EP0968010A2

EP0968010A2 - Bone substitute material with a surface coating of peptides having an rgd amino acid sequence

Info

Publication number: EP0968010A2
Application number: EP98905418A
Authority: EP
Inventors: Berthold Nies
Original assignee: Merck Patent GmbH
Current assignee: Merck Patent GmbH
Priority date: 1997-02-20
Filing date: 1998-02-11
Publication date: 2000-01-05
Also published as: ZA981393B; PL335103A1; AU6100498A; JP2001512344A; CN1247474A; HUP0000900A3; DE19706667A1; KR20000075463A; WO1998036782A2; WO1998036782A3; CA2281608A1; HUP0000900A2

Abstract

The invention relates to a bone substitute material based on a porous polymer material the surface of which is coated with peptides having an RGD amino acid sequence.

Description

Bone replacement material with a surface covering with peptides with RGD amino acid sequence

The invention relates to bone substitute material based on a porous polymer material which has a surface covering with peptides with RGD-

Has amino acid sequence.

Bone replacement materials are understood to be materials that serve as implants for the replacement or reconstitution of bone structures due to defects after surgical or illness-related surgery. Examples are implant shaped bodies such as various types of bone prostheses, bone connecting elements, for example in the form of medullary cavity nails, bone screws and osteosynthesis plates, implant materials for filling cancellous bone defects or tooth extraction cavities, and for plastic surgery treatment of contour defects in the jaw facial area.

For the healing process, such implant materials are regarded as particularly favorable if they have a high bioactivity, namely in that they are accepted in the organism and integrated into it. In the case of bone substitute material, this means that it should soon grow firmly and permanently with the body's own tissue, in particular with the bone.

It is known that the cheapest healing results have so far been achieved practically only with the body's own materials, ie with bone grafts. The availability of bone grafts is naturally limited. Autologous grafts, that is to say grafts from the same individual, can only be removed, if at all in a suitable form and quantity, by at least one additional surgical intervention, which in turn requires an additional healing process at the removal site. The same also applies in principle to homologous transplants, that is to say transplants from donor individuals of the same type. In addition, there are problems of tolerance as well as the risk of infection, which can still not be completely ruled out today Viruses, such as hepatitis and HIV viruses in particular. Furthermore, the storage of donor material in bone banks is complex and ultimately only limited in time.

Implant materials for bone replacement made of synthetic or non-body related materials can, depending on their nature and nature, show bioinert to bioactive behavior. However, the healing results of the body's own bone grafts have not yet been achieved by any synthetic implant material.

Recent findings show that the osseous integration of the implant material must first be preceded by cellular colonization of the surface. This is followed by deposition of extracellular matrix and the formation of new bone tissue. The entire process is multifactorial and is significantly influenced by the properties of the bone substitute material, the vitality of the bony bed and the biomechanical conditions.

The good to very good osteoconductive properties of calcium phosphate ceramics, hydroxyapatite-containing bone cement and special polymers are known, which are particularly notable for their hydrophilic surface. However, the good osteoconductivity of these materials often cannot be combined with optimized biomechanical properties, so the ceramics in particular are brittle and less adaptable to the elastic requirements in the bone.

One way to stimulate cellular adhesion to surfaces has been discovered with the discovery of integrins (proteins in the cell membrane). The integrins recognize and bind to amino acid sequences, for example the RGD sequence, on structural proteins. This controls the adhesion of cells in the body. The loading of implant surfaces with synthetically accessible peptides with RGD sequences with the aim of accelerating the healing of the implants is known. The previously known implants are mostly metallic prostheses, in particular made of titanium or titanium alloys. However, there are still no convincing implants with corresponding results that could approach the healing results of the body's own bone grafts.

The invention was therefore based on the problem of providing a bone replacement material which not only can bring about cellular adhesion, but which can also be integrated more quickly into the bone, and thus has a biological activity which comes as close as possible to that of the body's own bone grafts.

It has now been found that this is achieved by a bone substitute material which is essentially composed of a porous polymer material with a surface covering with peptides with an RGD amino acid sequence.

The loading of implant surfaces with synthetically accessible peptides with RGD sequences is known. The implants known and tested so far are mostly metallic prostheses, in particular made of titanium or titanium alloys. It is also known (for example from WO 91-05036) to treat surfaces such as those of polymers, metals or ceramic materials with peptides, which may also have RGD sequences. In this case, however, these peptides are specifically covalently bound. The surfaces mentioned are activated accordingly with reactive groups and reacted with the peptides with a coupling reagent, the peptides being covalently bound. However, there are no indications whatsoever which would lead to the porous polymeric bone substitute materials according to the invention which are loaded with peptides (that is to say no targeted creation of covalent bonds) which stimulate cell adhesion on the surface of the implants. The invention therefore relates to a bone substitute material based on a porous polymer material which has a surface covering with peptides with an RGD amino acid sequence.

The invention relates in particular to such a bone replacement material which is in the form of a shaped implant.

The invention also relates to an implantation set consisting of two or more separate components, one component of which contains a bone substitute material according to the invention and another component of which comprises a liquid preparation of a peptide with an RGD amino acid sequence.

The invention furthermore relates to the use of peptides with an RGD amino acid sequence for loading the surface of a porous and / or surface-structured polymer material for bone replacement, as a result of which biological activation takes place by stimulating the cell adhesion to this surface.

The invention furthermore relates to a method for the biological activation of bone replacement materials based on a porous polymer material by stimulating the cell adhesion to their surface, which is characterized in that the surface thereof is coated with a liquid preparation of a peptide with an RGD amino acid sequence.

Polymer materials are an otherwise less biocompatible material that, although their mechanical properties can be adapted to the bone, have not been used as a bone substitute so far because they do not bind to the bone.

The relevance of the present invention lies in the fact that this little biocompatible polymer material is used for mechanical reasons would be desirable as a bone substitute, optimized by loading with RGD peptides and achieving biocompatibility.

Preferred are porous polymeric materials, which are essentially polyacrylic and / or polymethacrylates, polymethyl methacrylates (PMMA),

Represent Polyethylene (PE), Polypropylene (PP) and / or Polytetrafluorethylene (PTFE). Of course, copolymers of the polymers mentioned with one another and copolymers of these polymers with other polymers can also be used. The production of such polymer materials ^is generally known to the person skilled in the art and need not be explained in more detail here.

In a preferred embodiment, the porous polymer material itself is present as a shaped implant body in the bone replacement material according to the invention, or in a further preferred embodiment it forms the surface or a surface coating of a shaped implant body.

In particular, shaped bodies according to the invention are preferred which have a partially or completely interconnecting pore system. Polymers with such pore systems can be produced, for example, analogously to the manner described in patent application EP 0 705 609. Furthermore, however, the general processes for the production of porous polymer materials are well known to the person skilled in the art and it is therefore not necessary to go into them here. Materials of this type are also commercially available. The skilled worker is familiar with their composition and the way in which they are processed.

Polymers or composites of polymers and mineral or metallic additives, in particular in particle or fiber form, are also preferred. If the polymer material itself is in the form of a porous implant, it can be produced, for example, by the processes described in EP 0 705 609 already mentioned, by selective fusion of polymethyl methacrylate particles (PMMA). This process is essentially carried out by mixing three different components together. The first component is a solid component, consisting of a finely divided polymer of acrylic and / or methacrylic acid esters (these polymers are commercially available) and optionally other additives such as polymerization catalysts, X-ray contrast agents, fillers and dyes. The second component is a liquid component consisting of acrylic and / or methacrylic acid ester monomers, optionally with additives such as polymerization accelerators and stabilizers. The third component consists of coarse granules, made of a biocompatible material with the largest particle diameter of 0.5 to 10 mm. Preferred materials are based on polyacrylates and / or polymethacrylates, polyolefins, copolymers of acrylates with styrene and / or butadiene and epoxy resins. The three main components are brought together and mixed together. After thorough mixing of the components, the polymerization begins due to the catalyst contained; The mass remains liquid to plastically deformable for a few minutes, after which the cured end product is available. Porous implants can thus be produced from bone cement particles, which preferably have an interconnecting porosity. According to the invention, these materials are then containing RGD

Load peptides. This porous bone substitute material can be used in the usual way during the liquid or plastic stage as bone cement for the implantation of bone prostheses. The surgeon can also process the mass into shaped bodies of any shape and size and, after hardening, implant them into the body areas to be treated for the reconstruction of bone defects or as local active substance depots. In a preferred embodiment, the porous polymer materials have an average pore size of 0.05 mm to 2.50 mm, particularly preferably 0.10 mm to 1.25 mm.

According to the invention, the surface of the shaped implant body must have a porous shape, which can be realized, for example, in the form that a composite material or a bone cement is provided with a porous surface coating or a corresponding roughened surface.

If the porous polymer material forms the surface or the surface coating of a shaped implant body, these can consist of all known and customary implant materials, provided that they can be coated with a layer of porous polymer. Implant materials can be divided into the classes mineral, in particular ceramic materials, physiologically acceptable metallic materials, physiologically acceptable polymer materials and composite materials made of two or more materials of the type mentioned.

Suitable mineral materials are, for example, materials based on materials containing calcium, such as, in particular, calcium carbonate, calcium phosphates and systems derived from these compounds. From the group of calcium phosphates, preferred are hydroxyapatite, tricalcium phosphate and tetracalcium phosphate.

However, mineral-based implant materials usually only guarantee high mechanical stability if they are used as ceramics, i.e. thus in the form of materials or workpieces sintered at sufficiently high temperatures.

More information on bone ceramics and particularly cheap methods for their production can be found, for example, in the patent documents DE 37 27 606, DE 39 03 695, DE 41 00 897 and DE 40 28 683 can be found.

Titanium or a titanium alloy is mainly used as the metallic material. Also of particular interest are composite materials that cover a much wider range in their mechanical properties than the pure polymers. In addition to the use of the polymers coated with RGD peptides as bone replacements, the combination of such materials with other implant components is of great importance.

An example of such a combination is the combination of a metal prosthesis (e.g. titanium) with a porous polymer treated according to the invention. For this, e.g. treated the titanium implant in a manner known per se for bonding with the polymer. This can be done, for example, using the Kevioc process or the Sulicoater process (described in DE 42 25 106). A layer of porous polymer is then applied to the pretreated titanium surface, for example analogously to the manner described in EP 0 705 609. The polymer-coated part of the prosthesis is then subsequently coated with the RGD peptide.

Another preferred embodiment of this invention represents the following implant material. Instead of the titanium implant, a corresponding implant made of a fiber composite material (carbon fiber and epoxy resin) is coated with a porous layer made of, for example, PMMA, and then the peptide coating takes place. Such an implant has the advantage of bone-adapted elasticity, creating a boundary layer-free bone-implant interface and achieving optimal force transmission from the implant into the bone. Clinically, bone resorption through "stress shielding" is avoided and the prosthesis lasts longer. The porous polymer layer that is applied to a corresponding implant material preferably has a layer thickness of 0.2 mm to 25 mm, particularly preferably from 2.0 mm to 20 mm.

The average pore size is preferably in the range from 0.05 mm to

2.50 mm, particularly preferred is the range from 0.10 mm to 1.25 mm.

Suitable peptides with RGD sequence that can be used according to the invention are all peptides and their compounds with non-peptide substituents that contain the amino acid sequence arginine-glycine-aspartic acid (RGD) and that can adhere to the polymer surfaces via their peptide and non-peptide substituents.

The following lists of preferred peptides or ^Pe Pt i i ^dverb inventions sol len merely exemplary and not limiting

Have character, using the following abbreviations:

Asp = aspartic acid

Gly = glycine

Arg = arginine

Tyr = tyrosine

Ser = serine

Phe = phenylalanine

RGD (Arg-Gly-Asp), GRGD (Gly-Arg-Gly-Asp), GRGDY (Gly-Arg-Gly-Asp-Tyr), RGDS (Arg-Gly-Asp-Ser), GRGDS (Gly-Arg- Gly-Asp-Ser), RGDF (Arg-Gly-Asp-Phe), GRGDF (Gly-Arg-Gly-Asp-Phe), compounds of peptides with fatty acids or acrylate-substituted RGD peptides. The peptides can be both linear and cyclic.

The coating of the bone replacement materials according to the invention with a peptide compound or a peptide with an RGD sequence is in itself no problem. It is expedient to start with a suitable liquid solution of the corresponding peptide, into which the material to be loaded is immersed. It could be shown that the final coverage of the implant surface is relatively independent of the concentration of the solution in a wide range. At very low

Concentrations can also be achieved by fully extending the exposure by appropriately extending the exposure time.

The preferred concentration range for the peptide solution is 10 ng - 100 μg / ml. The exposure time is preferably 10 minutes to 24 hours.

The surface coverage with peptides is preferably 50% to 100% of the free surface.

The peptide substance adheres firmly to the polymer surface without further treatment. The implants are sterilized in the usual way, for example by γ-radiation, heat or ethylene oxide, and are then ready for implantation.

In a preferred embodiment, the bone substitute material according to the invention is in the form of a ready-to-use implantation set consisting of two or more separate components, in which one component comprises a porous polymer material, preferably as a shaped implant, and another component comprises a liquid preparation of a peptide with an RGD sequence. Such an embodiment is particularly expedient in order to effectively counter possible stability problems which could arise in the case of long-term storage of ready-made bone replacement materials according to the invention. The bone substitute materials according to the invention are used in the form of such an implantation set in such a way that the porous material is inserted shortly before or during the surgical intervention for the implantation polymeric implant material loaded with the RGD peptide-containing solution in the prescribed manner.

Depending on the embodiment, the bone replacement material according to the invention thus represents an at least equivalent replacement for homologous and autologous bone grafts or is a significant improvement in healing behavior for other forms of bone replacement.

By immobilizing peptides with an RGD sequence on a porous polymer implant, the bone substitute materials according to the invention not only bring about cellular adhesion, but tests have shown that these implant materials integrate significantly faster than untreated implants.

As already mentioned, the positive influence of the RGD coating on the healing behavior of implants for bone replacement can be transferred to practically all types of bone replacement materials and implant materials, provided that they are of such a type or design that they are either entirely or partially made of porous Polymer material exist, or that the implants are coated with such a porous polymer layer. This requirement is also met, for example, by implants that have a porous structure or at least a roughened surface.

In principle, the bone replacement materials according to the invention can be present not only as shaped implants, but also in powder, granulate, particle or fiber form, depending on the location and the application.

Even without further elaboration, it is assumed that a person skilled in the art can use the above description to the greatest extent. The preferred embodiments are therefore only as descriptive disclosure, which is in no way limited in any way.

The full disclosure of all applications, patents, and publications listed above and below are incorporated by reference into this application.

The following examples are representative of the present invention.

example 1

a) Production of a porous polymeric molded body

A low-viscosity bone cement (composition: 31 g

Polymethyl methacrylate / polymethylacrylated (94/6) copolymer, 6 g of hydroxyapatite powder, 3 g of zirconium dioxide) is mixed with 30 ml of methyl methacrylate monomer in the usual way. The components contain the starter system dibenzoyl peroxide / dimethyl-p-toluidine. 100 g of pure, cylindrical shape are added to this dough

Add polymethyl methacrylate granules (diameter 2 mm, length 3 mm) and mix thoroughly with the bone cement dough. The mixed mass is placed in polypropylene molds and allowed to harden for about 15 minutes. The result is an interconnecting porous body with a porosity of 20%.

b) loading of the polymeric shaped body with RGD peptide

The shaped body obtained according to a) is immersed in a solution of the tetrapeptide GRGD concentration 100 μg / ml,

Exposure time approx. 60 minutes, loaded with this RGD peptide and finally dried. The success of the coating was then measured in a cell adhesion test. The results show that the unloaded cylinders are practically not colonized by cells, while the materials according to the invention show a dense cell turf deep into the pores.

Example 2

Experimental investigations

Species: rabbit

Implants: a) porous PMMA molded body b) PMMA molded body according to the invention coated with GRGD

Both implants were sterilized by γ-radiation and implanted in rabbit femora.

Implantation location: In the patellar sliding bearing of the femora, left and right.

After 2 weeks, new bone formation and mineralization are determined by histological examination.

Result:

a) PMMA

The implant site shows only a thin circular ring of newly formed bone trabecula, which is interspersed with connective tissue. A direct deposition of the bone trabeculae on the cement pearls cannot be seen. PMMA + GRGD

A massive trabecular new bone formation can be found here, which encompasses three quarters of the entire implant; the bone trabeculae are immediately superimposed on the cement pearls.

Claims

claims

1. Bone replacement material based on a porous polymer material, characterized in that it has a surface covering with peptides with RGD amino acid sequence.

2. Bone replacement material according to claim 1, characterized in that the porous polymer material is present as a shaped implant body.

3. Bone replacement material according to claim 1 or 2, characterized in that the porous polymer material forms the surface or a surface coating of a shaped implant body.

4. Bone replacement material according to one of claims 1 to 3, characterized in that the porous polymer material forms the surface or a surface coating of a shaped implant body which consists of a mineral or metallic material or of a composite material.

5. Bone replacement material according to one of claims 1 to 4, characterized in that the porous polymer material is essentially a polyacrylic and / or polymethacrylate, polymethyl methacrylate, polyethylene, polypropylene and / or polytetrafluoroethylene and / or a copolymer of these polymers with other polymers.

6. Bone replacement material according to one of claims 1 to 5, characterized in that these porous shaped bodies have a partially or completely interconnecting pore system.

7. Bone replacement material according to one of claims 1 to 6, characterized in that the surface coverage with peptides with RGD amino acid sequence is 50% to 100% of the free surface.

8. Implantation set of two or more separate components, one component of which contains a bone replacement material according to one of claims 1 to 7 and another component of a liquid preparation of a peptide with RGD amino acid sequence.

9. Use of peptides with RGD amino acid sequence for loading the surface of a porous and / or surface-structured polymer material for bone replacement, as a result of which biological activation takes place by stimulating cell adhesion on this surface.

10. A method for the biological activation of bone replacement materials based on a porous polymer material by stimulating the cell adhesion to the surface thereof, characterized in that the surface thereof is treated with a liquid

Preparation of a peptide with RGD amino acid sequence