KR20000075463A - Bone substitute material with a surface coating of peptides having an rgd amino acid sequence - Google Patents

Abstract

The present invention relates to a bone substitute based on a porous polymeric material coated with a peptide whose surface has an RGD amino acid sequence.

Description

Bone SUBSTITUTE MATERIAL WITH A SURFACE COATING OF PEPTIDES HAVING AN RGD AMINO ACID SEQUENCE}

By bone substitute material is meant a material used as an implant to replace or restore bone structures due to postoperative defects following a disease or accident. For example, molded implants such as various types of bone prostheses, for example bone-connected elements in the form of medullary nails, bone screws and bone joint plates, spongy bone defects or extraction cavity fillings. Plastic surgical implants for dragon and maxillary facial contour defects.

Implants that are particularly advantageous for the incorporation process are those that are highly bioactive, that is, tolerated and assimilate to the body. In the case of bone substitutes, this means a firm and permanent attachment to endogenous tissue, especially bone.

In fact, it is known that the most favorable binding results are obtained only by the endogenous substance, that is, bone graft. The availability of bone grafts is limited by their nature. Autologous grafts, i.e. grafts from the same individual, can be removed by at least one additional surgical procedure and require additional treatment of the removal site, even if they are available in virtually suitable shape and quality. The same is true for allografts, ie, transplants from allogeneic donors. In addition, in the case of allograft tissues, not only are there problems of suitability, moreover, the risk of viral infections, especially hepatitis and HIV viruses, can still not be completely excluded. In addition, the storage of donors in bone banks is expensive and, for final analysis, time is limited.

Implants for bone substitutes made of non-body-synthesized or body-related materials may function from bioinert to bioactive, depending on their nature and composition. However, to date no synthetic graft has achieved the result of endogenous bone graft binding.

Recent findings show that when implants are assimilated to bone, cell colonization on the surface proceeds first. Thereafter, extracellular matrix is deposited and new bone tissue is formed. The overall progression is affected by a number of factors and is significantly affected by the properties of the bone substitute, the viability of the stromal bones and the physiodynamic conditions.

Calcium phosphate ceramics, hydroxyapatite-containing bone cements and in particular certain polymers characterized by hydrophilic surfaces are known to have good or good osteoconductive properties. However, good bone conductivity of such materials often cannot be combined with optimized physiodynamic properties, so ceramics are particularly brittle and difficult to meet the elasticity required for bones.

The discovery of integrins (proteins in cell membranes) has revealed one possibility for stimulating surface cell adhesion. Integrins recognize and bind to amino acid sequences of structural proteins, eg, RGD sequences. Integrins regulate the attachment of cells in the body.

In order to facilitate binding of the implant, it is known to provide an implant surface having a synthetic peptide having an RGD sequence. The implants disclosed so far are mostly made of metallic prostheses, in particular titanium or titanium alloys. However, none of these types of grafts are yet to be available which show results that approximate the results of endogenous bone graft binding.

Therefore, the present invention is based on the problem of not only cell attachment but also assimilation into bone more quickly, thereby providing a bone substitute material with biological activity as close as possible to endogenous bone grafts.

It has been found that this problem can be achieved by bone substitutes consisting of a porous polymeric material coated with a peptide essentially having a RGD amino acid sequence.

It is known to provide an implant surface with a synthesizable peptide having an RGD sequence. Implants currently disclosed and tested are mostly made of metallic prostheses, particularly titanium or titanium alloys. It is also known to treat surfaces of polymers, metals or other ceramic materials, in particular with peptides having an RGD sequence (eg WO91-05036). In this case, however, these peptides are specifically covalently bound. The surface is properly activated into the reactor and reacts with the peptide by a coupling agent, where the peptide is covalently bound. However, there is no hint of the porous polymeric bone substitute according to the invention to which a peptide that stimulates cell adhesion to the implant surface is applied (ie no covalent bonds are specifically formed).

Therefore, the present invention relates to a bone substitute based on a porous polymeric material having a peptide surface coating having an RGD amino acid sequence.

The present invention relates in particular to bone substitutes in the form of molded implants.

The invention also consists of two or more separate components, one component comprising a bone substitute according to the invention, and another component comprising a liquid preparation of a peptide having an RGD amino acid sequence. It relates to a transplant kit.

The present invention also has an RGD amino acid sequence, which is used to stimulate and biologically activate cell adhesion to such surfaces by applying them to the surfaces of porous and / or surface-textured polymeric materials for bone substitutes. It relates to a peptide.

The invention also relates to a method for biologically activating bone substitutes based on porous polymeric materials by stimulating cell adhesion to the surface, wherein the surface is coated with a liquid formulation of a peptide having an RGD amino acid sequence.

Polymeric materials may be suitable for bone in mechanical properties, but biomaterials represent low materials. They could not be combined with bone and thus could not be used as a bone substitute.

The present invention is directed to optimizing and achieving biocompatibility by applying RGD peptides to such polymeric materials which are highly desirable as bone substitutes for mechanical reasons but are low in biocompatibility.

Preferred porous polymeric materials in this regard are polyacrylates and / or polymethacrylates, polymethylmethacrylates (PMMA), polyethylene (PE), polypropylene (PP) and / or polytetrafluoroethylene (PTFE). . Of course, copolymers of the aforementioned polymers with each other and copolymers of the aforementioned polymers with other polymers are also possible. The production of polymeric materials of this kind is generally known to those skilled in the art and is not described in detail herein.

In one preferred embodiment the porous polymeric material itself is in the form of a molded graft of bone replacement material according to the present invention, while in another preferred embodiment it forms a surface or surface coating of the molded graft.

The molded article according to the invention preferably has a pore system which is partially or completely interconnected. Polymers having such pore systems can be prepared, for example, in analogy to the process described in EP 0 705 609. However, those skilled in the art are also familiar with the general methods for preparing porous polymeric materials and need not be described further herein. In addition, materials of this kind are commercially available. Those skilled in the art are familiar with such compositions and methods for their preparation.

In this connection, preference is given to composites of polymers or polymers with mineral or metallic additives, especially in the form of particles or fibers.

If the polymeric material itself is in the form of a porous graft, it can be produced, for example, by the method described in EP 0 705 609, by spot fusion of polymethylmethacrylate (PMMA) particles. This method is carried out by mixing together three essentially different components. The first component consists of micro-particle polymers of acrylic acid and / or methacrylic esters (these polymers are commercially available) and, if appropriate, other additives such as polymerization catalysts, X-ray contrast media, fillers and dyes. It is a solid component. The second component is a liquid component consisting of additives such as acrylic ester and / or methacrylic ester monomers and, if appropriate, polymerization accelerators and stabilizers. The third component consists of coarse-particle granules of biocompatible material having a maximum particle diameter of 0.5 to 10 mm. Preferred materials are based on polyacrylates and / or polymethacrylates, polyolefins, copolymers of acrylates and styrene and / or butadiene and epoxy resins. Combine the three main ingredients and mix them together. After the components are well mixed, the polymerization is initiated due to the presence of the catalyst; The composition may remain liquid or plastically deform for several minutes, after which the cured final product is provided. Thus, it is possible in this way to produce a porous implant from bone cement particles which preferably have interconnecting porosity. This material is then subjected to RGD-containing peptides in accordance with the present invention. Such porous bone substitutes can be used as bone cement for implanting bone prostheses during the liquid or calcining step in conventional manner. The surgeon can also convert the composition into shaped articles of any shape and size and, after curing, can be implanted at the body treatment site, for restoration of bone defects or as a local active substance reservoir.

In a preferred embodiment, the porous polymeric material has an average pore width of 0.05 mm to 2.50 mm, preferably 0.10 mm to 1.25 mm.

Therefore, in the present invention, the surface of the molded implant must be in porous form, which can be achieved, for example, by providing a porous surface coating or a corresponding roughened surface on the composite material or bone cement.

When the porous polymeric material forms the surface or surface coating of the molded implant, it may be composed of known conventional implants so long as it can be coated with a porous polymeric layer. Implants can be divided into minerals, in particular ceramic materials, physiologically acceptable metal materials, physiologically acceptable polymer materials, and composite materials of two or more of the aforementioned materials.

Examples of suitable mineral materials include materials based on calcium-containing materials, in particular calcium carbonate, calcium phosphate and the like and systems derived from these compounds. In calcium phosphates, hydroxyapatite, calcium triphosphate and calcium phosphate are preferable.

However, mineral-based implants usually have high mechanical stability only when used in the form of ceramics, ie, materials or workpieces sintered at sufficiently high temperatures.

Detailed descriptions of bone ceramics and particularly advantageous methods for their preparation are described in the patent documents DE 37 27 606, DE 39 03 695, DE 41 00 897 and DE 40 28 683, for example.

The metallic material used is mainly titanium or titanium alloys. Also of interest are combination materials having a significantly wider range of mechanical properties than pure polymers. Therefore, in addition to using polymers coated with RGD peptides as bone substitutes, it is also very noteworthy to combine this kind of material with other implant components.

An example of such a combination is a combination of a porous polymer treated according to the present invention and a metal prosthesis (eg titanium). For this purpose, for example, titanium implants are treated by known methods to combine with polymers. For example, it can be carried out by Kevloc method or Sulicoater method (disclosed in DE 42 25 106). Then, for example, a porous polymer layer is applied to the pretreated titanium surface in a manner similar to that disclosed in EP 0 705 609. The polymer-coated portion of the prosthesis is then coated with RGD peptide.

Another preferred embodiment of the present invention is represented by the following implants. Instead of titanium grafts, grafts of the corresponding fiber composites (carbon fibers and epoxy resins) are coated with a porous layer, for example PMMA, followed by coating of the peptides. Since this type of implant has elasticity suitable for bone, the bone-graft interface without the boundary layer is created, with the advantage that optimal force is transferred from the implant to the bone.

Clinically, this prevents bone resorption through stress shielding, keeping the implant longer.

Preferably, the porous polymer layer applied to a suitable implant has a layer thickness of 0.2 mm to 25 mm, particularly preferably 2.0 mm to 20 mm.

The average pore width is preferably in the range of 0.05 mm to 2.50 mm, particularly preferably in the range of 0.10 mm to 1.25 mm.

Peptides having RGD sequences that can be used in the present invention include peptide and non-peptide substituents, including amino acid sequences, arginine-glycine-aspartic acid (RGD), which can be attached to the polymer surface via peptide and non-peptide substituents. Branch is suitable for all peptide compounds.

The following list of preferred peptides and peptide compounds is merely illustrative and is not intended to be limiting, with 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- Compounds of Gly-Asp-Ser), RGDF (Arg-Gly-Asp-Phe), GRGDF (Gly-Arg-Gly-Asp-Phe), peptides with fatty acids or other acylate-substituted RGD peptides. Peptides may be linear or cyclic.

Coating the bone substitute according to the invention with a peptide compound or peptide with an RGD sequence itself is not difficult. It is preferable to start with a suitable liquid solution of a suitable peptide to be immersed in the material to be applied. In this regard, it can be seen that the final coating of the implant surface is relatively independent of a wide range of solution concentrations. Properly extended exposure time allows complete application to the surface at very low concentrations.

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

The surface coated with the peptide is preferably 50% to 100% of the free surface.

Without further treatment, the peptide material adheres firmly to the polymer surface. The implant is sterilized by conventional methods such as, for example, gamma irradiation, heat or ethylene oxide, and then ready for transplantation.

In a preferred embodiment, the bone replacement material according to the invention comprises at least two individual components, preferably one comprising a porous polymeric material as a molded implant and a liquid formulation of a peptide having an RGD sequence. It consists of components and is in the form of ready-to-use implant kits. Embodiments of this kind are particularly advantageous in effectively preventing stability problems that may arise when long-term storage of the processed bone substitute material according to the invention. The bone replacement material according to the invention in the form of a graft kit of this kind is used by applying the RGD peptide-containing solution to the porous polymer implant just before or during surgery in the manner described above.

Therefore, according to this embodiment, the bone substitute according to the present invention is at least equivalent to that of allogeneic and autologous bone grafts, and is significantly improved in terms of binding action compared to other kinds of bone substitutes.

By immobilizing peptides with RGD sequences on porous grafts, bone substitutes according to the present invention not only result in cell adhesion but also experimentally demonstrate that these grafts in bone assimilate significantly faster than untreated grafts. can do.

The beneficial effect of the RGD coating on the binding action of the implant for bone substitutes is, as described above, if all or part of the composition is made of porous polymeric material, or if the implant is a natural product or a design coated with such porous polymeric layer, Applicable to a variety of bone substitutes and implants. This requirement is also met, for example, by implants whose surfaces are porous or at least roughened.

Theoretically, the bone substitute according to the present invention may be in the form of powder, granules, particles or fibers depending on the part to be inserted and the purpose of use as well as the molded implant.

Without further explanation, those skilled in the art will be able to use the above description in the broadest sense. Therefore, the preferred embodiment is merely illustrative and is not intended to be limiting in any case.

The complete description of all applications, patents, and publications mentioned above and below are hereby incorporated by reference.

The following examples are representative of the present invention.

Example 1

a) porous polymer moldings

Stir low viscosity bone cement (composition: 31 g of polymethylmethacrylate / polymethylacrylate (94/6) copolymer, 6 g of hydroxyapatite powder, 3 g of zirconium dioxide) with 30 ml of methyl methacrylate monomer in a conventional manner It was. The component consisted of a dibenzoyl peroxide / dimethyl-p-toluidine initiator system. To this paste 100 g of pure, cylindrical polymethylmethacrylate granules (diameter 2 mm, length 3 mm) were added and thoroughly mixed with the bone cement paste. The mixed composition was placed in a polypropylene mold and allowed to cure for about 15 minutes. As a result, an article having a pore interconnecting and having a porosity of 20% was obtained.

b) RGD peptide application to polymer moldings

In order to apply the RGD peptide, the molded article obtained in a) was immersed in a tetrapeptide GRGD-concentration 100 µg / ml solution at an exposure time of about 60 minutes and finally dried. The coating results were then measured by cell adhesion test. As a result, the material according to the present invention showed a dense cell lawn extending deeply into the pores, while the cells did not actually metastasize in the unapplied cylinder.

Example 2

Experimental investigation

Species: Rabbit

Implants: a) Porous PMMA moldings

b) PMMA molded articles according to the invention coated with GRGD

Both implants were sterilized by γ-ray irradiation,

The rabbit was implanted into the femur.

Implantation site: implanted in the patellar sliding bearing of the left and right femurs.

Two weeks later, histological examination evaluated new bone formation and mineralization.

result :

a) PMMA

The implant bed shows only a thin annular ring of newly formed bone ossein that has spread connective tissue. There is no evidence that bone shovel overlaps directly on cement beads.

b) PMMA + GRGD

A wide range of new bone ossein formations are found, including ¾ of the total implant; The bone shovel overlaps directly on the cement beads.

The present invention relates to a bone substitute based on a porous polymeric material coated with a peptide whose surface has an RGD amino acid sequence.

Claims (10)

A bone substitute based on a porous polymeric material, the bone substitute having a surface coating of a peptide having an RGD amino acid sequence. The method of claim 1, Bone replacement material, characterized in that the porous polymeric material is in the form of a molded implant. The method according to claim 1 or 2, Wherein said porous polymeric material forms a surface or surface coating of a molded implant. The method according to any one of claims 1 to 3, Wherein said porous polymeric material forms a surface or surface coating of a molded graft comprised of mineral or metallic material or composite material. The method according to any one of claims 1 to 4, The porous polymer material is essentially a polyacrylate and / or polymethacrylate, polymethylmethacrylate, polyethylene, polypropylene and / or polytetrafluoroethylene and / or copolymers of these polymers with other polymers Bone substitutes. The method according to any one of claims 1 to 5, Bone substitute material, characterized in that the porous shaped article has a pore system which partially or completely interconnects. The method according to any one of claims 1 to 6, Bone replacement material characterized in that the peptide surface coating having the RGD amino acid sequence is 50% to 100% of the uncoated surface. It consists of two or more separate components, one of which comprises a bone substitute according to any one of claims 1 to 7, wherein another component comprises a liquid formulation of a peptide having an RGD amino acid sequence. Implant kit comprising a. A peptide having an RGD amino acid sequence, used to stimulate and biologically activate cell adhesion to such a surface by applying it to the surface of a porous and / or surface-textured polymeric material for bone replacement. A method of biologically activating a bone substitute based on a porous polymeric material by stimulating cell adhesion to a surface, wherein the surface is coated with a liquid formulation of a peptide having an RGD amino acid sequence. .