CN1761531A - Methods and materials for nanocrystalline surface coatings and attachment of peptide amphiphile nanofibers thereon - Google Patents

Abstract

Biocompatible composites comprising peptide amphiphiles and surface modified substrates and related methods for attachment thereon.

Description

Methods and materials for nanocrystal surface coatings and peptide hydrophilic lipid molecule nanofibers attached thereto

This application claims the benefit of priority to US Provisional Application Serial Nos. 60/446,421 and 60/495,965, filed February 11, 2003, and August 18, 2003, respectively, each of which is incorporated herein by reference in its entirety.

The United States Government has certain rights in this invention pursuant to Grant Nos. DEFG02-OOER45810 and DMR0108342 awarded to Northwestern University from the Department of Energy and the National Science Foundation, respectively.

Background technique

Tissue engineering, the technique of using biocompatible scaffolds, offers a viable alternative to prosthetic materials currently used in restorative and reconstructive surgery (eg, craniomaxillofacial and spinal surgery). These materials also hold promise for forming tissue or organ equivalents for replacing diseased, defective or damaged tissue. Compatible biodegradable materials can be used as scaffolds that initiate and maintain tissue or bone growth, but which naturally degrade over time in the body. Such materials can also be used for the controlled release of therapeutic materials (eg, genetic material, cells, hormones, drugs or prodrugs) to predetermined areas. The polymers used to create these scaffolds, such as polylactic acid, polyorthoesters and polyanhydrides, are difficult to mold and lead inter alia to poor cell attachment and incorporation into the site of application of the tissue engineering material. With some exceptions, they also lack biologically relevant signals.

Self-assembling peptide-amphiphile nanofibers have been used to direct the growth of biominerals such as hydroxyapatite. These nanofibers comprise peptide-hydrophilic lipid molecules consisting of a hydrophobic aliphatic tail coupled to a relatively hydrophilic peptide headgroup. A peptide head group may comprise at least 2 parts: a structural part and a functional part. The structural part may include 2 to 4 cysteine residues, which can be used to covalently stabilize the self-assembled peptide hydrophilic lipid molecular structure through disulfide bond formation between individual peptide hydrophilic lipid molecules within the fiber. Alternatively, the moiety may comprise other residues such as, for example, serine, leucine, alanine or glycine. Although these residues may not contribute to the covalent stabilization of the nanofibers, they may be involved in the formation of structural organization in the assembled nanofibers, such as β-sheet formation. Functional headgroups may consist of different amino acid combinations including, for example, carboxyl, sulfhydryl, amine, phosphate and part of hydroxyl functional groups located near the terminus of the molecule furthest from the aliphatic tail of the molecule. Examples of carboxyl-containing residues include aspartic acid or glutamic acid. Examples of amine- or guanidinium-containing residues include lysine or arginine, respectively. When the peptide-hydrophilic lipid molecules self-assemble under aqueous conditions, it is expected that these functional residues will be displayed in self-assembled micelles (usually nanofibers) that they can react with other moieties to bind the peptide-hydrophilic lipid molecules. ) near the surface.

The versatility and functionality of these self-assembled nanofibrous materials prove useful for tissue repair, cell growth, or organ remodeling. The term tissue includes muscle, nerve, blood vessel and bone tissue, as well as other common understandings of tissue. The present invention is also applicable to the regulation, inhibition or promotion of axonal outgrowth of neurons, and the regulation, inhibition or promotion of cell-substrate adhesion between nerve cells. Coating these peptide-hydrophilic lipid molecular compositions onto the surface of stents and implants such as stainless steel stents, electrodes for electrical stimulation of nerves, or metal-based orthopedic implants can further enhance existing tissue engineering strategy. Importantly, multiple peptide signals are available for the same supramolecular self-assembling peptide-hydrophilic lipid molecule to achieve different and possibly synergistic effects.

The peptide-hydrophilic lipid molecule composition of this system may include a peptide component having residues capable of intermolecular cross-linking. The sulfhydryl moiety of cysteine residues can be used to form intermolecular disulfide bonds by introducing appropriate oxidizing agents or under physiological conditions. Conversely such bonds can be cleaved by reducing agents introduced into the system or under reducing conditions. The concentration of cysteine residues, when used, can also be varied to control the chemical and/or biological stability of the nanofiber system and thereby control the therapeutic delivery or release of cells or other beneficial agents using the nanofibers as carriers speed. For example, enzymes can be introduced into such nanofibers to control their biodegradation rate through the hydrolysis of disulfide bonds. This degradation and/or concentration of cysteine residues can be used in a variety of tissue engineering applications. The sulfhydryl functional groups of this peptide hydrophilic lipid molecule can also be used to attach supramolecular structures to surfaces. The complementary nature of the biological portion of the peptide hydrophilic lipid molecule mimics the amino acid sequence found in naturally occurring peptides. Self-assembling gels composed of peptide-hydrophilic lipid molecule nanofibers with RGD peptide sequences mimic the function of collagen fibrils to organize and guide the growth of hydroxyapatite crystals. Other potentially useful amino acid sequences in such peptides may include the YIGSR and IKVAV amino acid sequences. This amino acid sequence in the self-assembling peptide hydrophilic lipid molecule may have a synergistic effect on cell growth and nerve regeneration. The growth of cells on the matrix implanted or delivered to the body will benefit the implantation of artificial hearts, restore nerve function, and heal implanted blood vessels; by culturing epidermal cells on a fibrous grid to form skin implants and fabricate "Artificial skin".

Damage to the vascular endothelium and media, such as often occurs during balloon angioplasty and stenting procedures, has been found to stimulate neointimal proliferation leading to restenosis of atherosclerotic vessels. The normal endothelium, which lines blood vessels, is uniquely and completely compatible with blood. Endothelial cells initiate metabolic processes that actively prevent platelet deposition and thrombus formation in the vessel wall. Injured arterial surfaces within the vasculature are prone to thrombosis. Although systemic agents have been used to prevent coagulation and inhibit platelet aggregation, there is a need for direct treatment of damaged arterial surfaces to prevent thrombosis and subsequent intimal smooth muscle cell proliferation.

Stents composed of metals such as titanium and its alloys have been designed to promote organized endothelial cell growth. The stent includes a plurality of dimples on at least a part of the surface of the stent body, preferably arranged in a regular pattern on at least the inner surface of the stent body, such as a waffle. Other stents have surface features that include many folds, ridges, channels, or holes in the stent body, at least some of which pass through the interior and exterior sides of the stent body (i.e., through the stent body). tenter scaffold) and are sized to promote organized cell growth.

The growth of directed cells, such as neural cells and endothelial cells, on implantable surfaces and scaffolds would be desirable for regeneration and growth of cells, organs and tissues in the body. There is a need to provide surgical implants that promote the growth of tissue, vascular tissue, nerves and cells on or in the tissue surrounding the surgical implant. There is a need for new and better scaffolds, implants, stents and electrodes to be placed in the body that are suitable for promoting the growth of infiltrating cells into organized cellular structures such as occurs during angiogenesis and/or neovascularization process to help repair damaged body organs and blood vessels.

As part of related considerations, titanium and its alloys have been widely used as bone implant materials, where the metal's high strength-to-weight ratio, toughness, and bioinert properties of naturally formed oxide layers have led to widespread clinical success. But with the development of tissue engineering, researchers have investigated the use of calcium phosphate coating on the surface of titanium-based implants to introduce biologically active elements to the otherwise inert oxidized metal surface. In vitro tests have shown that calcium phosphate forms an osteoconductive coating that enhances cell attachment and proliferation. In vivo models have shown that the strength of the implant interface increases when titanium surfaces are coated with different calcium phosphate coatings, typically hydroxyapatite (Ca ₁₀ (PO ₄ ) ₂ (OH) ₂ ). The study also showed that the degradation of these calcium phosphate coatings at the implant-tissue interface contributed to the accelerated formation of new bone.

Common methods for coating Ti with these calcium phosphate coatings include plasma spraying, electrophoresis, sol-gel and solution-phase precipitation. Methods such as plasma spraying or sol-gel tend to produce a dense, often highly crystalline apatite phase with little or no phase selectivity, and some of these methods also fail to coat the inner surfaces of porous titanium structures . Many of these methods for growth involve extremely long growth times, weeks to months, low control over crystal size or shape, and lack any additional chemical functionality, such as those provided by organic macromolecules . Organic macromolecules are known to play a role in biomineral crystal modification. Additionally, where clusters form on porous surfaces, the surface coating is often less than 100%. However, solution-phase growth enables the nucleation of calcium phosphate coatings to occur directly on implant surfaces, even on porous surfaces. Furthermore, this wet chemical method allows the formation of not only hydroxyapatite but also other biologically relevant calcium phosphate phases such as octacalcium phosphate (Ca ₈ H ₂ (PO ₄ ) ₆ ·5H ₂ O) hydroxyapatite stone precursors. Solution-phase growth of these coatings also allows the incorporation of organic macromolecules into the coating, a feature not possible with some high temperature coating methods such as plasma spraying.

The interaction between different biological macromolecules and calcium phosphate coatings has been studied. The growth of calcium phosphate coatings was substantially inhibited in the presence of biomolecules such as albumin, fibronectin and poly(amino acids). For example, poly(L-lysine), a well-recognized cell adhesion promoter with excellent chemical functionality, was shown to inhibit the growth of apatite on titanium alloy surfaces. Poly(amino acid)s have been used as nucleating agents and macromolecular tethers to address this problem by growing poly(L-lysine)-containing organoapatite on poly(amino acid)-coated titanium-based surfaces. question. This method uses poly(amino acids) in several coating steps and layers; it also produces relatively large organoapatite clusters, which can be disadvantageous in coating structures with fine pore structures. An alternative approach to study is to grow an albumin-containing calcium phosphate coating on a pre-existing calcium phosphate layer.

There is a need to form polyamine-modified nanostructured calcium phosphate coatings on implantable metal surfaces. Grown onto calcium phosphate seeds, the new material combines the versatility and simplicity of solution-phase calcium phosphate growth on implantable surfaces with the chemical and biological functionality of poly(amines).

Coating material surfaces with biominerals is desirable so that substantially all of the surface is coated and the coating provides a favorable surface for chemical modification, peptide-hydrophilic lipid molecule nanofiber attachment, cell and tissue growth and adhesion. It would be more desirable if the coating could be used for materials suitable for implantation into a patient, and if the coating were degradable under physiological conditions.

overview

In part, embodiments of the invention relate to binding self-assembling peptide hydrophilic lipid molecules to other materials such as metals. The newly formed bonds will allow the assembly of the original self-assembling peptide-hydrophilic lipid nanofibers or spherical micelles to another material. Binding of suitable self-assembled nanofibers or micelles to a second surface can be used to further direct cell or tissue growth on the second surface. Alternatively, peptide-hydrophilic lipid molecules can be bound to the surface and used for directional growth of peptide-hydrophilic lipid molecule nanofibers, or can be used to initiate self-assembly of nanofibrous structures on the material surface. When the material of interest is a stent, this surface can be used for tissue repair, allowing cells to attach to the implant and minimizing conditions such as restenosis.

The binding of the peptide hydrophilic lipid molecules to the second surface can be carried out by physical adsorption, chemical adsorption or covalent attachment of the peptide hydrophilic lipid molecules, or self-assembled nanofibers or micelles comprising them, to the surface. Examples of such bonding include, but are not limited to, ionic, coordinative, chelating, amide or ester linkages between the self-assembled nanostructure and the surface. This conjugation scheme is expected to provide a stable mechanism for attaching peptide nanostructures to other materials, including metal surfaces, polymers, peptide-modified biomaterial coatings, or structures containing other peptides. This linkage will strongly stabilize the peptide-containing micelles on the surface of the material. This delivery scheme is suitable for applications ranging from modification of cell-specific behavior to drug delivery. In one embodiment, the peptide hydrophilic lipid molecule nanofibers comprise carboxyl-rich peptide sequences. This peptide hydrophilic lipid molecule is bound to the surface displaying free amines. Alternatively, the peptide hydrophilic lipophilic nanofibers may contain residues exhibiting free amines, while the second surface or structure will exhibit carboxyl functional groups.

With respect to such functionally modified surfaces, the embodiments of this paragraph through paragraph 0025, inclusive, are contemplated. One embodiment of the present invention is an organically modified biomineral coating on an implantable substrate whose surface has been pre-implanted with minerals. In a preferred embodiment, the organically modified coating comprises calcium phosphate coated on a metal substrate that has been pre-grafted with calcium phosphate. One embodiment of the invention is a method of coating a substrate with a biomineral coating.

Embodiments of the invention include a poly(L-lysine)-modified nanostructured calcium phosphate coating on a titanium surface grown on calcium phosphate seeds on the metal surface.

In one embodiment of the invention, the coating on the pre-seeded substrate consists of a (calcium-) metal deficient (octacalcium phosphate) mineral whose crystal growth is disrupted and modified by a polyamine, and preferably are polyamines including amino acids such as poly(L-lysine) present during mineralization. It is also believed that the (poly(L-lysine)) poly(amino acid) is intimately incorporated into the mineral phase.

One embodiment of the invention is a composition for coating a substrate with a modified crystalline material surface to promote cell attachment, tissue growth, or for delivery of a therapeutic composition. The coating solution comprises a solution in which the crystalline material is dissolved and a polyamine and preferably a polypeptide or a salt thereof. Compositions include a dissolved crystalline material of interest and a polyamine, where the polyamine may include amino acid monomers. The polymer preferably comprises amino acids having free functional groups for forming bonds with peptides, peptide hydrophilic lipid molecules, proteins and cells when they are incorporated into minerals. Preferred polymers include lysine monomers, more preferably polylysine or salts thereof.

In one embodiment, the coating is useful for cell growth and cell adhesion, and the coating is readily degradable under physiological conditions.

Another embodiment of the invention is a matrix for growing cells, tissue, or for releasing a therapeutic composition. This matrix can be used to grow cells or tissues in vitro, or it can be used to grow or culture cells or tissues such as bone in vivo. The matrix is made of a biocompatible material whose surface has been pre-seeded with mineral crystals and then coated with the mineral or material modified by its normal crystal structure by incorporation of polyamines, preferably polypeptides, into the material. The coating on the substrate can be further bonded to the peptide via another bond such as a disulfide or amide bond to the polyamine in the coating material, or to the crystalline material itself via other bonds.

Alternatively, the coating on the substrate may be bound to self-assembling peptide hydrophilic lipid molecules, or cross-linked self-assembling peptide hydrophilic lipid molecules, preferably via amide bonds. Substrate coating materials may also include oxides, hydroxides, phosphates, carbonates, oxalates and combinations of these ions which themselves bind to peptide or self-assembling peptide hydrophilic lipid molecules.

Another embodiment of the invention is a method for modifying the morphology of a material coated on a substrate. The method comprises, pre-seeding a biologically compatible matrix and then treating the pre-seeded crystal with dissolved crystalline material, or a solution composition of a biomineral with a polyamine or poly(amino acid) or an acid addition salt thereof Matrix, which will be incorporated into crystalline materials or biominerals to form nanocrystalline minerals. The morphology of the resulting coating can be controlled by the composition and method of coating the substrate. The method may further comprise the interaction of the binding molecule with the polyamine incorporated into the nanocrystalline material of the coating.

The morphology of the embodied coatings obtained according to the invention consists of irregularities 1-2 orders of magnitude smaller than pure inorganic mineral coatings. This enhanced structure and reduced morphological size will facilitate cell attachment, proliferation, and dissemination to monolithic substrates or surfaces coated with such organically modified materials. Furthermore, the disrupted poorly crystalline nature of the coating combined with the enzyme-vulnerable organic component of the mineral complex favors making the coating especially suitable for natural resorption and reconstruction processes. Finally, polyamino acids are incorporated into the coating to provide additional chemical functionality via free amine or sulfide groups located on the side chains of the lysine polymers. This chemical functionality can be used for incorporation or covalent attachment of biomolecules such as growth factors, biologically relevant peptide sequences or therapeutic drugs.

The new material combines the versatility and simplicity of solution-phase calcium phosphate growth on titanium with the chemical and biological functionality of poly(L-lysine).

Accordingly, embodiments of the present invention may also include self-assembling peptide hydrophilic lipid molecules coated on the surface of implantable stents, surgical devices, electrodes, stents, and other substrates. Coatings on these surfaces that include peptide hydrophilic lipid molecules can enhance the growth of cells and thus tissues in vivo.

One embodiment of the present invention provides a system of self-assembling peptide-hydrophilic lipid micelles, spherical or cylindrical, including one or more biological signals deposited on a substrate. Variation of the structural peptide sequence in the peptide-hydrophilic lipid molecule allows reversible crosslinking of the assembled nanofibers to the matrix for better or lower structural stability or controlled encapsulation in the hydrophobic inner core of the nanofibers The rate of delivery of molecules on or adsorbed on their hydrophilic surfaces.

In another embodiment, the peptide elements of the peptide hydrophilic lipid molecule are preferably carboxyl-terminated so that once assembled into fibers, these fibers can participate in further or urea conjugation to functionalize metal surfaces or some other type of surface.

Another embodiment of the present invention is a method for the preparation and utilization of self-assembling peptide-hydrophilic lipid molecule-coated surfaces of nanofibers as temporary scaffolds for cell growth and implantation.

Another embodiment of the present invention is a biodegradable, non-toxic self-assembling peptide hydrophilic lipid molecule nanofiber coating surface and scaffold, which can be used for in vitro and in vivo cell growth immediately after implantation, as endothelial cells, organs Supporting structure for tissues and nerve cells.

Another embodiment of the present invention is a method for assembling and constructing biodegradable self-assembling peptide hydrophilic lipophilic nanofiber coated surfaces and scaffolds, after surface or scaffold implantation, provides for cell growth support, but allows and enhances vascularization of growing cell clumps.

Another embodiment of the present invention is a self-assembling peptide hydrophilic lipid molecule coating surface with chemically distinct domains of the self-assembling peptide hydrophilic lipid molecule coating so that more than one type of cell can grow, Alternatively the growth rate of the cells on the matrix can be controlled.

Another embodiment of the present invention is an implantable self-assembling peptide-hydrophilic lipid molecule-coated stent adapted to facilitate intravascular or other tubular lumen in which the stent is implanted. Angiogenesis.

Another embodiment of the present invention is an implantable self-assembling peptide-hydrophilic lipid molecule-coated nanofiber stent adapted to enhance or stimulate neointimal infiltration, but with organization of infiltrating cells so that lead to the formation of new blood vessels.

Another embodiment of the present invention is an implantable self-assembling peptide-hydrophilic lipid molecule-coated stent when cultured in a cell-rich in vitro environment, or when implanted into a tubular body lumen For example in blood vessels, it is suitable for promoting the ingrowth of living cells.

Another embodiment of the present invention is a self-assembling peptide-hydrophilic lipid molecule-coated nanofiber-coated stent with growing through-holes of living cells and/or other cells designed for use when cells are implanted into tubular body lumens or A surface characteristic that promotes cell growth into organized cellular structures in organs.

Another embodiment of the present invention is a self-assembling peptide-hydrophilic lipid nanofiber coated stent in which living cells are genetically engineered to produce a therapeutic bioactive agent that will be extracted from the coated Layer nanofiber release, for example, is selected to inhibit or promote angiogenesis or intimal proliferation within an implanted stent.

Another embodiment of the present invention provides a technique by which functional cells from an organ in need are grown on a scaffold coated with nanofibers composed of self-assembling peptide hydrophilic lipid molecules. The coated scaffolds can be used in vivo or in vitro - using cell culture techniques followed by, after attachment and equilibration, transfer of the scaffold-cell complex to a site in the patient suitable for attachment, growth and function. During cell culture, nutrients and growth factors are supplied, allowing attachment, survival or growth as desired. Alternatively, nutrients and growth factors are encapsulated by self-assembling peptide-hydrophilic lipid micelles.

Scaffolds or surgical devices coated with self-assembling peptide-hydrophilic lipid nanofibers are beneficial for growing cells and tissues because their high surface area allows many sites for cell attachment and growth. The fibrous nature of the coating allows nutrients to penetrate the growing cell culture by diffusion until new blood vessels are formed. For an organ to be constructed in tissue culture and then successfully implanted, the matrix must have sufficient surface area and exposure to nutrients for cell growth and differentiation to occur prior to vascular growth after implantation. After implantation, the structure must allow the diffusion of nutrients and waste products, as well as continued vascular ingrowth, as cell proliferation occurs. Nanofibrous gels and micelles prepared from self-assembling peptide hydrophilic lipid molecules have high surface areas and are ideally suited to provide a good growth environment.

Description of drawings

Other aspects, features, benefits and advantages of embodiments of the present invention will be apparent, in part, with reference to the following specification, appended claims and drawings:

Figure 1A: Experimental setup (schematic) for calcium phosphate coating growth on titanium foil. Figure 1B illustrates a sample of the foil used, under which the solution was protected from precipitation.

Figure 2: Time-dependent pH change of the reaction solution during sample pre-seeding crystals and calcium phosphate coating growth.

Figure 3A-B: Scanning electron micrograph digital images comparing pure inorganic OCP (A) and pLys-CP (B) coatings on titanium foil. Inset (b) is a high-magnification image showing the nanoscale properties of the pLys-CP coating.

Figure 4: Powder XRD patterns of OCP and pLys-CP. The principal diffraction planes of the OCP are marked.

Figure 5: Reflectance FTIR spectra of OCP and pLys-CP coatings on Ti. The inorganic coating pattern showed the characteristic bands of OCP, while the pLys-CP coating showed the concomitant presence of poorly crystalline OCP and poly(L-lysine). The high frequency bands between 1350 and 2000 and above 3400 are believed to be due to reflections from the water surrounding the experimental equipment.

Figures 6A-B: A) _Scanning electron micrograph digital images of titanium surfaces previously seeded with _CaCl2 and _Na2HPO4 for 10 minutes. No calcium phosphate seeds are visible; B) Scanning electron micrograph digital image of _a titanium surface previously seeded with _CaCl2 and _Na2HPO4 for 2 hours. After 2 hours of growth, the seeds were clearly visible on the Ti surface.

Figure 7: Scanning electron micrograph digital images and corresponding EDS patterns showing the different degradation behavior of OCP coatings versus pLys-CP coatings. Scale bar line is 1 μm. The X-axis in the EDS graph represents energy (eV), and the EDS mode is normalized by the background intensity between 3000 and 3500 eV.

Figure 8: S:N ratios determined by XPS illustrating the binding affinity of cysteine to OCP and pLys-CP coatings. Sulfur and nitrogen were essentially not detected in the OCP samples. Error bars represent ±1 standard deviation from repeated measurements.

Figure 9 is an illustration of the schematic structure of a peptide that can be used for attachment to the coating of the invention, wherein (PO4) represents phosphorylated serine;

Figure 10A-B (A) digital images of scanning electron micrographs of self-assembling peptide hydrophilic lipid molecular nanofiber bundles attached to poly(L-lysine) modified calcium phosphate structural coatings of the present invention; ( B) Higher magnification scanning electron micrograph of the self-assembled nanofibers in (a), showing individual fiber layers;

Figure 11A-C Scanning electron micrographs of preosteoblast mouse calvarial cells spread onto pLys-CP coating on titanium foil after (A) 1 day, (B) 4 days and (C) 7 days of culture digital image.

Figure 12: Scanning electron micrograph digital image of nanofibers of peptide-hydrophilic lipid molecules covalently attached to amino-silanized titanium surface, showing low and high magnification images of these fibers covalently bound to Ti surface ;

A detailed description

Embodiments of the present invention generally involve bonding self-assembling peptide hydrophilic lipid molecule nanofibers or coatings of micelles to a second substrate to be placed within the body of a mammal. Such matrices may include porous scaffolds, electrodes and surgical implants such as stents. The self-assembling peptide hydrophilic lipid nanofiber coating is composed of peptide hydrophilic lipid molecules with amino acids that promote cell and tissue growth and adhesion to the matrix. Preferably, the design and function of the peptide-hydrophilic lipid molecule mimics naturally occurring structures such as proteins, cells and collagen. The matrix can be used outside the body to grow cells on the matrix and then placed in the body; alternatively, the coated matrix can be placed directly in the body and promote cell or tissue growth. Nanofibers or micelles can also encapsulate active compounds to promote the growth of such cells and tissues. Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular molecules, compositions, methods or protocols described, as these may vary. It is also to be understood that the terminology used in the specification is for the purpose of describing particular aspects or implementations only, and that it is not intended to limit the scope of the invention, which will be defined only by the appended claims.

It must also be noted that, as used herein and in the appended claims, singular forms include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "cell" is reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, the preferred methods, devices and materials are now described. All publications mentioned herein are incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular molecules, compositions, methods or protocols described, as these may vary. It is also to be understood that the terminology used in the specification is for the purpose of describing particular aspects or implementations only, and that it is not intended to limit the scope of the invention, which will be defined only by the appended claims.

The coupling agent used to bind the peptide hydrophilic lipid molecules to the second surface can be through physical adsorption, chemical adsorption or co-adsorption of the peptide hydrophilic lipid molecules, or their self-assembled spherical micelles or nanofibers with the second surface. price implant. Examples of coupling agent binding include, but are not limited to, ionic, coordinate, chelate, metal sulfide, amide or ester bonds between self-assembled nanofibers or micelles and the surface. This conjugation scheme is expected to provide a stable mechanism for attaching peptide nanostructures to secondary surface materials including, but not limited to, other self-assembling peptide hydrophilic lipid molecules, cell surfaces, proteins, Cartilage, metals, alloys, ceramics, glass, minerals, polymers and biocompatible implants such as stents, stents, electrodes and orthodentin. This attachment will allow robust stabilization of the peptide-containing micelles on the surface of the material. In one embodiment, peptide-hydrophilic lipid molecule nanofibers comprising carboxyl-rich peptide sequences are used. This peptide hydrophilic lipid molecule is bound to a surface displaying free amines.

In one embodiment of the coupling, the peptide-hydrophilic lipid molecule nanofibers are bound to an amino-silanized metal surface such as titanium or a metal alloy. The chemistries and methods used to form amide bonds between peptide hydrophilic lipid molecules and surfaces bearing such aminosilane surface groups are similar to those used in peptide synthesis (Knorr, et al; Fields et al; Wellings, et al.; the methods therein are incorporated herein by reference in their entirety). The reaction is carried out in a polar organic solvent such as, but not limited to, N,N-dimethylformamide (DMF) or N-methylpyrrolidone (NMP), both of which can dissolve amino acids. The method also includes the use of compounds such as O-benzotriazole-N, N, N', N'-tetramethyl-uronium-hexafluoro-phosphate (HBTU) as a catalyst to increase the concentration of the peptide on the hydrophilic lipid molecule. Reactivity of the carboxylic acid functional group. Other peptide coupling agents or activators include, but are not limited to: dicyclohexylcarbodiimide (DCC); O-(7-azabenzotriazol-1-yl)-1,1,3-,3 - tetramethyluronium hexafluorophosphate (HATU); and benzotriazol-1-yl-oxytripyrrolidinium phosphonium hexafluorophosphate (PyBOP). These reactive acid groups then react with free amines in the presence of a basic proton sink, diisopropylethylamine (DIEA), finally leading to a final elimination reaction to remove HBTU and water, leaving behind a stable amide bond.

Metal and metal alloy oxide surfaces can be modified with various amino-silanes for biological applications. These modifications can be used to attach different peptide hydrophilic lipid molecules or self-assembled micelles to oxide surfaces. For example, incubation of a TiOz-passivated titanium surface with the desired amino-silane generates Ti-O-Si bonds at the oxide-solvent interface, covalently linking the aminosilane to the oxidized metal surface. This arrangement leaves free amines exposed for standard amide-coupling reactions with suitable peptide hydrophilic lipid molecules; Covalently attached to the silanized Ti surface.

In one embodiment of the invention, standard amide coupling reactions are used for preassembled, cross-linked peptide nanofibers. For example, dilute solutions of peptide hydrophilic lipid molecules, which meet the compositional requirements as described above and are kept in a solution of a weak reducing agent such as dithiothreitol (DTT), self-assemble under acidic conditions to form peptide nanofibers . These nanofibers are crosslinked by the addition of non-destructive oxidizing agents, such as iodine, to form stable intermolecular and intrafiber disulfide bonds. The resulting suspension of these fibers was dialyzed against water to remove any reducing or oxidizing agents (such as DTT and iodine). This dialyzed suspension of crosslinked fibers is then lyophilized, and the dried fibers are resuspended in a peptide-soluble polar organic solvent such as DMIF or NMP by vigorous stirring and sonication. Covalent crosslinking of the fibers stabilizes them in non-aqueous environments.

The matrix is preferably a biocompatible material, which may include, but is not limited to, commercially pure titanium, titanium alloys, or other metals such as chromium and its alloys, stainless steels such as Hastalloy, 316L, and 304, and exhibit an oxide surface that can be used in Organic non-polar solvents, organic polar solvents and finally ultrasonic cleaning in distilled water. The cleaned metal or alloy is then etched, for example, in mild hydrofluoric acid, followed by repassivation in nitric acid. Passivated substrate samples were rinsed thoroughly in distilled water and dried. The cleaned passivated samples were then dehydrated by vacuum drying and stored above room temperature prior to aminosilylation. The dried passivated surface is introduced into a dilute solution of aminosilane, such as aminopropyltriethoxysilane (APTES) dissolved in an anhydrous hydrophobic organic solvent such as toluene, under a nitrogen atmosphere. Amino-silanized metal substrates are then thoroughly rinsed in organic non-polar solvents, organic polar solvents and finally in water before annealing at elevated temperature (eg 100°C) under inert gas. Matrices may also include, but are not limited to, biocompatible polymers, or various carbides, borides, and nitrides.

In another coupling agent embodiment, a metal with _an oxidized surface is immersed in a solution of _CaCl2 and _Na2HPO4 , or other similar salt, to pre-seede the surface with calcium phosphate. The matrix of pre-seeded crystals was then immersed in _a solution comprising poly(L-lysine), _CaCl2 and _Na2HPO4 . The samples were washed with water and dried at room temperature. Poly(L-lysine) was incorporated into the mineral phase of the resulting newly formed calcium phosphate coating, and free amines from the poly(L-lysine) side chains were displayed on the structured coating surface. Other minerals can be used in place of calcium phosphate, such as but not limited to calcium carbonate. Many different amines or polyamines, organic acids or polyorganic acids can be incorporated into minerals. Any such amines or polyamines (including poly(L-lysine)) can be physically adsorbed, chemisorbed or covalently grafted onto the passivated metal surface. See later discussion, Figures 1-11 and Examples 2-5 Amino acids and polyamic acids can also be used to treat the surface as described in U.S. Pat. Acids, can be used to oxidize the metal surface (whose oxide has been removed), thereby forming a direct bond there. This would be a way to couple amino acids, peptides, proteins or poly(amino acids) directly to the metal.

The second surface can be terminated with carboxylic acid groups as coupling agents. For example, 3-mercaptopropionic acid can be used to derivatize some metal surfaces with carboxylic acid groups. Alternatively, polymeric materials such as polyethylene can be oxidized to provide a carboxylic acid terminated surface. These carboxylic acid-terminated surfaces can react with amine- or hydroxyl-bearing peptide hydrophilic lipid molecules and bind them to a second surface.

In another embodiment, for example, two sets of peptide-hydrophilic lipid molecular fibers can be independently self-assembled, cross-linked, dialyzed, lyophilized and suspended in a solvent. One set of nanofibers is rich in carboxyl functional groups, while another set can be rich in free amines. These individual nanofibers can be bound together if combined in the presence of HBTU and DIEA. This application can be used to combine different amino acid sequences that can work synergistically with each other. This application can be further combined with metal surface modification, where a peptide-hydrophilic lipid molecular fiber type can be attached to the surface as described above, and a complementary fiber type can be attached to those attached fibers to form a bilayer of different covalent connected nanofibers. Another embodiment of the method described above includes the use of a metal surface other than titanium. It is reasonably contemplated that aminosilylation may be performed on any surface that presents a suitable oxide, including but not limited to titanium alloys, silicon, tantalum, chromium and alloys containing chromium (including stainless steel). Various ceramic secondary matrices are also useful in this regard, including alumina and various forms of silica.

Peptide-hydrophilic lipid molecules and their self-assembling nanofibers promote cell adhesion and growth on their surfaces. For example, the cell adhesion ligand RGD has been found elsewhere to play an important role in integrin-mediated cell adhesion. Peptide-hydrophilic lipid species with acidic amino acids and amino acids with RGD ligands can be used to mediate cell adhesion to peptide-hydrophilic lipid molecules, their self-assembled nanofibers or micelles or nanofibrous gels. The amino acid sequence IKVAV has been identified in other papers to be important for neuronal growth and development. Thus, peptide-hydrophilic lipid species with acidic amino acids and IKVAV sequences can be used to practice embodiments of the present invention to mediate neuronal growth to peptide-hydrophilic lipid molecules, their self-assembling nanofibers, micelles or nano Fiber gel. The amino acid sequence YIGSR has been identified in other papers to be important in promoting cell-matrix adhesion between nerve cells and may also play a role in axon guidance. Therefore, peptide-hydrophilic lipid molecular species with acidic amino acids and YIGSR sequences can be used to practice embodiments of the present invention to promote cell-matrix adhesion between nerve cells to peptide-hydrophilic lipid molecules, and their self-assembling nano fibers, micelles or their nanofibrous gels.

For example in dentin, the phosphoprotein family contains many repeats of the amino acid sequences Asp-Ser(P)-Ser(P) and Ser(P)-Asp-Ser(P). These massively phosphorylated proteins are suspected to play a role in hydroxyapatite mineralization. Thus, phosphoserine residues can be incorporated into peptide sequences that, after self-assembly, allow fibers to display a hyperphosphorylated surface similar to that presented by long peptide fragments. This peptide partially acquires the repeating organization of phosphate groups found in phosphoproteins.

Various C- or N-terminated peptide-hydrophilic lipid molecules useful in practicing embodiments of the invention can be prepared on an automated peptide synthesizer using standard fluorenylmethoxycarbonyl chemistry. A solution of peptide hydrophilic lipid molecules can be formed into nanofibers by changing the pH, adding salt, or by adding charged or chelated peptide hydrophilic lipid molecules. Typical peptide hydrophilic lipid molecules useful in embodiments of the invention are shown in Tables 1-3 below. Peptide hydrophilic lipid molecules such as those listed in Tables 1-3 form nanofibers as described in Hartgerink, et al, Science, 294, 1683-1688, (2001), and Hartgerink et al., PNAS, 99, 5133-5138 , (2002); the contents of which are included by reference in their entirety. Other peptide hydrophilic lipid molecules can be prepared as known to those skilled in the art, using known methods and synthetic techniques or directly modifying them depending on the desired hydrophilic lipid molecule composition or peptide sequence. For example, the peptide hydrophilic lipid molecules provided herein can be prepared as described in co-pending applications Serial No. 10/294,114 filed November 14, 2002 and Serial No. 10/368,517 filed February 18, 2003, characterization and/or assembly, each of which is incorporated herein by reference in its entirety. Without limitation, the peptide hydrophilic lipid molecules incorporated into the application, as described in their corresponding Tables, Figures and Examples, may also be used in the complexes and methods of the present invention. Table 1 PA N-terminal Peptides (N to C) C-terminal 1 C16 CCCCGGGS(P)RGD h 2 C16 CCCCGGGS(P) h 3 C12 CCCCGGGS(P)RGD h 4 C10 CCCCGGGS(P)RGD h 5 C14 CCCCGGGS(P)RGD h 6 C10 GGGS(P)RGD h 7 C16 GGGS(P)RGD h 8 C16 AAAAGGGS(P)RGD h 9 C10 AAAAGGGS(P)RGD h 10 C16 CCCCGGGS(P)KGE h 11 C10 AAAAGGGS(P)KGE h 12 C16 AAAAGGGS(P)KGE h 13 C22 CCCCGGGS(P)RGD h 14 C16 CCCCGGGSRGD h 15 C16 CCCCGGGEIKVAV h 16 C16 CCCCGGGS(P)RGDS h

Phosphorylated moieties may not be required depending on the desired cell or tissue growth. As discussed above, specific sequences of peptide components promote cell adhesion or interaction. Referring to 10-12 and 15 of PA, non-RGD sequences may be utilized depending on the cellular target. In particular, IKVAV sequences have been identified in other papers as being important for neuronal growth and development. Therefore, the hydrophilic lipid molecule composition of the present invention may include peptide components having such sequences for corresponding uses. Finally, according to Table 1, it should be noted that several PA compositions do not include cysteine residues. Although the cysteine amino acid can be used to enhance the stability of intermolecular nanofibers, it is not essential for the self-assembly of micelles or nanofibers, nor is it necessary for the binding of peptide-hydrophilic lipid molecules or their micelles to a second surface. not necessary. In a preferred embodiment, the cysteine amino acid is present to stabilize the self-assembling micelles or nanofibers during the peptide coupling reaction.

Triblock bola hydrophilic lipid molecules that self-assemble into fibers and micelles are also useful in the practice of the invention.

In one embodiment, an aqueous solution of one or more hydrophilic lipid molecule compositions described herein is added with a factor or agent sufficient to induce gelation under physiological conditions. Such gelation and/or self-assembly into nanofibers of various PA compositions can be achieved by drying at substantially neutral pH conditions, introducing multivalent, divalent or higher valent metal ions, chelating combined, and/or combined with differently charged hydrophilic lipid molecules. Table 2 PA N-terminal Peptides (N to C) C-terminal Net charge at pH7 17 C16 CCCCGGGS(P)RGD COOH -3 18 C16 AAAAGGGS(P)RGD COOH -3 19 C10 AAAAGGGS(P)RGD COOH -3 20 C16 CCCCGGGSRGD COOH -1 twenty one C16 CCCCGGGEIKVAV COOH -1 twenty two C16 CCCCGGGKIKVAV COOH ₂ +1

Electrodes, stents, stents, or surgical or other secondary surfaces can be coated in a variety of ways with nanofibers or micelles comprising peptide hydrophilic lipid molecules. The second surface, which contains amine or carboxylic acid groups on its surface, can be placed in a suspension of pre-self-assembled peptide hydrophilic lipid molecule nanofibers or micelles that have been dialyzed. Alternatively, a small sample of the nanofiber gel can be applied to an electrode, stent, stent or surgical device for a period of time and then washed with a solvent to remove excess gel. A solution of peptide hydrophilic lipid molecules can also be sprayed or aerosolized onto a substrate to coat the substrate, which is then exposed to acidic vapors to form nanofibers or micelles.

Alternatively, electrodes, stents, stents, and surgical devices are placed in a volume of peptide-hydrophilic lipid molecules, removed, and exposed to acidic vapors, immersed in a saline solution, or a solution containing peptide-hydrophilic lipid molecules to form nanofibers. Coatings on the second substrate can be performed in combination with these methods and can be repeated as necessary to ensure sufficient coating for the intended use. The coated substrate is then treated with eg HBTU and DIEA in NMP to couple the peptide hydrophilic lipid molecules to the second surface.

Exposure of this coating matrix with cysteine amino acids in nanofibers to oxidizing agents such as oxygen, iodine, hydrogen peroxide or ozone can be used for covalent capture and formation of disulfide bonds. This coating can provide thermal stability to nanofibers coated on scaffolds and devices, which can then be heated to enhance cell growth rates.

Other compounds can be incorporated into or encapsulated in the core of self-assembling peptide hydrophilic lipid molecules that make up the coating. These compounds enhance blood vessel ingrowth following implantation or delivery of the nanofiber-coated secondary matrix into the body. Nutrients, growth factors, inducers of differentiation or dedifferentiation, immunomodulators, inhibitors of inflammation, biologically active compounds that enhance or permit ingrowth of lymphatic networks or nerve fibers, and drugs, can also be incorporated into self-assembling peptides. Hydrolipid molecules in the nanofiber coating. A number of agents affecting cell proliferation have been tested as pharmacological treatments for stenosis and restenosis in an attempt to slow or inhibit smooth muscle cell proliferation. These compositions may include heparin, coumarin, aspirin, fish oils, calcium antagonists, steroids and prostacyclins. Such agents may be systemically encapsulated in fibers, or alternatively may be delivered on a more localized basis using drug delivery catheters. In particular, biodegradable peptide hydrophilic lipophilic nanofiber matrices containing one or more drugs can be implanted at the treatment site. Drugs are released directly at the treatment site as the nanofibers degrade.

Many cells can be grown on electrodes, stents, stents, surgical devices coated with self-assembling peptide-hydrophilic lipid molecule nanofibers. The stent or surgical implant coating consists of self-assembling peptide hydrophilic lipid molecules with peptides selected for optimal growth of that particular type of cell. For example, peptide hydrophilic lipid molecules with RGD, IKVAV, KGE, RGDS peptide sequences, and self-assembled nanofibers composed of them or their combinations may be optimal for cell growth.

Examples of cells suitable for implantation include, but are not limited to, hepatocytes and bile duct cells, islet cells of the pancreas, parathyroid cells, thyroid cells, cells of the adrenal-hypothalamic-pituitary axis including hormone-producing germ gland cells, epithelial cells, Nerve cells, cardiomyocytes, blood vessel cells, lymphatic vessel cells, kidney cells, intestinal cells, bone-forming cells, cartilage-forming cells, smooth muscle-forming cells, and skeletal muscle-forming cells.

The second surface should be shaped to maximize the surface area to allow adequate diffusion of nutrients and growth factors to the cells attached to the self-assembling peptide hydrophilic lipid molecules. Adequate diffusion through densely packed cells, where nutrients and oxygen diffuse from blood vessels into surrounding tissue, can occur in the range of approximately 200 to 300 microns under conditions similar to those found in the body.

In the present invention, cells may initially be cultured using techniques known to those skilled in the art of tissue culture. However, once the cells have begun to grow and cover the self-assembling peptide-hydrophilic lipid molecule-coated electrodes, stents, stents or surgical devices, they are implanted into the patient at sites suitable for attachment, growth and function. One advantage of the biodegradable SAP-lipophilic coating on scaffolds is that angiogenic compounds can be directly incorporated into the SAP-lipophilic nanofibers so that as the nanofiber coating degrades in vivo , they are slowly released. As the cell-self-assembling peptide hydrophilic lipophilic nanofibrous structure is vascularized and degraded, the cells will differentiate according to their intrinsic properties.

Secondary structures, such as porous scaffolds, can be coated with self-assembling peptide-hydrophilic lipid molecule nanofibrous compositions, which can be prepared in vitro for implantation to generate functional organ tissues in vivo. Scaffolds are three-dimensional structures coated with nanofibers of self-assembling peptide-hydrophilic lipid molecules, which can be biocompatible, biodegradable, or non-biodegradable. Examples of such scaffolds include porous ceramic materials available from Porex Corporation, Fairburn, GA; Mykrolis Corporation, Billerica, MA, and Robocasting, Albuquerque, NM. Nanofibers or micelles have peptide hydrophilic lipid molecules with amino acids that induce and support cell growth and attachment. Cells derived from different tissues attach to the surface of the fibers in vitro and uniformly throughout the nanofiber-coated scaffold in amounts effective to generate functional tissue, preferably in vivo. Alternatively, tissue or cells are grown in vitro on a scaffold coated with self-assembling peptide hydrophilic lipid molecule nanofibers in a nutrient solution to form a cell-scaffold composition that is implanted in a patient with sufficient vascularization, to allow blood vessels to grow into cell-scaffold compositions. Growth factors, compounds that stimulate angiogenesis, and immunomodulators can be bound to the nanofibers coating the cell-scaffold composition. Combinations of peptide-hydrophilic lipid nanofiber cell-scaffold compositions comprising different cell populations can be implanted.

If appropriate, immunosuppressant drugs may be injected at the site of the second surface or stent, implant or electrode. Alternatively, immunosuppressant drugs can be incorporated into self-assembling nanofibers or micelles that coat stents or surgical implants.

Under certain conditions, the body naturally produces another drug that, among its many effects, affects apoptosis. As explained by Amin et al. in U.S. Patent No. 5,759,836, which is incorporated herein by reference in its entirety, nitric oxide (NO) is produced by nitric oxide synthase, an inducible enzyme that is beneficial to arterial homeostasis. protein family. However, the role of nitric oxide in regulating apoptosis is complex. The proapoptotic effect appears to be associated with pathophysiological conditions in which inducible nitric oxide synthase produces large amounts of NO. Instead, the anti-apoptotic effect arises from the continuous low-level release of endothelial NO, which inhibits apoptosis and is thought to contribute to the anti-atherogenic function of NO. The pro- and anti-apoptotic effects of nitric oxide are discussed by Dimmeler in "Nitric Oxide and Apoptosis: Another Paradigm for the Dual Action of Nitric Oxide" (Nitric Oxide 14:275-281, 1997). Self-assembling peptide hydrophilic lipophilic nanofibers encapsulating nitric oxide synthase can be used to coat implanted surgical devices such as stents.

In one embodiment, a stent or surgical implant is coated with nanofibers composed of the peptide hydrophilic lipid molecules in Table 1 and Table 2. Stents, stents, electrodes, or surgical devices may be formed from any suitable substance, such as those known in the art, which may be adapted (e.g., molded, embossed, woven, etc.) to incorporate desired surface features . Preferred scaffolds and stents are formed from materials comprising metal, ceramic or polymeric fibers that are uniformly laid down to form a three-dimensional nonwoven matrix and sintered to form a labyrinth structure exhibiting a high porosity, typically in the order of about percent In the range of 50 to about 85 percent, preferably at least about 70 percent. Scaffold fibers generally range in diameter from about 1 micron to 25 microns. The average effective pore size in the secondary structure can be of a size to increase cell ingrowth into the pores and interstices, eg, the average diameter is in the range of about 1 micron to about 100 microns.

Substrate surfaces (i.e., electrodes, surgical devices or implants, stents or stents) coated with self-assembling peptide hydrophilic lipid nanofibers can be formed from biocompatible materials including metals and alloys , such as stainless steel, tantalum, nitinol, corrosion-resistant hairspring alloys; ceramics such as sapphire or silicon nitride, polymers such as polytetrafluoroethylene, PFA or polyethylene; or combinations of these materials. Scaffolds and/or nanofibers can be biodegradable or non-biodegradable. Scaffolds or stents can be made entirely of self-supporting and molded nanofiber gels; for suitable applications, the nanofiber gels can be degradable. Coated stents or implants can be coated with extracellular components such as collagen, fibronectin, laminin and complex mixtures of these components. Non-degradable materials are especially useful when cells are grown in culture for purposes other than implantation, as the preferred matrix structure can provide fixed cell densities higher than normally achievable when nutrients are supplied only by diffusion . Stents, stents, or surgical implants can be formed from biocompatible, non-porous polymers, or by incorporating soluble salt particles before they solidify, and then dissolving the salt particles to leave a vacuum and Gaps are made porous by polymer formation. Polymers can be biostable or bioabsorbable, such as many medical grade plastics including, but not limited to, high density polyethylene, polypropylene, polyurethane, polysulfone, nylon, and polytetrafluoroethylene. Porous polymeric stents can be formed by methods known in the art to have pores with an average diameter in the range of about 30 microns to about 65 microns.

The biological signal presented by the self-assembling peptide hydrophilic lipophilic nanofibers must be appropriate to the type of cell or tissue to be implanted and to maximize the exposure of the cell to the surrounding environment. It must also be designed to enhance the cells' ability to promote vascularization and invasion of scaffolds or tissues.

In one embodiment of the invention, a stent is coated with self-assembling peptide hydrophilic lipid molecule nanofibers. A coated stent body may be formed from a biocompatible polymer or biocompatible metal with surface features molded or molded into the surface. Suitable resiliency should be provided to the stent for in vivo manipulation, as is known to those skilled in the art. For example, the stents of the present invention may be formed from a porous biocompatible material, such as a porous matrix of sintered metal fibers or a polymer, wherein the pores are sized to promote tissue formation of ingrowth cells therein. Self-assembling peptide-hydrophilic lipophilic nanofibers are applied to the surface and/or through pores of polymers or metals.

When the coated stent is cultured in a cell-rich medium, or when the coated stent is implanted into a blood vessel or other tubular body cavity of an individual such as a mammal, the self-assembling peptide is hydrophilic The lipid nanofiber-coated stent body is designed to promote the infiltration and colonization of living cells on the stent. Furthermore, the surface features in the coated stent body were selected so that the living cells that infiltrated and colonized the self-assembling peptide hydrophilic lipid molecule nanofiber-coated stent underwent cell growth in a specific mode, It is determined by the placement and size of the surface features of the coated stent body. An example of such a predetermined pattern of cell growth is angiogenesis and/or neovascularization.

If, as known in the art, the coated surface is cultured in a cell-rich medium (e.g., 6-10 x ¹⁰⁴ endothelial cells in 0.8 ml medium) under cell culture conditions, perforated self- Assembled peptide-hydrophilic lipid-molecule nanofiber-coated surfaces (ie, stents, electrodes, or scaffolds) can readily grow living cells. Such cell culture methods are described, eg, in DADichek, et al., supra, which is hereby incorporated by reference in its entirety. The self-assembling peptide-hydrophilic lipid nanofiber-coated surface or matrix with such pores can be readily infiltrated by cells from the surrounding cellular environment to create an organized cellular structure similar to the surrounding body environment.

The surface of a matrix (ie, an electrode, stent, stent, or surgical device), which may contain a layer of biocompatible substance that expands or thickens in an aqueous environment to assume a three-dimensional form, wherein the layer covers the matrix at least a portion of the surface. For example, the biocompatible material can be or include one or more hydrogels such that when the hydrogel layer is exposed to an aqueous environment it absorbs water and swells to create a porous three-dimensional layer. Alternatively, the hydrogel may further comprise peptide hydrophilic lipid molecules or self-assembling peptide hydrophilic lipid molecules. In the case of a stent, expansion of the hydrogel and peptide nanofibers supports the surrounding tissue and provides sites for endothelial cell growth.

After placement in the host individual's body where it is needed, autologous cells naturally invade self-assembling peptide-hydrophilic lipid molecule nanofiber-coated matrices (stents, stents, electrodes, or surgical devices) and spontaneously generate organized The organized cellular structure changes according to the cellular composition of the body site where the matrix is implanted. For example, in a cell culture laboratory, prior to implantation, endothelial or other suitable cells can be invaded into self-assembling peptide hydrophilic lipid molecule-coated stent scaffolds to produce living nanoparticle scaffolds using methods known in the art. Fiber-coated stent. For example, a living peptide hydrophilic lipid molecule nanofiber coating matrix can be obtained according to the present invention, wherein the peptide hydrophilic lipid molecule nanofiber coating matrix grows cells selected from endothelial cells, smooth muscle cells, leukocytes, monocytes, epithelial cells, Polymorphonuclear leukocytes, lymphocytes, basophils, fibroblasts, stem cells, epithelial cells, eosinophils, etc. and living cells of combinations of any two or more thereof.

Typical intravascular stents have outer diameters in the range of about 2.0 mm to about 6.0 mm and wall thicknesses in the range of about 0.1 mm to about 12 mm, such as about 0.1 mm to about 1.0 mm. Of course, the particular size will depend on the anatomy where the stent is to be implanted. Stents may be expandable, such designs are disclosed, for example, in US Patent No. 5,059,211, incorporated herein by reference, which describes expandable stents made of porous polymeric materials. Stents are delivered through a catheter.

An advantage of this method is that it provides a method for the selective transplantation of parenchymal cells with the necessary biological functions without bypassing the transplantation of leukocytes and antigen presenting cells. The result is that the risk of tissue rejection is greatly reduced without the use of drugs. The present invention has another advantage over other methods for treating loss of organ function, because cells can be manipulated when in culture to introduce new genes to produce deficient protein products, or they can be modified to inhibit The antigen is expressed so that when cells of the same HLA histotype are not available, immunosuppression is not required.

The self-assembling peptide hydrophilic lipophilic nanofiber coated matrices (stents, electrodes, stents) of the present invention can be implanted using any surgical technique known in the art, depending on the particular body organ to be treated.

In embodiments of the therapeutic methods of the present invention, the ingrowth of living cells in the second matrix coated with the self-assembling peptide hydrophilic lipid molecule nanofibers may encapsulate beneficial bioactive agents. For example, coated nanofibers can encapsulate autologous cells of the individual implanted in the matrix, cells that naturally produce the desired bioactive agent seeded in the matrix prior to implantation, or genetically modified to produce the desired bioactive agent cells. Living cells capable of naturally producing one or more bioactive agents useful in practicing the methods of the invention include endothelial cells, smooth muscle cells, leukocytes, monocytes, polymorphonuclear leukocytes, lymphocytes, basophils, fibroblasts , stem cells, epithelial cells, eosinophils, etc., and suitable combinations thereof. Such cells may be donor or autologous cells.

Alternatively, nanofiber-encapsulated cells or compounds in coatings for use in embodiments of the therapeutic methods of the invention can be engineered to express and release bioactive agents in response to delivering the appropriate compound to the patient , so that the recombinant gene product is delivered to the site of the second substrate of the implant coating.

Nerve growth can also be promoted using, for example, electrodes or other surfaces coated with nanofibers of self-assembling peptide hydrophilic lipid molecules comprising the appropriate neuronal growth peptide sequence. After the nerve has grown along the length of the fiber, the construct is implanted at the appropriate location extending from the nerve's origin to the area where the nerve's function is desired.

In a variation of the method of using scaffolds or surgical implants with a single coating of nanofibers for attachment of one or more cell lines, the coated scaffold is constructed with a coating of different self-assembling nanofibers so that for each Population, initial cell attachment and growth occur separately. A single scaffold can also be formed from different materials to optimize the attachment of various cell types. Attachment is a function of cellular and structural composition. For example, coating surgical implants with nanofibers composed of collagen-like peptide hydrophilic lipid molecules with phosphorylated amino acid and RGD peptide sequences increases cell adhesion. In another example, self-assembling peptide hydrophilic lipophilic nanofibers (with phosphorylated amino acid and RGD peptide sequences), can be coated onto a biodegradable scaffold. After stent implantation and degradation, vascular cells form the appropriate junctions for delivering blood to the desired location. Pipelines for organ excretion can be constructed in a similar manner, often exploiting behaviors inherent to cells. Also promotes ingrowth of the lymphatic network and nerve fibers.

Optionally, the cells for growth on the nanofiber-coated surface or scaffold may be obtained from a donor or host individual, treated, and cultured in vitro on the nanofiber-coated scaffold prior to introduction into the individual. In presently preferred embodiments, the transplanted cells are "autologous" to the individual, meaning that the donor and recipient of the cells are the same.

According to embodiments of the methods of the invention, bioactive agents suitable for delivery by self-assembling peptide-hydrophilic lipid molecule nanofibers encapsulated in coated stents, electrodes, stents, or surgical devices, including those utilized by mammalian organisms Those bioactive agents that stimulate angiogenesis include those that modulate capillary formation in wounds, attracting smooth muscle to coat and support capillaries. Examples of such bioactive agents that can be encapsulated in coated nanofibers include vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), especially FGF-1, angiopoietin 1, thrombin ,etc. Additional examples of bioactive agents suitable for delivery according to the methods of the invention include antiproliferative, antirestenotic or apoptotic agents, such as platelet-derived growth factor-A (PDGF-A), transforming growth factor beta (TGF-beta) , nuclear factor-Kβ (NF-Kp), an inducible redox-controlled transcription factor, and the like.

The methods described in this specification can be used to deliver specific biofunctional peptide sequences to biomaterials or other surfaces, which can activate or modify different biological responses. Such responses may include selective binding to peptides bound to matrices or biomaterials, improved or increased cell proliferation, or even selective degradation of bioscaffolds. This scheme could even be used for drug delivery. Drugs, or other therapeutic molecules, can be incorporated into stable micelle assemblies, or they can be chemically bound to the nanofiber surface. It can be expected that these methods will have great potential for application in fields including bone repair, dental restoration, and cardiovascular stent modification.

As noted above, the methods and compositions of the present invention may also provide for the growth of nanocrystals or crystalline differential phases for normally crystalline materials in monolithic form or, more preferably, as coatings on substrates. The nanocrystalline phase is formed by contacting a substrate pre-seeded with a mineral onto its surface with a solution comprising dissolved crystalline material and an additive that is incorporated into the crystalline material of the coating and reduces the size of the crystalline domains of the material . The additives provide nanocrystalline morphology and also provide additional reactive functionality for chemically reacting the coating with other molecules. The compositions provide increased surface coverage of coated substrates, especially those with small features such as pores and channels. The coated substrate can be used for in vitro or in vivo cell growth on the nanocrystalline coated substrate material.

The composition of the present invention is preferably a solution which includes, but is not limited to, organic additives such as polyamines or acid addition salts thereof, and dissolved components of materials. The constituents of crystalline materials can be molecules or ions. The solution should be capable of dissolving the crystal components as well as additives. The solution may be an aqueous solution, an organic solution, or a combination thereof, which may include organic liquids such as ethanol, amines and their acid addition salts, amino acids, surfactants, and soluble components of crystalline materials.

Organic additives in the composition may include polyamines, acids or their salts, wherein the additives disrupt crystal growth and result in the formation of nanocrystalline phases of normally crystalline materials. The additive can be chosen to control its reactivity to coating degradation. Other additives may be poly(amino acids), or other polymers with side groups such as carboxylic acid, sulfonic acid, phosphoric acid, amine groups, thiols, hydroxyl groups, or combinations of these groups. These groups in the polymer can be used to conjugate other biologically relevant molecules such as peptides via disulfide, amide or peptide bonds. The concentration of polymer or its salt in solution may be less than about 100 millimolar, preferably 10-20 mM, and the concentration may be used to control the morphology of the coating. It is expected that lower concentrations of additives will cause less disruption of crystalline morphology than higher concentrations of organic additives. The polymers used in the present invention may be derived from natural sources, prepared by solid phase synthesis techniques known to those skilled in the art, or they may be purchased from suppliers such as Aldrich Chemical, Milwaukee WI.

Preferably, as shown in FIGS. 3A and 3B , a coating on a substrate in which organic additives are incorporated results in a material with a smaller feature than that deposited from a solution of the material onto a substrate without organic additives. morphological components formed above. Preferably, the coated features are less than about 2000 nanometers in size. The thickness of the coating on the substrate may be less than about 50 microns, preferably less than about 10 microns, more preferably less than 1 micron. Thinner coatings provide for cell attachment, and reduce clogging of small pore features in porous substrates such as titanium or tantalum biofoams.

Preferably, the addition of organic additives will affect crystal formation to produce nanocrystalline or poorly crystalline mineral phases. This property makes the coating material especially susceptible to acid degradation during cellular reconstitution. Alternatively, the coating material may be susceptible to enzymatic attack by biological enzymes such as but not limited to pronase and trypsin under physiological conditions. When the organic components of the mineral complex are digested by the enzyme, the enzyme destroys the coating. It is desirable that additives incorporated into the coating be sensitive to these two major degradation methods, acidic and enzymatic, in order to facilitate the natural bone remodeling process in vivo. Susceptibility of different organic additives in material coatings to acid or enzymatic digestion, which can be measured by coating morphology (e.g., by scanning electron microscopy) during treatment of substrates of prepared coatings with biologically active enzymes or physiological solutions and chemical (eg, X-ray photoelectron spectroscopy) changes over time. Mineral by-products from these degraded coatings are expected to be useful raw materials that can be used to form new mineralized structures.

The materials used for the coating are soluble in the solution. Inorganic materials useful for such coatings may include, but are not limited to, hydroxyapatite, fluoroapatite, fluoroapatite carbonate, hydroxyapatite carbonate, and combinations of these. Also useful are calcium phosphates, calcium oxalates, calcium carbonates and combinations of these inorganic materials. Calcium phosphates may include, but are not limited to, dicalcium phosphate dihydrate, octacalcium phosphate, magnesium substituted calcium phosphate. Inorganic ions such as, but not limited to, Zn ⁺² or Mg ⁺² , may also be associated with Ca ⁺² salts to be pre-seeded or incorporated into the coating. These inorganic materials, and salts of these materials, are available from natural sources or from chemical suppliers such as Aldrich Chemical, Milwaukee WI. The concentration of each component of the coating material in solution is preferably less than about 100 millimolar.

The temperature of the coating solution can be used to control the speed and morphology of the coating process. The temperature of the solution should not degrade the organic polyamine. The temperature may be below about 75°C, and is preferably in the range of about 5°C to 40°C.

The substrate to be coated is preferably a biologically compatible material and may comprise polymers, metals, metal alloys, ceramics or combinations of these. Before coating, the substrate preferably has the shape for its intended use. Examples of implants may include hip and knee implants, plates and nails for fractured bones, dental implants, and other reconstructions. The substrate used in the practice of the present invention may have an oxide surface, a hydroxide surface, or a combination of these groups coating at least a portion of the surface of the substrate. The coating preferably has a surface that contains functional groups that allow the nucleation of a seed layer of mineral or other material to be deposited onto the surface. Examples of functional groups in the surface include, but are not limited to, oxides, hydroxides, phosphates and carbonates. Metals and alloys useful in the practice of the present invention may include, but are not limited to, titanium and its alloys, surgical steel, amalgam, Co-Cr alloys, tantalum or silicon and silicon-based materials. The matrix is preferably an alloy of titanium alloys, an example of which is the titanium alloy known as Ti-6Al-4V, which is used in orthopedic and dental implants. The metal or alloy can be a bulk material, a porous foam, or a coating or deposit as an attached film on another substrate such as a ceramic. Suitable ceramic materials exhibit oxide and hydroxide functional groups, such as alumina, sapphire and calcium phosphate ceramics such as sintered apatite.

Pre-seeding of the substrate may be performed using a component of the coating composition or a substance structurally similar to it. The substrate may be pre-seeded with coating material by contacting the substrate with a coating solution without organic additives. For example, a solution of a planting composition _{of CaCl2} and _Na2HPO4 may be used to contact _the substrate, which is then coated with a solution comprising _CaCl2 , _Na2HPO4 and poly(L-lysine). The substrate is preferably contacted with CaCl ₂ and then with Na ₂ HPO ₄ . It is desirable to pre-seed the substrate to form a seed layer of coating material. The seed coating can also be formed by other methods including, but not limited to, chemical vapor deposition, atomic layer chemical vapor deposition, or spray coating.

A substrate coated with a coating material comprising an organic additive, useful for the growth or attachment of cells, tissue, or for the release of a therapeutic composition. Examples of tissues may include, but are not limited to, bone and dentin. The coated substrate can be used to culture cells or tissues in vitro by placing the coated substrate in a container containing appropriate cells, nutrients, and other reagents for the growth of the cell or tissue. The coated matrix, or matrix with cell culture thereon, can be used to grow or culture cells, tissue, dentin or bone in a patient after implantation. The matrix will be made of a biocompatible material coated with a material modified by incorporating organic additives such as poly(amino acids) into the material.

Substrates coated with materials and organic additives can be further modified to include other molecules, such as but not limited to amino acids, peptides or self-assembling peptide hydrophilic lipid molecules, in combination with the coating. For example, incorporation of pLys into the Ca-P layer also introduces valuable chemical ties for attaching functional biomolecules to the coating. The positively charged free amine side chains of poly(L-lysine) can be used as conjugation linkers, either through electrostatic interactions with negatively charged molecules, or by forming a link between the free amine of lysine and the carboxylic acid on the target molecule. between amide chains. The chemical functionality of the organic additives incorporated into the coating can be used to incorporate biomolecules such as growth factors, peptide sequences or therapeutic drugs. Peptide or self-assembling peptide hydrophilic lipid molecules can also be combined with reactive groups of organic additives such as poly(amino acids) incorporated into the coating material, or by binding molecules or self-assembling hydrophilic lipid molecules to the crystalline material itself. Such linkages include, but are not limited to, amide, ester and disulfide linkages. The peptides combined with the organic additives in the coating preferably comprise amino acid sequences that can be used for attachment of different types of cells. Examples of asymmetric peptides having amino acid sequences useful for attaching different types of cells include, but are not limited to, those in Table 3. Symmetrical peptide hydrophilic lipid molecules, such as those described in US Patent No. 5,670,483 and US Patent No. 5,955,343, which are hereby incorporated by reference in their entirety, may also be used in the practice of the present invention. Bola hydrophilic lipid molecules and self-assembling bola hydrophilic lipid molecules are also useful for incorporation into coatings of the invention. Examples of self-assembling peptide hydrophilic lipid molecules having amino acid sequences associated with the attachment of different types of cells can be prepared from the peptides of Table 3. Table 3 peptide hydrophilic lipid molecules; S (P) represents phosphorylated serine PA N-terminal Peptides (N to C) C-terminal 1 C16 CCCCGGGS(P)RGD h 2 C16 CCCCGGGS(P) h 3 C12 CCCCGGGS(P)RGD h 4 CI0 CCCCGGGS(P)RGD h 5 C14 CCCCGGGS(P)RGD h 10 C16 CCCCGGGS(P)KGE h 11 CI0 AAAAGGGS(P)KGE h 12 C16 CCCCGGGS(P)DS(P)D 13 C22 CCCCGGGS(P)RGD h 14 C16 CCCCGGGSRGD h 15 C16 CCCCGGGEIKVAV h 16 C16 CCCCGGGS(P)RGDS h

Alternatively, the coating on the substrate can also be bound to the peptide or self-assembling peptide hydrophilic lipid molecules via bonds to the coating. The self-assembling peptide hydrophilic lipid molecules bound to the coating on the substrate may further contain encapsulated drugs or therapeutic agents, drugs capable of promoting cell adhesion, growth factors or biologically relevant peptide sequences. The peptide hydrophilic lipid molecules may have amino acids such as sulfhydryl moieties or others for cross-linking to enhance the stability of the self-assembling peptide hydrophilic lipid molecules bound to the substrate coating.

The polymeric structure incorporated into the coating material on the substrate can further incorporate molecules such as but not limited to growth factors, therapeutic drugs, peptides and self-assembling peptide hydrophilic lipid molecules. Binding to molecules can be through van der Waals interactions, ionic bonding, hydrogen bonding or chelation. Alternatively, the coating material on the substrate can be bound to the peptide or self-assembling peptide hydrophilic lipid molecules via a variety of bonds including but not limited to disulfide bonds, preferably amide bonds between ester or polyamines and the peptide. Formation of amide bonds between the polymer and the peptide in the coating can be performed in polar organic solvents such as but not limited to N,N-dimethylformamide (DMF) or N-methylpyrrolidone (NMP) , both of which can dissolve amino acids. The method also includes, using a compound such as O-benzotriazole-N, N, N', N'-tetramethyluronium-hexafluoro-phosphate (HBTU) as a catalyst to enhance the peptide on the hydrophilic lipid molecule. The reactivity of the carboxylic acid functional group. Other peptide coupling agents or activators include, but are not limited to: dicyclohexylcarbodiimide (DCC); O-7-azabenzotriazol-1-yl)-1,1,3-3-tetra methyluronium-hexafluorophosphate (HATU); and benzotriazol-1-yl-oxytripyrrolidinium phosphonium hexafluorophosphate (PyBOP). These reactive acid groups then react with free amines, and then the presence of proton sink diisopropylethylamine (DIEA) facilitates the final elimination reaction to remove HBTU and water, leaving a stable amide bond. The self-assembling peptide hydrophilic lipid molecules can be cross-linked by disulfide bonds prior to binding or attaching to the coating on the substrate.

The material coating the substrate may consist of metal-deficient minerals incorporating additives. The additive incorporated into the coating may be present at up to about 25% by weight or less, and may reduce the size of the crystallites present in the coating compared to the crystallites present in the coating without the additive. Additives may include, but are not limited to, poly(amino acids). For example, the matrix may consist of a calcium-deficient octacalcium phosphate mineral whose crystal growth has been disrupted and modified by the polyamine, poly(L-lysine), present during mineralization. Without wishing to be bound by theory, it may be that poly(L-lysine) is intimately incorporated into the calcium deficient octacalcium phosphate material during the crystallization process. As shown by comparing Figures 3a and 3b, the morphology of the coating obtained from this organic modification consists of irregular features 1-2 orders of magnitude smaller than purely inorganic (octacalcium phosphate) mineral coatings. This increased structure and reduced component size are expected to have favorable effects on cell attachment, proliferation and spreading. Furthermore, the disrupted poorly crystalline nature of the coating, combined with the organic components of the mineral complex which are susceptible to enzyme attack, will make it especially suitable for natural resorption and reconstruction processes. Finally, incorporation of polyamino acids into material coatings provides additional chemical functionality through free amines or acids on side chains. This chemical functionality can be used to incorporate biomolecules such as growth factors, biologically relevant peptide sequences or therapeutic drugs.

One embodiment of the invention is a method of coating a material onto an implantable matrix. The method includes coating a biocompatible substrate having a coating-compatible seed layer with a solution composition of a dissolved coating material and an organic additive. The method may further comprise the steps or acts of preparing a seed layer on the substrate, and binding the molecular or self-assembling supramolecular structure to the coating material on the substrate. The coating composition can be applied to the pre-seeded substrate by methods known to those skilled in the art for contacting the substrate with the composition or coating the substrate with the composition. The substrate can be coated by dipping the substrate in a composition comprising _materials and polymers such as _CaCl2 , _Na2HPO4 and poly(L-lysine). For example, a pre-seeded substrate can be placed in a _CaCl2 solution supplemented with poly(amino acid) acid. A salt such as _Na2HPO4 is then added to this combination and _the sample is incubated. The coating step can be repeated one or more times. For substrates having pores or channels, the substrate is preferably coated by flowing the composition through the substrate, preferably for small pores, or through the substrate. Closed loop flow systems using pumps and composition reservoirs can be utilized with the flow rate controlled by pumps, valves or flow controllers. Alternatively, the substrate can be spray coated using an atomizer or other sprayer. Multi-layer coating of substrates with fresh coating composition produces increased coating thickness and uniformity on the substrate. The coating material preferably contains 1-15% by weight of organic additives.

Figures 3A and 3B illustrate the dramatic effect of polyamines such as poly(L-lysine) on the growth of octacalcium phosphate on pre-seeded biocompatible titanium substrates. The scanning electron micrograph in Figure 3A shows that the purely inorganic coating consists of large (>1 micron), well-formed, plate-like octacalcium phosphate crystals. In contrast, the poly(L-lysine)-modified coating shown in Fig. 3b consists of distorted, irregularly shaped, poorly crystalline components that are 1–2 orders of magnitude smaller than the purely inorganic form of the mineral. Many of the features that make up these materials are smaller than 100 nanometers in scale, giving the materials a truly nanoscale structure. This nanoscale structure, or morphology, facilitates cell attachment and dissemination onto coated substrates or coated monolithic samples. X-ray diffraction of this pLys-OCP coating further illustrates its poorly crystalline nature, which makes the material particularly sensitive to acid degradation pathways during cellular reconstitution. Treatment of pLys-OCP with biological enzymes such as pronase revealed by SEM and energy-dispersive X-ray spectroscopy (EDS) that the coating structure and morphology of the material were disrupted when the enzyme digested the organic components of the mineral complex. Coatings of poorly crystalline organic-material composites are particularly suitable not only for acid degradation but also for enzymatic digestion, the two main modes of natural bone resorption in vivo.

The presence of polyamines, such as poly(L-lysine), not only affects the coating morphology, crystallite size and reconstitution potential, but it also provides chemical functional elements to the system. The side chains of poly(L-lysine) incorporated into mineral coatings contain free amines available for chemical reactions. For example, these amines can form amide bonds with free acids on biologically relevant peptide sequences. Figure 9 illustrates a peptide hydrophilic lipid molecule that can self-assemble into nanofibers, where the aliphatic tail of the molecule is sequestered into the middle of the fiber, and the functional peptide sequence is exposed on the outside of the assembled nanofiber. Cysteine residues in the molecule can be exposed to oxidative conditions, thereby covalently stabilizing the nanofibers by forming intermolecular disulfide bonds. The carboxylic acid exposed on the outside of the molecule can react with free amines from poly(L-lysine) to form amide bonds, covalently linking the peptide hydrophilic lipid molecular nanofibers to the structured plys-OCP surface. Figure 10A shows nanofiber bundles attached to a poly(L-lysine) modified calcium phosphate coating on a titanium surface. In the higher magnification of Fig. 10B it is possible to resolve individual nanofibers (see arrows) which coat the structural components of the underlying (L-lysine) modified calcium phosphate coating. The peptide hydrophilic lipid molecule used in this example mimics phosphoprotein, a dentin-specific protein associated with the control of mineralization in teeth. Of course, almost any peptide-hydrophilic lipophilic nanofiber can be used for this application, as long as it exposes the necessary carboxylic acid for amide bond formation. Conversely, organic additives can be similarly incorporated into free acid exhibiting calcium phosphate coatings to bind free amine exhibiting PA nanofibers. Peptide hydrophilic lipid molecules are an example of supramolecular aggregates that can be attached to the surface of pLys-OCP, but this chemical functionality can similarly be used for the attachment of single molecules or peptide sequences. For example the peptide sequence arg-gly-asp (RGD), which is normally associated with cell attachment, can be coupled to the surface to increase cell attachment to the plys-OCP surface.

A variety of physical and chemical analyzes can be used to characterize coatings prepared by the methods and compositions of the invention. One skilled in the art can use methods such as XRD, RFTIR, XPS, TGA and elemental analysis to determine that additive modified coatings have reduced feature size compared to crystalline coatings without additives. These methods can also be used to determine the effect on morphology of using different amounts of additives. Incorporation of additives into the mineral phase can be evidenced, for example, by disruption of coating crystallinity as observed by XRD and FTIR, as well as by the coating's chemical reactivity or ability to promote cell attachment.

Example 1

Commercially pure titanium or any titanium alloy that presents a titanium dioxide surface is subjected to ultrasonic purification in organic non-polar solvents, organic polar solvents, and finally in distilled water. Purified titanium is etched in mild hydrofluoric acid, nitric acid solution before repassivation in nitric acid. Passivated samples were rinsed thoroughly in distilled water and dried. The cleaned passivated samples were then dehydrated by vacuum drying and stored at 120°C prior to amino-silylation. Under nitrogen, the dry passivated surface is introduced into a dilute solution of an aminosilane, such as aminopropyltriethoxysilane (APTES) in an anhydrous hydrophobic organic solvent such as toluene. Before annealing at 60°C for 1 hour under nitrogen, the amino-silylated titanium substrates were thoroughly washed in organic non-polar solvents, organic polar solvents, and finally in water.

PA nanofibers are covalently bonded to the amino-silanized _TiO2 surface. Add a solution of O-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate (HBTU) and diisopropylethylamine (DIEA) to the N,N - in a suspension of crosslinked nanofibers in dimethylformamide (DMF) to provide slightly less than 1 equivalent (0.95) of DIEA per free carboxylic acid on the nanofibers, and to amino-silanized titanium About 6 equivalents per estimated free amine on the surface. The solution was incubated for several minutes before exposure to the amino-silanized titanium surface. Once introduced, the amino-silanized titanium was shaken in the nanofiber reaction solution for at least 1 hour before being thoroughly rinsed with water and dried at room temperature. Figures 12A and 12B show low and high magnification images of these fibers covalently bound to a Ti surface.

Example 2

Covalent attachment of preassembled peptide nanofibers to poly(L-lysine)-modified calcium phosphate coatings on titanium surfaces. PA nanofiber preparation: Peptide nanofibers were assembled as above, cross-linked, dialyzed, lyophilized and resuspended in DMF.

Calcium phosphate coating preparation: Titanium foils were decontaminated, etched, passivated and cleaned as above. However, instead of drying it and _treating it with APTES, the titanium foil was immersed in a solution of _CaCl2 and _Na2HPO4 for at least 30 minutes to pre-seed the surface with calcium phosphate. This pre-seeded solution was replaced with a solution containing poly(L-lysine, CaCl ₂ and Na ₂ HPO ₄ ) for at least 3 hours before the samples were washed with water and dried at room temperature. It is believed that poly(L-lysine) is incorporated into the mineral phase of the resulting newly formed calcium phosphate coating and that free amines from the side chains of poly(L-lysine) appear on the structured coating surface .

PA nanofibers were covalently bonded to the amino-silanized poly(L-lysine) modified calcium phosphate-coated _TiO2 surface. Add a solution of O-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate (HBTU) and diisopropylethylamine (DIEA) to the N,N - in a suspension of crosslinked nanofibers in dimethylformamide (DMF) to provide slightly less than 1 equivalent (0.95) of HBTU per free carboxylic acid on the nanofibers, and for exposure to lysine-modified Approximately 6 equivalents of DIEA per estimated free amine on the calcium phosphate coated titanium surface. The solution was incubated for several minutes before being exposed to the coated titanium surface. Once introduced, the calcium phosphate-coated titanium was shaken in the nanofiber reaction solution for at least 1 hour before being thoroughly rinsed with water and dried at room temperature. Figure 10A is a scanning electron micrograph showing fiber bundles attached to the structured coating surface. Figure 10B is a higher magnification image revealing individual fiber layers on the architectural structure coated on the calcium phosphate coating.

Example 3

The method of pLys-OCP growth: the titanium surface was continuously purified in organic non-polar solvents, organic polar solvents and water. The cleaned titanium foils were briefly etched in a mild hydrofluoric, nitric acid solution to remove existing surface oxides before repassivation in the more concentrated nitric acid solution used for surface passivation. The acid-treated samples were then rinsed thoroughly with distilled water and placed in a pre-seeding solution consisting of 2 mM CaCl ₂ and 1.2 mM Na ₂ HPO ₄ for at least 30 min at room temperature. Longer exposure times (up to 24 hours) result in better coverage. After pre-seeding, samples were then placed in fresh mineralization solution consisting of 2 mM CaCl ₂ , 1.2 mM Na ₂ HPO ₄ , supplemented with 1 mM poly(L-lysine), and incubated at room temperature for at least 3 hours. This mineralization step can be repeated to increase the coating thickness. Mineralized samples were washed thoroughly with distilled water and dried at room temperature.

Example 4

Chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO). Solvents were obtained from Fisher Scientific, (Hanover Park, IL). Titanium foil was obtained from Goodfellow, Inc. (Berwyn, PA).

Commercially pure titanium (Ti) foil (0.032 mm) was cut into rectangular slices with a size of 5 x 8 mm. Bend one corner of each sample perpendicular to the foil side. The titanium foil was then ultrasonically cleaned in reagent grade methylene chloride, acetone and deionized water for 15 minutes each. The cleaned foils were then etched in 0.25% hydrofluoric acid (HF), 2.5% ( _HNO3 ) for 1 minute before being placed in 40% nitric acid ( _HNO3 ) for 40 minutes for surface passivation. The acid-treated samples were then rinsed thoroughly with deionized water. As shown in Figure 1, the decontaminated passivated samples were placed in the wells of a 24-well tissue culture polystyrene (TCPS) well plate with the folded corners facing down, effectively suspending the underside of the foil above the TCPS surface. This structure ensures that any coating observed beneath the titanium matrix grows directly on the foil surface and is not simply the result of attached precipitation out of solution.

Samples were then pre-seeded by placing them in a solution of 2 mM CaCl ₂ and adding Na ₂ HPO ₄ (final concentration 1.2 mM) at room temperature for a period of 10 minutes to 24 hours. Control pre-seeding solutions included 2 mM CaCl ₂ alone, 1.2 mM Na ₂ HPO ₄ alone, 1.2 mM NaCl alone, 2 mM CaCl ₂ with 1.2 mM Na ₂ HPO ₄ and 1 mM poly(L-lysine) (MW = 37,000). After pre-seeding, samples were then placed in 2 mL of fresh 2 mM CaCl ₂ solution supplemented with 1 mM poly(L-lysine). Na ₂ HPO ₄ was then added (final concentration 1.2 mM) and samples were incubated in this mineralizing solution at room temperature for at least 3 24 hours. Repeat the mineralization process one more time. The pH of these mineralization solutions was monitored with a Fisherbrand electronic pH meter. Mineralized samples were rinsed thoroughly with deionized water and dried by vacuum drying. Dried samples were then examined by X-ray photoelectron microscopy (XPS), reflected Fourier transform infrared spectroscopy (RFTIR), and scanning electron microscopy including energy dispersive X-ray analysis (EDS). XPS was performed using Omicron XPS at 15 kV and 20 mA and spectra were processed using EIS software (v 2.1.0). RFTIR was performed on the coated foil substrates using a Bio-Rad FTS-40 FTIR spectrophotometer (4000-700 cm ⁻¹ , 64 scans, 2 cm ⁻¹ resolution) using blank Ti foil as background. SEM samples were coated with 3 nm gold-palladium before examination in a Hitachi S4500 field emission scanning electron microscope at 20 kV with a Princeton Gamma Tech X-ray detector.

The non-attached pellet was then collected by a series of water washes, followed by centrifugation and lyophilization. The dried precipitate was tested by powder X-ray diffraction (XRD) using a Rigaku D-Max X-ray powder diffractometer at 40 kV and 20 mA. The water and organic content of the dried precipitate was determined by high-resolution thermogravimetric analysis (TGA) using a TA Instruments Hi Res TGA 2950. The sample was heated to 450°C at 3°C/min and held for 120 minutes.

Degradation experiments were performed on foil samples coated with OCP and pLys-CP grown as described above. The samples were rinsed thoroughly in Millipore water and placed in 1 ml of each individual degradation solution for 24 hours before being dried by vacuum drying. Enzyme-based solutions included 0.25% trypsin in Hanks' balanced salt solution (HBSS), and 0.2% pronase in HBSS. Coating degradation was performed by pH change using HBSS (pH 7.4) and citrate buffer solutions at pH 7, 6, 5, 4, 3 and 2. The processed samples were probed by EDS at 20 kV for 100 s in the SEM and then sputter coated with 3 nm Au-Pd for imaging at 20 kV.

Chemical functionality was measured by coupling Boc-S-tert-butylmercapto-L-cysteine (Boc-Cys(StBu)-OH) to free amines exposed on the pLys-CP coating. Both OCP and pLys-CP coatings were prepared as described above. The surface concentration of free amines was measured using a quantitative ninhydrin assay. Briefly, dried samples were treated with a mixture of phenol in ethanol and potassium cyanide in pyridine at 100 °C for 5 min before adding 60% ethanol and washing with tetraethylammonium chloride in dichloromethane. . The absorbance of the resulting purple solution was measured at 570 nm and compared to a standard curve measured from fractionated pLys solutions. Samples not used for the ninhydrin reaction were shaken overnight in 0.4 mL of dimethylformamide (DMF) containing 0.1% Boc-Cys(StBu)-OH, along with 0.95 molar equivalents of 1-H-benzo Triazolium, 1-[bis(dimethylamino)methylene]-hexafluorophosphate (1-), 3-oxide (HBTU) and 0.5 mM diisopropylethylamine (DIEA). Control samples were exposed to cysteine compounds in the absence of HBTU or DIEA. Half of the samples from each set of reaction conditions were rinsed thoroughly in deionized water, while the other half were washed in saturated NaCl solution for 10 minutes before being rinsed thoroughly in deionized water. The cleaned samples were dried and then examined by XPS at 225W (15kV and 15mW).

Calcium phosphate growth responses were followed visually as well as by monitoring the reaction pH. Calcium chloride solutions begin to clear and become colorless at approximately pH 5.8-5.9. Within seconds of adding the phosphate solution, the pH rose rapidly to approximately pH 7.8, producing a fine white precipitate. In a purely inorganic reaction, this suspended precipitate becomes coarser and coarser over the next 3-4 hours and settles in the reaction wells. However, the precipitates in solutions containing pLys remained extremely fine and had a low tendency to settle. Figure 2 shows the change in reaction pH, traced from the equilibrium point after phosphate addition (approximately 1 minute) to 24 hours. The pH trace was characterized by a relatively gradual decrease in pH, interrupted by a single abrupt decrease at exactly 1 hour from approximately pH 7.6 to pH 7.2. Notably, in the reaction solution containing pLys, the significant decrease in pH started earlier and the final pH remained slightly higher than that of the inorganic control.

SEM micrographs of the coatings produced by these reactions are shown in Figure 3. The coating in Figure 3A is purely inorganic, while the coating in Figure 3B has been modified by the incorporation of pLys. Purely inorganic coatings consist of large, thin, plate-like calcium phosphate crystals typically exceeding 1 micron in length and width, with a morphology consistent with octacalcium phosphate (OCP). The thickness of the coating was approximately 4-7 microns (2-3 crystal sizes), and the crystals were oriented both parallel and perpendicular to the sample surface throughout the thickness of the coating. In contrast, pLys-modified calcium phosphates (pLys-CP) consist of deformed, disrupted crystals that are 1 order of magnitude smaller than their inorganic counterparts. The high-magnification inset in Fig. 3B illustrates that these structures are further composed of substructures with sizes smaller than 100 nm, revealing nanoscale features in the modified coatings. The coating, also 2-3 parts thick, is typically 1 micron thick or less, but remains uniform over the entire foil surface.

TGA of OCP precipitation produced a mass change of approximately 9.5±0.2%, which is in good agreement with the expected water loss (9.2%) from hydrated OCP crystals. Analysis of the pLys-modified precipitate showed a similar amount of water loss, but produced a total mass loss of 23±1%, suggesting that the mineral consisted of 14% poly(L-lysine). Elemental analysis showed a total carbon, hydrogen and nitrogen content (by mass) of 14.2 ± 0.2%, confirming the lysine content derived from TGA. In addition, the mass ratio of carbon to nitrogen in the elemental analysis was 2.6, which was consistent with the expected carbon to nitrogen ratio of 2.57 in poly(L-lysine). This consistency rules out the possibility that pLys-CP contains significant amounts of any carbonized calcium phosphate species.

The X-ray diffraction pattern of the pLys-CP precipitate, shown in Figure 4, shows relatively weak, broad diffraction peaks consistent with poorly crystalline calcium phosphate. These broad peaks appear on the OCP crystal diffraction pattern obtained from the inorganic control. Figure 4 illustrates the difference in diffraction spacing for OCP (100), (010) and (002).

Examining the reflectance FTIR spectrum in Fig. 5, the inorganic coating produced bands corresponding to PO ₄ ^3- extensions at 963, 1025, 1037, 1078 and 1115 cm ⁻¹ . In addition, there are distinct bands characteristic of octacalcium phosphate, such as those at 873 and 917 cm ⁻¹ from the P-OH extension in HPO ₄ ²⁻ . In comparison, the pLys-CP spectrum better describes poorly crystalline or amorphous calcium phosphate, with broad PO ₄ ³ -bands at 963, 1025 and 1115 cm ^-1 . The clear HPO ₄ ^2- band in the inorganic sample has been replaced by a single, broad HPO ₄ ^2- band at about 880 cm ⁻¹ . Moreover, the pLys-CP spectrum clearly revealed the presence of poly(L-lysine) in the mineral, via _CH2 and _CH3 bands between 2990 and 2850 cm ^-1 , and a strong _NH2 deformation strip at 1650 cm ^-1 band and NH ₃ ⁺ band at 3073 cm ^-1 indicated. Together, these observations suggest that poly(L-lysine) has been incorporated into calcium phosphate mineral systems and has disrupted the crystallization that naturally forms the octacalcium phosphate phase. Coating and pretreatment analyzes by XPS are summarized in Table 4 below.

Table 4: XPS analysis of calcium phosphate pretreatment and coating coating Binding energy eV (±0.1) Ca:P ratio Ca 2p _3/2 Ca 2p _1/2 P2p Ols Nls Cls OCP 347.2 350.9 132.7 531.0 - 284.8 1.31±0.02 PLys-CP 347.2 350.8 132.8 531.0 400.2 284.8 1.15±0.02 Pre-seeded for 2 hours 347.2 350.8 133.2 531.1 - 284.9 1.30±0.06 Pre-seed for 10 minutes 347.0 350.6 133.2 531.5 - 284.8 1.55±0.06 Only _CaCl2 347.0 350.8 - 531.3 - 284.8 - Only Na ₂ HPO ₄ - - - 530.9 - - -

The calcium, phosphorus and oxygen binding energies for OCP and pLys-CP coatings are in good agreement with previously published values for calcium phosphates such as OCP. The nitrogen peak at 400.2 eV in the pLys-CP scan confirmed the presence of poly(L-lysine) in the modified coating. Determine the calcium to phosphorus ratio according to the expression in Equation 1:

$\mathrm{<span\; class="notranslate">\; Ca</span>}<span\; class="notranslate">\; :</span>\mathrm{<span\; class="notranslate">\; P</span>}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; Ca</span>}}<span\; class="notranslate">\; /</span>{\mathrm{<span\; class="notranslate">\; S</span>}}_{\mathrm{<span\; class="notranslate">\; Ca</span>}}}{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; P</span>}}<span\; class="notranslate">\; /</span>{\mathrm{<span\; class="notranslate">\; S</span>}}_{\mathrm{<span\; class="notranslate">\; P</span>}}}<span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; )</span>$

In Equation 1, Ix is the intensity of the XPS peak corresponding to element "x", and Sx is the sensitivity factor for element "x". The ratio of 1.31 in the inorganic coating is in good agreement with the expected value for OCP (1.33). A value of 1.14 in the pLys-CP coating is consistent with calcium-deficient OCP. The XPS data also provided some information about the pre-seeding process. First, the data show that, in the absence of phosphate, a small amount of calcium alone can be adsorbed on the Ti surface, while phosphate alone cannot significantly bind to the bare Ti surface after 24 hours. Alternatively, co-treatment of the surface with _CaCl2 and _Na2HPO4 results in the formation of relatively Ca-rich calcium phosphate complexes in as little _as 10 minutes. However, there was also no visible indication of crystal formation in SEM until about 1.5-2 hours. However, by 2 hours, the small crystallites visible in Figure 6 had decorated the metal surface, and the Ca-P ratio had dropped to 1.3.

The formation of such a seed layer on the Ti surface enables the successful growth of pLys-CP on the Ti surface. Table 5 below summarizes the coverage results from various pre-seeding treatments.

Table 5: Dependence of pLys-CP growth on Ti surface pre-seeding method Pre-seeded (24 hours unless otherwise stated) Subsequent pLys-CP coating growth 2mM CaCl ₂ +1.2mM Na ₂ HPO ₄ +pLys can be ignore 2mM _CaCl2 can be ignore _{1.2mM Na2HPO4} can be ignore 2mM NaCl can be ignore 2mM CaCl ₂ +1.2mM Na ₂ HPO ₄ (10 minutes) 25-50% coverage 2mM CaCl ₂ +1.2mM Na ₂ HPO ₄ (30 minutes) 75% coverage 2mM CaCl ₂ +1.2mM Na ₂ HPO ₄ (>3 hours) 100% surface coverage

From the table it is clear that the pre-seeding treatment including calcium and phosphate created a sufficient surface for pLys-OCP coating growth. Interestingly, the CaCl _pretreatment , which did result in calcium adsorption to the metal surface, was not sufficient to promote the subsequent growth of pLys-CP. Similarly, _Na2HPO4 treatment alone failed to promote subsequent pLys-CP formation on the metal _surface . A pre-seeded layer of calcium phosphate mineral complex successfully promoted the uniform growth of plys-CP.

It was also found that pLys-CP coatings are particularly sensitive to biologically relevant degradation conditions. Table 6 summarizes these observations, while Figure 7 illustrates SEM micrographs with corresponding EDS patterns to show the effect of degradation on the coating.

Table 6: Coating stability under acid and enzymatic degradation conditions Degradation solution Is the OCP coating stable? 1) Is the pLys-OCP coating stable? Hanks' Balanced Salt Solution (HBSS) buffered at pH 7.4 yes 2) yes pH 7.4 buffered MEM-medium containing 10% fetal bovine serum yes 3) yes pH7.0 citrate buffer yes 4) No (seed layer is stable) pH6.0 citrate buffer no 5) no 0.2% trypsin in HBSS, pH 7.4 yes 6) No (seed layer is stable) 0.2% Pronase in HBSS, pH 7.4 yes 7) No (seed layer is stable)

The test lasted 24 hours and both OCP and pLys-CP coatings were found to be relatively stable in pH 7.4 buffered media. However, when the pH was lowered to 7, the OCP coating was mostly stable, with slightly reduced Ca and P EDS intensities indicating very limited solubility. The structured pLys-CP coating dissolved visibly, leaving behind what appeared to be a residue of the inorganic pre-seeded layer, as evidenced by the structure shown in FIG. 7 . EDS analysis showed a large decrease in the intensity of the Ca and P peaks, but the peaks did not completely disappear due to residual inorganic seeds being stable at this pH. At slightly acidic conditions at pH 6, both coatings were completely dissolved. Micrographs of these matrices showed blanks, and EDS scans showed no evidence of calcium or phosphate. Treatment of the coating with an enzymatic solution of trypsin and pronase buffered at pH 7.4 revealed that purely inorganic coatings are stable, whereas pLys-CP coatings are unstable, after which only inorganic pre-seeded crystals are left layer and small EDS peaks for Ca and P.

Incorporation of pLys into the Ca-P layer also introduces valuable chemical bonds for linking functional biomolecules to the coating. The positively charged free amine side chains of poly(L-lysine) can be used as conjugation linkers, either by electrostatic interactions with negatively charged molecules, or by forming lysine free amines with carboxylic acids on target molecules between the amide chains. These binding schemes were confirmed by attaching cysteine molecules to the pLys-CP coating. Cysteine bound to the surface is shown by the appearance of a sulfur (ls) binding energy peak at 164 eV in the XPS spectrum of FIG. 8 . A comparison of the S:N molar ratios of these spectra, as shown in Figure 8, provides a semi-quantitative comparison of the sulfur content in the different samples. These data illustrate first that cysteines are only bound to pLys-CP and that significantly more cysteines are present when HBTU and DIEA are added to the reaction, perhaps due to increased bond stabilization of the amide bond formed sex. The S:N ratios of samples treated in HBTU and DIEA remained statistically indistinguishable when these samples were washed in saturated saline solution. When washed with saturated saline, the cysteines bound to pLys-CP in the absence of HBTU and DIEA were apparently displaced, as indicated by a large decrease in the S:N ratio.

The above observations describe a new calcium phosphate-organic composite coating on titanium surfaces. XRD, RFTIR, XPS, TGA, and elemental analysis collectively demonstrated that incorporation of pLys into this new coating produced a poorly crystalline, calcium-deficient complex of the octacalcium phosphate salt. SEM examination revealed a strong deformation effect of pLys on OCP crystal formation. The resulting coating consists of the irregular, nanoscale features shown on the clean, sharp crystals formed in the pure inorganic OCP coating. The incorporation of pLys into the mineral phase can be demonstrated by observing the breakdown of coating crystallinity by XRD and FTIR, as well as by enzymatic decomposition of the coating. If the polymer is coated only on the outer surfaces of small, modified crystals, those crystals would largely be expected to remain present, as in the inorganic controls. However, upon enzymatic degradation of the organic components in the pLys-CP coating, the pLys-modified coating disintegrated, leaving only inorganic seeds afterwards. This result strongly suggests that pLys is incorporated throughout the calcium phosphate structure. A very basic demonstration using cysteines demonstrated covalent amide coupling of biomolecules to the free amine linkages in pLys-CP. Naturally, the presence of an amide bond in the lysine polymer throughout the pLys-CP coating confounds the direct identification of the amide bond between cysteine and pLys. However, this coupling can be revealed by empirical extrapolation. Cysteine's sulfur content makes it a chemically unique marker of cysteine bound to the surface of a sample when examined by XPS. The selective appearance of the XPS sulfur peak in the pLys-CP sample suggests an interaction between cysteine and the pLys component of the coating. This interaction can take two forms: electrostatic and covalent. The electrostatic binding of materials involves the attraction between the negatively charged free acid of cysteine and the positively charged free amine on the side chain of pLys. It is possible that this electrostatic attraction binds cysteine to pLys-CP in the absence of HBTU and DIEA. Washing the samples undergoing this interaction with brine resulted in substitution of chloride ions for cysteines and a marked reduction in the amount of sulfur present on the samples. This substitution supports the electrostatic character of the binding. In contrast, when a cysteine was introduced to a free amine in the presence of the amide linking reagents HBTU and DIEA, the amide bond formed allowed the cysteine to reside on the pLys-CP surface. The dependence of this persistence on the presence of an amide coupling agent, and the insensitivity of binding to electrostatic displacement, strongly suggest covalent, amide coupling of cysteine to the pLys coating.

Although not wishing to be bound by theory, the mechanism by which pLys-CP coatings grow on oxidized titanium surfaces involves several sequential steps. The nucleation of calcium phosphate on titanium surfaces is thought to be related to the hydroxyl ions that decorate the surface of naturally occurring titanium dioxide (TiO ₂ ) at physiological pH. The XPS data presented above shows that Ca ²⁺ alone, but not PO ₄ ³⁻ alone, is moderately bound to the oxidized titanium surface during the pre-seeding stage. However, simultaneous introduction of Ca ²⁺ and PO ₄ ³⁻ , resulted in the rapid formation of calcium phosphate complexes with a Ca:P ratio of 1.55 consistent with amorphous calcium phosphate. These complexes nucleate on the metal surface, possibly through initial interactions between calcium ions and hydroxyl groups on the surface of the decorated oxide. Over time, these aggregates mature, reorganizing to incorporate additional phosphate into their structure. After a few hours, these aggregates grew into mineral parts (OCP) as shown in Figure 6, where the Ca:P ratio decreased from 1.55 to 1.33. However, when poly(L-lysine) was present during these phases of the nucleation phenomenon, calcium phosphate could not successfully nucleate directly on the metal surface, presumably due to the interference of the calcium-hydroxyl interactions by the positively charged side chains on pLys . It has been shown that positively charged pLys readily binds to titanium hydroxide oxide surfaces. It is reasonable to conclude that pLys blocks the necessary nucleating hydroxyl groups on the oxide surface. This phenomenon explains why pLys-CP cannot be directly grown on the bare Ti surface. However, when the surface is decorated with calcium phosphate seeds, there are many available nucleation sites, and pLys cannot completely inhibit the continued growth of the mineral phase. Obviously these calcium phosphate seeds promote the uniform growth of pLys-CP coating. Although the mechanism of this growth is not obvious, it is conceivable that new minerals grow as part of the disrupted epitaxy. New minerals nucleate and grow out of the calcium and phosphate already present on the surface, and as they are incorporated into the coating, pLys distorts the newly formed OCP crystals.

The calcium deficiency shown in the XPS Ca/P ratio of this pLys-CP coating illustrates that divalent calcium ions are excluded from newly formed crystals by positively charged polymer side chains through charge repulsion or crystallization site hindrance. . Also, there may be some preferential interactions between the positively charged side chains of pLys and the negatively charged phosphate ions. This situation may help to elucidate the early onset of mineral formation in the pH trace of pLys-CP. In the early stages of mineralization, this phosphate affinity will generate locally phosphate-rich Ca-P aggregates, which in turn trigger the early onset of crystallization. This phosphate-binding affinity of course disrupts proper crystal formation and produces phosphate-rich, or calcium-deficient OCP crystals. Either of the two interactions between plys and the Ca-P mineral component could be responsible for the formation of the twisted structure seen in the pLys-CP coating.

The pLys-CP coating of the present invention offers many advantages over other calcium phosphate coatings, especially from a clinical point of view. Solution phase growth of the coating makes its application suitable for all surface types, including porous surfaces, where currently employed calcium phosphate growth methods such as plasma spraying may not be feasible. The pLys-CP coating has a large surface area and the component size is fairly consistent with the apatite crystals found in natural bone. This nanoscale structure and high surface area are additional properties that are expected to promote initial cell adhesion, expansion and proliferation, which are important for forming a stable tissue-implantation interface. As an emphasis on this role, poly(L-lysine) has become a recognized promoter of cell adhesion, and its prominent presence in pLys-CP is expected to further enhance cell adhesion to implants coating. Not only does this pLys component increase the biological activity as a cell adhesion agent, but it also provides a chemically functional link for the attachment of other bioactive agents. This approach can be readily used to attach biologically relevant peptides, such as arg-gly-asp (RGD), therapeutic molecules such as bone morphogenetic proteins or anti-inflammatory drugs to implant coatings. Also advantageously, the coating is also sensitive to biodegradation through pH and enzyme-mediated mechanisms, the two main mechanisms for osteoclast resorption in native bone. Coating dissolution accelerates new bone formation and increases the strength of the implant interface. This coating has been engineered to act as an osteoconductive surface that can be easily recycled as a building block for new biogenetic mineralization.

The use of the pre-seeded layer can be used to promote the growth of other organically modified materials onto the surface. In the present invention, organic molecules incorporated into mineral coatings can exert effects on properties such as coating morphology and degradation. Organic influence on degradation can be used to engineer the time-dependent release of therapeutic molecules incorporated into minerals or chemically attached to pLys. Optional organic components can also be used to alter the impact of morphology or the rate at which the material degrades. Clearly this approach to surface coatings offers many broad and diverse potential applications which can greatly impact orthopedic and dental implant coatings.

Example 5

This example illustrates the chemical attachment of peptide-hydrophilic lipid nanofibers to a pLys-OCP coating on a titanium substrate: Peptide-hydrophilic lipid nanofibers and pLys-OCP are based on standard amide coupling reactions for pre-assembly , crosslinked peptide nanofibers. Specifically, a dilute solution of a peptide hydrophilic lipid molecule comprising a carboxylic acid near the C-terminus of the peptide portion, and at least 2 cysteines of the structural peptide portion (see Figure 9 and Hartgerink et al., PNAS, vol 99, pp 5133-5138, 2002 and references therein, referencing the methods and materials therein for the preparation of this peptide, which are hereby incorporated by reference in their entirety), were maintained in a weak reducing agent (such as dithiothreose Alcohol (DTT)) solution, self-assembled under acidic conditions to form peptide nanofibers. These nanofibers can be cross-linked by adding nondestructive oxidizing agents, such as iodine, to form stable intermolecular and intrafiber disulfide bonds. The resulting suspension of these fibers was dialyzed against water to remove any reducing or oxidizing agents (such as DTT and iodine). This dialyzed suspension of crosslinked fibers is then lyophilized and the dried fibers are resuspended in a peptide-soluble polar organic solvent such as N,N-dimethylformamide (DMF) or NMP by vigorous shaking and sonication . Covalent crosslinking of the fibers stabilizes them in anhydrous environments.

Add a solution of O-benzotriazole-N,N,N',N'-tetramethyluronium-hexafluoro-phosphate (HBTU) and diisopropylethylamine (DIEA) to the N,N - in a suspension of crosslinked nanofibers in dimethylformamide (DMF) to provide slightly less than about 1 equivalent (0.95) of HBTU per free carboxylic acid on the nanofibers, and for exposure to lysine modifications The calcium phosphate-coated titanium surface has approximately 6 equivalents of DIEA per estimated free amine. The solution was incubated for several minutes before being exposed to the coated titanium surface. Once introduced, the calcium-phosphate-coated titanium was shaken in the nanofiber reaction solution for at least 1 h before being thoroughly rinsed with water and dried at room temperature. Figure 10A is a scanning electron micrograph showing fiber bundles attached to the structured coating surface. Figure 10B is a higher magnification image showing the individual fiber layers of the architectural structure coated with a calcium phosphate coating.

Preliminary in vitro experiments with preosteoblast mouse calvaria cells demonstrated the biocompatibility of the pLys-CP coating. Titanium foil samples were coated with inorganic OCP and poly(L-lysine) modified calcium phosphate as described above. The matrices were autoclaved at 115°C for 30 minutes before being placed in sterile tissue culture polystyrene 24-well plates.

Immortal mouse precalvarial osteoblasts (MC3T3-E1 ) were cultured in T-75 flasks in MEM-α containing 10% fetal calf serum (Hyclone, Logan UT) and 1% penicillin/streptomycin. The medium was supplemented with 30 mM β-glycerol phosphate and 50 μg/mL ascorbic acid. At approximately 90% confluence, cells were removed from the T-flasks by treatment with 0.25% trypsin, 1 mM ethylenediaminetetraacetic acid (EDTA). Trypsinization was terminated by addition of medium and cells were pelleted by centrifugation. Cells were resuspended in culture medium and plated onto coated foil substrates at a density of 5 x ¹⁰³ cells/ ^cm2 . Add fresh medium to a total volume of 1 mL/sample. Cells were cultured in an incubator at 37°C and 5% _CO2 for 7 days with medium changes every 3 days.

Samples were removed from their culture wells at 1 day, 4 day and 7 day intervals and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer. After extensive washing in sodium cacodylate buffer, samples were post-fixed for 1 hour in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer. The fixed samples were then dehydrated in graded ethanol solutions (50%, 70%, 80%, 90%, 95%, 100%) and critical point dried by ethanol- _CO2 exchange. Dried samples were sputter-coated with 3 nm gold-palladium and examined by scanning electron microscopy.

The results of in vitro studies showed that cells cultured on these matrices remained viable, spread and proliferated, forming a confluent cell layer on the pLys-CP coating over the course of 7 days. Figure 1 IA shows a single cell spreading on the coating after 1 day, while Figure 1 IB shows multiple cells spreading on the surface after 4 days. In Figure 11C, after 7 days, a confluent cell layer formed by proliferating cells is visible. The experiments demonstrated that the material is nontoxic and indeed promotes cell adhesion and spreading, behaviors that are critical for normal osteoblast function.

The methods and materials of embodiments of the present invention would be well suited for coating titanium-based orthopedic implant materials with osteogenic calcium-phosphate coatings. As illustrated in the detailed examples above, this coating material composition is highly structured and can completely coat surfaces exposed to the reaction solution. Such coatings may have beneficial effects on cell attachment, spreading, proliferation, and possibly osteoblast differentiation. This effect can significantly improve the integration of the tissue with the implant surface. The incorporation of organic macromolecules within the coating increases the chemical functionality available for binding biologically functional materials to the coating surface, including peptide micelles, individual peptide sequences, or other therapeutic molecules such as drugs or growth factor. The low crystallinity of the material and the incorporation of macromolecules susceptible to enzyme attack may make the material a useful system for the slow release of these macromolecules. Similarly, the potential degradability of this coating makes it a ready source of calcium and phosphate materials for the subsequent biomineralization of the new bone matrix.

While the invention has been described in considerable detail with reference to certain preferred embodiments thereof, others are possible. For example, therapeutic macromolecules can be incorporated directly into the mineral phase instead of polyamines. Substitution of polyamines such as poly(L-lysine) need not be limited to therapeutic molecules, other amino acids, possibly including free acids such as glutamic acid or aspartic acid, can be incorporated into the mineral phase. These molecules will present different chemical functionalities on the material surface and may even change the way inorganic materials are modified. Furthermore, variations in calcium-phosphate ratio and concentration, different phases of calcium phosphate, such as hydroxyapatite, tricalcium phosphate, brushite or monetite, can be coated on substrates using the methods described here surfaces to produce coatings with different chemical, structural or material properties. Therefore, the spirit and scope of the appended claims should not be limited to the description and preferred versions contained in this specification.

Claims (20)

1. the bio-compatible compound of a nanostructured, it comprises biocompatible matrix, is positioned at the calcium phosphate component on this described matrix; With the mineral facies that are positioned at the nanostructured on the described calcium phosphate component, described mineral facies comprise calcium phosphate and poly-(L-lysine).

2. the compound of claim 1, the calcium content of wherein said mineral facies are less than stoichiometry, and described poly-(L-lysine) is incorporated in the described calcium phosphate.

3. react one of at least in the compound of claim 1, wherein said mineral facies and acid and digestive enzyme.

4. the compound of claim 1 also comprises and the described nanofiber that gathers the peptide amphipath of (L-lysine) coupling, and at least a described peptide amphipath comprises carboxyl functional group.

5. the compound of claim 4, wherein at least a described peptide amphipath comprises the RGD sequence.

6. the compound of claim 4 also comprises mammal preosteoblast cell culture.

7. the compound of claim 1, wherein said matrix comprises titanium.

8. method that promotes amine-modified calcium phosphate compositions growth, described method comprises: biocompatible matrix is provided; Basically single-phase calcium phosphate component is deposited on the described matrix; With introduce described matrix in the calcium phosphate culture medium, component that described culture medium comprises poly-(L-lysine).

9. the method for claim 7, wherein said matrix contact comprises the culture medium of reactive calcon and reactive phosphorus silicate reagent, and the time that described contact is carried out is enough to make described calcium phosphate component to be deposited on the described matrix.

10. the method for claim 8, wherein said calcium phosphate culture medium comprise at least a in reactive calcon and the reactive phosphorus silicate reagent.

11. the method for claim 10, wherein at least a described reagent comprise described poly-(L-lysine) component.

12. the method for claim 8, wherein said deposition comprises the formation crystallized calcium phosphate, and described introducing will gather (L-lysine) be incorporated into calcium phosphate mutually in.

13. the method for claim 12, wherein said introducing are induced the component that has produced the nanostructured that comprises calcium phosphate and poly-(L-lysine).

14. a method that makes peptide amphipath and biocompatible matrix coupling, described method comprises: biocompatible matrix is provided; Basically single-phase calcium phosphate component is deposited on the described matrix; Make mineral facies be deposited on described calcium phosphate and go up mutually, described mineral facies comprise calcium phosphate and poly-(the L-lysine) that mixes wherein; With described poly-(L-lysine) is contacted with the peptide amphipath, at least a described amphipath comprises carboxyl functional group.

15. the method for claim 14, wherein said peptide amphipath comprise the nanofiber assembling.

16. the method for claim 14, wherein said matrix contact comprises the culture medium of reactive calcon and reactive phosphorus silicate reagent, and the time that described contact is carried out is enough to make described calcium phosphate component to be deposited on the described matrix.

17. the method for claim 14, wherein said mineral facies are product of calcon and phosphate reagent, and introduce poly-(L-lysine) between the described stage of reaction.

18. the method for claim 14 also comprises making in described mineral facies and acid and the digestive enzyme contacting one of at least.

19. the method for claim 14, wherein at least a described peptide amphipath comprises the RGD sequence.

20. the method for claim 14 also is included on the described peptide amphipath and cultivates mammalian cell.

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