A mediator molecule is immobilized on the surface of a metallic or ceramic implant material. An anchor molecule (e.g., dialdehyde or cyanogen bromide) having a functional group that covalently binds the mediator molecule is covalently bound to the surface, and the mediator molecule is coupled to the functional group of the anchor molecule. The implant material may comprise titanium, titanium alloy, aluminium or stainless steel or hydroxylapatite. Oxide units on the implant material surface can be increased preferably by treating with hot chromic-sulphuric acid for 0.5 to 3 hours at a temperature between 100 to 250.degree. C. prior to binding the anchor molecule. Also, prior to binding the anchor molecule, the surface of the implant material can be activated by reacting with a silane derivative. Mediator molecules include BMP protein, ubiquitin and antibiotics, and the implant material may be an artificial joint or coronary vessel support such as a stent.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for the immobilization of mediator molecules on surfaces of metallic or ceramic materials which are used for implants such as artificial joints or also microimplants, for example so-called stents, as well as implants produced according to the method.

2. Description of the Related Art

The implantation of artificial joints or bones has gained increasing importance in recent years, for example in the treatment of joint dysplasias or joint dislocations or in sicknesses resulting from joint attrition as a result of improper joint positioning. The function of the implants and the materials used for their production, which, in addition to metals such as titanium or metal alloys, can also include ceramics or synthetic materials such as teflon, have been continually improved, so that following a successful healing process, implants exhibit lifetimes of 10 years in 90-95% of all cases. Yet despite this progress and these improved operational methods, an implantation still remains a difficult and strenuous operation, particularly since it is associated with a long process of healing-in of the implants, often including month-long stays in clinics and health resorts, including rehabilitation measures. In addition to the pain, the length of the treatment period and the separation from familiar surroundings represent heavy stresses for the affected patients. In addition, the long healing process incurs high personal and treatment costs due to the required intensive care.

The understanding of the molecular-lever processes required for a successful growing-in of an implant has markedly increased in recent years. Structural compatibility and surface compatibility are crucial for the tissue tolerability of an implant. Biocompatibility in a narrower sense depends only on the surface. Proteins play a crucial role at all levels of integration. These form an initially adsorbed protein layer as early as during the implantation operation and thus, as explained below, since the first cells will later colonize on this layer, determine the further progression of the healing-in of the implant.

In the molecular interaction between implant, also referred to as biomaterial, and tissue, a multitude of reactions take place which seem to be strictly hierarchically ordered. The adsorption of proteins on the surface of the biomaterial is the first biological reaction which takes place. In the resulting protein layer, single protein molecules are for example either transformed by conformational changes to signal substances which are presented on the surface, or protein fragments functioning as signal substances are released by catalytic (proteolytic) reactions. Triggered by the signal substances, cellular colonization takes place in the next phase, and can include a multitude of cells such as leucocytes, macrophages, immunocytes and finally also tissue cells (fibroblasts, fibrocytes, osteoblasts, osteocytes). In this phase other signal substances, so-called mediators such as for example cytokines, chemokines, morphogens, tissue hormones and true hormones play a decisive role. In the case of biocompatibility, there is a final integration of the implant into the entire organism, and one ideally obtains a permanent implant.

In light of work performed in recent years at the molecular level of osteogenesis, chemical signal substances, the so-called "bone morphogenic proteins" (BMP-1-BMP-13), which influence bone growth, have gained increasing importance. BMPs (in particular BMP-2 and BMP-4, BMP-5, BMP-6, BMP-7) are osteoinductive proteins which stimulate the formation of new bones and bone healing by effecting the proliferation and the differentiation of precursor cells to osteoblasts. Furthermore they promote the formation of hormone receptors, bone-specific substances such as collagen type 1, osteocalcin, osteopontin and finally mineralization. Here, the BMP-molecules regulate the three key reactions chemotaxis, mitosis and differentiation of the respective precursor cells. In addition, the BMPs play an important role in embryogenesis, organogenesis of bone and of other tissue, wherein osteoblasts, chondroblasts, myoblasts and vascular smooth muscle cells (proliferation inhibition by BMP-2) are known as target cells.

SUMMARY OF THE INVENTION

A particular aim in the immobilization method according to the invention is a degree of stimulation (that is, surface concentration of the immobilized protein) which allows a multivalent interaction between surface and cell and enables the effective control of bone and tissue formation.

To date, 13 BMPs including multiple isoforms are known. With the exception of BMP-1, the BMPs belong to the "transforming growth factor beta" (TGF-.beta.) superfamily, for which specific receptors on the surface of the corresponding cells have been found. As the successful use of recombinant human BMP-2 and/or BMP-7 in experiments on defective healing processes in rats, dogs, rabbits and monkeys has shown, no species-specificity seems to exist. Previous attempts to exploit the bone formation-triggering characteristics of the BMPs for implantation purposes, in which BMP-2 and/or BMP-7 were noncovalently applied to metallic or ceramic biomaterials, have however been largely unsuccessful.

The goal of the present invention is to produce improved biomaterials for use as implants.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention this goal is achieved by the provision of a method for the immobilization of mediator molecules on metallic and ceramic materials. In the method according to the invention, in a first step a chemical compound is covalently bound to the surface of the implant material as an anchor molecule, wherein this chemical compound has a functional group which can either be bound itself as a spacer molecule or to another compound serving as a spacer molecule. In a second step a mediator molecule such as a bone growth factor can be immobilized on the implant material via functional groups, for example free amino groups or carboxylate groups by means of a covalent bond. In this way it is possible to form a chemotactic and/or biologically active implant surface (a so-called juxtacrine surface), which leads to the colonization, proliferation and differentiation of bone cells.

The method according to the invention for the immobilization of the mediator molecules is distinguished by the fact that the implant material used is composed of metallic materials such as pure titanium or metallic titanium alloys such as chrome/nickel/aluminium/vanadium/cobalt-alloys (for example TiAlV4, TiAlFe2.5), stainless steels (for example V2A, V4A, chrome-nickel 316L) or ceramic materials such as hydroxylapatite, aluminium oxide or of a combination, in which for example metallic material is coated with ceramic material. Synthetic polymer materials are also suited for use as the implant material.

Further subject matter of the invention is the therapeutic prevention or alleviation of the late complication restenosis elicited by a proliferation of smooth vessel muscle cells by coating a coronary vessel support (so-called coronary stent, length approximately 10 mm) with the help of a biomolecule or a mediator, for example BMP-2, in order to promote healing-in and tolerability.

According to the invention the mediator molecules can be biomolecules which are advantageous for the biocompatibility of the implant in that they hinder a possible rejection of the implant and/or promote growing-in of the implant.

Preferred mediator molecules which can be used in the present method are bone growth-promoting proteins from the class of bone growth factors "bone morphogenic proteins" or also ubiquitin. It can be advantageous for the immobilization to use one protein of this class alone, in combination with other members of this class or also together with biomolecules such as proteins of other classes or low molecular weight hormones or also antibiotics to improve immunoresistance. Here, these molecules can also be immobilized on the surface via bonds which are cleavable in the biological environment.

According to the invention the surface of implant material is chemically activated, wherein the activation takes place via a silane derivative such as for example .gamma.-aminopropyltriethoxysilane or a trimethylmethoxy- or trimethylchlorosilane derivative or 3-glycidoxypropyltrimethoxysilane and the reaction is performed not only in an aqueous but also in an organic solvent. In a second step a spacer molecule serving as a spacer can be covalently coupled to the surface activated in this way. A dialdehyde such as glutaric dialdehyde, an isothiocyanate derivative or a triazine derivative can for example serve as the spacer. A dicarboxylic acid or a corresponding derivative such as succinic acid can be used as the spacer molecule. Following possible activation of the coupling group present in the spacer molecule, for example a carbonyl functionality, by way of a common method for this purpose, the bone growth-promoting protein is bound to the implant material via amino groups accessible on its surface.

According to the invention it is also possible to use an aryl amine as a spacer molecule. This can for example be obtained by reaction of the implant material activated by a silane compound with a benzoic acid chloride substituted with nitro groups such as for example p-nitrobenzoylchloride followed by reduction of the nitro group. In this case the covalent linking of the mediator protein takes place via three carboxyl groups which can be activated according to standard procedures for this purpose.

The present method further includes coupling of the mediator molecule via anchor molecules only, without prior activation of the implant surface by silane as described above by way of example, wherein cyanogen bromide can for example be used for this purpose. In this case the covalent immobilization of the mediator molecule can take place via three amino groups of the protein.

The method according to the invention includes the coupling of a bone growth factor to the surface of the implant via spacer molecules, the covalent bonds of which are not cleaved under physiological conditions. As an advantageous development, a bone growth factor is coupled to the surface of the implant via spacer molecules, the covalent bonds of which are cleavable under physiological conditions for a limited release of the mediator protein. Alternatively it is also possible to couple the bone growth factors without the help of the spacer molecule, for example by way of the carbodiimide method, to the activated surface of the implant.

According to another further development of the method, two or more spacer molecules are used for the immobilization of at least one bone growth factor.

The loading density of the mediator protein immobilized on the implant material according to the method of the invention is generally 0.03 to 2.6 .mu.g/cm.sup.2 (for example 1-100 pmol/cm.sup.2 BMP-2). In this loading range, a multivalent interaction between a cell (for example 10 .mu.m diameter) and the BMP-molecules on a biologicalized surface can be achieved, since approximately 10.sup.6-10.sup.8 immobilized protein molecules are located in the adhesion site.

The inventors have performed extensive experiments to elucidate the mechanism of the binding of the protein molecules to the surface. In the course of this, they found that with metallic surfaces such as for example with titanium the binding takes place via covalent bonds via the titanium dioxide molecules formed on the metal surface, which are preferably transformed into hydroxyl groups by treatment with dilute nitric acid.

In contrast to the methods known in the prior art, in which biomolecules are for example deposited onto polymer surfaces or inorganic bone materials and remain on the surface of the substrate only via affinity interactions with the polymer molecules, the inventors have been successful here in covalently anchoring the biomolecules to the surface and, in this way, providing them for a longer time on the surface of the implant.

Further investigations by the inventor have shown that the anchoring of the mediator molecules on the surface can be qualitatively and quantitatively improved by increasing the number of the accessible metallic oxide units on the surface. It was found by the inventors that the number of oxide groups can surprisingly be increased by treating the surface of the metal with hot, preferably sediment-free chromic-sulfuric acid. In contrast to the expectation that the metal dissolves under these conditions, a relatively uniform oxide layer is generated on the surface of the metal by the use of this acid. The method is so mild that even coronary vessel supports, so-called stents (which can for example be fashioned from stainless steel or titanium) can be coated without destroying the thin sensitive meshing (50-150 .mu.m diameter). In this way the oxide layer can reach a thickness of 10 .mu.m up to 100 .mu.m and can be relatively "smoothly" constructed without pits or holes. Pure titanium or titanium alloys (for example TiAlV4, TiAlFe2.5), aluminium or stainless steel (for example V2A, V4A, chrome nickel 316L) can be used as the metal for the implant. A common commercial chromic-sulfuric acid of 92% by weight H.sub.2SO.sub.4, 1.3% by weight CrO.sub.3 and with a density of 1.8 g/cm.sup.3 as for example available from the company Merck is preferably used to achieve a thin smooth layer of metal oxide. In order to achieve this, the metal substrate is placed in the chromic-sulfuric acid and is treated over a time span of 1 up to 3 hours at 100 to 250.degree. C., preferably 30 min at 240.degree. C., is subsequently carefully rinsed with water, is boiled in water or in a solution of 1-4 mM EDTA (ethylenediaminetetraacetate), preferably 4 mM EDTA for 30 min, in order to remove the chrome ions remaining on the surface, and is then dried.

If a thicker metal oxide layer and/or an oxide layer with small micro- and nanopores is to be provided on the metal surface, the chromic-sulfuric acid described above is diluted with water to a density of 1.5 to 1.6 g/cm.sup.3. In a subsequent treatment of the surface of the metal implant as described above with the acid diluted in this way, a "rough" surface layer with pits and pores is formed, so that the surface available for loading with mediator molecules is increased. It is therefore possible to apply a multitude of different oxide layers with different characteristics to metal surfaces with high adhesion by tuning to various densities of chromic-sulfuric acid. The invention is therefore also directed to such a method for forming a thermodynamically unified metal oxide layer (no contact angle hysteresis) on the implant material by means of hot chromic-sulfuric acid.

The metal oxide layer on the implant material made of the materials cited above can then be activated via treatment with dilute nitric acid (approximately 5% by weight) and subsequent coupling of a silane derivative, optionally additionally of a spacer molecule, as described above. The mediator molecules can then be anchored via the molecules of the silane derivative or of the spacer via coupling methods such as for example by way of carbonyldiimidazole on the implant surface.

In order to exclude the nonspecific adsorption of the mediator molecules, which can be up to 30% of the adsorbed mediator molecules on the metal surface, it is further preferred in the scope of the present invention to first couple an adsorption-preventing layer of spacer molecules such as for example agarose to the surface of the implant on which the metal oxide layer is provided, to which adsorption-preventing layer the mediator molecules can then be coupled. A prevention of nonspecific adsorption can make sense in order to for example preclude a blocking of BMP-receptors as a result of conformational changes of the BMP-proteins following nonspecific adsorption to the surface. The invention is therefore also directed to such a method for the formation of a nonspecific binding-preventing coating on the metal oxide layer and subsequent coupling of the mediator molecules. The use of a coating of agarose for this purpose is preferred.

A ceramic material such as for example hydroxylapatite can be used as the implant material. Here, the hydroxylapatite should first be activated by treatment with aminoalkylsilane and then reacted with a coupling agent such as carbodiimidazole. In the next step a coupling of the mediator molecules such as BMP or ubiquitin to the surface can take place. When using hydroxylapatite, the use of spacer molecules is not necessarily required.

In the case that the mediator molecules used are not easily soluble under the coupling conditions, the solubility can be increased by addition of surfactants/detergents and the reaction can be performed. In this way, difficultly soluble bone growth factors and other mediators can be kept in solution at pH-values>6 without losing biological activity by ionic and nonionic detergents in the concentration range of 0.05-10%, preferably 1-5% by weight, in particular 0.066% SDS at pH-values>6, in particular pH 8-10 for the covalent coupling method at alkaline pH.

The influence of materials modified by the method of the invention on bone cells or on osteoblast cell lines (MC3T3-E1) were studied in cell culture systems, wherein the modified materials were presented in flake form for this purpose. It was observed that, following application of the cells, confluent cell lawns formed and functional changes by BMP-2 (for example synthesis of alkaline phosphatase) on the materials took place.
 

Claim 1 of 9 Claims

1. A method of immobilization of a mediator molecule on an implant material, comprising: covalently binding an anchor molecule to a chemically activated surface of the implant material, wherein the anchor molecule has a functional group having sufficient reactivity to allow covalent binding of a chemical compound; binding an agarose spacer molecule to the anchor molecule, wherein the agarose spacer molecule has an additional functional group having sufficient reactivity for covalent binding of the mediator molecule; covalently immobilizing a mediator molecule on the implant material using the additional functional group; wherein the mediator molecule comprises a biomolecule that at least one of (a) reduces rejection of the implant material, and (b) promotes growing-in of the implant material; and wherein said implant material comprises at least one component selected from the group consisting of a metal, a metallic alloy, and a ceramic material.
 

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