JP2010088933A - Peptide-coated implant and method for preparing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an implant of a general nature for the human and animal bodies, which is coated with peptide that can selectively mediate adhesion of specific cells in the particular environment of the implant. <P>SOLUTION: The invention describes possibility of biofunctionalization of a biomaterial, in particular the implant, by coating it with synthesized cell- or tissue-selective RGD peptide which in vitro stimulates the adhesion of mainly cell species intended to accomplish the tissue integration of the suitable biomaterial in each case while, at the same time, not stimulating well the adhesion of cell species contrary to the process in vitro. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to implants of general properties for the human and animal bodies that are coated with a peptide that can selectively mediate the attachment of specific cells in the specific environment of the implant. In particular, the present invention relates to RGD peptide coated implants and methods for their preparation.

  The present invention specifically targets the surface coating of general biomaterials and implants for the purpose of promoting tissue selective enhanced integration after surgical insertion into the appropriate tissue. Based on the principle of cell type attachment stimulation.

  In this way, taking into account the specific tissue environment into which the implant is inserted, various surface portions of the implant can be coated and utilized with various peptides that mediate cell attachment, particularly RGD peptides. be able to.

  In this method, in addition, with respect to tissue processing, the generation of “informatized” biohybrid organs with biological information for organ regeneration can be achieved by different peptides that occupy different areas of the implant surface, and are specific to different cell types. It is also possible to use self-formation by a method of mechanical activation.

  Hereinafter, the term “peptide of the present invention” includes all peptides that can mediate cell attachment unless otherwise stated or appended. Among these, in particular, an arginine (R), glycine (G) and aspartic acid (D) amino acid sequence (RGD peptide) is considered. Examples of suitable RGD peptides as well as suitable peptides that do not include RGD will be further referred to later. In addition, similar peptides that do not contain the RGD sequence but nevertheless affect cell attachment are also included. In the broadest concept, the present invention also includes non-peptide compounds that qualitatively have the same biological activity as the peptide compound.

  In the context of the present invention, a biomaterial or implant is defined as a material that can be introduced into the human or animal body to restore the function of the corresponding naturally damaged tissue. These include, for example, hip internal prosthetic organs, artificial knee joints, jaw implants, alternative tendons, alternative skins, artificial blood vessels, cardiac pacemakers, artificial heart valves, chest implants, stents, catheters and shunts.

  Implant integrity in the body has been found to still have problems. Tissue integration of materials often proceeds too slowly and too incompletely to obtain sufficient mechanical stability of the tissue / biomaterial bond to perform its function. Due to its poor interfacial or biocompatibility, the composition of the implant surface that prevents active absorption of the surrounding healthy tissue or cells often causes this. This complicates the formation of a stable tissue-implant boundary layer and causes incomplete tissue integration, which turns around and relaxes, tissue reabsorption, infection, inflammation, allergies, microthrombosis ( Cause restenosis). As a result, corrective and new surgical interventions for replacement of implants (eg hip endoprostheses, jaw implants, catheters and extraosseous fixation) are required (Malchau and Herberts, 1996, Prognosis of the Total Hip Arthoplasty, Vol. 63. Annual Meeting of the American Academy of Orthopedic Surgeons, Atlanta; Haddad et al., 1996, The Journal of Bone and Joint Surgery, Vol. , Pin Tract Infections).

  Furthermore, especially in hip endoprostheses, so-called sterile implant relaxation occurs where bone cells and tissue do not form a direct bond to the biomaterial as desired, but instead fibroblasts and connective tissue occur as interfering elements. It turns out to be a problem. As a result, this prosthesis is associated with connective tissue instead of bone tissue, so that the stability of the prosthesis-connective tissue tissue bond is not sufficient to meet the mechanical requirements for force transmission at the hip prosthesis Is. Eventually, this leads to relaxation of the prosthesis (Pilliar et al., 1986, Clin. Orthop., 208: 108-113), which also requires corrective surgery. A further example of an undesirable cell type that attaches to implants is platelets, which also induce the formation of microthrombi and inhibition of implant integration (Phillips et al., 1991, Cell, 65, 359).

  The lack of ability to integrate biomaterials or implants into the body has a particularly serious impact in the case of fully replacement organs, as different cell types come into contact with the implant and the required integrity is targeted. In order to avoid extremely complicated transplantation procedures with the help of other patients, in the field of tissue processing, for example, so-called biohybrid organs consisting of carrier materials that are covered with living cells and can be transplanted as functional units With more and more attempts are being made to achieve treatment of functional disorders of the liver, pancreas, kidney and spleen. In most cases, for this purpose, functional healthy cells are included in or encapsulated in a resorbable or non-resorbable membrane in vitro and then delivered to the patient as an artificial biohybrid or virtual organ. Transplanted (eg: Lim et al., 1980, Science, 210, 908-912; Altman et al., 1982, Horm. Met. Res. Suppl., 12, 43-45; Zekorn et al., 1989 , Transplantation Proceedings, Vol. 21, 2748-2750; Altman et al., 1982, Horm. Met. Res. Suppl., Vol. 12, pp. 43-45: EP 0 504 781 B1). However, here too, problems such as fibrosis with a lack of nutrient supply to the graft, immune defense reaction due to cell release from the capsule, and thrombus formation due to blood clotting ability on the material surface are very frequent. To happen.

It is known to cause biointegration / implant tissue integration by coating it with a peptide that mediates cell attachment. To this end, on the one hand, a peptide comprising the tripeptide amino acid sequence arginine-glycine-aspartic acid (RGD), or a non-peptide analogue thereof, and on the other hand, a non-RGD-containing peptide that mediates cell attachment (e.g. Or non-peptide analogs thereof, as is known, are integral components of many proteins, especially extracellular matrices (eg, collagen type I, fibronectin, laminin, vitronectin, entactin, osteopontin, thrombosponge Or the blood coagulation cascade (fibrinogen, von Willebrand factor) functions as a central recognition pattern for eukaryotic cell attachment (eg: Pierschbacher and Ruoslahti, 1984, Nature, 309: 30) ~ 33; Yamada, 1991, J. Biol. Chem., 266: 12809-12812 page). The sequences defined by the present invention are recognized and bound by integrins, which are receptors on the cell surface. Since cell attachment to the corresponding proteins is mediated by a number of different integrins, the integrin expression pattern of the cell type is critical for the attachment properties to these proteins. Generation of most short-chain peptides with appropriate sequences that can selectively and specifically bind only to specific integrins—measurement design and synthesis targets and activates only those cell types that express these integrins Make it possible to do. That is, for example, it cannot simultaneously stimulate the attachment of unwanted cell types such as α _IIb β ₃ bearing platelets and selectively binds to α _v integrin receptors, ie α _v β ₃ / α _v β ₅ RGD peptides that can selectively stimulate the attachment (adhesion) of carrier cells (osteoblasts, osteoclasts, endothelial cells) are known (Haubner et al., 1996, Vol. 7, Am. Chem. Soc. 118: 7461). In contrast, other RGD peptides have the opposite effect and selectively bind to the α _IIb β ₃ integrin receptor, ie, for example, selectivity for platelets (Phillips et al., 1991, Cell # 1). 65, 359).

  In the present invention, it is known to attach a peptide obtained by synthesis to the surface of an implant. In this case, the peptide is more or less attached to the surface by adsorption or by covalent bonds. For example, DE 1 97 06 667 describes biomaterials for bone replacement members based on porous polymer materials having a surface coating of peptides having the RGD amino acid sequence by adsorption. WO 91-05036 further discloses a prosthesis made of metal, in particular titanium or a titanium alloy, in particular a peptide which may also have the RGD sequence is covalently bound to its surface. Valentini et al. (1997, May, Transactions of the 23rd Annual Meeting of the Society for Bomaterials, New Orleans, USA) describe the covalent attachment of RGD peptides to titanium screws with fluorinated ethylene propylene interlayers. Yes. Rezania et al. Reported a silicon dioxide or titanium dioxide surface coated with an amino-functional organosilane by covalent bonding at the same annual meeting, and eventually covalently bound the thiol-containing RGD peptide by a heterobifunctional crosslinker Has been achieved.

  However, these technical solutions address implants that are specifically coated on the surface with a peptide as defined by the present invention, prepared to selectively match the particular cell type of the tissue surrounding the implant in question. Or it does not answer the request to make biomaterials available.

  Therefore, after inserting the implant into the body, these tissues or cells that need to function actively with them, such as bone cells in hip endoprostheses or epithelial cells for skin, hair or tooth replacement Species are each arranged exclusively or selectively for their tissue integration and at the same time interfere with this process, such as platelets or fibroblasts that promote the formation of thrombus or connective tissue capsules, for example. It is desirable that the cell type can modify the biomaterial so that it is prevented from undergoing selective interaction with the implant.

  Furthermore, according to the present invention, which exclusively or at least preferentially stimulates the attachment of these selected cell types that retain the corresponding complementary integrin, resulting in the promotion of in vivo synthesis of the corresponding selected tissue. It is a desirable and attractive method to coat an implant with a defined peptide (or non-peptide analog thereof).

  With respect to the development of complete biohybrid organs (skin, blood vessels, urinary tract, bladder, esophagus, pancreas, liver, spleen, kidney), it is targeted and spatially limited so that different cellular in vivo processes can be performed. And, as desired, the various cell-selective peptides defined by the present invention activate the various cell types desired for each specific organ case by coating different surface parts of the implant. Getting is a decisive advance.

  The present invention now mainly stimulates the attachment of cell types that will complete the tissue integration of the corresponding biomaterial in each case, while at the same time cell types that are contrary to the process in the test tube. By coating with a synthetic, cell- or tissue-selective RGD peptide as defined by the present invention that does not well stimulate the attachment of all biomaterials for organs, especially implants Describe the possibility of adding biological functions. By using such a coating, an accelerated and improved integration of various biomaterials / implants can be achieved with improved long-term stability after insertion into the body.

  Furthermore, using this idea of coating different material surface parts of the implant with different peptides defined by the present invention, all possibilities appear in the development of “informatized” biohybrid organs (“tissue processing”). , It can carry biological information for the selective activation of various target tissues or target cells and can therefore be integrated into the body by self-organization, in this sense Can improve or even enable it for the first time.

  That is, the present invention essentially comprises a carrier matrix and a sequence that recognizes a binding site on an integrin receptor that governs attachment on human or animal cells, and the target human or animal somatic cell. An implant suitable for different human and animal organs comprising a peptide coating surrounding the matrix, comprising the same or different peptides for adhesion stimulation, wherein the carrier matrix is an appropriate functional reaction of the peptide layer Implants into cells derived from cell attachment stimulating activity, which has a reactive group capable of binding on the surface, capable of forming a stable covalent bond with the functional group and related to its corresponding different structure In the specific region in which they are to be inserted, the pattern and specificity of the natural different complementary integrins of the tissue cells adjacent to them In which the peptide is locally distributed in different ways on the surface of the implant so that a locally differentiated, selective, biologically active coating pattern of the implant surface exists. It is related with the implant characterized by being.

  The present invention further provides an organ / tissue based on an inorganic carrier matrix having a surface coated with a cell adhesion stimulating peptide that is selective for the complementary integrin pattern of tissue cells immediately adjacent to the implant. A method for preparing a suitable implant, known per se, (i) determining in vitro integrin receptor structures of target cells or target tissues into which the implant is introduced in vivo; (ii) It relates to a method comprising selecting or synthesizing a peptide having an appropriate complementary structure, and (iii) binding the peptide to an appropriate surface of an implant.

In particular, the invention relates to methods and implants / biomaterials having the following characteristics: said peptides, in particular RGD peptides, are optionally shared via branched, surface-enhancing molecules and / or molecular anchors. Is attached to the implant surface by binding; preferably it can stimulate α _v β ₃ / α _v β ₅ bearing cells, ie, in particular, stimulates the attachment of eg osteoblasts, osteoclasts, endothelial cells RGD peptides are used that can, but cannot stimulate the attachment of platelets or fibroblasts at the same time; the carrier matrix used can be ceramic, polymeric material or metal shaped or non-shaped sites, or biohybrid organs or It is a virtual organ.

At the molecular level, the peptides defined by the present invention are essentially designed from the following elements:
An amino acid sequence suitable for attachment that selectively recognizes and binds to a selected cell type (eg, the RGD sequence) carrying domain;
A spacer for presenting cell recognition and recognition sequence-carrying domains to cells so that cell binding is possible only from a steric point of view,
A molecular anchor for stable binding of the peptide derivative to the biomaterial or implant surface,
-Prior to binding to the surface of the biomaterial, cell attachment is arbitrarily increased by binding the peptide defined by the present invention to a branched molecular structure (so-called dendrimer or tentakel) that exerts an effect of expanding the surface area. Can be made.

  The surface of the biomaterial or implant is to be understood in the context of the present invention not only as a direct surface of the carrier matrix, but also as a further coating which may be, for example, a polymer material, natural or artificial bone material, protein or protein derivative. It is.

  Suitable carrier matrices are in particular members made of ceramic, metal, polymeric material (eg PMMA) or preferably resorbable bone replacement material. Recovery of the initial tissue state can be achieved, for example resorbable or biodegradable materials consisting of polylactides, in particular racemic D, L polylactide compounds or resorbable calcium phosphate or hydroxyapatite mixtures, and for example WO 96 / Particularly suitable are materials as disclosed in 36562 or EP 0 543 765. Depending on the field of use, collagen or agar may also be suitable as a carrier matrix.

  The term “biohybrid organ” is understood to mean a normal inorganic matrix to which biological cells have been added or bound in any way (see above). In the present invention, this does not include cells or includes only different types of corresponding peptides defined by the present invention on different implant surface portions, and selectively inserts surrounding cells when inserted into a defective tissue. It is also understood to mean the corresponding sequence that can be activated. The advantage of such cellular biohybrid organs is that “informatized” biocompatible implants that can be manufactured in a cost-effective and controllable manner carry biological information for organ regeneration It is. Secondly, the integration of such biohybrid organs into the body, eg, self-reliant by an endogenous regenerative process that can avoid immune defense reactions that frequently occur due to transplanted foreign cells or foreign proteins. Completed by configuration.

  In accordance with the present invention, the implant generally takes the form of a shaped body or prosthesis, where the shaped body is prepared to fit a particular tissue / bone defect. In the case of a biohybrid organ, the prosthesis is a membrane or coating coated with or without corresponding cells, and a peptide as defined by the present invention, as well as others such as disclosed in eg EP 0 504 781 It can only be configured by an array.

  Suitable peptides that can be used in the present invention include all peptides and non-peptides that contain regions or amino acid sequences that govern cell attachment and that can be bound to the implant surface via peptide and non-peptide substitutes. It is a compound having a peptide substitute. In particular, possible corresponding peptides are those having the RGD sequence.

The following list of preferred peptides and peptide compounds is intended to be merely exemplary but not of any limiting nature, and the following abbreviations are used:
AsP (D) = aspartic acid
Gly (G) = Glycine
Arg (R) ＝ Arginine
Tyr (Y) = Tyrosine
Ser (S) = Serine
Phe (F) = Phenylalanine
Lys (K) = Lysine
DPhe (f) = D-phenylalanine
Pro (P) = Proline
Leu (L) = Leucine
Ile (I) = Isoleucine
Val (I) = Valine
Glu (E) = glutamic acid
Thre (T) = Threonine
Ala (A) = Alanine

(A) Examples of suitable RGD-containing peptides:
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),
Cyclo-RGDfK (Arg-Gly-Asp-DPhe-Lysine),
Cyclo-RGDfKG (Arg-Gly-Asp-DPhe-Lys-Gly).

(B) Examples of suitable non-RGD containing peptides:
LDV (Leu-Asp-Val),
LGTIPG (Leu-Glu-Thr-Ile-Pro-Gly),
REDV (Arg-Glu-Asp-Val),
IKVAV (Ile-Lys-Val-Ala-Val),
YIGSRG (Tyr-Ile-Gly-Ser-Arg-Gly),
LRE (Leu-Arg-Glu),
PDSGR (Pro-Asp-Ser-Gly-Arg),
DGEA (Asp-Gly-Glu-Ala),
RYVVLPR (Arg-Tyr-Val-Val-Leu-Pro-Arg).

  The peptides defined by the present invention may be either linear or cyclic. The peptides and peptide sequences described above may be present in longer peptides having a total of about 4-20 amino acids, depending on the peptide of the invention selected. Similarly, amino acids that adopt the D or L configuration or are C- and / or N-alkylated are also included in the invention. According to the invention, a cyclic peptide is taken to mean a peptide that closes to form a ring via an amide bond and preferably has no free carboxyl or amino group in the molecule. In the present invention, RGD peptides, particularly those selected from the above list, are particularly preferred, and among these, a cyclic one is disclosed in DE-A-1 95 38 741, and osteoblasts The pentapeptide RGDfK, which is specific for, and the hexapeptide RGDfKG, which also exists in its cyclic state and is specific for platelets, are preferred.

Corresponding linear and cyclic peptides defined by the present invention are disclosed, for example, in the following patent applications: EP 0 632 053, EP 0 655 462, EP 0 578 083, EP 0 770 622, DE 1 95 38 741 ing. In particular, although it selectively binds to α _v β ₃ / α _v β ₅ integrin-expressing cell types (eg, osteoblasts, osteoclasts, endothelial cells), for example, α _IIb β ₃ carrying cell types (eg, Peptides that do not bind simultaneously to platelets) are preferred. Peptides and corresponding derivatives can be readily synthesized by standard methods if not otherwise obtained.

  In principle, the peptides defined by the present invention can be attached to the surface of a biomaterial by adsorption or covalent bonding. The adsorption method can be applied to a plurality of different peptides for one and the same implant, as the locally selective different coating of the surface in the present invention can only be done in an almost unsatisfactory manner using this technique. Is not very suitable.

  The binding of peptides or their non-peptide analogues to the carrier surface, mainly by covalent bonds using so-called molecular anchors, is well known per se and is described, for example, by Singer et al. (1987, J. Cell. Biol., 104, 573); Brandley, Schnaar (1989, Develop. Biol., 135, 74); Massia, Hubbell (1990, Anal. Biochem., 187) Hirano et al. (1991, J. Biomed. Mat. Res., 25: 1523); Lin et al. (1992, Biomaterials, 13: 905); Nicol et al. (1992, J. Biomed. Mat. Res., 26: 393); Dee et al. (1995, Tissue Engin., 1: 135), but in this case, the covering of the implant is common. In particular, any method can be used.

Furthermore, the present invention is, for example, the peptide to the surface containing the acryloyl or methacryloyl anchor component (or its non-peptide analogs) for binding, were applied beginning in the present invention, "Kevloc ^(R)" process ( EP 0 712 621) or the corresponding peptide to the corresponding carrier matrix using an acryloyl or methacryloyl anchor component, generally via an acryloyl / methacryloylsilane derivative intermediate layer (eg 3-methacryloxypropyltrimethoxysilane) of the binding, as in the used in the present invention "Silicoater ^(R)" process (DE-a 42 25 106) , for an implant preparation according to the invention, is per se novel use of known coating methods . A further possibility of attaching the peptides defined by the present invention to the surface of a carrier matrix or implant originally explains the technical teaching for pearlescent pigment coatings in water-based coating systems for metals and plastics in the automotive and plastics industry. It constitutes a use similar to the silanization process described in DE-A 43 21005. Furthermore, a method of coating a gold surface with a thiol group-carrying peptide, which was originally described in another relationship (Heuvel et al., 1993, Analytical Biochem, Vol. 215: 223), is also suitable for the present invention.

  The method outlined has never been used to coat implants intended for biological activation.

  The binding of the corresponding peptide defined by the present invention to the implant surface takes place in the present invention via a suitable anchor molecule, i.e. the peptide is generally not directly attached to the implant surface. The insertion of such molecules, as defined in more detail below, has as a point to consider the steric requirements of the biological receptor on the target cell, especially in relation to the binding of the corresponding peptide.

  For this purpose, the implant surface must carry suitable functional groups or reactive units that allow binding with the corresponding functional group of the anchor molecule. The functional groups that are made available on the implant surface depend on the actual carrier matrix composition (metal, plastic, bone material) that changes as needed. In the case of metal implants, it is possible to produce a surface layer reactive to the SH groups of the anchor molecules, for example by depositing gold. Silanization of metal surfaces by known methods (see above) also forms compounds with suitable anchor molecules according to the invention, using silane-containing adhesion promoters (see below) where appropriate. To a reactive surface that can Implants made of natural bone or natural-like bone material (eg calcium phosphate cement) can be combined with anchor molecules containing reactive phosphonic acid groups (Principle: Chu, Orgel, 1997, Bioconjugates Chem., Vol. 8) : Described on page 103). Also, as part of itself, anchor molecules according to the invention having reactive acrylic acid groups are attached to implants made of acrylate-based plastics (eg PMMA) or other materials with a suitable plastic coating. be able to.

Thus, anchor molecules meaning of the invention, at least two different functional groups, i.e., usually a peptide defined by the present invention, particularly the free NH ₂ groups in the side chain of RGD peptides (free carboxyl group) One functional group that is a free carboxyl group (free NH ₂ group) that generates an amide bond (—CO—NH—), and preferably localized to the other end of the carbon chain of the anchor molecule, Depending on the composition or requirements, a modified or substituted alkyl chain or carbonized with other functional groups, preferably (meth) acrylate-containing groups or mercapto groups, which directly or indirectly bond to the implant surface A molecule based on hydrogen chains. In principle, it is also possible to use other functional groups that can react directly with the corresponding reactive groups on the implant surface or on suitable intermediate layers to give stable bonds.

  As already mentioned above, the anchor molecule of the present invention has the function of a spacer at the same time, i.e., in addition to what has been outlined, the selection of the ligation can improve cell binding or is steric. As may be possible from a point of view, the domain that governs cell adhesion stimulation is appropriately selected and has a specifically regulated length to allow the appropriate distance from the target cell.

Cell recognition and biological function of the domain that retains the corresponding amino acid sequence can be selected, for example, from α _v β ₃ / α _v β ₅ integrin-expressing cell types (eg, osteoblasts, osteoclasts, endothelial cells) Was confirmed by a synthetic peptide (Haubner et al., 1996, J. Am. Chem. Soc., 118: 7461-7472).

The anchor molecule of the present invention preferably has the following linear structure, and the peptide defined by the present invention corresponds to the corresponding anchor via one of its amino acid side chains, preferably the NH ₂ group of the lysine side chain. Bound to the free carboxyl terminus of the molecule.
(I) Mercapto (amide) carboxylic acid derivative:
-CO- (CH ₂ ) _k -X-SH,
Where X is a single bond or -CO-NH- (CH ₂ ) _l-
k = 2-12 and l = 2-4
(Ii) Acrylamide carboxylic acid derivative:
-CO- (CH ₂ ) _m- [NH-CO- (CH ₂ ) _n ] _p -NH-CO-CH = CH ₂ ,
Where m, n = 2-8; p = 0-2,
(Iii) Acrylamide-amide triethylene glycolic acid derivative:
-(CO-CH ₂ -O-CH ₂ -CH ₂ -O-CH ₂ -CH ₂ -NH) _q -CO- (CH ₂ ) _r -NH-CO-CH = CH ₂ ,
Where q = 1-3 and r = 2-8.

In particular, the following types of specific anchor molecules are preferred.
(Ia) -CO-CH ₂ -CH ₂ -SH (mercaptopropionic acid)
(Ib) -CO-CH ₂ -CH ₂ -CO-NH-CH ₂ -CH ₂ -SH (mercaptoethylamide succinic acid)
(Iia) -CO- (CH ₂ ) ₅ -NH-CO-CH = CH ₂ (Acrylamide hexanoic acid)
_{(Iib) -CO- (CH 2)} 5 -NH-CO- (CH 2) 5 -NH-CO-CH = CH 2 ( acrylamido hexanoic acid - amide hexanoic acid)
_{(Iiia) -CO-CH 2 -O} -CH 2 -CH 2 -O-CH 2 -CH 2 -NH-CO- (CH 2) 5 -NH-CO-CH = CH 2 ( acrylamido hexanoic acid - Amidotori Ethylene glycolic acid)
(Iiib) - (CO-CH 2 -O-CH 2 -CH 2 -O-CH 2 -CH 2 -NH) 2 -CO- (CH 2) 5 -NH-CO-CH = CH 2 ( acrylamide hexanoic acid -Diamide triethylene glycolic acid)
Usually, according to the present invention, any anchor molecular structure having at least 6 carbons in a linear carbon chain is preferred. In fact, it has surprisingly been found that anchor molecules of this length are particularly preferred in order to achieve the best results with respect to the promoted and enhanced tissue integration of the implant. The notation of at least 6 carbon atoms in the linear chain is in the present invention the total length of the molecule between the peptide and the implant surface. That is, anchor molecules of the structure shown above with shorter chains (eg, type ia) are also suitable, unless otherwise specified, a chain-extending binding component is inserted between the peptide and the implant surface.

The anchor molecule is linked to the peptide defined by the present invention in the form of an amide via a carboxyl functional group by standard methods, but by this method the structure of the peptide-NH-CO-anchor type molecule And, as indicated, attaches to the implant, thereby also building the following type of product: peptide-NH-CO-anchor molecule-implant (surface). Preference is given to a corresponding implant structure consisting of one of the above-mentioned definition peptides, in particular one of the RGD peptides, one of the general and specifically defined anchor molecules individually mentioned above, and a suitable surface-reactive implant. The following implants are particularly preferred:
Cyclo-RGDfK NH-CO-thiol derivative (type: i) -implant,
Cyclo-RGDfK NH-CO-acrylate derivative (type: ii) -implant,
Cyclo-RGDfK NH-CO-acrylate-glycol derivative (type: iii) -implant,
Cyclo-RGDfKG NH-CO-acrylate-glycol derivative (type: iii) -implant,
Here, the linear carbon chain of all anchor molecules has at least 6 atoms.

Particularly preferred among these are:
Cyclo-RGDfK NH-CO-thiol derivative (type: ib) -implant,
Cyclo-RGDfK NH-CO-acrylate derivative (type: iia) -implant,
Cyclo-RGDfK NH-CO-acrylate derivative (type: iib) -implant,
Cyclo-RGDfK NH-CO-acrylate-glycol derivative (type: iiia) -implant,
Cyclo-RGDfKG NH-CO-acrylate-glycol derivative (type: iiia) -implant,
Cyclo-RGDfK NH-CO-acrylate-glycol derivative (type: iiib) -implant.

  The preparation of these preferred structures is carried out by standard methods, or further described below, or in Applicant's co-filed parallel applications relating to such peptide anchor structures.

As already explained in detail above, there are essentially three alternative pathways for tethering the described cell or tissue-selective peptide derivatives to the surface of the biomaterial in the present invention, each RGD sequence being The molecular recognition pattern of the retained domain can be maintained unchanged selectively for certain cell types and spacers, while molecular anchors can change, for example, depending on the binding deformation described below :
Binding of a thiol peptide derivative to a gold-coated biomaterial surface (eg, type (i) anchor molecule);
The binding of (meth) acryloyl peptide derivatives to acrylate or methacrylate coated biomaterials (eg type (ii) or (iii) anchor molecules);
A silane-coated biomaterial surface (eg type (ii) or (iii) anchor molecule) using (meth) acryloylsilane derivatives (eg 3-methacryloxypropyltrimethyloxysilane) as adhesion promoter or intermediate layer Binding of (meth) acryloyl peptide derivatives to

For various peptide bond deformations to the biomaterial surface, the determination of the critical minimum length of the anchor molecule preferably exhibits hydrophobic / hydrophilic properties that are alternatively different from the chain length of 6 to 24 carbon atoms. together with the anchor molecules defined by the present invention having, carried out by synthesizing a peptide defined by the present invention (e.g., by itself standard methods, different number of -CH ₂ - units and / or amide hexanoate or ethylene By using glycols, followed by testing of biological activity by determining cell attachment in vitro after coating of the appropriate biomaterial).

In the foregoing method, depending on the material properties of the implant, a suitable coating method can be selected to condition the surface before actual binding with the peptide derivative according to the invention. In addition, depending on the tissue type or cell type intended to achieve biomaterial / implant integration, for example, α ₆ β ₄ integrin from epithelial cells (eg bone, jaw, skin or hair implant applications) Or coating of other peptides that activate the integrin of the corresponding target cell type, such as α _IIb β ₃ integrin from platelets. α _v β ₃ specific RGD peptides have selectivity for endothelial cells and osteoblasts, resulting in, for example, suitable for coating artificial blood vessels or bone implants. This makes it possible to realize a living surface coating suitable for almost any desired organ for implants in the field of bone, blood vessels, teeth, skin and hair replacement.

  Before a suitable implant or biohybrid organ according to the present invention is made available, a specific cell type can be applied so that a specific and selective coating of the implant to be inserted into the selected tissue can be performed later. Suitable peptides must be pre-tested and determined for biological activity in an in vitro test system.

  The analysis of the integrin receptor structure of the target tissue or target cell into which the implant is to be inserted, which is necessary for this purpose, can be performed using conventional, known immunohistochemistry, such as eg immunofluorescence or immunohistochemistry of tissue samples. Implement by process. The antibodies against the various integrin receptors or subunits thereof necessary for this purpose are known and available on the one hand, or are known per se, for example suitable immunizations. It can be suitably manufactured by various methods.

  The peptides defined by the present invention are covalently bound at various concentrations, for example, to the surface of polystyrene media coated with bovine serum albumin (BSA). This test carrier material does not play any essential role in determining a suitable peptide. Similarly, the bonding method used here is also of little importance. For practical reasons, the coupling in these determinations can also be effected by incubation and adsorption of the peptide on a test carrier.

Subsequently, an appropriately coated surface for attachment of selected tissue cell cultures (eg, osteoblasts) whose attachment characteristics can correspond to cells in the natural tissue to be activated in vivo. Find out above. Criteria for selecting an appropriate cell culture for the adhesion test are, for example, its integrated expression comparable to target cells in vivo after transplantation, such as its α _v β ₃ / α _v β ₅ or α _IIb β ₃ expression It is a fluorescently labeled antibody against α _v β ₃ , α _v β ₅ or α _IIb β ₃ integrin, or against the α _v , α _IIb , β ₃ or β ₅ subunit of the integrin receptor By using a fluorescence activated cell sorter (FACS). In the case of other target cell types in vivo with different integrin receptor patterns, other antibodies must therefore be used. These are known and available on the one hand, or can be produced by known standard methods, for example by appropriate immunization.

  Various selected cell types are inoculated and incubated on the BSA pretreated polystyrene media surface coated with the peptide under consideration. Thereafter, non-adherent cells are washed away.

  If the binding properties of the different selected cell types on the test surface coated with different peptides as defined by the present invention are positive, i.e. there is an appropriate specificity, in each case about 60 of the inoculated cells. Corresponding to an infection curve with a maximum adsorption ratio of ˜100%, and at a concentration of RGD peptide in the coating solution of about 5 nM to 5 μM, half of the maximum number of cells binds.

Similarly, various adhesion-promoting interlayers can be used for immobilization on a modified or modified biomaterial surface, as described in Binding appropriate peptides to BSA pre-coated polystyrene surfaces. The following can be stated as:

Prior art implants have the following disadvantages:
Integration of incomplete and slow implants into the tissue,
・ Limited tolerance within the organization,
Improper functional stability of the implant / tissue boundary layer,
Lack of stimulatory action of the implant on tissue formation,
• Non-physiological properties of the implant surface.

The consequences of this further problem are:
Aseptic implant relaxation (eg, fibrous capsule formation),
・ Local formation of microthrombus,
·infection,
·inflammation,
・ Tissue resorption,
-Corrective surgery.

These problems are largely eliminated by the method that can be used according to the present invention or the implant produced thereby. The subject of the present invention is characterized as follows:
Generation of adherent peptides that are complementary to the integrin expression pattern of the target tissue / target cell—measurement design;
Selective stimulation of cell attachment of target cells to achieve tissue formation without simultaneously causing cell attachment that inhibits the process;
More stable coating with new peptide anchor molecules;
-Promote and improve the process of implant integration into tissue.

  It could be shown that the critical steric absolute minimum distance between the cell recognition sequence on the peptide and the surface of the uncoated material is between 2.0 and 3.5 nm, preferably between 2.5 and 3.5 nm. Maximum coverage (80-100%) can be achieved at minimum distances of 3.0-5.0 nm.

BRIEF DESCRIPTION OF THE FIGURES: FIG. 1: Analysis of integrin composition of primary human osteoblasts by FACS (fluorescence activated cell sorter) using fluorescent conjugated antibodies against α _v β ₃ and α _v β ₅ integrins.

x-axis: fluorescence intensity (count)
y-axis: cell count
M21: α _v β ₃ and α _v β ₅ positive control
M21: α _v β ₃ and α _v β ₅ positive control
HOB: Primary human osteoblast culture
ROB: Primary rat osteoblast culture
Figure 2: Attachment of MC3T3H1 osteoblasts to polystyrene test surfaces coated with various RGD peptides via BSA.

x-axis: concentration of RGD peptide in coating solution (μM)
y axis: Cell coverage (%)
Upper curve: cyclo-RGDfK NH—CO—CH ₂ —CH ₂ —S— (sulfo SMPB)
( “Thiol peptide 1-SMPB derivative” ), SMPB = succinimidyl 4- (p-maleimidophenyl) butyric acid;
Nakagami Curve: cyclo _{-RGDfK NH-CO-CH 2 -CH} 2 -CO-NH-CH 2 -CH 2 -S- ( sulfo SMPB) ( "thiol peptide 2-SMPB derivative"),
Under Medium Curve: cyclo _{-RGDVE CO-NH-CH 2 -CH} 2 -CH 2 -CH 2 -CH 2 -CO-NH-CH 2 -CH 2 -S- ( sulfo SMPB) ( "thiol peptide 3-SMPB derivative " ),
Lower curve: Thiol peptide Target: Cyclo-R βA DfK-NH-CO—CH ₂ —CH ₂ —S— (sulfo SMPB)
Figure 3: Osteoblast attachment to the test surface coated with the thiol peptide 1-SMPB derivative (see footnote in Figure 2) x-axis: peptide concentration in coating solution (μM) y-axis: cell coverage (%) Top Curve: MC3T3 H1 mouse osteoblasts Middle upper curve: primary human bone ancestor cells Middle curve: primary human osteoblasts Middle lower curve: α _v β ₃ negative control cell M21L Lower curve: primary rat osteoblasts Figure 4: Osteoblast attachment to test surface coated with thiol peptide 1-SMPP derivative (10 μM in coating solution) (see Figure 2) x-axis: dissolved cyclo-RGDfK (no molecular anchor) in the attachment medium Concentration (μM) y-axis: Cell coverage (%) Upper curve: MC3T3 H1 mouse osteoblast Middle curve: primary human osteoblast Lower curve: primary rat osteoblast Figure 5: Osteoblast adhesion to test surface coated with thiol peptide 1-SMPB derivative (10 μM in coating solution) (see Figure 2) x axis: number of inoculated cells / cm ² y axis: number of adherent cells / cm ² Upper curve: Primary rat osteoblast Middle curve: MC3T3 H1 mouse osteoblast Middle lower curve: Primary human osteoblast Lower curve: BSA negative coating FIG. 6: Adhesion of osteoblasts to the test surface coated with the thiol peptide 1-SMPB derivative (10 μM in coating solution) (see FIG. 2) x axis: number of inoculated cells / cm ² y axis: calculated required surface area / Cells (μm ² ) Upper curve: primary human osteoblasts Middle curve: MC3T3 H1 mouse osteoblasts Lower curve: primary rat osteoblasts Fig. 7: Adhesion of osteoblasts to the test surface coated with thiol peptide 1-SMPB derivative (10 µM in coating solution) (see Fig. 2) x-axis: time (min) y-axis: cell coverage (%) Upper side Curve: MC3T3 H1 mouse osteoblasts Middle curve: primary human osteoblasts Lower curve: primary rat osteoblasts Figure 8: Bone cement coated with various RGD peptides-MC3T3 H1 osteoblast adhesion to PMMA surface: x axis: concentration of RGD peptide in coating solution (μM) y axis: cell coverage (%) Upper curve: Cyclo-RGDfK NH-CO-acrylate derivative (type: iiia, acrylate peptide 3), Upper curve: Cyclo-RGDfK NH-CO-acrylate derivative (type: iib, acrylate peptide 2), Lower curve: Cyclo-RGDfK NH -CO-acrylate derivative (type: iiib, acrylate peptide 4), lower curve: cyclo-RGDfK NH-CO-acrylate derivative (type: iia, acrylate peptide 1), Figure 9: Attachment of MC3T3 H1 osteoblasts to porous PMMA / PHEMA surfaces coated with various RGD peptides: x-axis: concentration of RGD peptide in coating solution (μM) y-axis: cell coverage (%) Upper curve : Cyclo-RGDfK NH-CO-acrylate derivative (type: iiia, acrylate peptide 3), middle curve: cyclo-RGDfK NH-CO-acrylate derivative (type: iiib, acrylate peptide 4), lower curve: cyclo-RGDfK NH -CO-acrylate derivative (type: iib, acrylate peptide 2), Figure 10: MC3T3 H1 osteoblast attachment to porous PMMA / Plex Y7H surfaces coated with various RGD peptides: x-axis: RGD peptide concentration in coating solution (μM) y-axis: cell coverage (%) Curve: cyclo-RGDfK NH-CO-acrylate derivative (type: iiia, acrylate peptide 3) Middle curve: cyclo-RGDfK NH-CO-acrylate derivative (type: iiib, acrylate peptide 4), lower curve: cyclo-RGDfK NH-CO-acrylate derivative (type: iib, acrylate peptide 2), Figure 11: Maximum adhesion of MC3T3 H1 osteoblasts to bone cement-PMMA or polystyrene-BSA-SMPB surfaces coated with various RGD peptides of various molecular lengths: x-axis: RGD peptide molecular length (nm) y-axis: Maximum coverage (%) Figure 12: Attachment of MC3T3 H1 osteoblasts to a V4A stainless steel surface coated with RGD peptide cyclo-RGDfK NH-CO-acrylate derivative (type iiia, acrylate peptide 3). The stainless steel surface was pre-coated on the part in various ways.

x-axis: RGD peptide concentration in the coating solution (μM)
y axis: Cell coverage (%)
Upper curve: Kevloc ^(R) coating,
Medium curve: Silicoater ^(R) coating,
Lower curve: pigment coating,
Figure 13: Proliferation of MC3T3 H1 osteoblasts on bone cement-PMMA surface coated with RGD peptide cyclo-RGDfK NH-CO-acrylate derivative (type iiia, acrylate peptide 3).

x-axis: culture period (days)
y-axis: cell count Upper curve: 100 μM peptide in coating solution,
Middle top curve: 1 μM peptide in coating solution,
Middle lower curve: 0.1 μM peptide in coating solution,
Lower curve: uncoated control.
Figure 14: Attachment of MC3T3 H1 osteoblasts and platelets to bone cement-PMMA surface coated with RGD peptide cyclo-RGDfK NH-CO-acrylate derivative (type iiia, acrylate peptide 3).

x-axis: osteoblasts alone and platelets alone seeded on surfaces with peptide concentrations in various coating solutions of 100 μM, 10 μM and 1 μM y-axis: cell count (absorption at 405 nm)
Figure 15: Attachment of MC3T3 H1 osteoblasts and platelets to bone cement-PMMA surface coated with RGD peptide cyclo-RGDfKG NH-CO-acrylate derivative (type iiia, acrylate peptide 5).

x-axis: osteoblasts alone and platelets alone seeded on surfaces with peptide concentrations in various coating solutions of 100 μM, 10 μM and 1 μM y-axis: cell count (absorption at 405 nm)
Figure 16: Adhesion of MC3T3 H1 osteoblasts, platelets and osteoblast / platelet mixture to bone cement-PMMA surface coated with RGD peptide cyclo-RGDfK NH-CO-acrylate derivative (type iiia, acrylate peptide 3).

x-axis: osteoblasts alone, platelets alone and osteoblast / platelet mixture seeded on the surface with peptide concentration in 100 μM coating solution y-axis: cell count (absorption at 405 nm)
Figure 17: Adhesion of MC3T3 H1 osteoblasts, platelets and osteoblast / platelet mixture to bone cement-PMMA surface coated with RGD peptide cyclo-RGDfKG NH-CO-acrylate derivative (type iiia, acrylate peptide 5).

x-axis: osteoblasts alone, platelets alone and osteoblast / platelet mixture seeded on the surface with peptide concentration in 100 μM coating solution y-axis: cell count (absorption at 405 nm)

  The following examples further illustrate the invention but do not limit the invention.

Example 1:
(A) Synthesis of cyclo-RGD peptide (Arg-Gly-Asp-DPhe-Lys) having an anchor mercaptopropionic acid (→ cyclo-RGDfK NH-CO-thiol derivative type: ia) by the method of DE 1 95 38 741 Went according to. The synthesis of biologically inactive corresponding cyclo-R βA DfK derivatives as well as cyclo-RGDfK derivatives without anchor molecules was carried out as well.

  (B) Cyclo (R (Pbf) GD (tBu) fK (Z)) (Pbf = pentamethylbenzofuransulfonyl; tBU = tert-butyl) was synthesized by solid phase peptide synthesis (Merrifield, Angew. Chem. 1985, 97th). Volume: 801) and subsequent cyclization (eg, according to Zimmer et al., 1993, Leibigs Ann. Chem: 497). After selective removal of the Z-protecting group by standard methods, cyclo (R (Pbf) GD is obtained in 5 ml of dimethylformamide to give cyclo (R (Pbf) GD (tBu) fp [sa-K]). The lysine side chain can be extended by reaction with (tBu) fK) 0.1 mmol succinic anhydride (sa) 0.2 mmol.

(C) Synthesis of cyclo-RGD peptide (Arg-Gly-Asp-DPhe-Lys) having anchor mercaptoethylamide succinic acid (→ cyclo-RGDfK NH-CO-thiol derivative type: ib = cyclo (RGDf [thiol-sa -K]) was carried out as follows: 10 mmol of cysteamine hydrochloride and equimolar amount (eq.) Of triphenylmethanol were dissolved in glacial acetic acid at 60 ° C. and treated with 1.1 equivalents of BF ₃ etherate with stirring. After stirring for 50 minutes, the mixture was neutralized with saturated NaHCO ₃ solution and extracted with ethyl acetate, the residual oil from the extract was dissolved in tert-butanol, brought to pH = 2 with dilute hydrochloric acid to the hydrochloride salt. The resulting structural unit was converted to cyclo (R (Pbf) GD (tBu) fp [sa-K]) using EDClxHCl by standard methods. And removes the side chain protecting group.

(D) Synthesis of cyclo-RGD peptide (Arg-Gly-Asp-DPhe-Lys) having anchor acrylamide hexanoic acid (→ cyclo-RGDfK NH-CO-acrylic acid derivative type: iia = cyclo (RGDf [acryl-ahx- K]) was performed as follows:
An acrylic anchor system was synthesized separately and attached to the peptide side chain as an active ester. To that end, 10 mmol of 6-aminohexanoic acid and 1.8 equivalents of calcium hydroxide were suspended in water and 1.2 equivalents of acryloyl chloride were added at 0 ° C. Insoluble calcium hydroxide was removed by filtration, and the filtrate was acidified to pH 2 using concentrated hydrochloric acid. The precipitated product was recrystallized from ethyl acetate. Yield: 65%. 10 mmol of crystalline 6-acrylamidohexanoic acid was suspended in 50 ml of dichloromethane, treated with 1 equivalent of N-hydroxysuccinimide, and 1.2 equivalent of EDClxHCl was added at 0 ° C. After stirring for 1 hour, 10 μl of glacial acetic acid was added to stop the reaction. This was extracted several times with ice, saturated sodium carbonate solution and water, and the extract was dried over sodium sulfate. Yield: 52%. The active ester present here was coupled to cyclo (R (Pbf) GD (tBu) fK) in DMF and the side chain protecting group was removed by standard procedures.

(E) Synthesis of cyclo-RGD peptide (Arg-Gly-Asp-DPhe-Lys) having anchor acrylamide hexanoic acid-amido hexanoic acid (→ cyclo-RGDfK NH-CO-acrylic acid derivative type: iib = cyclo (RGDf [ Acrylic-ahx-ahx-K]) was performed as follows:
For the synthesis of the acrylic anchor system, 10 mmol of aminohexanoic acid was dissolved in aqueous sodium phosphate buffer (pH 8) and cooled to 0 ° C. 2 mmol of acrylamide hexanoic acid active ester (see (d)) was dissolved in ethanol / CHCl ₃ and added slowly. The pH was maintained at 8 using dilute NaOH. After distilling off the organic solvent, the aqueous phase was acidified to pH 2.6 using concentrated hydrochloric acid, the precipitated solid was filtered off, washed with water and dried over phosphorus pentoxide. Yield: 70%. The acrylic anchor system present here is attached to the peptide side chain using EDClxHCl by known methods to remove the protecting group.

(F) Synthesis of cyclo-RGD peptide (Arg-Gly-Asp-DPhe-Lys) having anchor acrylamide hexanoic acid-amido triethylene glycolic acid (→ cyclo-RGDfK NH-CO-acrylic acid derivative type: iiia = cyclo ( RGDf [acrylic-ahx-tEG-K]) was performed as follows:
An acrylic anchor system was prepared by solid phase peptide synthesis (see above). Acrylamide hexanoic acid (see above) was attached as a terminal component. Yield: 97%. The acrylic anchor system present here is attached to the peptide side chain using EDClxHCl by known methods to remove the protecting group.

  (G) Synthesis of cyclo-RGD peptide (Arg-Gly-Asp-DPhe-Lys) having anchor acrylamide hexanoic acid-amido triethylene glycolic acid-amido triethylene glycolic acid (→ cyclo-RGDfK NH-CO-acrylic acid derivative) Type: iiib = cyclo (RGDf [acryl-ahx-tEG-tEGK]) was performed as in (f) Yield: 85%.

  (I) Synthesis of cyclo-RGD peptide (Arg-Gly-Asp-DPhe-Lys) having anchor acrylamide hexanoic acid-amido triethylene glycolic acid (→ cyclo-RGDfK NH-CO-acrylic acid derivative type: iiia = cyclo ( RGDf [acrylic-ahx-tEG-KG]) was carried out as in (f) Yield: 81%.

Example 2:
Covalent bond formation of peptides according to the present invention using sulfosuccinimidyl 4- (p-maleimidophenyl) butyric acid on a polystyrene medium surface pre-coated with bovine serum albumin (BSA) by Singer et al., 1987 , J. Cell. Biol., 104: 573 or Ruoslahti et al. (1982, Methods. Enzymol., 82: 803-831).

Therefore, in order to generate a BSA layer on polystyrene, 48-well plates (Costar, “Non-tissue medium treatment”, Art. No. 3547), 250 μl of PBS (phosphate buffered saline), pH, per well = 8.3, coated with 2% BSA and incubated overnight at room temperature. The plates were then washed with 250 μl PBS per well, pH = 8.3 and incubated with 250 μl each of PBS, pH 8.3 solution containing 100 μg / ml SMPB for 1 hour at room temperature. Next, the wells were washed three times with 250 μl of PBS, pH 8.3 each time. For the preparation of coating solutions, specific thiol-RGD peptides were prepared in PBS, pH 8.3 to the following final concentrations: 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM and 1 mM. After adding 250 μl of each coating solution per well, the plates were incubated overnight at room temperature and then washed three times with PBS, pH 8.3. Nonspecific cell binding sites were blocked by adding 250 μl each of a 5% solution containing BSA in PBS, pH 7.4, followed by incubation at room temperature for 2 hours and washing with PBS, pH 7.4. A polystyrene surface treated with a solution containing 5% BSA in the same manner as the positive sample for coating suppression was used as a negative coating control. Next, the adhesion of four cell cultures obtained from three different species was studied on the above-mentioned surface coated with RGD peptide:
-Primary human osteoblasts derived from the apex of the corpus cavernosum of adult patients (Siggelkow et al., 1997, Bone, 20: 231);
-Primary human bone progenitor cells derived from bone marrow of adult patients (Vilamitjana-Amedee et al., 1993, In vitro Cell Dev. Biol., 29: 699);
Primary osteoblasts derived from calvaria of rat neonates prepared and cultured according to the method of Yagiela and Woodbury (1977, The Anatomical Record, 188: 287-305);
-Osteoblast line MC3T3 H1 obtained from calvaria of newborn mouse (Heermeier et al., 1995, Cells and Materials, Vol. 5: pp. 309-321), and-α _v β ₃ / α _v β ₅ integrin negative Human melanoma cell line M21L as a control.

Prior to using these cell types for attachment experiments, their integrin expression was assessed for α _v β ₃ or α _v β ₅ receptors and integrin subunit α using the Becton-Dickenson fluorescence activated cell sorter (FACS). _v Verified with fluorescently labeled antibodies against β ₃ and β ₅ . In this case, strong expression of the integrin was observed (FIG. 1).

The described cell types are seeded at a seeding density of 48000 cells / cm ² into Costar-48 well plates coated with RGD peptide as described above, then 37 ° C., 95% atmospheric humidity and 5% CO _2. Incubated for 1 hour under the following conditions. Cells that did not adhere during the experiment were washed twice with PBS, pH 7.4.

  Adherent cells are quantified by determining the activity of the cellular enzyme N-acetylhexosaminidase using an appropriate calibration curve according to Landegren's method (1984, J. Immunol. Methods, 67: 379-388). Indirectly.

Adhesion of cultured mouse MC3T3 H1 osteoblasts by coating the surface of BSA pretreated polystyrene cell culture medium with α _v β ₃ or α _v β ₅ selective thiol peptide 1-, 2- (sulfo-SMPB) derivatives Leads to a strong and dose-dependent stimulus (see Figure 2). In contrast to this, the biologically inactive thiol peptide control has no significant effect, resulting in high selectivity and high specificity of the integrin-RGD peptide interaction. Inferior α _v β ₃ / α _v β ₅ selective thiol peptide 3- (sulfo-SMPB) (cyclo-RGDVE-aminocysteine-amidohexanoic acid: Delforge et al., 1996, Anal. Biochem., 242 , P. 180) also show only significantly weaker effects than the corresponding thiol peptide 1 and 2 derivatives.

The binding properties of different cell types to BSA pretreated polystyrene surfaces coated with thiol peptide 1- (sulfo-SMPB) derivatives or thiol peptide 2- (sulfo-SMPB) derivatives are about 70% of the inoculated cells (primary rat bone Blast cells), infection curves with a maximum adsorption rate of about 80% (primary human osteoblasts and primary human bone progenitor cells) and about 90-100% (MC3T3 H1 mouse osteoblasts), and concentrations in the coating solution of 50- One half of the maximum number of cells at 1000 nM corresponds to binding to the RGD peptide (FIG. 3). In contrast, α _v β ₃ / α _v β ₅ negative control cells (M21L) do not show any significant cell attachment. The cell adsorption rate described above is increased by about 20-40 times compared to a 5% BSA coated surface that does not result in any significant cell attachment for all osteoblast cultures used.

  The similar behavior of the osteoblast culture described above confirms that the adhesion stimulating effect is specific to osteoblasts but is species independent.

  Binding of the osteoblast medium to the BSA pretreated polystyrene surface coated with the thiol peptide 1-sulfo-SMPB derivative can be completely inhibited by adding cyclic RGDfK dissolved in the adherent medium (Fig. 4). This result proves that the observed cell attachment phenomenon is mediated only by the RGD peptide.

The dependence of cell attachment of primary human osteoblasts or MC3T3 H1 cells on a BSA pretreated polystyrene surface coated with the thiol peptide 1,2 (sulfo-SMPB) described above depends on the saturation curve. Draw (Figure 5). However, no significant cell attachment is observed on the BSA negative coating. An average surface area per adherent cell of about 200-500 μm ² calculated for the maximum cell seeding density gives an average cell-cell distance of about 15-25 μm on this surface (FIG. 6). Since the osteoblast diameter is about 10-15 μm, the peptide coating is so numerous that a densely coated surface with osteoblasts can be achieved in which the cells are arranged immediately adjacent to each other. Clearly, cell attachment sites are available. The time course of the cell attachment process on the surface is dependent on the cell type, indicating rapid cell attachment that is completed after about 45-60 minutes (FIG. 7). Cell lines (eg MC3T3 H1) here show a faster rate of attachment than primary cell cultures (human, rat). No significant differences between the two thiol peptide derivatives used can be seen in these experiments.

Example 3:
Four different acrylate RGDs with different molecular spacer distances to study the effect of different anchor molecule lengths, ie the distance between the RGD sequence of cell recognition and the material surface, on the degree of osteoblast attachment Peptides (cyclo-RGDfK NH-CO-acrylate derivatives (type: iia, iib, iiia, iiib) were prepared.

  Synthesis of the cycloRGD peptide was performed according to the disclosure of Example 1, or by Pless et al., 1983, J. Biol. Chem., 258: 2340-2349 or Currath et al., 1992, Eur. J. Biochem., Volume 210: Performed according to the steps on pages 911 to 921.

  The corresponding anchor lengths of these peptides are approximately 2.6 nm (acrylate peptide 1, type iia), 3.5 nm (acrylate peptide 2, type iib), 3.7 nm (acrylate peptide 3, type iiia) and 4.2 nm (acrylate peptide). 4. Type iiib).

Due to the covalent attachment of these peptides to the polymerized PMMA (polymethylmethacrylate) surface, suitable shaped articles (diameter: 10 mm; height: 2 mm) with different porosity were prepared from three different PMMA components.
-PMMA-based bone cement (Merck KGaA, Germany, certification number 5181.00.00) corresponding to product instructions (40g powder + 20ml liquid).
・ 10g of PMMA / PHEMA (polyhydroxyethyl methacrylate) particles (manufactured by Biomet) with a particle size of 0.5 to 0.6mm were added to a HEMA solution (68.6% HEMA, 29.4% TEGMA [triethylene glycol dimethacrylate], 2% tBPB [tert-butyl]. Peroxybenzoate]) 1.3 ml and adhered to the porous carrier material.
・ PMMA particles (Plex Y7H, Rohm, Germany) 10g with particle fragments 0.7-2mm are mixed with 1.5 ml of methyl methacrylate (MMA) solution (68.6% MMA, 29.4% TEGMA, 2% tBPB) to form a porous carrier material. This was used for attachment.

  In order to covalently bond the acrylate peptide to the prepolymerized PMMA molded article, a stock solution of acrylate peptides 1, 2, 3 and 4 is added in each case to a final concentration of 100 μM in DMSO / 0.2% camphorquinone. Dissolved and prepared in (w / v). In addition, a series of concentrations were prepared in each case with final peptide concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM and 10 μM by dilution with isopropanol / 0.2% camphorquinone (w / v). Molded articles were incubated for 2 hours at room temperature under sunlight and stored dry at 4 ° C. Prior to use, the samples were washed with PBS, pH 7.4 to remove unbound peptides, and left overnight at 4 ° C. in PBS, pH 7.4.

  Nonspecific cell binding sites were blocked by adding 250 μl each of BSA 5% added solution of PBS, pH 7.4 per molded article, followed by incubation for 2 hours at room temperature and washing once with PBS, pH 7.4. .

  A PMMA molded article treated with a solution of isopropanol / 0.2% camphorquinone (w / v) instead of the peptide solution was used as a negative control.

  Subsequently, MC3T3 H1 osteoblasts obtained from the calvaria of newborn mice (Heermeier et al., 1995, Cells and Materials, Vol. 5: pp. 309-321) were examined for adhesion to the peptide-coated surface. .

MC3T3 H1 osteoblasts were seeded in Costar 48-well plates as described above with PGD moldings coated with RGD peptide at an inoculation density of 48,000 cells / cm ² at 37 ° C., 95% atmospheric humidity and 5% Incubated for 1 hour under CO ₂ conditions. Cells that did not adhere during the experiment were washed twice with PBS, pH 7.4.

  Adherent cells are quantified by determining the activity of the cellular enzyme N-acetylhexosaminidase using an appropriate calibration curve according to Landegren's method (1984, J. Immunol. Methods, 67: 379-388). Indirectly.

  The binding properties of MC3T3 H1 osteoblasts to different PMMA molded articles coated with the acrylate RGD peptides 1 (type iia), 2 (type iib), 3 (type iiia) and 4 (type iiib) Both correspond to the infection curve. The maximum adsorption rate is about 20% for acrylate peptide 1 and about 80-100% for acrylate peptides 2, 3 and 4. The half-maximal binding of cells occurs for acrylate peptides 2, 3 and 4 at RGD peptide concentrations in coating solutions of about 1 to 10,000 nM.

  The cell adhesion rate on the bone cement shaped article coated with RGD peptide is increased about 1.5 times (acrylate peptide 1) and about 5 to 15 times (acrylate peptide 2, 3 and) compared to the uncoated surface. (Figure 8).

  The cell attachment rate on porous PMMA / PHEMA particle shaped articles coated with RGD peptide is increased by about 2 to 5 times (acrylate peptides 2, 3 and 4) compared to the uncoated surface ( Figure 9).

  The cell attachment rate on the porous Plex Y7H particle shaped article coated with RGD peptide is increased about 2-3 times (acrylate peptide 2, 3 and 4) compared to the uncoated surface (Fig. Ten).

  For PMMA / PHEMA or PMMA-Plex Y7H samples, there is a greater variation in cell attachment values and a constant maximum adherence rate to samples coated with RGD peptide than for PMMA bone cement models, but the coating control Both stronger attachment of osteoblasts to the sample is observed.

  For acrylate peptides 2, 3 and 4, a very similar stimulation of cell attachment is observed both in the comparison with each other and with thiol peptides 1 and 2 coated on the test BSA control, but the shortest peptide Acrylate peptide 1 is almost inactive. From this it can be concluded that a critical minimum distance of about 2.5-3.5 nm is required between the RGD cell recognition sequence and the material surface in order to be able to achieve sterically satisfactory cell attachment ( Figure 11).

  The higher activity of acrylate peptide 3 on the bone cement PMMA carrier points to an optimal molecular structure for cell attachment, both in terms of molecular length as well as any hydrophilic / hydrophobic distribution or spacer ratio.

Example 4
To test osteoblastic cell adhesion to peptide-coated metal surfaces, V4A stainless steel shaped articles (7 x 7 x 1 mm ³ ) were subjected to three different coating modifications, in each case followed by RGD peptide. Covered. For this purpose, first, a stainless steel shaped article is incubated for 15 minutes at 60 ° C. in an ultrasonic bath for cleaning, rinsed with deionized water, incubated for 30 minutes in acetone, and again with deionized water. Rinse and incubate at 60 ° C. for 24 hours for drying.

Subsequently, stainless steel platelets were coated with three modifications.
a. Kevloc ^(R) process (Heraeus Kulzer GmbH, Welheim , Germany):
Kevloc the ^(R) primer solution, thinly applied and incubated at room temperature for 3 minutes. Next, the Kevloc ^(R) Bond solution, likewise thinly applied, and 20 minutes activation at 180 ° C. in a furnace.

This coating deformation directly creates available free acrylate groups on the metal surface, so that the acrylate peptide 3, type iiia is then directly covalently bonded as described in Example 3 for polymerized PMMA bone cement. It became possible.
b. Silicoater ^(R) process (Heraeus Kulzer GmbH, Welheim , Germany):
Siliseal the ^(R) solution, thinly applied, dried for 5 minutes at room temperature. Next, the Sililink solution was similarly applied thinly and activated in an oven at 320 ° C. for 3 minutes.

  In order to make available acrylic acid groups on the coated metal surface, a pretreated stainless steel shaped article was immersed in deionized water heated to 75 ° C. and the pH was adjusted to about 8.00 using NaOH. A 1% 3-methacryloxypropyltrimethoxysilane solution (Huels AG, Germany) was then added dropwise over 1 hour with stirring until a pH of about 6.50 was achieved. The mixture was then stirred at 75 ° C. for 1 hour; at this time the pH was about 6.35.

Coupling of acrylate peptide 3, type iiia was performed as described in Example 3 for polymerized PMMA bone cement.
c. Pigment coating (Merck KGaA, Darmstadt, Germany)
This coating variant originally describes the technical teaching for the coating of pearlescent pigments for water-based coating systems for metals and plastics in the automotive and plastics industry, as described in DE-A 4321005 It went by.

For this reason, the stainless steel shaped article was immersed in deionized water heated to 75 ° C., the pH was adjusted to about 8.00 using NaOH, and the AlCl ₃ solution was added dropwise over 2 hours with stirring. Next, a 5% sodium silicate solution was similarly added dropwise over 2 hours with stirring. In the following steps, a 1% 3-methacryloxypropyltrimethoxysilane solution (Huels AG, Germany) was added dropwise over 1 hour with stirring. The mixture was then stirred at 75 ° C. for 1 hour; at this time the pH was about 6.40.

  Coupling of acrylate peptide 3, type iiia was performed as described in Example 3 for polymerized PMMA bone cement.

  After coating a stainless steel shaped article with acrylate peptide 3, type iiia by three different methods, adhesion of MC3T3 H1 osteoblasts was examined. For this, the procedure described in Example 3 was used.

  The binding properties of MC3T3 H1 osteoblasts to V4A stainless steel shaped articles pretreated using three different methods and coated with the acrylate peptide 3 correspond in each case to the infection curve (FIG. 12). .

Maximum deposition rate is about 60% for pigment coated, and Kevloc ^(R) or Silicoater ^(R) method was about 80% for. Half of the maximum cell binding occurs at a concentration of RGD peptide in the coating solution of about 1-100 nM for all three coating variants. The cell attachment rate on RGD peptide-coated stainless steel shaped articles is increased about 3-8 times compared to the uncoated control.

For the Kevloc ^(R) or Silicoater ^(R) method, in each case, one coating solution (Kevloc ^(R) primer alone, Kevloc ^(R) bond alone, Siliseal ^(R) alone, Sililink ^(R) alone Only) was used to form the coating. In this case, each of the double coated compared to ^{(Kevloc (R)} primer / ^{bond, Siliseal (R) / Sililink (} R)), no significant differences in cell attachment was observed.

Example 5:
To supplementarily study not only osteoblast attachment but also growth on the RGD peptide surface, the procedure was as follows:
The coating method described in Example 3 was selected for covalent attachment to polymerized PMMA bone cement shaped articles of acrylate peptide 3, type iiia.

Next, we performed MC3T3 H1 mouse osteoblasts with two changes, inoculating 12,000 cells / cm ² instead of 48,000 cells / cm ² and attaching 2 hours instead of 1 hour. In the same manner as in Example 3, it was adhered to the RGC peptide-coated bone cement shaped article. In addition, three different peptide concentrations of 0.01 μM, 1 μM and 100 μM were also examined in the coating solution.

After washing away non-adherent cells, MC3T3 H1 osteoblasts were cultured in serum-containing (10%) medium for 15 days under conditions of 95% atmospheric humidity, 5% CO ₂ and 37 ° C. During this period, the medium was changed twice per week, and after 1, 2, 3, 4, 5, 10 and 15 days, cell counts were determined by the WST-1 test from Boehringer Mannheim (Germany). In each case, a typical log phase of cell growth of cultured osteoblasts was seen here (FIG. 13). The maximum achieved cell count was directly dependent on the RGD peptide concentration in the coating solution. That is, after culturing for 15 days, about 1,000,000 cells (100 μM peptide), about 550,000 cells (1 μM peptide), about 300,000 cells (0.01 μM peptide) or about 230,000 cells (control without peptide) were achieved per 1 cm ² . That is, at the highest peptide concentration of 100 μM, an approximately 4-5 fold increase in cell proliferation was observed compared to uncoated samples.

Example 6:
The prototype used for material coating with different cell-selective RGD peptides to obtain implants with locally differentiated biologically active coating patterns to stimulate the attachment of different cell types is polymerized PMMA It was a bone cement model. To this, two RGD peptides with different cell selectivity were covalently linked. In this case, acrylate peptide 3, which has selectivity for α _v β ₃ / α _v β ₅ integrin-bearing osteoblasts, type iiia, and acrylate peptide 5, which has selectivity for α _IIIb β ₃ positive crystal plate, type iiia Was bonded to a PMMA shaped article as described in Example 3.
a. In the first experiment, PMMA shaped articles were coated with acrylate peptides 3 and 5 at concentrations in the coating solution of 100 μM, 10 μM and 1 μM, respectively. Next, these RGD peptide-coated members were inoculated in parallel with osteoblasts and platelets to determine their cell attachment.

MC3T3 H1 osteoblasts (350,000 cells / cm ² ) and human platelets (50,000,000 cells / cm ² ) are inoculated in parallel on PMMA shaped articles coated with acrylate peptide 3 and acrylate peptide 5, and their cell adhesion is inoculated. Determined as described in Example 3.

  Human platelets were prepared according to the method of Shattil and Brass (J. Biol. Chem., 262: 992-1000, 1987). This experiment was able to verify the cell selectivity of the two RGD peptides.

_{α v β 3 / α v β} 5 positive osteoblasts, α _v β ₃ / α _v Compared with beta ₅ negative platelets, acrylate peptide _{3 (α v β 3 / α} v β 5 selective) coated PMMA molding It preferentially adhered to the product (Figure 14). Here, for osteoblasts, a converted Abs (405 nm) of about 6.0 (100 μM peptide in the coating solution), about 2.0 (10 μM peptide) or about 1.8 (1 μM peptide) was found. However, for platelets, very low cell attachment values of about 3.0 Abs (100 μM peptide in the coating solution), about 1.5 Abs (10 μM peptide) or about 0.2 Abs (1 μM peptide) were seen. However, the opposite results were seen with the acrylate peptide 5 (α _IIIb β ₃ selective) coated PMMA shaped article. In this case, α _v β ₃ / α _v β ₅ positive platelets adhered preferentially compared to α _IIb β ₃ negative osteoblasts (FIG. 15).

Here, calculated Abs (405 nm) of about 4.0 (100 μM peptide in coating solution), about 0.5 (10 μM peptide) or about 0.2 (1 μM peptide) was seen for platelets. However, for osteoblasts, very low cell attachment values of about 1.5 Abs (100 μM peptide in coating solution), about 1.0 Abs (10 μM peptide) or about 0.2 Abs (1 μM peptide) were seen.
b. Also in the second experiment, PMMA shaped articles were similarly coated with acrylate peptides 3 and 5 although the concentration in each coating solution was only 100 μM. These RGD peptide-coated members were then inoculated in parallel with osteoblasts, platelets or osteoblast / platelet mixtures to determine their cell attachment. MC3T3 H1 osteoblasts (350,000 cells / cm ² ), human platelets (50,000,000 cells / cm ² ) and cell mixtures (350,000 osteoblasts / cm ² and 50,000,000 platelets / cm ² ) with acrylate peptide 3 and acrylate peptide 5 Inoculated in parallel on the PMMA shaped article coated with, and its cell attachment was determined as described in Example 3.

α _v β ₃ / α _v β ₅ positive osteoblasts (approximately 6.0 Abs) are preferred to acrylate peptide 3 coated PMMA shaped products compared to α _v β ₃ / α _v β ₅ negative platelets (approximately 3.0 Abs) (Fig. 16). When both cell types were inoculated as a mixture, a result approximately equivalent to the osteoblast-only result (approximately 6.5 Abs) was obtained.

However, for acrylate peptide 5 (α _IIb β ₃ selective) coated PMMA shaped articles, the opposite results were seen. Here, α _IIb β ₃ positive platelets (about 4.0 Abs) preferentially adhered as compared to α _IIb β ₃ negative osteoblasts (about 1.5 Abs) (FIG. 17). Inoculation of both cell types as a mixture gave results approximately equivalent to the sum of the individual results for platelets and osteoblasts (approximately 6.5 Abs).

These results indicate that coating of the implant surface with various cell-selective RGD peptides can be used for the generation of implants that mediate the preferred attachment of selected cell types. In this case, a comparison between osteoblasts and platelets indicates that:
Surface coverage with integrin-selective RGD peptides will prevent the attachment of these cell types with the addition of complementary integrins compared to cells that do not have these special integrins or only to a minor extent. Can be significantly irritating.
This effect is maintained even when using a cell mixture that is very close to the in vivo state.

Claims (14)

Essentially a carrier matrix and
Consists of peptide coatings with sequences that recognize binding sites on integrin receptors that govern adhesion on human or animal cells, the same or different to stimulate targeted attachment of human or animal somatic cells Comprising a peptide coating surrounding the matrix, containing peptides,
Different human and animal organs on the surface of which the carrier matrix has a reactive group capable of binding capable of forming a stable covalent bond with a compatible functional reactive group of the peptide layer An implant suitable for
Derived from the cell attachment stimulating activity related to its corresponding different structure, the specific different integrin patterns and uniqueness of the tissue cells adjacent to them in the specific area where the implant should be inserted into the cell The peptide is arranged on the surface of the implant such that there is a locally differentiated, selective, biologically active coating pattern of the implant surface. And The implant according to claim 1, wherein the peptide is attached to the surface of the implant by an anchor molecule. 3. Implant according to claim 2, characterized in that the anchor molecule consists of one of the following structures:
-CO- (CH ₂ ) _k -X-SH, (i)
Where X is a single bond or -CO-NH- (CH ₂ ) _l- ,
k = 2-12 and l = 2-4;
_{-CO- (CH 2) m - [} NH-CO- (CH 2) n] p -NH-CO-CH = CH 2, (ii)
Where m, n = 2-8; p = 0-2,
-(CO-CH ₂ -O-CH ₂ -CH ₂ -O-CH ₂ -CH ₂ -NH) _q -CO- (CH ₂ ) _r -NH-CO-CH = CH ₂ (iii),
Where q = 1-3 and r = 2-8. 4. Implant according to claim 3, characterized in that the anchor molecule is selected from one of the following structures:
-CO-CH ₂ -CH ₂ -SH; (ia)
_{-CO-CH 2 -CH 2 -CO-} NH-CH 2 -CH 2 -SH; (ib)
-CO- (CH ₂ ) ₅ -NH-CO-CH = CH ₂ ; (iia)
-CO- (CH ₂ ) ₅ -NH-CO- (CH ₂ ) ₅ -NH-CO-CH = CH ₂ ; (iib)
_{-CO-CH 2 -O-CH 2} -CH 2 -O-CH 2 -CH 2 -NH-CO- (CH 2) 5 -NH-CO-CH = CH 2; (iiia)
-(CO-CH ₂ -O-CH ₂ -CH ₂ -O-CH ₂ -CH ₂ -NH) ₂ -CO- (CH ₂ ) ₅ -NH-CO-CH = CH ₂ (iiib) The peptide is bonded to the implant surface through a carboxyl group of an anchor molecule by an amide bond, and the anchor molecule is bonded to the implant surface through a mercapto or acrylic acid group. The implant according to one item. 6. Peptide anchor molecules are attached to the implant surface such that the steric critical, absolute minimum distance between the cell recognition region of the peptide and the surface of the uncoated material is between 2.0 and 3.5 nm. The implant according to 1. Implant according to any one of claims 1 to 6, characterized in that the peptide is capable of binding to α _v β ₃ / α _v β ₅ expressing cells. The implant according to any one of claims 1 to 7, comprising a peptide having the amino acid sequence RGD. 9. Implant according to claim 8, characterized in that it comprises a cyclic RGDfK sequence peptide. Implant according to any one of the preceding claims, comprising an adhesion promoting interlayer and / or a branching molecule that provides a surface area expansion effect between the peptide or peptide anchor molecule and the surface of the carrier matrix. . The carrier matrix is a ceramic, a polymer material, a suitable metal shaped article, or a structure made of these materials, which can be formed as a biohybrid organ by in vivo or in vitro colony formation using cells. The implant according to any one of claims 1 to 10. A method for preparing an implant according to claim 1-11,
(I) determine the integrin receptor structure of the target tissue where the implant is to be inserted in vivo with an in vitro test system;
(Ii) selecting or synthesizing the peptide having an appropriate complementary structure;
(Iii) the peptides are covalently linked directly to each arbitrarily modified surface of the implant or via the anchor molecule, and the local arrangement of different peptides on the surface of the implant should be inserted into the implant; A method characterized in that it corresponds to a specific predetermined local sequence of a target cell having a complementary integrin pattern. 13. The method according to claim 12, wherein the in vitro determination of the integrin receptor structure of the target tissue is performed with an immunohistologically active antibody. 14. A method according to claim 12 or 13, characterized in that the binding of the peptide or peptide anchor molecule to the implant surface is carried out by known methods for other types of coatings.

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