METHOD FOR PRODUCING A MATERIAL FOR A BONE IMPLANT

Abstract

A material for a bone implant contains: (a) a carrier structure having a surface that has at least one biocompatible material; (b) a matrix covalently bound to the surface; and (c) calcium phosphate embedded in the matrix. A medically acceptable, highly compatible and versatile material can be provided, if the matrix has at least one polysaccharide.

Claims

1. A method for producing a material for a bone implant, which comprises the steps of: providing a carrier structure having a surface formed from a biocompatible material; coupling a covalent coupling of a matrix having at least one polysaccharide to the surface; and mineralizing the matrix with calcium phosphate.

2. The method according to claim 1, which further comprises performing the coupling step by the following steps in any desired order: covalent coupling of a linker molecule selected from the group consisting of a diamine linker, a diamine linker and a succinic acid linker, a UV-grafted polyacrylic acid, a photocoupleable linker, and an azidoaniline linker, to an activated surface; and covalent coupling of the polysaccharide with carboxylic acid groups to a diamine linker molecule, or a hexamethylene-diamine-modified polysaccharide to succinic acid linkers, or an unmodified polysaccharide via ester bonds to a polyacrylic acid linker or a photocoupleable linker.

3. The method according to claim 2, which further comprises carrying out the covalent coupling of the photocoupleable linker to the activated surface at a wavelength with a range of 200 nm to 400 nm.

4. The method according to claim 2, which further comprises carrying out the covalent coupling of a carboxy-functionalized polysaccharide by means of amine and carboxyl group coupling to the photocoupleable linker.

5. The method according to claim 2, which further comprises using an azidoaniline linker as the photocoupleable linker.

6. The method according to claim 5, which further comprises carrying out the covalent coupling of the the azidoaniline linker to the activated surface at a wavelength with a range of 200 nm to 300 nm.

7. The method according to claim 3, which further comprises carrying out the covalent coupling of the photocoupleable linker to the activated surface at the wavelength with a range of 240 nm to 260 nm.

8. The method according to claim 3, which further comprises carrying out the covalent coupling of the photocoupleable linker to the activated surface at the wavelength of 254 nm.

9. The method according to claim 4, which further comprises using an azidoaniline linker as the coupleable linker.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0094] The FIGURE of the drawings shows correspondence between x-ray powder diffractogram produced with scratched-off deposition on sample plate IS019 and signal of hydroxyapatite from the literature.

DETAILED DESCRIPTION OF THE INVENTION

[0095] The methods mentioned and used in the following text (ATR-IR analysis, scanning electron microscopy, confocal laser scanning microscopy, fluorescence spectrometry, NMR measurements, thermogravimetric analysis (TGA), UV tests, contact angle measurement, nuclear magnetic resonance spectroscopy) were carried out according to the principles and methods known to the person having ordinary skill in the art using known devices.

[0096] It is possible by means of wet chemical modification of the PEEK surface to achieve a reduction in the carbonyl groups of the PEEK framework by treating the film with NaBH.sub.4 in DMSO at 120° C.:

##STR00002##

[0097] The reaction can be carried out without exclusion of oxygen under stirring in a 500 milliliter (mL) flask. 10 PEEK plates (1 cm.sup.2 (square centimeter) each) were placed in 20 milliliters (mL) of DMSO and stirred. Heating was carried out to 120° C., and after 20 min, 13 mmol (490 mg) of NaBH.sub.4 was added. The reaction time was 4 h 30 min. The PEEK was washed for 15 min in 20 mL of MeOH, 10 min in 20 ml of H.sub.2O and 35 min in 20 mL of 1 M HCl. After rinsing in EtOH, the PEEK was dried in a vacuum drying oven for 2 h at 40° C. and 50 mbar. The reaction was verified by means of an ATR-IR spectrum (ATR-IR: v 3400 cm.sup.−1 (m) (cm.sup.−1: wave number), strong attenuation of the 1647 cm.sup.−1 (w) C═O stretching vibration, not shown).

[0098] The reduced PEEK films can be further converted to the succinic acid ester. The esterification can be carried out by means of succinic anhydride in acetone at room temperature:

##STR00003##

[0099] 10 PEEK plates (1 cm.sup.2 each) were placed in 30 ml of acetone and heated to 40° C. 1 gram (g) (10 mmol) of succinic anhydride was added. The reaction time was 5 h 35 min. The plates were washed with 20 mL of acetone, H.sub.2O and ethanol each and dried in a vacuum drying oven for 2 h at 40° C. and 50 mbar. The reaction was verified using an ATR-IR spectrum. ATR-IR: v 3400 cm.sup.−1 (m), 2924 cm.sup.−1 (w), 2861 cm.sup.−1 (m) spa CH.sub.2 stretching vibrations, 1705 (w) COOH stretching vibration (not shown).

[0100] Purified PEEK films can be converted to pure diamine (ethylenediamine (EDA) and 1,3-diaminopropane). Imines (Schiff bases) are formed on the PEEK surface. The reaction can be carried out for 3 h under reflux of the diamine and stirring of the mixture:

##STR00004##

[0101] 5 PEEK plates (1 cm.sup.2 each) were placed in 10 mL of ethylenediamine or 1,3-diaminopropane. The reaction mixture was heated under reflux while stirring for 3 h. The reaction mixture was cooled to RT, and the PEEK plates were thoroughly washed with acetone. The modified films were dried in the vacuum drying oven for 2 h at 40° C. and 50 mbar. The reaction was verified using an ATR-IR spectrum ATR-IR: v 2925 cm.sup.−1 (m), 2854 cm.sup.−1 (m) spa CH.sub.2 stretching vibrations, 1620 cm.sup.−1 (w) C═N stretching vibration (not shown).

[0102] We were also able to introduce a succinic acid linker into the amine-functionalized PEEK samples: the PEEK films were added to 10 mL of dry 10 millimole (mM) succinic anhydride DMF-solution (DMF: dimethylformamide). After 10 h, the films were carefully washed with MilliQ (ultrapure water) and examined by ATR-IR spectroscopy (not shown). ATR-IR: v 2925 cm.sup.−1 (m), 2854 cm.sup.−1 (m) spa CH.sub.2 stretching vibrations, 1706 cm.sup.−1 (w) C═O stretching vibration.

[0103] The modified PEEK films were subjected to contact angle measurement. The modified PEEK films were treated with buffer prior to measurement, rinsed with MilliQ and thoroughly dried. The contact angle measurements can be carried out 5 seconds (s) after drop application. Contact angle measurements with ultrapure water (MilliQ) showed significantly increased hydrophobicity of 66° in ethylenediamine-modified PEEK compared to unmodified PEEK films (84.5°). In the variant modified with 1,3-diaminopropane, the contact angle also decreased, to 70°, as the surface was also more hydrophilic. The increase in hydrophilicity indicates a successful course of the reaction in the conversion of PEEK with diamines.

[0104] Untreated PEEK films can be coated with polyacrylic acid using a grafting-from polymerization method (UV-light-induced PEEK-modification):

##STR00005##

[0105] The test specifications used can be carried out in one step. One can work with degassed aqueous solutions of distilled acrylic acid. An OSRAM Vitalux 300 without further filters can be used as a UV light source.

[0106] 4 PEEK plates (1 cm.sup.2 each) were placed in a Schlenk flask and degassed in 3 vacuum/nitrogen cycles. The corresponding amount of degassed MilliQ water (4 freeze-pump-thaw cycles) was added. After adding the degassed acrylic acid (30 min nitrogen purging), the flask was irradiated under stirring with the UV lamp from a distance of 15 cm. The reaction time was between 15 min and 75 min. The concentration of the acrylic acid solution was between 5 wt % and 25 wt %. ATR-IR: v 1705 cm.sup.−1 (m) COOH stretching vibration.

[0107] The respective possible conditions and results are shown in Table 1.

TABLE-US-00001 TABLE 1 Reaction batches of UV-grafting with acrylic acid. Acrylic acid Reaction Gel concentration time layer on Item [wt %] [min] surface MG10 20 75 yes MG11 10 70 yes MG13 7.5 70 yes MG19 5 30 yes MG20 5 45 yes MG12 5 60 yes

[0108] The PEEK films grafted with polyacrylic acid films were examined by scanning electron microscopy (SEM) (not shown).

[0109] With each batch, an essentially homogeneous layer of polyacrylic acid was formed. With increasing polyacrylic acid concentration and reaction time, an increasing amount of polyacrylic acid was deposited on the surface, essentially in bead form, or the layer thickness increased. The lower the concentration of polyacrylic acid (30 micrometers (μm) for MG10 as compared to 3 μm for MG12), the smaller the beads were.

[0110] Samples that were polymerized for 30 min with an acrylic acid content of 5 wt % were used for further surface functionalization. Under these conditions, the coating was still thick enough to be described as a homogeneous coating (not shown), and at the same time it can be assumed with this layer thickness that the mechanical properties of the bulk material are not adversely affected. Because of the thick gel cushion of up to 5 millimeters (mm), the polyacrylic acid layers produced at higher acrylic acid concentrations are not suitable for the target application as bone implant material, as a gel cushion on the surface sharply impairs the quality of the mechanical contact with the surrounding tissue.

[0111] Under slight magnification, one can see that the polyacrylic acid has formed linear structures with the beads. The formation of these lines could be due to the drying methods (vacuum oven), but could possibly also be attributable to the hydrophobicity of the PEEK surface. The acrylic acid molecules diffused on the active site have a greater affinity for a growing polyacrylic acid layer than for the hydrophobic PEEK surface. This could also explain the line structures composed of PAA beads. The average bead size under these reaction conditions is 1.7 μm and is thus again approximately half as large as in 60 min polymerization. The coating results were verified by means of ATR-IR spectra (not shown).

[0112] The coating produced with 5 wt % of acrylic acid and 30 min UV treatment was found to be particularly suitable. Under these conditions, as a thin layer could be applied to the PEEK, no excessively large gel cushion was deposited.

[0113] UV-induced grafting polymerization is thus particularly well suited for the coating of PEEK with polyacrylic acid, as significant amounts of the PAA were detected on the PEEK surface.

[0114] It was also possible to carry out polymerization in pure acrylic acid, and as comparison, in pure methyl acrylate, i.e. polymerization in the pure monomer. The results were verified by means of ATR-IR spectra (not shown).

[0115] Coupling of 1,4-diaminobutane to the PAA-coated PEEK surface:

[0116] The polyacrylic acid layer can be modified by coupling of diamine linkers, so that amide bonds can later be formed with organic acids. Coupling of the diamine species to the carboxyl groups can be carried out using the modern coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). The activation and coupling can be carried out with DMT-MM at a buffered pH of 9:

##STR00006##

[0117] Quantification of the amino groups on the surface of the linker-functionalized PEEK film (substrate) can be carried out in a manner known to the person having ordinary skill in the art by means of the fluorescent dye C-coumarin:

##STR00007##

(star=7-hydroxicoumarin fluorophor, Sub=substrate, cf. S. Shiota; S. Yamamoto; A. Shimomura; A. Ojida; T. Nishino; T. Maruyama, Langmuir 2015, 31, 8824-8829):

##STR00008##

[0118] Based on the fluorescence intensity, the concentration of the dye in solution can be determined, thus allowing conclusions to be drawn as to the number of surface amino groups. In the functionalized PEEK samples (with ethylenediamine, 1,3-diaminopropane 4 and tetramethylenediamine (TMDA)), a higher NH.sub.2 density than in a non-functionalized PEEK sample was detected (data not shown).

[0119] Synthesis of the modified polysaccharides:

[0120] Two strategies can be used for modifying the PEEK films with polysaccharides: introduction of the linker molecule (diamine) on the carboxylate PEEK surface and introduction of the linker on the polymer. In the first case, unmodified polysaccharide is coupled to free amino groups on the substrate in a polymer coupling step, while in the second case, amine-functionalized polysaccharides are anchored to carboxyl groups on the substrate.

[0121] In one batch, unmodified hyaluronic acid:

##STR00009##

or alginic acid (shown as structural sections of alginic acid with the various poly-G, poly-M and alternating blocks. Depending on the origin of the alginic acid, the ratio of G to M varies):

##STR00010##

can be functionalized with an amine and then coupled to carboxyl groups on the PEEK surface. Here, it may be necessary to carry out deacetylation before the functionalization.

[0122] Deacetylation of hyaluronic acid:

[0123] Unmodified hyaluronic acid is composed of a D-glucuronic acid and an N-acetylglucosamine unit. It therefore has one free carboxyl functional group and one acetylated amine functional group per disaccharide monomer. In addition to substitution reactions on the OH groups, two suitable common methods for introducing amine functionalities are functionalization of the carboxyl groups with diamine linkers or the deprotection of the N-acetyl group to form a free amine.

[0124] The deacetylation can be carried out in an aqueous hydrazine solution under hydrazine sulfate catalysis:

##STR00011##

[0125] 50 mL of hydrazine monohydrate and 0.5 g of hydrazine sulfate were added to 1 g of sodium hyaluronate in order to prepare a 2 percent by weight (wt %) solution based on the polymer. After stirring for 72 h at 55° C., the polymer product was precipitated in cold ethanol, filtered and vacuum-dried (24 h). The residue was taken up in 20 mL of 5% acetic acid. Aqueous iodic acid solution (10 mL, 0.5 M) was added to this solution, wherein the temperature was maintained for 1 h in an ice bath at 4° C. Aqueous hydrogen iodide solution (57%, 3 mL) was added. After 15 min, the violet solution was extracted five times in a separating funnel with 25 mL each of diethyl ether until the aqueous phase was colorless. The pH of the solution was adjusted with a 0.2 M NaOH solution to 7-7.5. The polymer was precipitated in 1 volume equivalent of ethanol, dissolved in H.sub.2O and dialyzed against deionized water. The dialysis water was changed twice daily. After dialysis for 3 days, the solution was freeze-dried and the deacetylated hyaluronic acid was obtained as a product.

[0126] The free amino groups could be used as anchor groups for coupling to the carboxyl groups of the PEEK-PAA surface. The polymer was examined by NMR spectroscopy to determine the degree of deacetylation (not shown).

[0127] Modification of hyaluronic acid by means of hexamethylene diamine and adipic acid dihydrazide:

[0128] The free carboxyl groups of the hyaluronic acid can be suitable for a variety of possible modifications of the polymer. For example, there have been reports in the literature on amidation, esterification or Ugi condensation.

[0129] Hexamethylene diamine (NMDA) can be used to synthesize the amidated hyaluronic acid. The coupling of the amine can be carried out via classical EDC/NHS-coupling (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide coupling) in an aqueous medium. The reaction is divided into an activation phase in slightly acidic buffer solution and a coupling phase in slightly basic buffer solution. The pH of the reaction had to be continuously monitored and readjusted:

##STR00012##

[0130] A

[00001]$3\frac{\mathrm{mg}}{\mathrm{mL}}$

aqueous sodium Hyaluronate solution was prepared (500 milligrams (mg) in 167 mL of MilliQ). Based on the number of carboxyl groups in the polymer, 30 eq. of hexamethylene diamine (HMDA, 30 eq., 39.6 mmol, 4.6 g) were added. The pH of the solution was adjusted to 7.5 (0.1 M NaOH, or 0.1 M HCl). EDC (4 eq., 5.28 mmol, 0.9 g) and NHS (4 eq., 5.28 mmol, 0.607 g) were dissolved in 10 mL of water and then added to the reaction solution. The pH of the mixture was maintained at 7.5 by adding 0.1 M NaOH. The reaction solution was stirred overnight. The pH was adjusted to 7, and the polymer was precipitated in ethanol (3 volume equivalents). The polymer was dissolved in MilliQ

[00002]$\left(5\frac{\mathrm{mg}}{\mathrm{mL}}\right)$

and dialyzed for 6 days against FDI water (fully deionized water). The FDI water was changed twice daily. The purified product was freeze-dried for 4 days. The degree of HMDA functionalization was determined by means of .sup.1H-NMR spectroscopy. 46% HMDA functionalization.

[0131] The reaction was carried out with a very large excess of diamine (30-fold, based on the number of carboxyl groups in the polymer) in order to prevent crosslinking of the hyaluronic acid. The successful coupling of the HMDA linker was confirmed by .sup.1H-NMR spectra, and the successful functionalization of the carboxyl groups by forming amide bonds with the HMDA linkers was confirmed by ATR-IR spectra (not shown).

[0132] Modification of hyaluronic acid with adipic acid diazide:

[0133] The synthesis specifications were modified for synthesis of the adipic acid dihydrazide (ADH) derivative. As ADH shows lower basicity than HMDA, coupling is already possible in the acidic pH range of 4.8. This made it possible to dispense with the addition of NHS:

##STR00013##

[0134] A

[00003]$3\frac{\mathrm{mg}}{\mathrm{mL}}$

sodium hyaluronate solution was produced by dissolving 500 mg sodium hyaluronate in 170 mL of H.sub.2O. Based on the number of carboxyl groups in the polymer, a 40-fold molar excess of adipic acid dihydrazide (ADH, 52.8 mmol, 9.2 g) was added. The ADH was given the time to completely dissolve (15 min). The pH of the reaction mixture was adjusted to 4 using 1 M HCl solution. Ethanol (50 mL, 50 percent by volume (vol %)) was added, and stirring was carried out for 30 min. 4 eq. of EDC-HCl (5.3 mmol, 0.9 g) were added. The pH was maintained for 2 h at approx. 4.8 using 1 M HCl. After 2 h, the reaction was stopped. Neutralization of the solution by means of 1 M NaOH (pH=7). The reaction solution was added to a pre-washed dialysis membrane tube

[00004]$\left(\mathrm{MWCO}=3500\frac{g}{\mathrm{mol}}\right)$

and dialyzed for 9 days. Dialysis was carried out one day against a 100 mM NaCl, followed by alternating dialysis against a 25 vol % ethanol solution one day and against DI water the next. The ethanol/DI water cycle was repeated 4 times. The polymer solution was finally freeze-dried for 3 days. The degree of ADH functionalization was determined by means of .sup.1H-NMR spectroscopy. 53% ADH functionalization.

[0135] The synthesis can be carried out with a large excess of ADH in order to again prevent crosslinking of the hyaluronic acid. In both synthesis processes, the product had to be extensively dialyzed in order to remove the large excess of HMDA or ADH. The successful coupling of the ADH-modified hyaluronic acid was confirmed by .sup.1H-NMR spectra, and the successful functionalization of the carboxyl groups by forming amide bonds with the ADH was confirmed by ATR-IR spectra (not shown).

[0136] HMDA-Modified Alginic Acid:

[0137] Analogously to hyaluronic acid, alginic acid can also be functionalized with HMDA. The coupling can be carried out according to a specification for octylamine functionalization:

##STR00014##

[0138] 30 mL of 3 wt % aqueous sodium alginate (1 g sodium alginate) was placed in a flask, and the pH was adjusted using 0.1 M HCl to 3.4. The polymer solution was thus diluted to 50 mL (2 wt %). 797.5 mg (4.16 mmol) of EDC-HCl were dissolved in 4 mL of H.sub.2O and added to the polymer solution. The ratio of the EDC to the carboxyl functionalities was thus 0.7. The concentration of the EDC was determined by the molar frequency of the sodium alginate monomers

[00005]$\left(M=168.11\frac{g}{\mathrm{mol}}\right)$

in the polymer. After a reaction time of 5 min, 10 eq. of hexamethylene diamine (7.05 g) were added. The solution was stirred for 24 h at room temperature. The polymer was precipitated in acetone, dissolved in H.sub.2O after drying, and dialyzed against H.sub.2O. The water was changed twice daily. After dialysis for 9 days, the polymer solution was freeze-dried for 4 days. 938 mg of product was obtained. The degree of HMDA functionalization was determined by means of .sup.1H-NMR spectroscopy. 31.5% HMDA functionalization.

[0139] The synthesis can be carried out with a large excess of HMDA in order to prevent crosslinking of the alginic acid. The product can be extensively dialyzed in order to remove the excess HMDA. The successful coupling of the HMDA linker was confirmed by .sup.1H-NMR spectra, and the successful functionalization of the carboxyl groups by forming amide bonds with the HMDA linkers was confirmed by ATR-IR spectra (not shown).

[0140] Functionalization of the PEEK surface with polysaccharides:

[0141] Various strategies can be used for further functionalization of the PEEK surface. On the one hand, various modified polysaccharides can be provided with amine functionalities and in the next step coupled to the carboxyl groups on the PEEK surface. The other approach starts with modification of the carboxyl groups on the surface by diamines, in order to carry out coupling of unmodified polysaccharides in the next step.

[0142] Coupling of amine-functionalized polysaccharides:

[0143] In order to allow the polysaccharides to be applied to the polymer substrate, a peptide bond should be formed. On the one hand, one can use classical EDC/NHS coupling chemistry. On the other, one can also work with the modern coupling reagent DMT-MM. Classical EDC/NHS coupling can be carried out in two stages. After 20-minute activation of the PEEK film in slightly acidic MES buffer, coupling with the modified polysaccharides can be carried out overnight in slightly basic phosphate buffer:

##STR00015##

[0144] As an alternative coupling reaction, single-stage coupling with the modern coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) can be selected. The activation mechanism and the subsequent formation of the peptide bond can take place as follows:

##STR00016##

[0145] This option is advantageous in that the reaction can be carried out at a constant pH of 9, and the reaction vessel does not have to be changed between activation and coupling. The coupling at a pH of 9 is significantly more rapid than coupling to the NHS ester at a pH of 7.3, because the amines at a pH of 9 are predominantly in the form of free amines, which is important for the nucleophilic attack on the activated carbonyl center. NHS coupling cannot be carried out at such high pH levels, because the NHS ester would be hydrolyzed too quickly in the aqueous solution:

##STR00017##

[0146] Modification of the imine-functionalized PEEK surface:

[0147] The imine-functionalized PEEK surface can be reacted under identical reaction conditions with native hyaluronic acid and alginic acid.

[0148] Direct modification of the iminated PEEK surface with native hyaluronic acid. The coupling was carried out by means of DMT-MM in aqueous phosphate buffer at pH=8:

##STR00018##

[0149] Direct modification of the iminated PEEK surface with native alginic acid. The coupling was carried out by means of DMT-MM in aqueous phosphate buffer at pH=8 (aminated PEEK film was shaken overnight with polysaccharide in PBS buffer (PBS: phosphate-buffered saline) at pH=8 (67 mM) in 1 mM DMT-MM solution. Concentration of hyaluronic acid: 0.1 mg/mL; alginic acid: 0.05 mg/mL. Washing with MilliQ water was carried out three times):

##STR00019##

[0150] Modification of the PEEK surface coated with polyacrylic acid:

[0151] Coating of the PEEK substrates with polyacrylic acid, as described above, was highly successful. The extremely large number of carboxyl groups that were introduced onto the PEEK surface in this manner served as a point of departure for further functionalization with different polysaccharide derivatives. Coupling reactions were thus carried out with ADH-hyaluronic acid, HMDA-hyaluronic acid, deacetylated hyaluronic acid and HMDA-alginic acid.

[0152] Modification of the PEEK surface coated with polyacrylic acid with ADH-hyaluronic acid. The coupling was carried out by means of DMT-MM in aqueous phosphate buffer at pH=8:

##STR00020##

[0153] The reaction was carried out under the same conditions as the previous couplings.

[0154] Modification of the PEEK surface coated with polyacrylic acid with HMDA-hyaluronic acid. The coupling was carried out by means of DMT-MM in aqueous phosphate buffer at pH=8:

Modification of the PEEK surface coated with polyacrylic acid with HMDA-alginic

##STR00021##

acid. The coupling was carried out by means of DMT-MM in aqueous phosphate buffer at pH=8:

##STR00022##

[0155] Modification of the PEEK surface coated with polyacrylic acid with deacetylated hyaluronic acid. The coupling was carried out by means of DMT-MM in aqueous phosphate buffer at pH=8:

##STR00023##

[0156] Overall, significantly more coupling tests were carried out on the PEEK substrate. An overview of the reactions is shown in Table 2. The couplings marked with an X were carried out. All reactions were carried out under the same basic conditions using DMT-MM as a coupling reagent.

TABLE-US-00002 TABLE 2 Overview of polysaccharide couplings to PEEK substrates. Deac.- ADH- HMDA- HMDA- hya Alg hya hya hya Alg PEEK-imine X X PEEK-imine- X X X X COOH PEEK-PAA X X X X PEEK-PAA- X X amine

[0157] The reaction diagrams for the individual reactions are as follows: successful functionalization was confirmed by ATR-IR spectra (not shown).

[0158] Direct modification of the iminated PEEK surface with native alginic acid. The coupling was carried out by means of DMT-MM in aqueous phosphate buffer at pH=8:

##STR00024##

[0159] Modification of the iminated and carboxylated PEEK surface with ADH-hyaluronic acid. The coupling was carried out by means of DMT-MM in aqueous phosphate buffer at pH=8:

##STR00025##

[0160] Modification of the iminated and carboxylated PEEK surface with HMDA-hyaluronic acid. The coupling was carried out by means of DMT-MM in aqueous phosphate buffer at pH=8:

##STR00026##

[0161] Modification of the iminated and carboxylated (succinic acid) PEEK surface with HMDA-alginic acid. The coupling was carried out by means of DMT-MM in aqueous phosphate buffer at pH=8:

##STR00027##

[0162] Modification of the iminated and carboxylated PEEK surface with deacetylated hyaluronic acid. The coupling was carried out by means of DMT-MM in aqueous phosphate buffer at pH=8:

##STR00028##

[0163] Modification of the PEEK surface coated with polyacrylic acid with ADH-hyaluronic acid. The coupling was carried out by means of DMT-MM in aqueous phosphate buffer at pH=8:

##STR00029##

[0164] Modification of the PEEK surface coated with polyacrylic acid with HMDA-hyaluronic acid. The coupling was carried out by means of DMT-MM in aqueous phosphate buffer at pH=8:

##STR00030##

[0165] Modification of the PEEK surface coated with polyacrylic acid with HMDA-alginic acid. The coupling was carried out by means of DMT-MM in aqueous phosphate buffer at pH=8:

##STR00031##

[0166] Modification of the PEEK surface coated with polyacrylic acid with deacetylated hyaluronic acid. The coupling was carried out by means of DMT-MM in aqueous phosphate buffer at pH=8:

##STR00032##

[0167] Modification of the PEEK surface coated with polyacrylic acid and then treated with tetramethylene diamine with native hyaluronic acid. The coupling was carried out by means of DMT-MM in aqueous phosphate buffer at pH=8:

##STR00033##

[0168] Modification of the PEEK surface coated with polyacrylic acid and then treated with tetramethylene diamine with native alginic acid. The coupling was carried out by means of DMT-MM in aqueous phosphate buffer at pH=8:

##STR00034##

[0169] Modification of the iminated PEEK surface with coupled succinic acid with native alginic acid. The coupling was carried out by means of DMT-MM in aqueous phosphate buffer at pH=8:

##STR00035##

[0170] Mineralization tests with hydroxyapatite:

[0171] In addition, three different batches for the mineralization of hydroxyapatite per PEEK-PAA sample were tested. First, two tests were carried out with calcium prestructuring and one with phosphate prestructuring.

[0172] Here, the following Ca prestructuring would be possible:

[0173] An established synthesis route for hydroxyapatite can be modified and used for the mineralization of PEEK-PA films. The PEEK-PA film was placed in 0.3 M calcium chloride solution at a buffered pH of 9 and then stirred for 30 min. A disodium hydrogen phosphate solution, also at a buffered pH of 9, was now added at a rate of

[00006]$3\frac{\mathrm{mL}}{\min }.$

After addition was completed, the mixture was stirred overnight.

[0174] Ca prestructuring and phosphate prestructuring were also carried out:

[0175] Simpler variants of the mineralization can also be carried out. PEEK-PAA samples were placed for 72 h in diammonium hydrogen phosphate (phosphate prestructuring, 6 mL vial with 1 M aqueous (NH.sub.4).sub.2HPO.sub.4 solution) or calcium nitrate Cal (6 mL vial with 1 M aqueous Ca(NO.sub.3).sub.2 solution). After this rest time, each of the samples was placed in the other solution respectively (0.6 M (NH.sub.4).sub.2HPO.sub.4 or 0.6 M Ca(NO.sub.3).sub.2 solution) and left therein for one week in order to also allow the counterions to diffuse into the gel.

[0176] Photoinducible Coupling

[0177] According to a further example, azido-functionalized hyaluronic acid was bonded by light-induced coupling to a PEEK surface. The reaction sequence of the light-induced coupling of azidoanilines with hyaluronic acid to PEEK could be as follows:

##STR00036##

[0178] Azidoaniline groups were thus coupled to the carboxy groups of polysaccharides such as e.g. alginic or hyaluronic acid (see below). The coupling product of polysaccharides and azidoaniline linkers was then bonded with light to the PEEK surfaces.

[0179] Coupling of the azidoaniline linkers to the polysaccharides takes place in a first step by means of standard EDC-mediated amine and carboxyl group coupling to the carboxyl-functionalized polymer. Large polymers such as high-molecular-weight hyaluronic acid form strong secondary structures (such as helix structures, etc.) that also cause high viscosity. This makes the diffusion of reagents to the functional groups poor and hinders accessibility. For this reason, the hyaluronic acid can be pre-treated if necessary. A suitable means for this would be for example a cleaved hyaluronic acid, such as an enzymatically cleaved or ultrasound-cleaved hyaluronic acid. In enzymatic cleavage, for example with hyaluronidase, the hyaluronic acid is cleaved into fragments of approx. 15 kilodaltons (kD), and in ultrasound treatment into fragments of approx. 300 kD.

[0180] Production of an azido-functionalized hyaluronic acid could be carried out according to Eychenne, Romain, et al. “Rhenium Complexes Based on an N20 Tridentate Click Scaffold: From Synthesis, Structural and Theoretical Characterization to a Radiolabeling Study.” European Journal of Inorganic Chemistry 2017.1 (2017): 69-81), or commercially obtained hyaluronic acid could be used.

[0181] Synthesis of photoreactive hyaluronic acid (hya-N3) (cf. Chen, Guoping, et al. “Photoimmobilization of sulfated hyaluronic acid for antithrombogenicity.” Bioconjugate Chemistry 8.5 (1997): 730-734.):

[0182] Material: 100 mg (1 equivalent) of the (cleaved) hyaluronic acid; 45 mg (1 equivalent) of 4-azidoaniline; 58.2 mg (1.15 equivalents) of EDC-CI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl);

dissolve all three reactants in 110 mL of MilliQ water;
adjust pH to 7;
stir overnight away from light;
purify by dialysis unit UV signal in dialysis water is no longer detectable at 255 nm;
and
freeze-dry.

[0183] Coating of polyether ether ketone (PEEK) with azido-functionalized hyaluronic acids:

wash PEEK substrate with ethyl acetate and acetone respectively for approx. 1 min;
prepare solution of azido-functionalized hyaluronic acids (1 mg/mL) (enzymaticallycleaved azido-functionalized hyaluronic acid “hya-enz-N3” and ultrasound-cleaved azido-functionalized hyaluronic acid “hya-US-N3”) in MilliQ-water and 3 mL vial with snap-on cap by mixing and shaking;
apply 50 μL of the respective solution to washed PEEK substrates with a pipette;
dry overnight in the air (cover the dabbed PEEK films, for example with a 96-well plate, in order to protect the respective substrate type from dust deposits. A snap-on cap placed on the cover provides the necessary ventilation. Also cover with aluminum foil for protection from light.).

[0184] Observation 1: A spot of a drop-shaped material deposit is clearly visible after drying (not shown):

spread out the PEEK plates with dried azido-functionalized hyaluronic acid on a cloth; and
irradiate with short-wave UV (254 nm) for 100 min at a distance of approx. 1 cm.

[0185] Observation 2: On both substrate types (hya-Enz-N3; hya-US-N3), one can see a round (drop-shaped), brownish deposit that appears darker than the deposit after the drying process (cf. Observation 1). The substrates with hya-US-N3 are somewhat darker colored than the substrates with hya-Enz-N3.

[0186] Wash PEEK substrates with hyaluronic acid derivatives after drying and exposure to light for 24 h on a shaker table in MilliQ and a 50 mL Falcon tube.

[0187] Dry washed PEEK substrates for 24 h in a Falcon tube with a perforated parafilm cover in the vacuum oven.

[0188] Observation 3: The spot of the material deposit is still clearly visible on the washed, irradiated PEEK film substrate treated with a hyaluronic acid derivative. On a similarly washed non-irradiated film, in contrast (negative sample), no spot can be seen. Therefore, material was successfully coupled to the PEEK substrate in a wash-resistant manner. More for hya-US-N3 than for hya-Enz-N3) (not shown).

[0189] After this, mineralization tests were carried out with the PEEK substrates coated with hyaluronic acid derivatives produced as described above.

[0190] Mineralization Solution:

[0191] 40.5 g of NaCl; 1.8475 g of CaCl.sub.2); 0.735 g of Na.sub.2HPO.sub.4; 8.875 g of HCl (conc.); 500 mL of MilliQ

[0192] Preparation: prepare MilliQ water, then dissolve salts therein and add HCl (weighed out in a syringe). Store in a laboratory flask until use.

[0193] Procedure:

[0194] Prepare dilution of the mineralization solution depending on the desired pH range

a) pH 7: mineralization solution to MilliQ water in a ratio of 2:8
b) pH 8: mineralization solution to MilliQ water in a ratio of 1:9
c) pH 9: mineralization solution to MilliQ water in a ratio of 1:18
Adjust pH immediately before use with 1 M Trizma® base (Sigma), as this “activates” the solution and initiates the mineralization.

[0195] Then immediately add 25 mL of activated mineralization solution to the substrates to be mineralized to 30 mL vials with snap-on caps (1 substrate per vial). Ensure that the coated side faces upward and the plate does not float on the solution, but is fully immersed.

[0196] Then place the room temperature samples on a shaking table. Place the samples at elevated temperatures (approx. 37° C.) in a corresponding water bath (raised, so that they are immersed but not completely under water).

[0197] After the desired period of time, remove the substrates, wash them in 10 mL of MilliQ in a 15 mL Falcon tube on the shaking table for 30 min, and then dry overnight in a vacuum drying oven.

[0198] After this, conduct analysis by scanning electron microscopy (SEM) for surface structures, energy-dispersive x-ray analysis (EDX) for elemental composition, and then x-ray diffraction (XRD) for mineral phase (partially not shown).

[0199] Different test batches were carried out (cf. Table 3). PEEK was used as a substrate in all cases, either enzymaticallycleaved azido-functionalized hyaluronic acid (abbreviated as Enz) or ultrasound-cleaved azido-functionalized hyaluronic acid (abbreviated as US) was used as a coating, incubation was carried out at a pH of 7, 8 or 9 (see above for dilutions), and coupling was carried out in all batches by means of UV light. In addition, mineralization blank tests were conducted at all three pH levels and at both temperatures (RT/37° C.) without the presence of a substrate, with no mineralization being detected in any cases (not shown).

TABLE-US-00003 TABLE 3 Mineralization test batches P B pH T A E T IS005 Enz 7 37 d1, 15:10 d2, 14:30  23 h, 20 min, 1 d IS006 Enz 7 37 d1, 15:10 d5, 13:30 118 h, 20 min, 5 d IS008 Enz 7 RT d1, 15:10 d2, 14:30  23 h, 20 min, 1 d IS009 Enz 7 RT d1, 15:10 d5, 13:30 118 h, 20 min, 5 d IS019 Enz 8 37 d1, 13:45 d2, 14:15  24 h, 30 min, 1 d IS020 Enz 8 37 d1, 13:45 d5, 12:40 118 h, 55 min, 5 d IS022 Enz 8 RT d1, 13:45 d2, 14:15  24 h, 30 min, 1 d IS023 Enz 8 RT d1, 13:45 d5, 12:40 118 h, 55 min, 5 d IS033 Enz 9 37 d1, 14:05 d2, 13:20 2 3 h, 15 min, 1 d IS036 Enz 9 RT d1, 14:05 d2, 13:20  23 h, 15 min, 1 d IS011 US 7 37 d1, 15:10 d2, 14:30  23 h, 20 min, 1 d IS012 US 7 37 d1, 15:10 d5, 13:30 118 h, 20 min, 5 d IS014 US 7 RT d1, 15:10 d2, 14:30  23 h, 20 min, 1 d IS015 US 7 RT d1, 15:10 d5, 13:30 118 h, 20 min, 5 d IS025 US 8 37 d1, 13:45 d2, 14:15  24 h, 30 min, 1 d IS026 US 8 37 d1, 13:45 d5, 12:40 118 h, 55 min, 5 d IS028 US 8 RT d1, 13:45 d2, 14:15  24 h, 30 min, 1 d IS029 US 8 RT d1, 13:45 d5, 12:40 118 h, 55 min, 5 d IS039 US 9 37 d1, 14:05 d2, 13:20  23 h, 15 min, 1 d IS042 US 9 RT d1, 14:05 d2, 13:20  23 h, 15 min, 1 d IS043 US 9 RT d1,14:05 d4, 13:00  94 h, 55 min, 4 d Legend: P = name of sample, B = coating, pH = pH value, T = temperature in [°C.] (RT = room temperature), A = beginning of incubation (d = day, XX:YY = time), E = end of incubation, t = time in hours [h] and minutes [min], (corresponds to X) day(s) [d].

[0200] Observation 4:

[0201] In samples IS005, 006, 008, 009, 011, 012, 014, 015, 022, 028, 036, 042 and 043, the hyaluronic acid ring or spot is recognizable on SEM examination. This finding is consistent with signals in the EDX analysis of carbon and oxygen. However, no wash-resistant film or deposit can be seen on the hyaluronic acid coating, nor is any calcium or phosphorus detectable in the EDX analysis that would indicate mineralization.

[0202] In samples IS019, 020, 023, 025, 026, 029, 033 and 039, on the other hand, a wash-resistant film or deposits can be seen in the areas of the hyaluronic acid ring or spot. On EDX analysis, in addition to carbon and oxygen, calcium and phosphorus are also observed, which indicates mineralization.

[0203] In the following, results are presented by way of example for the samples IS019 (enzymatically cleaved hyaluronic acid coating, pH 8, 37° C., 1 day embedding time) and IS025 (ultrasound-cleaved hyaluronic acid coating, pH 8, 37° C., 1 day embedding time).

[0204] In the SEM analyses, deposits are clearly visible on all of the plates. The deposits are not homogeneously, but irregularly distributed. Many areas are covered by a filmlike layer, and other areas show spongelike bead material accumulations. In IS019, the hyaluronic acid coating is a ring (as was the case for all previous samples with enzymatically cleaved hyaluronic acid) and is not completely but partially covered with the deposited material. The agglomerations show no preference for the hyaluronic acid coating or PEEK, but appear to be distributed equally heterogeneously at all sites (not shown). In IS025, in contrast, the hyaluronic acid coating is a filled spot (as was the case for all previous samples with hyaluronic acid cleaved by means of ultrasound) and is covered with an extremely thick layer of the deposited material. There are signs of preferential mineralization of the hyaluronic acid coating and weaker signs of covering of the uncoated PEEK areas (not shown).

[0205] EDX analysis of the respective foamlike beaded deposits clearly shows the presence of phosphorus and calcium in addition to carbon and oxygen. The content percentages for IS019 are oxygen (O) 44.6%, carbon (C) 21.8%, calcium (Ca) 20.5% and phosphorus (P) 13.1% and those for IS025 are oxygen (O) 45.2%, carbon (C) 40.9%, calcium (Ca) 9.0% and phosphorus (P) 4.9%. Carbon is attributable to PEEK, the hyaluronic acid and the carbon adhesive tape with which the sample was attached to the sample carrier for the test. Calcium, phosphorus and oxygen indicate the possible presence of calcium phosphate compounds. Mapping was carried out, with the following results (not shown): the calcium and phosphorus signal distribution is consistent with the beadlike material deposits. Carbon is primarily located at sites where the substrate or the hyaluronic acid coating is not covered by the beadlike material. Oxygen is relatively regularly distributed, but particularly at sites where the beadlike material was deposited (and more strongly at sites where the hyaluronic acid layer is present, as said layer contains more oxygen than PEEK).

[0206] Initial measurements from XRD analysis of the IS0019 sample indicate calcium metaphosphate. However, the signal of the coating is very difficult to read out, because PEEK is polycrystalline and thus emits extremely strong, sharp signals that mask all other signals, and in addition, the hyaluronic acid is only minimally visible. Accordingly, parts of the white deposit were scratched off so that they could be measured individually, i.e. without substrate signals. The x-ray powder diffractogram of the scratched-off deposit is shown in FIG. 1 (dashed graph). In addition, the signal of hydroxyapatite (R060180 from the RRUFF database) was included in the diagram (black graph). It can be seen that there is a high degree of agreement between the signal of the sample IS019 and the literature signal of hydroxyapatite. It can therefore be assumed that in mineralization, hydroxyapatite is produced on the hyaluronic acid coating of PEEK.

[0207] The following conclusions can be drawn from the tests:

[0208] With respect to the effect of pH, the general trend appears to be that the higher the pH of the mineralization solution, the more rapidly deposition occurs on the substrates. The optical impression currently confirms this for all of the mineralization tests observed so far from pH 7 to pH 9.

[0209] A higher temperature (here: 37° C. in the water bath) compared to room temperature (approx. 21° C.) appears to promote the deposition of material on the substrates. It was possible to observe this macroscopically in all previous mineralization tests.

[0210] In addition, the deposited materials appear to be rather coarse at RT, while a temperature of 37° C. appears to promote the formation of fine deposits.

[0211] Example: At elevated temperatures, deposits were clearly visible after only one day at pH 8 (e.g. IS019 and IS025) and pH9 (e.g. IS033 and IS039), while at RT (IS022, IS036, IS028, IS042), virtually no deposition or no deposition was seen after one day under these conditions.

[0212] A longer embedding time should increase the amount of precipitated material, or in the case of extremely slow precipitation, a longer embedding time should be required for deposition to occur at all. Contrary to this expectation, the largest deposition amounts were observed for the samples with an embedding time of 24 h. It may be that with a longer embedding time, conversion or diffusion processes take place that decrease the visible deposits compared to samples with shorter embedding times. In-depth analyses could provide more detailed information on the distribution of elements in the mineralized substrate.

[0213] It appears at this point that a uniform coating is best achieved using the ultrasound-cleaved hyaluronic acid solution, as this solution forms a filled-in material spot on the PEEK substrate. In IS025 and IS026, preferential material deposition appears to take place on this coating. Enzymatically cleaved hyaluronic acid appears to leave only a ring of coating material on the substrate and shows no preferential material deposition areas, with the exception of a minimal area in the sample IS020.

SUMMARY

[0214] A study was conducted on the surface functionalization of the bone implant plastic polyether ether ketone in order to allow improved incorporation into the treated bone area. Polyether ether ketone surfaces were successfully subjected to chemical modification by different methods. The surface properties were modified using small molecules, and hydroxyl, carboxyl- and imine functionalities were obtained on the surface. The modified surface was analyzed and characterized by means of ATR-IR spectroscopy (not shown). Moreover, functional polymers such as polyacrylic acid, but also polymethyl acrylate (PMA), were deposited on the polyether ether ketone surface by means of UV-induced grafting polymerization. The polyacrylic acid layer was examined by different surface analysis methods, such as ATR infrared spectroscopy, scanning electron microscopy and confocal laser scanning microscopy in order to collect spectroscopic data on the surface and obtain a precise picture of its topography (not shown). The polyacrylic acid layer was modified by coupling of diamine linkers so that amide bonds could layer be formed with organic acids. In order to allow quantitative conclusions on the degree of surface functionalization with amino groups to be drawn, the cleavable fluorescent dye C-coumarin, with which it was possible to indirectly quantitate the accessible amino groups on the surface, was synthesized. Quantitation was successfully carried out in the samples that had been directly imine-functionalized with diamines.

[0215] Hyaluronic acid with adipic acid dihydrazide, hexamethylene diamine and alginic acid was modified only with the diamine in order to couple amine linkers for subsequent anchoring to the various polyether ether ketone substrates. Hyaluronic acid was also deacetylated in order to introduce amine functionalities onto the polysaccharide in this manner. The modified polysaccharides were characterized by means of NMR and ATR infrared spectroscopic methods (not shown).

[0216] The numerous modified and unmodified polysaccharides were coupled to the complementary PEEK substrates. The coupled samples were examined by means of ATR infrared spectroscopy, scanning electron microscopy and in some cases thermogravimetry (not shown).

[0217] In the present invention, azidoaniline groups as photocoupleable or light-inducible linkers were coupled to the carboxy group of polysaccharides such as e.g. alginic or hyaluronic acid, and the latter were then bonded with light to a PEEK surface. Coating of the polyether ether ketone with azido-functionalized hyaluronic acid was successfully demonstrated. Moreover, clear indications were seen that mineralization of the PEEK surface coupled with hyaluronic acid derivatives takes place.

[0218] Outlook

[0219] As the surface-induced radical polymerization was highly successful, there are several approaches, particularly in this area, on which further studies could be based. Even though the introduction of polysaccharide structures on the polyether ether ketone surface was found not to be trivial, the polymerization with acrylic acid functioned extremely well, indicating that a highly promising approach would be to directly coat the PEEK surface with polymers of modified acrylic acid derivatives. Examples of suitable alternative acrylic-acid-based monomer units include acrylic acid derivatives modified with sugar molecules, which are known to play an important role in the cellular adhesion of osteoblasts. It would also be of interest if the monomer units carried oligosaccharides of hyaluronic acid, or also short adhesion-mediating RGD peptide sequences, etc.:

##STR00037##

[0220] A highly sensitive surface analysis method is XPS (x-ray photoelectron spectroscopy). This method might make it possible to detect polysaccharides on the surfaces of the produced substrates.

[0221] Research on the swelling behavior of the polyacrylic acid layer on the polyether ether ketone substrate could be an approach for optimizing the coupling conditions so that it would be possible to successfully carry out polysaccharide detection even with simple analysis methods. Coupling in non-aqueous media would also be conceivable, but this approach would certainly be problematic as well due to the poor solubility of the polysaccharides.

[0222] Further studies of precipitating hydroxyapatite in the polyacrylic acid layer should also be carried out, as it has been established that the hydroxyapatite coating has positive effects on acceptance in the body.