biomaterial (2) a chromatography step involving adsorption of proteins on the biomaterial (3) the high-resolution separation of eluted proteins by 2-DE gel and (4) the identification of proteins by mass spectrometry. Experiments were performed with proteins from platelets rich plasma (PRP) adsorbed on a biomaterial which consist in titanium bioactivated with PolyNaSS. Our results show that chromatographic approach combined to 2-DE gels and mass spectrometry provides a powerful tool for the analysis and identification of proteins adsorbed on various surfaces.

Bioactive polymer grafting onto titanium alloy surfaces.

Bioactive polymers bearing sulfonate (styrene sodium sulfonate, NaSS) and carboxylate (methylacrylic acid, MA) groups were grafted onto Ti6Al4V alloy surfaces by a two-step procedure. The Ti alloy surfaces were first chemically oxidized in a piranha solution and then directly subjected to radical polymerization at 70 degrees C in the absence of oxygen. The grafted surfaces were characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS) and the toluidine blue colorimetric method. Toluidine blue results showed 1-5microgcm(-2) of polymer was grafted onto the oxidized Ti surfaces. Grafting resulted in a decrease in the XPS Ti and O signals from the underlying Ti substrate and a corresponding increase in the XPS C and S signals from the polymer layer. The ToF-SIMS intensities of the S(-) and SO(-) ions correlated linearly with the XPS atomic percent S concentrations and the ToF-SIMS intensity of the TiO(3)H(2)(-) ion correlated linearly with the XPS atomic per cent Ti concentration. Thus, the ToF-SIMS S(-), SO(-) and TiO(3)H(2)(-) intensities can be used to quantify the composition and amount of grafted polymer. ToF-SIMS also detected ions that were more characteristic of the polymer molecular structure (C(6)H(4)SO(3)(-) and C(8)H(7)SO(3)(-) from NaSS, C(4)H(5)O(2)(-) from MA), but the intensity of these peaks depended on the polymer thickness and composition. An in vitro cell culture test was carried out with human osteoblast-like cells to assess the influence of the grafted polymers on cell response. Cell adhesion after 30min of incubation showed significant differences between the grafted and ungrafted surfaces. The NaSS grafted surfaces showed the highest degree of cell adhesion while the MA-NaSS grafted surfaces showed the lowest degree of cell adhesion. After 4 weeks in vivo in rabbit femoral bones, bone was observed to be in direct contact with all implants. The percentage of mineralized tissue around the implants was similar for NaSS grafted and non-grafted implants (59% and 57%). The MA-NaSS grafted implant exhibited a lower amount of mineralized tissue (47%).

Bone tissue response to titanium implant surfaces modified with carboxylate and sulfonate groups.

The present study assessed in vivo new bone formation around titanium alloy implants chemically grafted with macromolecules bearing ionic sulfonate and/or carboxylate groups. Unmodified and grafted Ti-6Al-4V exhibiting either 100% carboxylate, or 100% sulfonate, or both carboxylate and sulfonate groups in the percent of 50/50 and 80/20 were bilaterally implanted into rabbit femoral condyle. Neither toxicity nor inflammation were observed for all implants tested. After 4 weeks, peri-implant new bone formation varied as a function of the chemical composition of the titanium surfaces. The percent bone-implant contact (BIC) was the lowest (13.4 +/- 6.3%) for the implants modified with grafted carboxylate only. The value of BIC on the implants with 20% sulfonate (24.6 +/- 5.2%) was significantly (P < 0.05) lower than that observed on 100% sulfonate (38.2 +/- 13.2%) surfaces. After both 4 and 12 weeks post-implantation, the BIC value for implants with more than 50% sulfonate was similar to that obtained with the unmodified Ti-6Al-4V. The grafted titanium alloy exhibiting either 100% sulfonate or carboxylate and sulfonate (50% each) groups promoted bone formation. Such materials are of clinical interest because, they do not promote bacteria adhesion but, they support new bone formation, a condition which can lead to osseointegration of bone implants while preventing peri-implant infections.

[New antitumor agent: In vitro activity on breast carcinoma cells]. / Nouvel agent antitumoral : activité in vitro sur les cellules de carcinomes mammaires.

Previous work showed that established interactions between water-soluble polymers and cell membrane receptors can lead to modulate cell proliferation and differentiation in vitro. These polymers can be considered as bioactive. The aim of this work was to establish the consequences of the interactions between human breast cancer cells MCF7 and polymers of various chemical compositions regarding cell adhesion and proliferation onto tissue culture plate. Water soluble copolymers were synthesized by radical polymerization and are composed of methacrylic acid and sodium styrene sulphonate units. The modulation of the MCF7, biological-induced by these polymers of various compositions, was evaluated. The influence of the polymers chemical composition on the kinetics of cell proliferation, as well as cell morphology and spreading, were studied. A polymer concentration-dependent inhibition effect was observed. One hundred microgram per liter polymers solutions induced strong inhibition of cell proliferation, as well as a change of the MCF7 cells morphology, which can be related to an inhibition of cell spreading. The polymers/MCF7 cells interactions are modulated by the chemical composition of the copolymers and then the respective rate in sulphonate and carboxylate groups distributed along the macromolecular chain.

Grafting of bioactive polymers onto titanium surfaces and human osteoblasts response.

In this paper, the adhesion of human osteoblast-like cells (line MG63) onto functionalized pure Titanium (Ti cp) has been studied. The Titanium surfaces were functionalized by grafting bioactive polymers bearing anionic groups such as sodium sulfonate. The grafting was achieved under inert atmosphere, by radical polymerization of sodium styrene sulfonate NaSS after the activation of the surface. ATR/FTIR and XPS were used to analyse the chemical composition of the grafted and non grafted titanium surfaces. The efficiency of the grafting was evidenced by the high amounts of grafted polyNaSS (5 microg/cm(2)), measured by Toluidin Blue colorimetric method. Biological tests have been investigated to highlight the influence of the grafting polymer on the cell response. Human osteoblast-like cells were cultured on titanium surfaces. Differences in the adhesion strength of cells were observed. Mineralization of osteoblast-like cells was studied after 28 days of culture and the amount of calcium formed were evaluated. Surface modification by bioactive polymers bearing anionic groups appears as an effective way to stimulate the bone regeneration over that, as provided by titanium as suggested by basic studies and in vitro results.

Surface modification of hydrogel intraocular lenses to prevent cell proliferation.

Intraocular lenses made from a hydrogel matrix were coated with a bioactive polymer exhibiting sulfonate and carboxylate groups. The anchorage of the macromolecular chains bearing the anionic groups to the hydrogel implant surface was obtained by an intermolecular reaction of the photosensitive groups attached to the ionic polymer, in order to obtain a pseudo-interpenetrated network. Cell proliferation assays performed on coated and uncoated hydrogel lenses showed an inhibiting effect of the bioactive polymer coating by up to 40% at day 6. The inhibiting effect was due to the presence and the distribution of both sulfonate and carboxylate groups along the macromolecular chains, which led to the appearance of ""bioactive sites"" allowing controlled interactions of surface, adhesive proteins and cells. (Journal of Applied Biomaterials & Biomechanics 2004; 2: 183-9).

Bioactive polymers grafted on silicone to prevent Staphylococcus aureus prosthesis adherence: in vitro and in vivo studies.

As joint prostheses become infected preventive strategies are needed. Silicone prostheses were coated with a COO - and SO3 - bearing bioactive copolymer, Q5, synthesized by radical polymerization and the adherence of Staphylococcus aureus (S. aureus)to them was evaluated in vitro and in vivo. Copolymer Q5 contains tris(trimethylsiloxy) methacryloxy propyl silane favoring the compatibility with the silicone matrix, cinnamoyl ethyl methacrylate allowing a network formation at the surface of the silicone prostheses, two ionic monomers: methacrylic acid and sodium styrene sulfonate. In vitro experiments were conducted on Q5-coated silicone lenses and on Q5-coated silicone prostheses. In both cases, materials were incubated with fi-bronectin (Fn) because of its important role in S. aureus adherence to implant surfaces. The percentage of adhesion inhibition was observed at approximately 40% for the coated materials compared to the untreated silicone. Rabbits underwent double-blind partial knee replacements with Q5-coated or control implants fitted into the intramedullary canal of the tibia, and 10 7 bacteria were injected into the knees. The number of bacteria adherent on the prostheses was determined 24 hr later. Signifi-cantly fewer bacteria adhered to Q5-coated than control prostheses (2.26 +/- 0.76 vs 3.86 +/- 0.54 log10 CFU/ml; p < 0.0035). Bioactive polymer coating could provide a new method of preventing joint-prosthesis infections. (Journal of Applied Biomaterials & Biomechanics 2003; 1: 178-85).