CA2281608A1

CA2281608A1 - Bone substitute material with a surface coating of peptides having an rgd amino acid sequence

Info

Publication number: CA2281608A1
Application number: CA002281608A
Authority: CA
Inventors: Berthold Nies
Original assignee: Individual
Current assignee: Merck Patent GmbH
Priority date: 1997-02-20
Filing date: 1998-02-11
Publication date: 1998-08-27
Also published as: EP0968010A2; KR20000075463A; CN1247474A; AU6100498A; WO1998036782A2; HUP0000900A3; ZA981393B; PL335103A1; HUP0000900A2; WO1998036782A3; DE19706667A1; JP2001512344A

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 coating of peptides with the RGD amino acid sequence The invention relates to bone replacement material based on a porous polymer material which has a surface coating of peptides with the RGD amino acid sequence.
Bone replacement materials mean materials used as implants for replacing or reconstituting bony structures on account of defects after surgical operations occasioned by disease or accident. Examples which may be mentioned are shaped implants such as bone prostheses of a wide variety of types, bone-connecting elements, for example in the form of medullary nails, bone screws and osteosynthesis plates, implant materials for filling in spongy bone defects or dental extraction cavities, and for plastic surgical treatment of contour defects in the maxillofacial region.
Implant materials which are regarded as particularly favourable for the incorporation process are those having a high bioactivity, namely such that they are accepted by the body and integrated into it. In the case of bone replacement material, this means firm and permanent adhesion to endogenous tissue, in particular to bone, should take place soon.
It is known that hitherto the most favourable incorporation results are in practice achieved only with endogenous materials, that is to say with bone transplants. The availability of bone transplants is limited by their nature. Autologous transplants, that is to say transplants from the same individual, can, if in fact available in a suitable shape and quantity, be removed only by at least one additional surgical operation, in turn occasioning an additional healing process at the site of removal. The same also applies in principle to homologous transplants, that is to say transplants from donor individuals of the same species.

With the latter there are also problems of compatibility and moreover the risk of infection with viruses such as, in particular, hepatitis and HIV
viruses, which still cannot be completely precluded.
Furthermore, storage of donated material in bone banks is costly and, in the final analysis, only limited in time.
Implant materials for bone replacement made of synthetic materials not related to the body or of materials related to the body may show a behaviour ranging from bioinert to bioactive, depending on their nature and composition. The results of incorporation of endogenous bone transplants have not, however, to date been achievable by any synthetic implant material.
Recent findings show that integration of the implant material into bone must be preceded by cellular colonization of the surface. This is followed by deposition of extracellular matrix and formation of new bone tissue. The complete process is multifactorial and is considerably influenced by the properties of the bone replacement material, the vitality of the substrate bone and the biomechanical circumstances.
It is known that good or very good osteoconductive properties are possessed by calcium phosphate ceramics, hydroxyapatite-containing bone cement and specific polymers which are distinguished, in particular, by a hydrophilic surface. However, the good osteoconductivity of these materials often cannot be combined with optimized biomechanical properties, so that the ceramics in particular are brittle and have low adaptability to the elastic requirements in the bone.
One possibility of stimulating cellular adhesion to surfaces was found with the discovery of integrins (proteins in the cell membrane). Integrins recognize amino acid sequences, for example the RGD sequence, on structural proteins and bind thereto. This controls the adhesion of cells in the body.
The furnishing of implant surfaces with synthetically available peptides with RGD sequences with the aim of speeding up the incorporation of implants is known.
Implants disclosed to date are mostly metallic prostheses, in particular made of titanium or titanium alloys. However, no convincing implants of this type with results which might approach the results of incorporation of endogenous bone transplants are yet available.
The invention was therefore based on the problem of providing a bone replacement material which is able not only to bring about cellular adhesion but also to be integrated into bone more quickly, and thus has a biological activity which comes as close as possible to that of endogenous bone transplants.
It has now been found that this is achieved by a bone replacement material which is essentially composed of a porous polymer material with a surface coating of peptides with the RGD amino acid sequence.
The furnishing of implant surfaces with synthetically available .peptides with RGD sequences is known. The implants disclosed and tested to date are mostly metallic prostheses, in particular made of titanium or titanium alloys. It is furthermore known (for example from WO 91-05036) to treat surfaces, such as those of polymers, metals or else ceramic materials, with peptides which may, inter alia, also have RGD
sequences. In this case, however, these peptides are specifically covalently bonded. The said surfaces are appropriately activated with reactive groups and reacted with the peptides using a coupling reagent, whereupon the peptides are covalently bonded. However, it contains no hints which would lead to the porous polymeric bone replacement materials according to the invention, which are loaded (that is to say no specific formation of covalent bonds) with peptides which stimulate cell adhesion on the surface of the implants.
The invention therefore relates to a bone replacement material based on a porous polymer material which has a surface coating of peptides with the RGD amino acid sequence.
The invention relates in particular to a bone replacement material of this type which is in the form of a shaped implant.
The invention also relates to an implantation kit consisting of two or more separate components, one component of which comprises a bone replacement material according to the invention and another component of which comprises a liquid preparation of a peptide with the RGD amino acid sequence.
The invention furthermore relates to the use of peptides with the RGD amino acid sequence for loading the surface of a porous and/or surface-textured polymer material for bone replacement, whereby biological activation takes place through stimulation of cell adhesion to this surface.
The invention further relates to a method for the biological activation of bone replacement materials based on a porous polymer material by stimulating cell adhesion to the surface thereof, which is characterized in that the surface thereof is coated with a liquid preparation of a peptide with the RGD amino acid sequence.
Polymer materials represent a material which is otherwise of low biocompatibility and, although their mechanical properties can be adapted to that of bone, they have not to date been employed as bone replacement because they do not unite with the bone.
The relevance of the present invention is now that this polymer material of low biocompatibility, which would be very desirable as bone replacement for mechanical reasons, is optimized by the loading with RGD peptides and biocompatibility is achieved.
Porous polymeric materials preferred in this connection are essentially polyacrylates and/or polymethacrylates, polymethylmethacrylates (PMMA), polyethylenes (PE), polypropylenes (PP) and/or polytetrafluoroethylenes (PTFE). It is, of course, also possible to employ copolymers of the said polymers with one another, and copolymers of these polymers with other polymers. The production of polymer materials of these types is generally known to the skilled person and need not be explained in detail here.
In a preferred embodiment, the porous polymer material itself is in the form of a shaped implant in the bone replacement material according to the invention or, in another preferred embodiment, it forms the surface or a surface coating of a shaped implant.
Shaped articles according to the invention which are particularly preferred are those having a partly or completely interconnected pore system. Polymers with such pore systems can be produced, for example, in analogy to the procedure described in the patent application EP 0 705 609. However, the skilled person is furthermore well aware of the general process for producing porous polymer materials, and there is therefore no need to pursue this any further here. In addition, materials of this type are also commercially available. The skilled person is familiar with their composition and the way of processing them.

Also preferred in this connection are polymers or composites of polymers and mineral or metallic additives, in particular in particulate or fibrous form.
If the polymer material itself is in the form of a porous implant, it can be produced, for example, by the process described in the abovementioned EP 0 705 609 by spot fusion of polymethylmethacrylate (PMMA) particles.
This process is essentially carried out by mixing three different components together. The first component thereof is a solid component consisting of a fine-particle polymer of acrylic and/or methacrylic esters (these polymers are commercially available) and, where appropriate, other additives such as polymerization catalysts, X-ray contrast media, fillers and dyes. The second component is a liquid component consisting of acrylic and/or methacrylic ester monomers, where appropriate with additives such as polymerization accelerators and stabilizers. The third component consists of coarse-particle granules, of a biocompatible material with a maximum particle diameter of from 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 combined and mixed together. After the components have been intimately mixed, the polymerization starts owing to the catalyst which is present; the composition remains liquid or capable of plastic deformation for a period of some minutes, after which the cured final product is present. It is thus possible in this way to produce porous implants from bone cement particles which preferably have an interconnecting porosity.
These materials are then loaded according to the invention with RGD-containing peptides. This porous bone replacement material can be used in a conventional way during the liquid or plastic stage as bone cement for implanting bone prostheses. The surgeon is also able to convert the composition into shaped articles of any shape and size and, after curing, implant them for reconstruction of bone defects or as local active substance depots into the body regions to be treated.
In a preferred embodiment, the porous polymer materials have an average pore width of from 0.05 mm to 2.50 mm, particularly preferably 0.10 mm to 1.25 mm.
It is thus necessary according to the invention for the surface of the shaped implant to have a porous form, which can be achieved, for example, by providing a composite material or a bone cement 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, they can consist of all known and conventional implant materials as long as they can be coated with a layer of porous polymer.
Implant materials can be divided into the classes of mineral, in particular ceramic, materials, physiologically acceptable metallic materials, physiologically acceptable polymer materials and composite materials of two or more materials of the type mentioned.
Examples of suitable mineral materials are materials based on calcium-containing materials such as, in particular, calcium carbonate, calcium phosphates and systems derived from these compounds. Ones to be mentioned as preferred from the group of calcium phosphates are hydroxyapatite, tricalcium phosphate and tetracalcium phosphate.
However, mineral-based implant materials usually ensure high mechanical stability only if they are employed as ceramics, that is to say in the form of materials or workpieces which have been sintered at sufficiently high temperatures.

_ g _ Details on bone ceramics and particularly favourable processes for producing them 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.
The metallic material employed is mainly titanium or a titanium alloy. Also of particular interest are combined materials whose mechanical properties cover a considerably wider range than do the pure polymers.
Thus, besides the use of polymers coated with RGD
peptides as bone replacement, combination of materials of this type with other implant compone [sic] is also very important.
An example which may be mentioned of such a combination is combining a metal prosthesis (for example titanium) with a porous polymer treated according to the invention. For this purpose, for example, the titanium implant is treated in a manner known per se for combining with the polymer. This can take place, for example, by the Kevloc 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 in a manner analogous to that 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 is represented by the following implant material. In place of the titanium implant, a corresponding implant made of a fibre composite material (carbon fibre and epoxy resin) is coated with a porous layer of, for example, PMMA, after which the peptide coating takes place. An implant of this type has the advantage of elasticity adapted to bone, creating a bone-implant interface without boundary layer, and achieving optimal force transmission from the implant into the bone.

_ g _ Clinically, this avoids bone resorption through stress shielding, and the prosthesis is held for longer.
The porous polymer layer applied to an appropriate implant material preferably has a layer thickness of from 0.2 mm to 25 mm, particularly preferably from 2.0 mm to 20 mm.
The average pore width is preferably in the range from 0.05 mm to 2.50 mm and the range is particularly preferably from 0.10 mm to 1.25 mm.
Suitable peptides with the RGD sequence which can be employed according to the invention are all peptides and their compounds with non-peptide substituents which contain the amino acid sequence arginine-glycine-aspartic acid (RGD) and which are able to adhere via their peptide and non-peptide substituents to the polymer surfaces.
The following lists of preferred peptides and peptide compounds are intended to have merely an exemplary and by no means limiting character, with the following abbreviations being used:
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 else acrylate-substituted RGD peptides. The peptides can be either linear or cyclic.

The coating of the bone replacement material according to the invention with a peptide compound or a peptide with the RGD sequence is not difficult per se. It preferably starts from a suitable liquid solution of the appropriate peptide, into which the material to be loaded is immersed. It has been possible to show in this connection that the eventual coating of the implant surface is relatively independent of the concentration of the solution over a wide range. It is possible with very low concentrations to achieve just as complete loading of the surface by appropriate prolongation of the exposure time.
The preferred concentration range for the peptide solution can be stated to be 10 ng - 100 ~g/ml. The exposure time is preferably from 10 minutes to 24 hours.
The surface coating with peptides preferably amounts to 50~ to 100 of the free surface.
The peptide substance moreover adheres firmly to the polymer surface without further treatment. The implants are sterilized in a customary way, for example by 'y irradiation, heat or ethylene oxide, and are then ready for implantation.
In a preferred embodiment, the bone replacement material according to the invention is in the form of an implantation kit which is ready for use and consists of two or more separate components, in which one component comprises a porous polymer material, preferably as shaped implant, and another component comprises a liquid preparation of a peptide with the RGD sequence. An embodiment of this type is particularly advantageous for effectively countering possible stability problems which might occur on long-term storage of finished bone replacement materials according to the invention. The bone replacement materials according to the invention in the form of an implantation kit of this type are used by loading the porous polymeric implantation material with the RGD
peptide-containing solution in the prescribed manner shortly before or during the surgical operation.
Thus, depending on the embodiment, the bone replacement material according to the invention represents an at least equivalent substitute for homologous and autologous bone transplants, or is a considerable improvement in respect of the incorporation behaviour for other types of bone replacement.
Not only do the bone replacement materials according to the invention bring about cellular adhesion, owing to the immobilization of peptides with the RGD sequence on a porous polymer implant, but it has also been possible to demonstrate in experiments that integration of these implant materials in bone takes place significantly faster than with untreated implants.
The beneficial effect of the RGD coating on the incorporation behaviour of implants for bone replacement is, as already mentioned, applicable to virtually all types of bone replacement materials and implant materials as long as they are of such a nature or design either that they consist wholly or partly of porous polymer material, or that the implants are coated with such a porous polymer layer. This requirement is also met by, for example, implants whose surface has a porous structure or is at least roughened.
It is possible in principle for the bone replacement materials according to the invention to be in the form not only of shaped implants but also of powders, granules, particles or fibres, depending on the requirements of the site of insertion and the purpose of use .

It is assumed even without further explanation that a skilled person is able to utilize the above description in the widest sense. The preferred embodiments are therefore to be interpreted merely as a descriptive and by no means as in any way a limiting disclosure.
The complete disclosure of all the applications, patents and publications mentioned hereinbefore and hereinafter is incorporated into this application by reference.
The following examples are representative of the present invention.
Example 1 a) Production of a porous polymer shaped article A low-viscosity bone cement (composition: 31 g of polymethylmethacrylate/polymethylacrylt [sic]
(94/6) copolymer, 6 g of hydroxyapatite powder, 3 g of zirconium dioxide) is stirred with 30 ml of methylmethacrylate monomer in a conventional way.
The components comprise the dibenzoyl peroxide/dimethyl-p-toluidine starter system.
100 g of pure, cylindrical polymethylmethacrylate granules (diameter 2 mm, length 3 mm) are added to this ,paste and thoroughly mixed with the bone cement paste. The mixed composition is put in polypropylene moulds and left to cure for about 15 min. The result is an article with inteconnecting [sic] pores and a porosity of 20~.
b) Loading of the polymeric shaped article with RGD
peptide The shaped article obtained as in a) is immersed in a solution of the tetrapeptide GRGD-concentration 100 ~.g/ml, exposure time about 60 minutes, for loading with this RGD peptide, and is finally dried. The success of the coating was then measured in a cell adhesion test. The results show that the unloaded cylinders are virtually not colonized by cells, whereas the materials according to the invention show a dense cell lawn extending deep into the pores.
Example 2 Experimental investigations Species: Rabbit Implants: a) porous PN~IA shaped article b) PMMA shaped article according to the invention coated with GRGD
Both implants were sterilized by y irradiation and implanted into rabbit f emora .
Implantation site: Into the patellar sliding bearing of the left and right femora.
After 2 weeks, the new bone formation and the mineralization are assessed by histological examination.
Result:
a) PMMA
The implant bed shows only a thin circular ring of newly formed trabecular bone permeated with connective tissue. There is no evident direct superimposition of the trabecular bone on the cement beads.

b) PMMA + GRGD
Extensive new trabecular bone formation can be found in this case and envelops three quarters of the entire implant; the trabecular bone is directly superimposed on the cement beads.

Claims

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

2. Bone replacement material according to Claim 1, characterized in that the porous polymer material is in the form of a shaped implant.

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.

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

5. Bone replacement material according to any of Claims 1 to 4, characterized in that the porous polymer material is essentially a polyacrylate and/or polymethacrylate, polymethylmethacrylate, polyethylene, polypropylene and/or polytetrafluoroethylene and/or a copolymer of these polymers with other polymers.

6. Bone replacement material according to any of Claims 1 to 5, characterized in that these porous shaped articles have a partly or completely interconnecting pore system.

7. Bone replacement material according to any of Claims 1 to 6, characterized in that the surface coating of peptides with the RGD amino acid sequence amounts to 50% to 100% of the free surface.

8. Implantation kit consisting of two or more separate components, one component of which comprises a bone replacement material according to any of Claims 1 to 7 and another component of which comprises a liquid preparation of a peptide with the RGD amino acid sequence.

9. Use of peptides with the RGD amino acid sequence for loading the surface of a porous and/or surface-textured polymer material for bone replacement, whereby biological activation takes place through stimulation of cell adhesion to this surface.

10. Method for the biological activation of bone replacement materials based on a porous polymer material by stimulating cell adhesion to the surface thereof, characterized in that the surface thereof is coated with a liquid preparation of a peptide with the RGD amino acid sequence