A bioactive polymer grafted on titanium oxide layer obtained by electrochemical oxidation. Improvement of cell response.

The anchorage failure of titanium implants in human body is mainly due to biointegration problem. The proposed solution is to graft a bioactive polymer at the surface of the implant in order to improve and control the interactions with the living system. In this paper, we describe the grafting of poly sodium styrene sulfonate on titanium surface by using a silanization reaction. The key point is to increase the TiOH content at the surface of the implant which can react with methoxy silane groups of 3-methacryloxypropyltrimethoxysilane (MPS). Two procedures were used: chemical oxidation and electrochemical oxidation. The last oxidation procedure was carried out in two different electrolytes: oxalic acid and methanol. These different oxidation methods allow controlling the roughness and the depth of the oxide layer. The methacryloyl group of MPS grafted at the titanium surface by silanization reaction is copolymerized with sodium styrene sulfonate using a thermal initiator able to produce radicals by heating. Colorimetric method, ATR-FTIR, XPS techniques and contact angle measurements were applied to characterize the surfaces. MG63 osteoblastic cell response was studied on polished, oxidized and grafted titanium samples. Cell adhesion, Alkaline Phosphatase activity and calcium nodules formation were significantly enhanced on grafted titanium surfaces compared to un-modified surfaces.

A new approach to graft bioactive polymer on titanium implants: Improvement of MG 63 cell differentiation onto this coating.

Integration of titanium implants into bone is only passive and the resulting fixation is mainly mechanical in nature, with anchorage failure. Our objective, to increase the biointegration of the implant and the bone tissue, could be obtained by grafting a bioactive ionic polymer to the surface of the titanium by a covalent bond. In this paper, we report the grafting of an ionic polymer model poly(sodium styrene sulfonate) (polyNaSS), in a two-step reaction procedure. Treatment of the titanium surface by a mixture of sulfuric acid and hydrogen peroxide allows the formation of titanium hydroxide and titanium peroxide. In the second reaction step, heating of a metal implant, placed in a concentrated solution of sodium styrene sulfonate monomer (NaSS), induces the decomposition of titanium peroxides with the formation of radicals capable of initiating the polymerization of NaSS. Various parameters, such as temperature of polymerization and time of polymerization, were studied in order to optimize the yield of polyNaSS grafting. Colorimetry, Fourier-transformed infrared spectra recorded in an attenuated total reflection, X-ray photoelectron spectroscopy techniques and contact angle measurements were applied to characterize the surfaces. MG63 osteoblastic cell response was studied on polished, oxidized and grafted titanium samples. Cell adhesion, alkaline phosphatase activity and calcium nodules formation were significantly enhanced on grafted titanium samples compared to unmodified surfaces.

Osteoblast functions on functionalized PMMA-based polymers exhibiting Staphylococcus aureus adhesion inhibition.

Staphylococcus aureus adhesion and osteoblast functions were assessed on functionalized poly(methyl methacrylate)-based terpolymers bearing randomly distributed carboxylate and sulfonate groups. These terpolymers were synthesized by radical polymerization, characterized by nuclear resonance spectroscopy and classified by the ratio R=[COO(-)/COO(-)+SO(3)(-)] in the range 0.5-0.8. Bacterial adhesion study showed that fibronectin-coated terpolymers with R varying from 0.5 to 0.8 exhibited inhibition rate of S. aureus adhesion from 90% to 98% as compared to the adhesion on unfunctionalized poly(methyl methacrylate). In contrast, the adhesion of osteoblasts onto the same functionalized terpolymers was decreased by 20% when compared to the results obtained on poly(methyl methacrylate). While the amount of attached osteoblasts are similar onto all the functionalized terpolymers whatever its R value, the cell proliferation was different and was found to vary with R in the range 0.5-0.8. Osteoblast proliferation, alkaline phosphatase activity and accumulation of calcium in the extracellular matrix of these cells, cultured on the functionalized terpolymers with R equal to 0.7-0.8 were similar to that observed onto non-functionalized poly(methyl methacrylate). In contrast, osteoblast proliferation was inhibited on terpolymers with an R value around 0.6. These results provide evidence that functionalized poly(methyl methacrylate)-based terpolymers with R ratio equaling 0.7-0.8 simultaneously inhibit bacteria adhesion and support osteoblast functions pertinent to new bone formation. These functionalized polymers could, therefore, be used as coating or grafted on orthopedic and dental implants to render them both bone compatible and able to prevent bacterial infection.

Surface modification of silicone intraocular implants to inhibit cell proliferation.

Photo-cross-linkable polymers bearing cinnamic, sulfonate, and carboxylate groups were synthesized by radical polymerization leading to randomly distributed copolymers. These polymers were used to coat silicone intraocular lenses in order to reduce posterior capsule opacification, also named "secondary cataract". We previously demonstrated that polymers containing both carboxylate and sulfonate groups inhibit cell proliferation, and formulations with the ratio R = COO-/(COO- + SO3-) equal to 0.64 provided the highest inhibitory effect. Ionic polymers with this formulation were synthesized to contain a monomer with pendant siloxane groups in order to get compatibility with the silicone matrix of the intraocular lenses. Anchorage of the ionic polymer at the surface of the silicone implant was achieved by a cycloaddition reaction of the photosensitive groups according to two options. These modified silicone surfaces grafted onto intraocular lenses were shown to inhibit cell proliferation to 60%.

Monitoring cell adhesion processes on bioactive polymers with the quartz crystal resonator technique.

The Thickness Shear Mode (TSM) quartz crystal resonator has been extensively used as sensitive sensor in various electrochemical and biological applications. This technique based on the propagation of an ultrasonic shear wave generated by a sinusoidal electric field through a piezoelectric quartz resonator, provides a non-destructive and powerful means to probe changes at solid-solid or solid-liquid interfaces. In this study, TSM was used to characterize cell-polymer interactions developing during the cell adhesion process. TSM sensing was used to monitor the inhibiting properties of bioactive polymers towards fibroblast McCoy adhesion processes. For this purpose, thin films of various bioactive polymers exhibiting either carboxylate or/and sulfonate functional groups were deposited onto the TSM. Measurements of the time variation of the electrical motional resistance in the vicinity of the mechanical sensor resonant frequency were performed as the quartz crystal resonator was either coated with the continuous polymer phase or polymer plus cell suspensions. Cell adhesion processes on these surfaces was investigated by cell counting and the quartz resonator-based technique. Inhibition of fibroblast McCoy adhesion onto thin polymer films of various chemical compositions was analyzed and discussed in the perspective of a possible application of these bioactive polymers to fabricate intraocular lenses able to prevent secondary cataract phenomena.

Vitronectin is significant in the adhesion of lens epithelial cells to PMMA polymers.

A major complication of intraocular lens surgery is diminished visual acuity caused by the regrowth of lens epithelial cells (secondary cataract). Polymethylmethacrylate (PMMA) is a commonly used intraocular lens material. This study addresses the mechanisms underlying the initial adhesion of lens epithelial cells to PMMA and a functionalized PMMA-based terpolymer known to inhibit cell proliferation. Rabbit lens epithelial cells were cultured on the test polymer surfaces in medium containing serum depleted of either fibronectin or vitronectin (or both) to identify the role of these proteins in the initial process of cell adhesion. Adherent cells were quantitated after 60 min, and the actin cytoskeleton and focal contact formation were compared in each serum treatment on both polymers. Vitronectin was significantly more effective for initial cell attachment to both polymers than fibronectin. Normal cell spreading on PMMA required vitronectin and was independent of fibronectin, whereas cell spreading on the terpolymer was abnormal and required the presence of fibronectin and vitronectin together. Together, these results help to explain the inhibition of cell proliferation previously shown on the functionalized PMMA. This work contributes to the design of a polymer for use in intraocular lenses that inhibits proliferation of the target cells.

Alternative intracellular signaling mechanism involved in the inhibitory biological response of functionalized PMMA-based polymers.

A PMMA-based polymer previously shown to inhibit cell proliferation was compared to untreated PMMA. Conformation of adsorbed proteins, cell adhesion, cytoskeleton formation, and integrin activation were examined. Fibronectin adsorbed in a different conformation on the PMMA-based polymer exposing a different balance of the heparin-binding domains. Fibroblasts attached in equal numbers to both surfaces over a 4-h period, but the integrins involved in the adhesion process elicited different intracellular signaling pathways. Cells attached to PMMA showed activation of FAK and MAP as they spread using an assembled actin cytoskeleton. Cells attached to the polymer showed early and strong MAP activity that resulted in nonassembly of the actin cytoskeleton and sub-optimal cell spreading. We conclude that the chemistry of the polymer surface dictated a different conformation of the adsorbed proteins that resulted in alternative cell signaling and diminished cell spreading. This accounted for the biological inhibition previously reported on the PMMA-based polymer.

Biomimetic poly(methyl methacrylate)-based terpolymers: modulation of bacterial adhesion effect.

Adherence of Staphylococcus aureus, responsible for major foreign body infections, was assessed onto functionalized poly(methyl methacrylate)-based terpolymers bearing sulfonate and carboxylate groups and onto poly(methyl methacrylate) as control. These terpolymers, have been synthesized by radical copolymerization of methyl methacrylate, methacrylic acid, and sodium styrene sulfonate by varying the ratio R = [COO(-)]/[COO(-) + SO(3)(-)] from 0 to 1 and keeping ionic monomer content between 7 and 18%. Adsorption of fibronectin onto poly(methyl methacrylate) was shown to dramatically promote bacterial adherence, whereas a strong inhibition of bacteria adherence was observed onto functionalized terpolymers containing both carboxylate and sulfonate groups. When terpolymers were predominantly functionalized by carboxylate groups, bacteria adherence was favored and reached values close to those obtained for poly(methyl methacrylate). These results have been related to the distribution of the anionic groups along the macromolecular chains, creating active sites responsible for specific interactions with fibronectin and inducing modifications of its conformation. The conformation of the adsorbed adhesive protein was then suggested to have an influence on the availability of its interaction sites to bacteria adhesins and therefore on modulation of bacteria adherence. Inhibition of Staphylococcus aureus adherence by functionalized poly(methyl methacrylate)-based terpolymers is of great interest in the field of biomedical implants and especially in the case of ophthalmic applications.

Modulating fibroblast cell proliferation with functionalized poly(methyl methacrylate) based copolymers: chemical composition and monomer distribution effect.

Poly(methyl methacrylate)-based terpolymers bearing sulfonate and carboxylate groups have been synthesized by radical copolymerization leading to polymers with random distributions of ionic monomer units. Fibroblast cells were seeded on terpolymers of various molar compositions of ionic groups. Kinetics of the cell proliferation were examined and systematically compared to the nonfunctionalized control polymer, poly(methyl methacrylate). Modulation of cell proliferation was observed on 15% ionic monomer content copolymers of various compositions (R = COO(-)/(COO(-) + SO(3)(-)) and varies from 0 to 1). The inhibition percentage of cell proliferation calculated for each polymer by comparison to the cell proliferation on the control was plotted against R and gave a maximum value for R close to 0.55. Copolymers with ionic group contents higher or lower than 15% exhibit inhibition percentages fitting with those previously observed for the same R values, showing that the hydrophilic properties are not sufficient to explain the modulation effect of this material toward cells. Moreover, for each polymer tested, cells, even if inhibited in growth, were shown to be viable, indicating that the synthesized terpolymers exhibit cytostatic properties excluding any cytotoxic effect. Such polymers may be used for the fabrication of biocompatible intraocular lenses and prevent secondary cataract.