FR2850107A1 - Grafting a bioactive polymer to a polymer material, useful for making biomedical materials, comprises distending the surface of the polymer material before ozonization and radical polymerization - Google Patents

Abstract

Grafting a bioactive polymer to a polymer material by ozonization before radical polymerization of a monomer solution comprises distending the surface of the polymer material before ozonization. An Independent claim is also included for a biomedical material or ligament prosthesis comprising a polymer material grafted as above.

Description

The present invention relates to a method of

  treatment of polymeric material as well as biomimetic prostheses.

  The rupture of the anterior cruciate ligament (ACL) of the knee is observed in four out of every thousand subjects. Therapeutics have evolved a lot in recent years. At present, polyethylene terephthalate (PET) prostheses are the most widely used, chemical treatments as well as the specific braiding of the fibers which has made it possible to arrive on the market with high performance ligaments.

  For this kind of PET prostheses, it is constantly sought to improve the fibroblastic rehabitation, in order ultimately to improve the viability of the implant. Indeed, PET is in particular a synthetic material which is solid and well tolerated biologically. However, prosthesis rupture problems are conventionally encountered, which can be explained by incomplete recolonization. Biocompatible materials are now being sought, particularly in the form of fibers, the surface of which is grafted, and is capable of promoting fibroblastic regrowth and of improving the organization of collagen.

  It has now been shown that certain suitable surface treatments of the polymeric material make it possible to obtain biomimetic materials, that is, capable of mimicking living materials and systems.

  In this regard, processes for grafting functional polymers to the surface of polymeric materials are already known. Some of these processes consist in generating peroxide radicals on the surface of the material by plasma, irradiation, electron beams or ozonation, and then radically polymerizing a solution of monomers. The ozonation technique is known to uniformly generate peroxides even if quantitatively the plasma is more efficient. However, this ozonation technique gives mediocre results on PET surfaces, in comparison with the levels of peroxides generated under the same conditions on a polyurethane (PU) surface.

for example.

  The present invention therefore aims to overcome these disadvantages by providing a method of surface treatment of a polymeric material for notoriously and unexpectedly graft performance with performance of bioactive polymers or copolymers on PET. The surface is pretreated by ozonation prior to radical polymerization of a solution of at least one monomer.

  According to an essential characteristic of the invention, the ozonation is preceded by a step of preparing the surface of the polymeric material consisting of a distension of said surface. This distension is preferably obtained with a solvent capable of modifying the surface by swelling. It is well understood that such a preparation makes it possible to increase the level of peroxides, whatever the nature of the polymeric material.

  The invention also relates to biomedical materials, and in particular ligamentous prostheses, obtainable by implementing the method of the invention. Indeed, the polymerization conditions, the pretreatment (s) carried out according to the invention, as well as the flexibility of choice of the monomers and thus the graftings obtained by the implementation of the method make it possible to obtain materials with very advantageous properties, as will appear in the examples.

  The invention will be better understood on reading the detailed description and examples below and in view of the appended figures - FIG. 1 illustrates the grafting obtained by the process of the invention on a semi-crystalline PET film ( surface density in grafted monomer units) as a function of the duration of the ozonation and the concentration of monomers.

  FIG. 2 illustrates the grafting obtained by the process of the invention on semicrystalline PET woven fibers (degree of grafting expressed in molar concentration of grafted monomer units per unit area) as a function of the ozonation and polymerization times.

  FIG. 3 illustrates the adsorption of collagen I in the case of PET grafted with the biomimetic polymers according to the invention, compared to ungrafted PET.

  As a polymeric material whose surface is modified by grafting according to the invention, it is possible to use any type of biocompatible polymer that can be used to produce biocompatible articles whose surface properties are to be improved in terms of biological activities such as adhesion and cell proliferation, and in terms of lubrication, hydrophilicity, for example. Thus, polyurethane (PU), polystyrene (PS), silicone, poly (methyl methacrylate) (PMMA) or polyesters are well known.

  In particular, among the polyesters, mention may be made of the PET usually used for the manufacture of implants 20 and prostheses. More particularly, PET may be used in the form of knitted or non-knitted fibers, or of implantable artificial ligaments, in particular as a ligamentous prosthesis.

  According to a variant of these ligament prostheses 25 disclosed in patent EP 0,561,710, for example, a polyester ply, base of the ligament, is composed of longitudinal technical polyester poly (ethylene terephthalate) weft fibers and a transverse yarn. textured polyester. Longitudinal technical fibers 30 can be united by a knitted transverse structure, a part of the longitudinal fibers can be left free. The shape and dimensions of the ligaments vary according to the joint to be repaired. The diameter of the ligament varies with the number of longitudinal fibers constituting it, this number varying according to the desired resistance as well as the corporeality and the activity of the patient to be treated.

  PET can also be used in other forms (pellets, films) necessary for and / or compatible with their subsequent use, especially in all biomedical applications of polymeric materials.

  Preferably, according to the invention, semicrystalline PET, which is fibrous or non-fibrous, and whose crystallinity level is 35% 10%, is used. For example, PET fiber is used having a glass transition temperature at 750C 50C, a melting temperature at 260C 50C, and a crystallinity level of 40% 5%. By way of non-limiting example, the process of the invention was applied to PET film, having a thickness of the order of 200 micrometers, to PET in the form of knitted fabric having a thickness of 1 μm. order of 1 mm and PET in the form of fibers of diameter of the order of 25pm.

  As a monomer to be grafted onto the surface in order to modify its properties, hydrophilic monomers capable of radical polymerization and copolymerization giving rise to biocompatible polymers grafted onto the surface of the polymer material, these grafted biocompatible polymers, may be mentioned according to the invention. are said to be bioactive.

  Such monomers, such as carboxylates, phosphates, sulphonates and sulphates are described in US Pat. No. 6,365,692, and they can be used according to the invention, alone or in mixtures. For example, methacrylic acid and styrene sulfonate and mixtures thereof may be mentioned.

  This (or these) monomer (s) are, according to the invention, dissolved in a suitable solvent for the radicular polymerization reaction, for example water, preferably bi-distilled water.

  As regards the concentration of monomer (s) in the solution, according to the invention, it is possible to choose any concentration compatible with the implementation of the radical polymerization reaction. Concentrations close to the limiting monomer solubility in the solvent, however, preferably between about 5% and the limiting monomer solubility in the solvent are preferred. Thus, in the case of sodium sulphonate (NaSS) and methacrylic acid (AM), concentrations of between 5% and 20% by weight in water, for example of the order of 15%, will be preferred. by weight in water.

  It is preferred according to the invention to operate with solutions of monomers close to saturation, or to operate with concentrations of monomers in the solvent such that the solvent medium of (or) monomer (s) is viscous. Near solubility or saturation is meant any lower concentration of 1 to 7% by weight of the limiting solubility or the saturation concentration.

  According to the invention, prior to contacting the polymer material with the monomer solution (s), and subsequent radical polymerization, the surface of the polymeric material is subjected to a surface modification chemical treatment by ozonation. According to an essential characteristic of the invention, the surface of the polymer material is, prior to this ozonation, treated by washing and placed in the presence of a solvent to rid the surface of fats and impurities. This pretreatment also has the function and effect of distending the surface of the polymer material by swelling.

  The polymer material incorporates, especially at the surface, the molecules of the solvent, it swells on the surface, which facilitates and improves the fixation of the peroxides resulting from the subsequent ozonation, and consequently the subsequent grafting.

  According to the invention, any ethereal solvent of toxicity compatible with the use of the final biocompatible material is suitable, and in particular solvents of the cyclic ether and aliphatic ether type. Solvents with strong dielectric constants, preferably greater than 30, are preferred according to the invention. Among the preferred solvents, there may be mentioned tetrahydrofuran (THF), dimethylsulfoxide (DMSO), N, N-dimethylformamide (DMF), N N, N-dimethylacetamide (DMA), and N-methylpyrrolidone (NMP). THF is particularly preferred.

  The solvent pretreatment pretreatment is carried out at a suitable temperature in view of the solvent used, for example the ambient temperature. The duration of the pretreatment can be of the order of 5 minutes to 1 hour, preferably between about 10 and 25 minutes. Thus, the polymeric material may be washed and swelled by immersion in THF, for example, for about 15 minutes at room temperature.

  Optionally, the distillation step is followed by a treatment to remove traces of low molecular weight organic acids such as terephthalic acid and higher oligomeric acids from the surface of the polymeric material. FART.

  According to this variant, at least the surface of the polymeric material is treated with an aqueous solution of hot sodium carbonate or other alkaline earth or alkaline carbonates, such as CaCO 3 or K 2 CO 3. For example, in order to neutralize the acid traces mentioned above, the surface can be treated for about 10 minutes with a solution of Na2CO3 at about 5%, at a temperature of 1000C 50C.

  Optionally, the polymeric material is further, as needed, washed at least once with water, preferably bi-distilled water, between the pre-treatment steps above. For example, before being contacted with the solution of the monomer (s), the polymer material treated by ozonation, and optionally treated with Na2CO3, or the like illustrated above, can be washed several times at water, preferably bi-distilled, until the pH of the rinse water is neutral.

  In addition, after this pretreatment, the surface of the polymeric material may be rinsed with ethereal solvent, for example with THF, and then dried in a vacuum oven.

  According to the invention, the ozonation prior to the application of the monomer solvent solution is carried out under conditions of duration which depend on the amounts of ozone used. For example, the ozonation time is of the order of 20 to 90 minutes for amounts of ozone of the order of 50 g / m3.

  The polymerization conditions depend on the nature of the monomer (s) chosen. With regard to the necessary polymerization time, according to the invention, the gelification time of the solvent medium of the monomer (s) at the reaction temperature is comparable. Thus, for example, for NaSS, the polymerization time of sodium polystyrene sulfonate (PSSNa) is of the order of 1 h at 70 ° C., and of the order of 1 h 30 at 650 ° C.

  In addition, at the end of the polymerization, the grafted polymeric materials can be washed, in particular to remove unreacted monomer residues.

  Thus, it is possible to wash the functionalized surface several times, with a suitable solvent of the monomer or monomers, for example, double distilled water, and the washing can be optionally finished with any suitable solvent, absolute ethanol. for example, to eliminate any traces of ungrafted polymer.

  According to the invention, it has thus been possible to obtain surface densities of graft polymer on the surface of the polymer material, in the case of PSSNa on PET, of the order of 0.4 g / cm 2 (ie 2.109 mol / cm 2) on a film, and of the order of 20 g / cm 2 (or 107 mol / cm 2) on a fabric as described above. Such results can be obtained for fibrous or non-fibrous materials pretreated by immersion in THF prior to ozonation. For surfaces not pretreated with a solvent capable of distending them, the surface densities are about five times lower than those described according to the invention, namely of the order of 0.1 μg / cm 2 film and 4 μg / cm 2 for a fabric. constituting an artificial ligament 30 such as that of patent EP 0,561,710. This factor 5 reflects the difference in peroxide density, the polymerization conditions being equal elsewhere.

  As will become apparent below, the functionalized polymer materials make it possible to provide the surfaces with interesting biological properties, especially for prostheses, and particularly artificial ligaments.

  Indeed, the choice and the dosage of the functionalization monomers allow adaptation to the biological conditions required by the subsequent use of the functionalized material.

  The applications are not however limited to ligament prostheses, but include any material that can be used to produce any type of biomedical material such as implants, prostheses, suture material, or even cell culture supports, immunological test supports or Protein purification, for example, where the properties mentioned above, including biocompatibility, cell proliferation, adhesion and lubrication properties are required.

or desirable.

  Examples 1 to 3 given below illustrate the implementation of the treatment method according to the invention, applied to PET materials, while Examples 4 and 5 make it possible to better understand the performance of the materials thus treated.

EXAMPLE 1

  1) Pre-treatment 16 mm diameter pellets were cut from semi-crystalline commercial PET sheets 0.2 mm thick from Bayer Company (PET Ga). The pellets were dipped for 15 minutes in THF, then rinsed with double-distilled water and then rinsed with absolute alcohol. They were then dried in a vacuum oven for 30 minutes.

  2) Monomer Styrene sodium sulphonate (NaSS) was purified by recrystallization from a mixture of bi-distilled water (10/90 by volume) and dissolved at 70 ° C. in this solution. It was then vacuum filtered with a sintered glass disk 3 of porosity index 3, and stored at 4 C.

  The formed NaSS crystals were recovered by filtration and the resulting solid was dried under vacuum at 500C until a constant weight was obtained. - 9

  3) Ozonation Six PET pellets were placed in a tubular reactor (500 cm3) containing bi-distilled water (100 cm3), equipped with an ozone supply dip tube.

  The ozonation was carried out with a gas flow equivalent to ozone at 50 g / m 3 of oxygen. The pellets were rinsed three times with double distilled water, three times with absolute alcohol, and three times with THF, and then dried in a vacuum oven for 30 minutes at 25 ° C.

  4) Grafting A 15% by weight solution of NaSS in bidistilled water was prepared, and the solution was added to a vessel where the pellets were added, and the oxygen was removed by bubbling with water. argon. The container was sealed and heated in a water bath at 65 ° C. for 90 minutes. The pellets were recovered at the end of the reaction and were washed several times for one hour with double-distilled water, then rinsed three times with absolute alcohol, then three times with THF, and then dried under vacuum. during one hour.

  5) Demonstration of grafting by visible UV spectroscopy with desiccated toluidine.

  The sulphonate groups present on the surface of the grafted samples are complexed with a solution of toluidine blue (5.10-4 M) at a pH = 10 and at a temperature of 30 C for 6 h. A buffer solution of 2-amino-2-methyl-propanol (buffer solution which maintains the pH at 10, at 37 ° C.) is heated. 0.2 ml of buffer solution is introduced into a 50 ml flask containing the toluidine blue solution. The samples are rinsed for 20 minutes in a pH 9 solution of NaOH. A) Complexation: The quaternary amine group of toluidine complexes with the surface-grafted sulfonate groups of the PET, and this complexation gives the samples a blue color. .

  b) Decomplexation The colored samples are then immersed in a 50% by volume aqueous solution of acetic acid in order to decomplex and extract toluidine from the grafted layer. Toluidine contains an amine group available for quaternization with the carboxylic group. acetic acid. The quaternary amine acetate thus formed then passes into the acetic solution.

  Then, the concentration of toluidine extracted in acetic solution is evaluated by U.V.Visible spectroscopy at its absorption maximum (615 nm). This makes it possible to determine the concentration of styrenic units which have been fixed on the surface (assuming the existence of a 1/1 complex between dye molecule and motif). After calibration of a Perkin Elmer spectrometer controlled by the Lambda 12 software, the spectra of the different samples in solution are recorded.

  It is verified that the polymerization of the monomers has taken place and the concentrations of adsorbed toluidine blue and therefore of grafted monomers are determined.

  The spectra obtained have an absorption peak, the maximum of which is 615 nm, characterizing toluidine in 50% acetic acid solution. The height of this peak varies in proportion to the concentration of Toluidine Blue solution. The molecular extinction coefficient, E by the Beer-Lambert relationship, was determined from a calibration line.

  An S value of 5.10-4 L.mol-1.cm-1 was deduced for toluidine in 50% acetic acid solution.

  X-ray Photoelectron Spectroscopy (X.P.S.) X-ray photoelectron spectroscopy (XPS) analyzes of the grafted surfaces were performed with a Surface Science X-probe spectrometer. A filtered X-ray source using KOE radiation from aluminum was used to stimulate photoemission. The energy of emitted electrons was measured with a hemispheric energy analyzer, at 50eV for high spectral resolution and at 150eV for chemical element analysis.

  Service Physics software has been used for the spectral resolution as well as for the determination of the elemental composition of the surface. During the spectral acquisition, the pressure in the analysis chamber was maintained at 10-9 torr. The spot size of the X-ray used was 800 μm and the angle of incidence 55, the penetration depth being 50 A. The binding energies of the elements are referenced with respect to the carbon ls (CC and CH). ) at its maximum at 285 eV. Three measurements are performed on each sample.

  6) Results Pellets were ozonated for durations (d) of 2, 5, 10, 20 and 40 min. The grafting rate results, expressed in the density of grafted monomer units (10-1 μg / cm 2) for monomer concentrations of 5% (0) and 15% (o) by weight and variable ozonation times. are shown in Figure 1 which illustrates the influence of the ozonation time and the monomer concentration on the grafting rate of poly (NaSS) on semi-crystalline PET films (radical polymerizations carried out at 65 C and stopped after 90 min of reaction).

  Moreover, the results of XPS of the various pellets for ozonations of variable duration have shown that an optimal level of grafted polyNaSS is obtained for ozonation times of the order of 30 to 60 min, the sulfur levels and of atomic sodium being maximal. In addition, it has been observed in particular that the C-H (Cls) carbon content increases as expected, as a result of the grafting of sodium polystyrene sulphonate hydrocarbon macromolecular chains onto PET. The level of C-HCls increased from 63% for crude PET to 72% for PET grafted with polyNaSS.

  Finally, the results obtained in terms of grafting (surface quantity) were of the order of 0.4 μg / cm 2 for a monomer concentration of 15 wt% and of the order of 0.025 μg / cm 2 for a monomer concentration. from 5% by weight, - 12 for ozonation times of 30 min and a polymerization time of 1 h 30, at a temperature of 65 ° C.

EXAMPLE 2

  The same treatment was carried out on rectangular samples of knitted fabric (3 cm 2 of surface and 1 mm thick) consisting of semi-crystalline PET ligament fibers of the type of those of patent EP 0 561 710.

  Each fiber comprises about fifty yarns of 20 μm diameter each, has a crystallinity level of 40% + 5% and a melting temperature of 260 ° C.

  The ozonation was carried out on six rectangles of tissue under the same conditions as those described in Example 1. The grafting conditions were identical to those of Example 1 but applied to the only 15% NaSS concentration. in weight.

  The results obtained concerning the degree of grafting, expressed in molar concentration of monomer units grafted per unit area (108 mol / cm 2) for a monomer concentration of 15%, as a function of the polymerization time (dp) at 65 ° C. and for varying ozonation times (o: 30min; 60min;: 120min) appear in Figure 2, which illustrates the influence of ozonation and polymerization times.

  These results have shown that an optimum is reached in terms of grafting rate and measurement accuracy, for an ozonation time of 60 min and polymerization times of 90 min. Under these conditions, the surface density of graft polymer was 20 g / cm 2 for an ozonation time of 60 min and 12 g / cm 2 for an ozonation time of 30 min.

EXAMPLE 3

  The same treatment as that described in Example 2 was carried out on rectangular samples of knitted fabric of fibers of the same dimensions and characteristics as previously mentioned in FIG.

Example 2. - 13

  The same ozonation conditions and grafting conditions were used, but this time applied to an aqueous solution of an even-weight mixture of 15% by weight sodium styrene sulphonate and 15% distilled methacrylic acid (MA). % by weight, ie an AM / NaSS composition of 70/30 mole percent.

  Under these conditions, the results obtained showed that the molar concentration of grafted monomeric units is 1.6 × 10 -7 mol / cm 2. Assuming that an AM-NaSS random copolymer of the same composition as that of the monomer mixture (i.e., 70/30 mole percent AM / NaSS) was formed, the surface density of the graft copolymer was of 19.5 g / cm 2.

  EXAMPLE 4: Adsorption Characteristics of Type I Collagen Molecules on Films and PET Fibers Rat type I collagen (Sigma C7661) was used for all experiments; it was radiolabeled with I (125) -Na (Amersham) using the chloramine T method. Modified PET and PET discs (diameter 6 mm) or fibers were incubated for one hour with different concentrations. of collagen in solution. The disks or fibers were then washed with culture medium (DMEM, Gibco) to remove all unadsorbed collagen molecules, and the radioactivity emitted by the collagen I molecules adsorbed on the copolymer was measured. using a counter.

  The adsorption results of 125 I-radiolabelled collagen I molecules on PET-based surfaces: Adsorbed Collagen I (CA) (mole / L) / introduced Collagen I (IC) (mole / L) (*) PET disc, O: PET-NaSS disc) which appear in Figure 3 show that the adsorption of collagen I is markedly superior in the case of PET grafted by biomimetic polymers.

  The analysis of results according to the Langmuir model made it possible to determine Ka and Bmax as follows.

  Ka (M) Bmax (M) B max (mg / cm 2) PET 3.3.107 l, 6.l10- 424 PET-NaSS 2.7 x 10 2, 3.10-9 610 The results show that the adsorption of Collagen 1 is significantly higher on PET grafted with PSSNa (610 g / cm 2) compared to adsorption on non-grafted PET (424 μg / cm 2) since it corresponds to an increase of 43%.

  EXAMPLE 5 Adhesion and Proliferation of Human Fibroblasts on Modified PET Surfaces

  The FHPA cells used for these experiments come from a primary culture of human fibroblasts.

  In order to preserve a homogeneous phenotypic expression, these cells are used between the 5th and the 10th passage.

  They are cultured in the complete culture medium (DMEM supplemented with 10% FCS, 1% L-Glutamine and 1% antibiotic).

  PET and PET films grafted with PSSNa were placed at the bottom of the 24-well plate culture wells (Costar). The FHPA cells were inoculated (10,000 cells per well) in a complete culture medium. The number of cells on the different supports was determined every day (from 1 to 10 days of culture) after enzymatic detachment, by direct counting under an optical microscope using a particle counter (Coulter). The proliferation parameters of the FHPA cells are then calculated: doubling time, latency time, number of confluent cells. The values obtained, number of cells with identical confluence 1.105 / well on grafted and ungrafted PET, show that the modified PET surface does not exhibit a cytotoxic effect or an inhibitory effect on FHPA cells.

  The adherence of FHPA cells was studied on pre-or non-adsorbed grafted PET surfaces of collagen I. After incubation for 20 to 40 minutes at 370 ° C., the nonadhered cells were removed by renewal. from the culture medium. Physical force was then applied to cells that were placed under orbital shaking (60 rpm) for 10 min. The results of these experiments show that after 40 minutes of adhesion, the application of an orbital force did not induce the detachment of cells, regardless of the type of pre-adsorbed proteins on PET. of pre-adsorbed collagen, adhesion forces on grafted PET are substantially (5 to 8 times) greater than on ungrafted PET. - 16

Claims (14)

  1 - Process for the surface treatment of a polymeric material by grafting bioactive polymers or copolymers, in which the surface is pretreated by ozonation, before a radical polymerization of a solution of at least one monomer, characterized in that the ozonation is preceded by a step of preparing the surface of the polymeric material consisting of a distension of said surface.   2 - Process according to claim 1, characterized in that the distension is obtained with a solvent capable of modifying the surface by swelling.   3 - Process according to claim 2, characterized in that the solvent is of the cyclic or aliphatic ether type.   4 - Process according to claim 2 or 3, characterized in that the solvent has a dielectric constant greater than 30.   Process according to one of Claims 2 to 4, characterized in that the solvent is chosen from tetrahydrofuran (THF), dimethylsulfoxide (DMSO), N, N-dimethylformamide (DMF) and N, N-dimethylacetamide (DMA). ), and N-methylpyrrolidone (NMP).   6 - Process according to one of claims 1 to 5, characterized in that the monomer solution has a concentration of monomer (s) close to the saturation monomer (s) in the solution of the monomer (s).   7 - Method according to one of claims 1 to 6,   characterized in that the monomer is of the carboxylate or sulfonate type.   8 - Process according to one of claims 1 to 7,   characterized in that the monomer is sodium styrene sulfonate.   9 - Process according to one of claims 1 to 8, characterized in that the polymeric material is polyethylene terephthalate.   - Process according to claim 8, characterized in that the polyethylene terephthalate is semi-crystalline.   11 - Method according to one of claims 1 to 10, characterized in that the polymeric material is fibrous.   12 - Process according to one of claims 1 to 11, characterized in that the polymeric material is treated by extracting traces of organic acids of low molecular weight before the radical polymerization.   13 - Process according to one of claims 1 to 12, characterized in that the polymeric material is treated with an aqueous solution of alkali or alkaline earth carbonate, especially sodium carbonate.   14 - Method according to one of claims 1 to 13, characterized in that the duration of the ozonation is of the order of 20 to 90 min. 15 - Method according to one of claims 1 to 14, characterized in that the ozonation is carried out at ozone levels of the order of 50g / cm3.   16 - Biomedical material comprising a graft polymer material obtainable according to one of claims 1 to 15.   17 - ligament prosthesis comprising a graft polymer material obtainable according to one of the Claims 1 to 15.