CN101410144A - Modified surfaces for attachment of biological materials - Google Patents

Abstract

Translated fromChinese

本发明涉及沉积在选定的基体上的生物活性表面涂层。可以设计出沉积在大多数金属或非金属基体上用来提供表面的表面纳米结构的膜涂层以促进增强的组织/细胞粘附。附着的细胞，包括成骨细胞、成纤维细胞以及内皮细胞，保持可存活性并将在适合的条件下容易分化和增殖。除了在纳米表面结构的硅上，在大部分涂覆的基体上，成纤维细胞和内皮细胞表现良好的附着和生长。

The present invention relates to bioactive surface coatings deposited on selected substrates. Surface nanostructured film coatings that are deposited on most metallic or non-metallic substrates to provide surfaces can be engineered to promote enhanced tissue/cellular adhesion. Attached cells, including osteoblasts, fibroblasts, and endothelial cells, remain viable and will readily differentiate and proliferate under suitable conditions. Fibroblasts and endothelial cells showed good attachment and growth on most of the coated substrates except on nanostructured silicon.

Description

Be used for the improved surface that biomaterial adheres to

The cross reference of related application

The application requires the priority of No. the 60/786th, 118, the provisional application sequence submitted on March 27th, 2006, and its full content is hereby expressly incorporated by reference.

Technical field

The present invention relates to provide the improved coating of adhering substrate for cell and other biological material.The coating surface of the nanometer structure of selecting (nano-textured) promotes cell growth and propagation and can be deposited as stable coating on metal or nonmetal basal body.

Background technology

The rejection of implantable device is a main difficult problem, because body self is not accepted (recognize) external source foreign body.Therefore, the various medical treatment devices that use at present all can not promote rehabilitation and may usually cause high infection rate.For example, in conduit, joint replacement, soft tissue repair and tooth plantation, usually find these problems.Thereby implant should promote cell attachment to reduce to infection minimum and the raising cure rate under the perfect condition.

The material that forms implanting device is normally attempted to promote or is improved cell attachment and the surface of carrying out modification.Regrettably, even there is cell attachment, yet there is not fully to promote the device of the long-term indwelling of tissue growth.Some schemes are used somatomedin and are come aid in tissue to connect on the surface of device, yet the result is always not satisfactory, and somatomedin can not normally use.Almost also do not have surface that the trial of success can improve implant so that the new organization cell easily adheres to and grows, and prior art fails to develop the surface of remarkable enhancing tissue apposition.

The bioactivity coatings that can improve the traditional titanium base material performance that is used for plastic surgery's application has obtained people's attention.At present the device that uses is to make by traditional metallurgical technology, promptly strengthens bone as face coat and adheres to by apply hydroxyapatite on titanium.Commercial, by the spray deposited technology of high-temperature plasma hydroxyapatite is coated on the titanium-based metal, this technology is converted into the nanocrystal hydroxyapatite hydroxyapatite of the micron particle size that contains less crystal phosphoric acid calcio matter.The plasma spray coating deposition of hydroxyapatite is a kind of painting method commonly used; Yet this has caused phase transformation, and it may cause the formation of highly soluble calcium phosphate, and this can cause being coated with delamination layer (Furlong, etal., 2001 in the process of clinical practice; Baker, et al., 2006).

Recently, developed multiple technologies and be used to obtain the nanoscale surface roughness, these technology comprise superfine metal coating (Webster et al., 2004 of use such as titanium; Valiev, etal., 2004).And the chemical etching of having used anodic titanium and titanium deposition is attempted to produce and is used for that osteoblast adheres to and osteoplastic surface adhesion force subsequently (Yao, et al., 2005).

It is believed that the material with Nanosurface feature compares with the material with micrometer-class, can strengthen formation (Sato, et al., 2005 of bone; Popat, et al., 2005).Regrettably, for the implant of present use, traditional coating processes can not provide effective osteanagenesis needed nanostructured surface.

Be intended to strengthen the major part of having done adhering to of tissue and implantable device at present and make great efforts to concentrate on pressed metal implant that exploitation is made of nanometer powder and plastics with nanometer structure.The problem that these two kinds of methods all have is to have lost a large amount of intensity owing to surfaction makes material.

Recently, the method that designs surgical plastic implant of future generation concentrates on synthetic implant surface and the specific nanometer configuration of finding in osseous tissue that is produced by the natural fine extracellular matrix protein is complementary.Nanostructured and the molecule found in osseous tissue show, the bone formation cell usually with the surface interaction with nanometer roughness, yet the traditional synthetic metal that uses at present has little rough surface, and is level and smooth (Kaplan on nano-scale, et al., 1994A; Kaplan, et al., 1994B).

Inorganic mineral granule (ore particle, the mineral grain) mean size that braiding (immaturity) bone has is 10-50nm.The inorganic mineral granule mean size that the active lamellar bone that replaces woven bone (woven bone) has is that long 20-50nm and diameter are 2-5nm.Yet in nano-grade size, the implant surface that uses also is that majority all is level and smooth all even be not at present.Shown that such smooth surface helps " fiber healing " (fibro integration, fibrointegration) (cell formation), it finally utilizes the connective tissue of not expecting of layering (stratified) will place the implant of bone to encapsulate (encapsulate) (Webster, et al., 2004).

The cell attachment coating on making great efforts the exploitation orthopedic devices, also need be such as the adhering coating on those devices that are used for the tooth plantation.Hydroxyapatite or ACTIPORE^TMCoating is difficult for being deposited on the typical medical treatment device matrix such as CoCrMo.Even deposition is fine, but adhesion is usually relatively poor and delamination can take place.

Exist two kinds of major techniques to be used to form the surface that promotes tissue apposition at present.A kind of method is with the nano metal powder press forming, so that obtain certain surface roughness; Another kind method is to produce the nanometer rough surface by moulding process on plastics.

The press forming of metal dust and sintering have at low temperatures produced the surface that promotes the tissue ingrowth on the matrix surface.Regrettably, this combination can not be used for many plastic surgery uses, thereby because the plastic surgery use desired intensity need powder at high temperature sintering obtain required intensity.The micro structure of the sintering temperature deface that improves also thereby has been lost any advantage for tissue apposition.

Polymer surfaces is shaped to the main main points that the nanometer structure has become the surface of making great efforts design promotion tissue growth.This method has obtained limited success, and partly cause is that mold is being given suitable nanometer structure and obtained only have limited ability among the concordance result for frosting.Quality control in manufacturing process is normally underproof, because flow into the restive and parts scrappage (part rejection rate) of the plastics of coarse mold up to 50%.

The defective of prior art

Defective in surface compacting and the forming technique shows, there is demand in the method that has the tissue adherence energy of remarkable improvement and be suitable as the face coat of the coating on the medical implant for preparation.

Thereby, for adhering to metal or nonmetallic surface, have more superior tissue apposition performance and having a kind of demand for the avirulent coating of living cells.Adhering coating with these characteristics can be deposited on the various matrix surfaces ideally by continuous and economically viable technology.

Summary of the invention

The present invention is based in part on for can accurately controlling nano-particle (granule reaches and is not more than about 100nm) sedimentary understanding and unpredictable discovery, promptly, tissue apposition can be increased to significantly big degree by ion deposition technology (IPD) coating deposited on selected matrix that improves than by traditional plasma vapor deposition method coating deposited.This discovery has caused the development to the method for the coating that is used for the production nanostructured, and this nano-structured coating is as biocompatibility (biocompatible, biocompatible) support (scaffold) of osteanagenesis.

Especially surprising discovery is to observe the sedimentary metal coating of IPD on the silicon, and comparing based on the IPD plated metal on the matrix of other metals and polymer, does not promote the cell attachment of some type, for example, and fibroblast or endotheliocyte.Yet most of subject cells show as adhering to and breed enhancing on the sedimentary metal surface of IPD on metal or the number of different types polymer.When needs selectivity osteoblast adheres to so that during promote osteogenesis, it may be very favorable using silicon substrate, because fibroblast promotes soft tissue and callus to form.Be intended to comprise on the implant of promote osteogenesis that osteanagenesis and osteanagenesis can delay or suppress to restore.

The cell that has been found that some kind can easily be attached to the coating of nanostructured and cell that discovery is adhered to will be bred under the environment that is fit to.The immature cell that adheres to, for example fibroblast and osteoblast will break up on the support coating as the nanostructured of support or matrix and breed.As already discussed, when the IPD plated metal of coated with nano structure on silicon substrate was surperficial, adhering to of fibroblast or endotheliocyte was an exception.

The IPD method that is used to prepare nano-structured coating is based on the IPD technology of improvement, and it is adjusted into the nano-particle output of increase and controlled deposition.The IPD plated metal preferably the switching rate of about 500Hz (switching rate) from the plasma arc target (plasma arc target) of controlled speed and be deposited to obtain the nano-structured coating of expectation.

Have been found that and use controlled IPD technology plated metal on plastics or metallic matrix, the adhesion that can improve nano-particle material and matrix surface.Compare with the film or the surface that prepare by other technologies of using in industry, coating deposited has promoted higher histiocyte adhesion rate.IPD technology directly is deposited on nano-particle material on the matrix, does not need special or primer (primer) " seed " applies.

In order to improve cell adhesion and the tissue growth on the implanting device, do not expect the size and the density of deposition materials on matrix surface of strict control nano-particle at first.For many years, for technicians, thereby the major trend of plasma deposition process is to reduce the quantity preparation cleaning film more uniformly more deposit nano-particle from the teeth outwards.Even the common practise in the industry is that the granule in 1 micron magnitude range also is destructive for the quality of deposited film usually, thereby deposited film should be level and smooth as much as possible and do not have a granule.

Therefore, sprayed and in fact the size that is deposited on the matrix has strengthened the tissue apposition quality of coating rather than made its minimizing less than the nano-sized particles of about 100nm by target, this againsts one's expectation.Therefore, an importance of the present invention is the method that exploitation is used to improve rather than reduce nano-particle output and controls nanoparticle size.As everyone knows, compare with plasma gas phase deposition (PVD) technology of other types, ion plasma deposition technology can obtain higher sedimentation rate and be easy to produce more bulky grain, and more rather than less nanoparticle deposition will improve the tissue apposition performance of sedimentary nanostructured surface, and this point is gone over not known or admitted by the people.

Therefore, by being devoted to reduce rather than improve bulky grain output, other people have obtained some successes improving for the effort of the plasma arc deposited method and apparatus of improvement on the tissue apposition.Result described in the present invention shows that the deposited film with super nanoparticle density has improved the tissue apposition characteristic of the thin film of IPD production, and this film is particularly advantageous for the application on implanting device.Can on purpose prepare the bulky grain (it has been generally acknowledged that it does not have help to performance improvement) in selected nanoparticle size range, thereby strengthen the tissue apposition coating.

Develop the multiple method that is particularly suitable for fast deposition nanometer structure coating, for depositing at a high speed not only in order to obtain good adhesive force, and be used for increasing nanoparticle deposition.Therefore, the present invention includes a kind of output (production) and sedimentary method that is used to improve the intensive coating of nano-particle of biocompatible materials.This coating shows the tissue apposition and the adhesion characteristics of improvement.The result that using modified IPD nano-particle coating processes obtains obtains intensive, highly homogeneous, highly adherent, thick coating, and it is very suitable for promoting the tissue apposition on the employed implantable medical device in human body and the veterinary's application.

The coating that is produced by the IPD method is not subjected to the restriction of substrate types and can be applied to various materials, comprises such as the non-conducting material of plastics and pottery and such as the conductive material of metal.The method that forms controlled nanometer structured surface can be used to sedimentary organism compatibility film on medical treatment device, and it is at the implantation site healing acceleration.

Therefore an object of the present invention is to provide a kind of IPD technology of improvement of utilizing deposits adhering coating on matrix, thereby forms the method for the intensive tissue apposition coating surface of controlled nanometer.

Utilize the improvement IPD deposition of attaching surface, on matrix, prepare coating.The target that will comprise the combination of potential adhesion metal or metal places vacuum chamber and target is applied electric energy to produce electric arc, makes the target metal ion turn to the plasma of ionized particles.With reactant gas,, optionally introduce in the vacuum chamber this gas and ionixedpiston granule reaction like this as oxygen or nitrogen.In deposition process, by in a controlled manner changeably control put on the electric energy of target and/or optionally mobile matrix and make it near target or away from target, control the deposition of plasma particle on matrix.

Whether the IPD method provides attaching surface on medical treatment device or material, with traditional medical treatment device and comparing that material is provided, no matter applied routinely, all promotes healing faster in the body.This is by depositing metal on polymer or the metallic matrix, realizing so that form the surface of highly homogeneous nanostructured on matrix.

Dispersive metal, metal nitride or metal oxide particle can be deposited on the various matrix materials by IPD, comprise metal, plastics, glass, soft, porous paper, pottery, their combination etc.Though matrix can comprise any multiple device, but medical treatment device be particularly preferred and can comprise conduit, implant, support, endotracheal tube (airway, trachealtube), plastic surgery's fixed shunt (pin shunt), drainage tube, prosthetic device, tooth implant, dressing and wound closure.Should be understood that the present invention is not limited to these devices and can expands to the device that other are used for medical field, for example face shield, medicated clothing (clothing), surgical instrument and surface.

Target can be any solid material or the combination with material of adhesion property, as long as target material can the ionizing by the arc plasma body technology.Preferable material has potential adhesion property and is biocompatibility; Promptly do not damage the metal of FUTURE ENVIRONMENT (intendedenvironment).Such material comprises alloy and metal, comprises zinc, niobium, tantalum, hafnium, zirconium, Nitinol, titanium, titanium 6-4, chromium, cobalt, nickel, copper, molybdenum, ferrum/chromium/nickel (rustless steel), platinum and gold, is commonly referred to " adhesion metal " here.

The invention provides deposition on matrix surface of gold, titanium, Nitinol or other metal ions, soak into or layering, to form by intensive nanostructured greater than the granulometric composition of 5nm.Nanostructured surface provides attachment site for cell or other biological substance.Cell is incorporated on the solid-state structure of millimicro-micromicron and micron order crystalline metal and metal-oxide compound, and it can be used as and diffuses in the surface or diffuse to the combination of lip-deep unit price, two valency and multivalence oxide and deposit.

Generally speaking, the present invention relates to prepare a kind of biological matrix that applies, it is included in plated metal ion plasma on the matrix forming the particle metal coating of nanostructured dense distribution, and make this coating and one or more cells contacting a period of times so that this one or more cell attachment to coating surface.One or more cells that are attached to deposited coatings have formed the matrix of biological coating, and it is keeping the biology performance of attached cell.Biological coating effectively is attached to substrate or support (scaffolding), make cell or tissue (no matter be under artificial culture environment or under natural environment, as may be under the residing situation of medical implant) under the condition that is fit to easily adhere to and grows.When immature cell adhered to, biological coating can allow differentiation, and for example osteoblast is reached maturity and is osteocyte.

In fact, almost any cell all can be attached to the face coat of nanometer structure; Generally speaking be any mononuclear cell.Example comprises leukocyte, lymphocyte, neutrophil, oxyphil cell, Monocyte etc.Particularly preferred cell comprises osteoblast, fibroblast and endotheliocyte.And the mixture that reckons with different cells also can easily be attached to these cells and can growing and breed.

Use ion plasma deposition (IPD) technology on multiple matrix surface, to produce biological coating.Selection is used as target as the metal of metallizing, and when producing ion beam or electric arc between target and matrix anode, its generation is deposited on the metal ion on the plate target.When metal ion in the generation at target place by handling that arc speed is controlled and when the deposition of anode substrate is controlled by the relative distance of itself and target, can producing the nano grain surface of highly dense.These nano-particle embed in matrix surfaces, make them relatively firmly and highly anti-peel off (peeling).Importantly, they are as the friendly type substrate of cell, and making them is ideal for the coating on the medical implant.

The IPD deposited metal ions can deposit thick and fast preferably as nano-particle, rather than near the larger particles of micron-scale (micro size).The most preferably size range of nanoparticle size is about 1 to about 100 nanometers, is particularly preferred for two kinds of more general metallizing titaniums and the about 15nm of Jin Eryan wherein.About 10³Granule/cm²To about 10⁴Granule/cm²Nanoparticle density provide the typical density of good biological coating.Coating thickness preferably about 0.1 is to about 3 microns.

The target that is used for the IPD method can be any metal, yet consider and be used in the living body biological thing or on it, Nitinol, CoCrMo, gold, platinum, copper, tantalum, titanium, zirconium, hafnium, zinc or their combination are preferred, and wherein Nitinol, gold and titanium are particularly preferred.

Matrix can be any multiple material, no matter is metal or comprises plastics and pottery nonmetal.Exemplary matrix material comprises UHMWPE, EPTFE, PTFE, PEEK, polypropylene, polyurethane, polyimides, polyester, nylon, titanium, ferrum/chromium/nickel (steel), cobalt, chromium, zirconium, nickel, Nitinol and their alloy and combination.

The present invention also comprises compositions, and it comprises (bioviable) cell that one or more biologies that are attached to the nanostructured metal film can be survived.This film is that (about 1 micron size is with about 10 by the metallic particles of ion plasma deposition³To 10⁴/ cm²Density Distribution) be prepared from.The typical sedimentary metal comprises Ag, Au, Ti, CoCrMo and their mixture.

But the biology survivaling cell can be any mononuclear cell.The contacted cell of medical treatment device such as implant that particularly preferred cell is those and the most probable surface-coated that has fibroblast, osteoblast or endotheliocyte.

Preferred embodiment comprise the osteoblast that is attached to the nanostructured titanium surface that is deposited on the UHMWPE.For endotheliocyte, preferred surface is the titanium that is deposited on UHMWPE or the PTFE.Coating for metal surfaces will comprise usually size reach 15nm, with about 10³To about 10⁴/ cm²Density Distribution on matrix surface and have about 0.3 a granule to about 1nm thickness.Alternative nanometer structure metal surface comprises gold, titanium and Nitinol.

Nanostructured surface coating by IPD method preparation is high stability, because coating is impregnated into metal or about 10 nanometers of the polymeric matrix degree of depth to about 100 nanometers.Exemplary preferred ion plasma deposition metal surface can comprise size about 1 micron to about 100 microns, area density about 10³To 10⁴/ cm², about 10 to the 100 microns nano-particle of thickness.

Definition

Ion plasma deposition (IPD) is to use cathode arc to discharge in target material (being generally solid metal) to produce the method for energetic plasma.The electric arc laser printer produces the plasma of pilot arc on the metal and the high-energy-density on the electric arc makes metal gasification and ionizing.Vacuum arc is different from high-voltage arc, because metal vapors itself is ionized, rather than ambient gas is ionized.

Plasma gas phase deposition (PVD) is the thin film deposition processes in gas phase, and wherein source material physically is transferred to matrix in a vacuum, does not relate to any chemical reaction.Such deposition comprises thermal evaporation, electron beam deposition and sputtering sedimentation.IPD technology is a subclass of physical vapour deposition (PVD).

Macroscopic view or bulky grain are to the particulate explanation by the target emission, and the granule that is meant greater than about 100nm of the present invention, and nano-particle is the granule that big or small maximum is about 100 nanometers.

" adhesion property " and " potential adhesion property " is intended to understand following true term: some metals, with its simple substance attitude (elemental state) inertia and can not be used as effective attachment site too usually, but when being ionized the much better than adhesion effect of Shi Zeke performance.Therefore, comprise that the adhesion metal of target has potential adhesion property, this knows on the metal ion basis in many cases.When being ionized, adhesion metal also can combine to form oxide or nitride and their combination with multiple reactant gas such as oxygen or nitrogen.

The biomaterial that the present invention uses comprises component of organization such as cell, such as the mineralising inorganic matter of hydroxyapatite and such as the bio-matrix material of collagen.

Unless otherwise mentioned, otherwise Nitinol is defined as about 55/45 compositions of the nickel that has specific grain structure respectively and titanium.

Term " about " used herein is intended to represent that specific numeral may not be accurate, but can be higher or lower in 10% scope, is determined by particular procedure that uses or method.

The term that uses in the claim " a kind of " be not to be restricted to a kind of separately.

The known abbreviation of multiple polymers comprises: PEEK (polyether-ether-ketone); PTFE (politef); EPTFE (expanded PTFE, or expanded polytetrafluoroethylsealing); And UHMWPE (ultra-high molecular weight polyethylene).

CoCrMo is the alloy of cobalt, chromium (chrome) and molybdenum (usually respectively with about ratio of 64%, 28% and 6%); Ti-gal-4V is the alloy that uses in surgical implant, comprises 89% titanium, 6% aluminum and 4% vanadium.

KSI is the standard tension test, and it applies 1000psi with the test adhesion to the surface.

" biology can be survived " used in the present invention is meant that biomaterial keeps the exemplary term of its natural biotic potential; This means the ability of keeping growth and propagation for cell.

Biological coating is the film that adheres to base material or " matrix ", and it has such as cell, tissue, cellular matrix (cell matrices) and such as the performance of the biomaterial of the inorganic structure component of hydroxyapatite and bone.

Description of drawings

Fig. 1 shows the general features of the cathode arc IPD device of improvement:target 1;Matrix 2; Movably thematrix holder 3;Vacuum chamber 4; Thepower supply 5 of target; And theelectric arc controller 6 of adjusting arc speed.

Fig. 2 shows the comparison of titanium coated substrates (titanium) and uncoated matrix.Seen in the low amplification SEM photo of uncoated titanium on UHM and PTFE and IPD titanium deposition, the titanium deposition causes nano level surface roughness.For two uncoated samples, the Bars=10 micron, for the UHMWPE that is coated with titanium, the Bars=20 micron, and for the PTFE that is coated with titanium, the Bars=10 micron.

Fig. 3 A shows with the cell density that is attached to the titanium rod and compares, UHMWPE that is coated with Ti after 1 day and the increase on the PTFE osteoblastic density, N=3,^*P＜0.01 and^*PP＜0.01.

Fig. 3 B shows with the cell density that is attached to the titanium rod and compares, UHMWPE that is coated with Ti after 3 days and the increase on the PTFE osteoblastic density, N=3,^*P＜0.01 and^*PP＜0.01.

Fig. 3 C shows with the cell density that is attached to the titanium rod and compares, UHMWPE that is coated with Ti after 5 days and the increase on the PTFE osteoblastic density, N=3,^*P＜0.01 and^*PP＜0.01.

Fig. 4 compared after 1,3 and 5 day, the PTFE of uncoated titanium and be coated with increase on the PTFE of Ti the fluorescence microscope images of osteoblast density.Bars represents 100 microns.

Fig. 5 showed after 7,14 and 21 days, the increase on the UHMWPE of solid titanium metal, UHMWPE, PTFE, coating and the PTFE that applies the formation (formation) of osteoblast (calcification).The N=3 sample is compared with corresponding uncoated sample^*P＜0.01 and compare with the solid titanium metal bar^*PP＜0.01.

Fig. 6 show silicon that Ti applies, polyethylene and

Cell adhesion, N=3; Compare with uncoated sample separately^*P＜0.01.

Fig. 7 show the silicon of coating and uncoated silicon, polyethylene and

Fluorescence microscope images, the difference of cell number on the more different surfaces.

Fig. 8 shows with uncoated sample separately and compares, and UHMWPE that titanium applies and the fibroblast of PTFE adhere to contrast.Also show among the figure, compare, fibroblastic cell adhesion that has reduced on the silicon that titanium applies with uncoated silicon.Data are the meansigma methods of three samples, n=3, wherein^*Representative is compared P＜0.01 with uncoated counterpart.

Fig. 9 A compares with uncoated sample separately, the chart that the fibroblasts proliferation on silicon, UHMWPE and PTFE that titanium applies after 1 day is compared with uncoated sample separately.Each post is represented the meansigma methods of 3 samples; Compare with uncoated matrix⁺P＜0.01.

Fig. 9 B compares with uncoated sample separately, the chart that the fibroblasts proliferation on silicon, UHMWPE and PTFE that titanium applies after 3 days is compared with uncoated sample separately.Each column is represented the meansigma methods of 3 samples; Compare with uncoated matrix⁺P＜0.01.

Fig. 9 C compares with uncoated sample separately, the chart that the fibroblast proliferation on silicon, UHMWPE and PTFE that titanium applies after 3 days is compared with uncoated sample separately.Each post dress thing is represented the meansigma methods of 3 samples; Compare with uncoated matrix^*P＜0.01.

Figure 10 show silicon that titanium applies, polyethylene and

The fluoroscopic image of matrix has shown and has compared different in these lip-deep fibroblastic quantity with uncoated matrix.

Figure 11 shows with uncoated sample and compares, after 7,14 and 21 days, and the block diagram that the protein level of measuring by the absorbance of silicon, UHMWPE and PTFE sample that titanium is applied changes.Compare with previous (previous) time point with uncoated sample separately^*P＜0.01.

Osteoblastic propagation after Figure 12 A shows 1 day, silicon, UHMWPE and PTFE that titanium is applied compare with uncoated matrix separately.Each column is represented the meansigma methods of three samples;^*P＜0.01.

Osteoblastic propagation after Figure 12 B shows 3 days, silicon, UHMWPE and PTFE that titanium is applied compare with uncoated matrix separately.Each column is represented the meansigma methods of three samples;^*P＜0.01.

Osteoblastic propagation after Figure 12 C shows 5 days, silicon, UHMWPE and PTFE that titanium is applied compare with uncoated matrix separately.Each column is represented the meansigma methods of three samples;^*P＜0.01.

Figure 13 be after 1,3 and 5 day relatively titanium that apply with uncoated PTFE on the photo of fluoroscopic image of osteoblastic proliferation.

The specific embodiment

The invention provides the adhering coating that is better than prior art and be used to deposit a plurality of advantages of other states of adhering coating technology.The IPD deposition process that is used to prepare the biological coating of improvement can be controlled particulate size, comparing volume of production, process efficiency, the scalability that significantly improves than low-running-temperature, with traditional plasma arc technology and can be applicable to multiple matrix material for certain material.The key property of deposition materials is the high surface adhesive to matrix, and partly cause is that ionized particles is embedded in the matrix surface.The IPD deposition surface comprises the nano-particle of dense arrangement, and it has certain contribution for the surface character that remarkable enhancing cell/tissue adheres to, breaks up and breeds.

Disclosed IPD technology is carried out under vacuum, and is used to produce the nanostructured surface that promotes cell attachment.According to target material (be preferably nickel, titanium, gold and/or contain the alloy or the compositions of these metals), suitably control the energy level (energy level) commonly used of 150eV to 500eV.Energy level also depends on the size of target, if thereby target bigger, can need higher energy input.This technology allows to deposit being low to moderate at least under about 30 ℃ temperature, and this temperature is a sedimentary preferred range on heat-sensitive resin and plastic substrate.

Generally speaking, this method requires in vacuum chamber selected matrix to be placed between target and the anode, and described target comprises the ionizable metal.Between target and anode, produce arc discharge.The electric energy that is applied to target is carried out variable control, make to produce to have the bulky grain of about 100 nanometers to about 5 microns sizes.Alternatively, or additionally, in the process of arc discharge, under the temperature between about 25 ℃ to about 75 ℃, in one section preset time, can adjust the mobile of matrix to about 30 inches scope at about 10 inches and make it more near target or away from target.This will produce the about 1nm of thickness and film to about 50 microns, high density, bulky grain, adhesive attach on matrix.

Use traditional vacuum arc deposition (VAD) method to be difficult to obtain superior coating and obtained, comprise being coated with not being considered as medical treatment device usually or the surface of exogenous (exotic) nickel/titanium alloys, exogenous CoCrMo alloy and other alloys of the coating in using.When the technology that adopts based on the IPD of improvement, can realize thinner coating and shorter processing time, adhesive attractioin is identical or better simultaneously.Higher output is possible, and it can cause product cost to be saved, and particularly is a remarkable advantages in medical industries.

According to disclosed method, by the palladium metal ion being turned on the surface that plasma is deposited into adhesion metal matrix or in the surface.Exist the different kinds of ions plasma deposition apparatus, those that in International Application No. WO 03-044240, describe for example, its content is hereby expressly incorporated by reference.These basic devices can be improved and be used for selected metal is carried out controlled deposition, to be used as the coating that is suitable for cell attachment.

When being coated with when being deposited upon on the matrix, can control the oarse-grained relative populations that penetrates by target.Bulky grain is to be penetrated and the molten drop (moltenblob) of the metal that is not gasified totally by target.This molten drop is intensive and is made up of purified target material.The surface of molten drop normally has electric charge, and massive material then is electroneutral.

A key character of the IPD technology of improvement is metal or metal/oxide coating can be embedded in the matrix surface, thereby compares the more excellent adhesiveness of acquisition with the coating that obtains by other deposition.Can control embedding technology by electric arc being adjusted to apart from a specified distance of target.Can obtain such embedding coating: for plastic cement, embedding reaches 100nm at least; For metal and ceramic matrix, embedding reaches 10nm at least.

The suitable device of the plasma arc depositing operation that is used to improve is an IPD technology shown in Figure 1.As shown in fig. 1, theanode 1 of target material places vacuum chamber 4.Thereby produce electric arc at the target place by electric energy and make target ionization bypower supply 5supplies.By control machinery 3make matrix 2 towards or away from target motion, the composition of plasma is selected, control or be oriented to this matrix.Power-supply controller ofelectric 6 is used to control arc speed.

IPD needs not to be line-of-sight deposition method (line of sight deposition method).Rotation and transfer (racking) are necessary for the geometry of complexity, and transfer and rotation are complicated as other PVD technologies hardly usually.In addition, this technology surpasses 10 microns Kong Eryan for any size, produces 5: but the length-width ratio that 1 repeating hole penetrates (repeatable hole penetration aspect ratio).Because bulky grain is assembled, so be difficult to detect hole less than 10 microns.

For for golden or silver-colored material, use the common coating speed that obtains of IPD technology in the present invention in about 100nm to the 5 micron scope of per minute.For these metals, obtain per hour to exceed 45000 square inches coated area with coating speed greater than per minute 200nm.Except the coating speed that improves and bigger volume, IPD technology because only need single layer coating, this means less work and higher processing speed/output for needing less processing per square inch.

The effectiveness that adheres to response also depends on the process time that is used to form attaching surface.Formed and had the attaching surface that difference is adhered to response the long process time from 5 seconds to a few minutes.

The granular size of IPD deposited coatings is preferably controlled by the electric energy that adjustment puts on target, make granular size in about 100 nanometers to about 5 microns scope, wherein for the coating on the medical treatment device that wherein needs tissue apposition, the preferred granule in nanometer range.Can be controlled as the diameter of granular size less than 100nm by sedimentary titanium of method or the gold grain that discloses.

Unexpectedly, the surface of using this IPD technology to apply is the surface of the compatibility for cell proliferation and growth.Compare with uncoated surface, the polytype cell will adhere to the matrix of washing and show remarkable enhanced growth.Illustrate, for osteoblast, fibroblast and endotheliocyte, tissue growth strengthens on the IPD plated metal on the nonmetal basal body.This is obviously indicating can use these biocompatible coatings in such as the medical applications of hip replacement (hip replacement) and other orthopaedic implants.

Although known osteoblast adheres to the polymer of gold or titanium coating at least at first, but the present invention shows on the polymer of the several types sedimentary gold of IPD or titanium can significantly strengthen the long-time growth that adheres to and continue, especially it should be noted that on the UHMWPE that titanium applies, wherein the cell adhesion of Ti Gaoing after 5 days, can improve about 600%, even after 21 days highly significant still.PTFE that PEEK that applies for gold or titanium and gold apply also observes the cell adhesion that has improved, although the latter is for the relatively low adhesion of osteoblast performance.

Endotheliocyte on the UHMWPE that titanium applies is observed similar effect, wherein compares with uncoated sample, and the cell adhesion of observing on the UHMWPE that titanium applies improves 500%, improves 100% on the PTFE that titanium applies.

As if fibroblast has identical situation, compares with uncoated sample, cell density improves 78% on the PTFE that titanium applies, and improves 90% on UHMWPE.

In the sharp contrast of the silicon that applies for titanium, to compare with independent silicon or titanium, fibroblast shows obviously less adhesiveness trend.

By following non-limiting example the present invention further is described.

Materials and methods

The human osteoblast

End user's analogy osteoblast in the cell adhesion experiment in this research (CRL-11372, U.S. typical case DSMZ (American Type Culture Collection), population number (population numbers) 2-4).Before inoculating cell, the matrix paid close attention to some extent all use phosphate buffer (PBS) (1X concentration (strength)) rinsing.Be supplemented with the Da Shi improvement Yi Geer culture medium of 10% hyclone (Hyclone) and 1% penicillin/streptomycin (Hyclone) (Dulbecco ' s Modified Eagle Medium) (Hyclone) in, with 3500 cells/cm²The initial inoculation density of matrix, cultured cell on matrix.Then at cell culture condition (37 ℃ of temperature, the 5%CO of standard₂With 95% dampness) under, cell was bred onmatrix 1,3 and 5 day; Every other day change a subculture.After the time cycle that limits, sucking-off cell culture medium from hole (well), then with PBS gently rinsingmatrix 3 times to remove any not adherent cell.Use fixing (fix) cell of formalin (Fisher) of 4% then and use DAPI (Sigma) dyeing.In fluorescence microscope (Swiss) counting cells number and photograph down.

For long cell experiment, osteoblast is inoculated with the cell density of 50,000 cells/support, and is being supplemented with 10%PBS, 1%P/S, 2.16 * 10^-3G/ml beta-glycerophosphate and 5 * 10^-5Cultivate 1,14 among the DMEM of g/ml Ascorbate, reach 21 days.When the time cycle that limits finishes, use three freeze-thaw circulations to make cytolysis.In order to measure, subsequently matrix to be immersed in the hydrochloric acid (J.T.Baker) of 1N and to spend the night with dissolving calcium mineral deposit by the quantity of the sedimentary calcium mineral of osteoblast.Collect these supernatant then and be used for by using calcium meter (Calcium assay) (Sigma Diagnostics; Procedure No.587) measures the content of calcium according to the description of manufacturer.All experiments are all triplicate, and repeat at least three times.

Endotheliocyte

(Greenbush NY) buys the rat aorta endotheliocyte, and grows in the DMEM that contains 10%FBS and 1%P/S to assemble (confluence) from Vec Technologies.Before cell experiment, sample is carried out supersound process (sonicated) and high temperature sterilize.

With endotheliocyte with 3500 cells/cm²Be seeded on each matrix.At first sample is placed the Tissue Culture Dish in 12-hole and 24-hole.Add the celliferous drop of 175 μ l of culture medium in the hand-hole and at 37 ℃ at 5%CO₂Under cultivated 4 hours.Clean sample 3 times with PBS, fixing 10min in formaldehyde, and then in PBS, clean 3 times.Use fluorescence microscope and DAPI dyestuff pair cell counting.And acquisition cellular morphology image.Experiment is carried out in triplicate, and respectively repeats (each average data point is six samples altogether) twice.Use student t-to check to determine difference between the matrix.

Fibroblast

In cell experiment, use fibroblast (CRL-2317, U.S. typical case DSMZ, population 2-4) and osteoblast (CRL-11372, U.S. typical case DSMZ, group number 2-4).Before inoculating cell, with phosphate buffer (PBS) (1X concentration) rinsing matrix.Be supplemented with the Da Shi improvement Yi Geer culture medium of 10% hyclone (Hyclone) and 1% penicillin/streptomycin (Hyclone) (Dulbecco ' s ModifiedEagle Medium) (Hyclone) in, with 3500 cells/cm²The initial inoculation density of matrix, cultured cell on matrix.Some experiments are undertaken by fibroblast separately, and some are to inoculate fibroblast and osteoblast (carrying out pre-staining with different fluorescent markers) simultaneously to determine competitive cell adhesion.Then at cell culture condition (37 ℃ of temperature, the 5%CO of standard₂With 95% dampness) under, cell was adhered onmatrix 4 hours.After the predetermined time cycle, sucking-off cell culture medium from the hole, then with PBS gently rinsingmatrix 3 times to remove any notadherent cell.Use 4% formalin (Fisher) fixed cell then and use Hoescht 33528 dyestuffs (Sigma) dyeing.In fluorescence microscope (Swiss) counting cells number and photograph down.

The surface characterizes

Under the 5kV accelerating potential, use field emission scanning electron microscope to learn (LEO) JEOLJSM-840 scanning electron microscope the sedimentary coating surface of IPD is carried out scanning electron microscope (SEM) analysis.Use (JEOL) software records digital picture of digital scan generator adduction (Digital Scan GeneratorPlus).Obtain fluorescence microscope images with the Leica fluorescence microscope, excitation wavelength 365nm, absorbance is measured at the 400nm place.

Statistics

Use standard variance analysis (ANOVA) technology, carry out statistical analysis in conjunction with Duncan multiple-range test (Duncan ' s Multiple Range) experiment.All experiments are all carried out in triplicate, and triplicate at least; P＜0.01 is considered to have statistical significance.

Embodiment

The IPD depositing metallic films that embodiment 1-is controlled

Fig. 1 shows the plasma-deposited device on selected matrix (2) that is applicable to that control is penetrated from cathode arc target source (1).The size of deposited particles and therefore the degree of the nanometer structure of deposition surface control by the movably matrix holder (3) in the vacuum chamber (4), or control by the power supply (5) and the arc speed actuator (6) of target.Matrix is more near arc source, and it is big more and extrude intensively more to be deposited on granule on the matrix.

In order to prepare the coated substrates that is used for cell adhesion, relative with target by matrix is placed than distant location, thus the matrix deposition is not contained king-sized particulate film.This forms the adhesiveness film.Then by matrix being placed the position of approaching target, thereby the matrix deposition obtains the intensive film of larger particle.

The control of the distance of matrix and target

With reference to Fig. 1,, matrix (sample 1) is placed movably matrix holder (3) in the distance of 30 inches of distance targets.Chamber (4) bled reach the level of 5E-4 holder.Under 100 amperes electric currents and 16 volts, start electric arc.With per 15 seconds speed of one inch matrix (2) is transferred to more near target, and lasts till between matrix and the target to be 8 inches (30min) always.

In the vacuum chamber of the level that is pumped to the 5E-4 holder, matrix (sample 2) is placed apart from 30 inches distances of target.Under 100 amperes electric currents and 16 volts, start electric arc.Matrix is kept matrix in 30 inches distances of distance target, keep 30min.

The cross section ofsample 1 andsample 2 uses sem analysis to detect.Oarse-grained amount and size increase along with the thickness of film insample 1; That is, less and less near matrix place bulky grain, and along with the growth of film thickness, quantity and size increase.On the contrary, the cross section insample 2 is that wherein bulky grain is considerably less uniformly.

The control of electric arc electric energy

Nanoparticle deposition and size can also be used for controlling by making of controlled IPD power supply, and this power supply can be set to fully slow down or quicken the speed of electric arc.Electric Arc Motion speed is directly related with the oarse-grained quantity of generation.The speed of the lip-deep electric arc of target of slowing down causes it to produce more bulky grain, and it can be used for increasing oarse-grained density.The film density of the increase that obtains has also improved the ability of tissue apposition in film.On the contrary, the speed that improves electric arc on the target will reduce oarse-grained output.This will produce more energetic ions, and its surface that can embed matrix is interior to produce good adhesive force.

Sample 3 does not use the electric arc controller, and matrix is placed apart from 12 inches distances of target.It is indoor that two samples all place, with the different time, and operation respectively, and be pumped to the 5E-4 holder.For power supply, electric arc is set to 100 amperes.Each target has two power supplys, is total up to 200 amperes when making beginning.Do not using under the electricarc controller sample 3operations 5 minutes.

Sample 4 moves with the switching current of the best with 300 hertz of speed.

Control this conversion so that on target, be maintained 200 amperes, but each power supply all can rise (ramp up) or descend (ramp down) thus it all is unequal causing the electric current on the power supply whenever.This forces the electric arc one section specific range that moves in preset time, thereby controls oarse-grained density and size.

Sample 3 and 4 is carried out the SEM cross-sectional analysis.Exceptsample 4 has the much bigger average bulky grain size and density thansample 3, run through whole thickness, film is homogeneous (consistent).Oarse-grained average-size in thesample 3 is approximately one micron, and density is 10³Granule/cm²Oarse-grained average-size in thesample 4 is approximately three microns, and density is 10⁴Granule/cm²

Embodiment 2-IPD coating deposited

See Fig. 1,vacuum chamber 4 is pumped to suitable operating pressure, usually in the scope of 0.1mT to 30mT; Yet the ability of the effective attaching surface of IPD prepared (having lasting rate of release) does not rely on any particular job pressure in the scope of 0.1mT to 30mT.Equally, IPD technology does not rely on operative temperature.Common operative temperature is in 25 ℃ to 200 ℃ scope, but lower or higher temperature also can be used.The temperature section ground that is adopted is determined by matrix.Temperature between about 20 ℃ to about 40 ℃ in the scope is suitable for preparing most of attaching surface.

Can use and come rotary substrate, or make the matrix lift-over pass through the deposition region with any direction with respect to the track of the deposition materials of introducing as turntable.Target is applied electric energy to produce electric arc at the target place.Be applicable under the voltage of source material, the electric energy scope can be several amperes to the hundreds of ampere.Voltage normally in 12 to 60 volts scope, and according to interior source material determine voltage with being in proportion, size of source material can be in length is several inches to several feet scope in this.

The exemplary coatings of the IPD titanium deposition on UHMWPE and PTFE matrix has been shown among Fig. 2.From the SEM photo as seen, sedimentary metal is that the surface structure becomes more nanometers coarse (more nano-rough) surface.

Nitinol coating on the steel

The Nitinol target is placed the vacuum chamber of ion plasma deposition device together with selected matrix.Electric arc turns to Nitinol metallic target ion the plasma of Nitinol ion, uncharged particle and electronics.Control Nitinol granule is to have scope less than the granular size of 1 nanometer in to about 50 microns.

The Nitinol target is preferably medical grade.Although use the low-purity metal can obtain gratifying result in some cases, recommend highly purified target material, thereby avoid to have toxic impurities.Can also use different alloys; For example, CoCrMo.

Use described depositing operation, with the Nitinol surface deposition of routine on steel matrix.Nickel adopts identical electric energy so that produce 50/50 nickel/titanium mixture with the titanium target.This mixture is deposited on the steel sample and by SEM and EDX analyzes.The oarse-grained average-size that SEM scanning illustrates in the sample is approximately one micron, and density is 10⁴Granule/cm²EDX illustrates about 51% titanium, 49% nickel mixture is evenly distributed on the surface.The pull test of standard illustrates the adhesion strength greater than 1KSI (1000psi).

Gold plating on the Nitinol

Use the depositing operation that discloses, 5 microns gold platings are deposited on the Nitinol pipe of commerciallyavailable diameter 1/8 thickness 0.005.By this Seed Layer of sem analysis (seed layer).SEM scanning illustrates about one micron average bulky grain size, density about 10⁴/ cm²The standard tension test shows the adhesion strength greater than 1KSI (1000psi).

Al₂O₃On the titanium Seed Layer

Utilize the depositing operation ofembodiment 1 and 2, apply Al as Seed Layer with three microns titaniums₂O₃Dish.With this Seed Layer of sem analysis.The oarse-grained average-size that SEM scanning illustrates in the sample is approximately one micron, and density is 10⁴Granule/cm²The standard tension experiment shows the adhesion strength greater than 1KSI (1000psi).

In further testing, titanium flame-spraying is also carried out pull test once more to Seed Layer.Coating illustrates the intensity greater than 1KSI once more.

Nitinol coating on the support

Use the depositing operation that discloses that Nitinol is deposited on the support.Be deposited as 1 micron thickness with being coated with, wherein average bulky grain is of a size of 1 micron, and density is 10⁴Granule/cm²The pull test of standard illustrates the adhesion strength greater than 1KSI.Coating shows as to have for vascular tissue and is attached to surperficial necessary characteristic, thereby expects that it can suppress restenosis.

The osteoblast of embodiment 3-on the polymeric matrix that applies adheres to

The polymeric matrix that preparation titanium and gold apply.Matrix is PEEK, UHMWPE and PTFE, is coated with gold, titanium or uncoated respectively.

With all substrates place 12-hole tissue culture ware (Corning, NY) in and with the sterilization phosphate buffer (PBS) (1X concentration contains 8gNaCl, 0.2g KCl, 1.2g Na in the 1000ml deionized water₂HPO₄And 0.2g KH₂PO₄, pH transfers to 7.4 (all chemicals are available from Sigma)) and rinsing.Be supplemented with among the DMEM (Hyclone) of 10%FBS (Hyclone) and 1%P/S, at 2ml then with 2500 cells/cm²Concentration osteoblast is seeded on the briquetting of being paid close attention to (compact), and subsequently at 37 ℃ of temperature, 5%CO₂With cultivate under the standard cell lines condition of culture of 95% dampness.Behind the 4hr, from the hole sucking-off cell culture medium and with PBS rinsing matrix 3 times to remove not adherent cell.(PA) the secure attachment cell is also with Hoechst 33258 dyestuffs (Sigma) dyeing for Fisher Scientific, Pittsburgh for formaldehyde with 4%.At fluorescence microscope (Leica) down observation of cell nuclear and counting, under 365nm, excite, launch under the 400nm.Cell is expressed as the average number of cell on 8 random areas of each matrix.All experiments are all carried out in triplicate and are assessed cell adhesion based on the average number of adherent cell.Utilize standard variance analysis (ANOVA) to come analytical data.P＜0.01 is considered to have statistical significance.

Utilize SEM to check osteoblastic form and attachment sites on the matrix of paying close attention to.When adhering to the mensuration end, pair cell dewaters by continuous washing in 50%, 60%, 70%, 80% and 90% alcoholic solution.Under ar gas environment, in 100 person of outstanding talent's holder vacuum and under the electric current at 10mA, use Hummer I spray etch instrument (Sputter Coater) (Technics) to utilize gold-palladium thin layer to spray coated sample three minutes then.Identical with the sample that does not have cell, use JEOL JSM-840 scanning electron microscope, under the accelerating potential of 5kV, take pictures.Applied Digital sweep generator adduction (Digital ScanGenerator Plus) (JEOL) software comes record digital image.

The result shows, compares with uncoated sample separately, is being coated with osteoblast adhesion enhancing (PEEK＜UHMWPE and PTFE) on three kinds of polymeric matrixs of nano-particle Ti or Au.Compare with the Ti of the micron particle size of present use, be coated with on the sample of nano-particle Ti at all, osteoblast adheres to better.

The PTFE that is coated with nano-particle Ti or Au is more superior than PEEK that is coated with nano-particle Ti or Au and UHMWPE respectively.The PTFE that is coated with Ti shows as best osteoblast and adheres to.Table 1 shows with coated substrates and compares, the result that the osteoblast of uncoated matrix is cultivated.

Table 1

Sample	Matrix	Coating	Number	Change relatively	% changes
						1	PEEK	Do not have	49.6	1.00	0
2	PEEK	Ti	83.2	1.68	67.74
						3	PEEK	Au	71.7	1.45	44.56
4	PTFE	Do not have	70.5	1.00	0
						5	PTFE	Ti	82.5	1.17	17.2
6	PTFE	Au	73	1.04	3.55
						7	UHMWPE	Do not have	27.3	1.00	0
8	UHMWPE	Ti	56.6	2.07	107.33
						9	UHMWPE	Au	65.8	2.41	141.03

The morphocytology result conforms to the result who quantitatively obtains; That is, compare with uncoated sample, on the polymer that is coated with Ti or Au, osteoblast shows as the cellular invasion (spreading) of increase.

Embodiment 4-is at the UHMWPE of titanium coating and the osteoblastic proliferation on the PTFE

Use titanium coated with PTFE and UHMWPE matrix as described.With the uncoated PTFE of razor finishing and UHMWPE sample to form smooth adhesive surface.Before inoculation, in 70% ethanol, sample carried out supersound process and high temperature sterilize or be exposed to 20min under the ultraviolet light of 120-350nm.Make osteoblast (ATCC CRL11373) in the DMEM that is supplemented with 10%FBS and 1%P/S incubation growth up to gathering.

With 3500 cells/cm²Osteoblast is seeded in places 12-hole and 24-porocyte culture dish on each matrix then.175 μ, 1 celliferous drop in the culture medium is placed on the sample then under 37 ℃ at 5%CO₂The middle 4hr that cultivates.Clean sample 3 times with PBS, fixing 10min in formaldehyde washes 3x again in PBS.Utilize fluorescence microscopy and DAPI dyestuff pair cell counting then.The pair cell form is taken pictures.Experiment is carried out in triplicate, and repeats (each average data point is six samples altogether) separately twice.The statistical analysis (student t-check) that utilizes standard is to determine the difference between the matrix.

The result shows, compares with uncoated sample separately, and on UHMWPE and PTFE matrix, titanium Nanosurface coating has significantly improved the propagation of osteocyte.The statistical significance of one group of sample can not obtain, may be because each sample applies the difference of density; Yet the difference between each coating and the uncoated sample is significant.Fig. 3 A has compared UHMWPE that uncoated and titanium apply and PTFE at the 1st day cell proliferation by the cell of measuring every square millimeter; Fig. 3 B be titanium that apply with uncoated UHMWPE and PTFE cell proliferation at the 3rd day; Fig. 3 C be titanium that apply with uncoated UHMWPE and PTFE cell proliferation at the 5th day.As shown in the table 2, the UHMWPE that titanium applies is superior to the PTFE matrix.Be initially only about half of that the comparative of observing increases at the osteoblastic proliferation that increases on the PTFE that titanium applies on the UHMWPE that titanium applies.At the 3rd day and the 5th day, compare with uncoated matrix, the PTFE that titanium applies shows as the growth less than 2 times in cell proliferation, and the UHMWPE that titanium applies compares even still kept the propagation of the raising that surpasses 5 times after 5 days with its uncoated counterpart.Compare with uncoated sample separately, the statistical analysis of UHMWPE measurement result (N=6) is p＜0.1.

Table 2

The growth of cell proliferation

Matrix	1day*	3days*	5 days*
				The UHMWPE that Ti applies	5.3	8.8	5.8
The PTFE that Ti applies	2.7	1.9	1.5

^*Compare with corresponding uncoated matrix

The fluorescence micrograph of the proliferative cell of 10X amplification has been shown among Fig. 4, has compared the PTFE that applies at 1,3 and 5 day titanium.Fig. 5 showed at 1,3 and 5 day, and osteoblastic breeding ratio on the UHMWPE that titanium applies.

The endotheliocyte of embodiment 5-on titanium adhesion property

In this embodiment, the Ti 6-4 with 200nm applies three kinds of matrixes.The average nanoparticle size of coating is 30 to 40 nanometers, and confirms by sem analysis.

The result shows that (shown in Figure 6) cell adhesion on the silicon part that applies reduces by 25%, cell adhesion on the UHMWPE that applies increase by 500% and on thePTFE sample 100% cell adhesion increase by 100%.Fig. 7 show that apply and uncoated silicon, polyethylene andOn the fluorescence microscope images of endothelial cell density.

The fibroblast adhesiveness of embodiment 6-on the matrix that titanium applies

With 3500 cells/cm²Fibroblast is seeded on each matrix.Sample is placed 12 holes and 24 porocyte culture dishs.Will be in culture medium the celliferous drop of 175 μ l place on the sample and at 37 ℃, 5%CO₂Under cultivate 4h.When the limiting time end cycle,clean sample 3 times with PBS, fixing 10min in formaldehyde cleans 3x again in PBS.Then with fluorescence microscopy and DAPI dyestuff pair cell counting.The pair cell form is taken pictures.Experiment is carried out in triplicate and is repeated (each average data point is six samples altogether) separately twice.The statistical analysis (student t-check) that utilizes standard is to determine the difference between the matrix.

Shown in measuring by cell density, compare with uncoated sample, the fibroblast adhesiveness significantly increases on the coated sample of PTFE and UHMWPE, and showing as respectively increases by about 78% and 90% (Fig. 8).UHMWPE and PTFE for titanium applies also observe the fibroblast quantity of increase and the diffusion of increase.

Embodiment 7-fibroblast adheres to/repels

In this embodiment, the Ti 6-4 with 200nm applies three kinds of matrix UHMWPE, silicon and PTFE.The average nano-particle size of coating is 30 to 40 nanometers and confirms by sem analysis.

Fibroblast is bought from ATCC (CRL-2317) and has among the DMEM of 10%FBS and 1%P/S incubation growth until gathering.As the Material Used sample that is provided.Before cell experiment, sample is carried out supersound process and high temperature sterilize.

With 3500 cells/cm²Fibroblast is seeded on each matrix.At first sample is placed 12-hole and 24-porocyte culture dish.Will be in culture medium the celliferous drop of 175 μ l add in each sample and at 37 ℃, 5%CO₂Under cultivate 4h.Clean sample 3 times with PBS then, fixing 10min in formaldehyde cleans 3 times in PBS again.Then with fluorescence microscopy and DAPI dyestuff pair cell counting.And obtain the cellular morphology image.Experiment is carried out in triplicate and each sample repeats twice (each average data point is six samples altogether).Check to determine difference between the matrix with student t-.

The result of this research shows for the first time, compares with other samples of test in this research, and external fibroblast adhesiveness reduces (Fig. 8) on the silicon that titanium applies.For other all matrixes, to compare with uncoated sample, the fibroblast adhesiveness on the coating increases.1,3 and 5 day fibroblasts proliferation shows in the test cultures, compares with uncoated sample separately, and fibroblastic adhesiveness increase of the PTFE that titanium is applied is more obvious, then has less adhesiveness on silicon that titanium applies and UHMWPE.In 1,3 and 5 day test result shown in Fig. 9 A, 9B and the 9C.Each column is represented n=3, wherein each contrast^*P＜0.01.This is a promising result because around the plastic surgery or blood vessel implant that the titanium that applies on by silicon constitutes, fibroblastic less adhesion be converted into less (less) soft, scar tissue.For other all matrixes, to compare with uncoated sample, the fibroblast on coating adheres to be increased.

The less adhering quantitative data of fibroblast conforms on fibroblast morphological images and the silicon that applies at titanium qualitatively.By the fluorescence microscopy analysis, as shown in Figure 10, compare with other the matrix that tried, on the silicon that titanium applies, almost do not observe the cell of good diffusion.

The albumen that embodiment 8-increases on that apply and uncoated sample is synthetic

In this embodiment, the Ti 6-4 with 200nm applies three kinds of matrixes.The average nanoparticle size of coating is 30 to 40 nanometers, and passes through sem analysis and confirm.Osteoblast is cultivated (culture) growth until polymerization (confluence) available from ATCC (CRL-11372) and in having 10%FBS and 1%P/S.

The material sample of the coating of using as being provided.With razor uncoated sample is repaired so that adhesive surface is smooth.Before cell experiment, in 70% ethanol, carry out supersound process and high temperature sterilize or carry outUV processing 20 minutes.

With 3500 cells/cm²Osteoblast is seeded on each matrix.At first sample is placed 12-hole and 24-porocyte culture dish.Will be in culture medium the celliferous drop of 175 μ l place on each sample and at 37 ℃, 5%CO₂Cultivate 4h under the atmosphere.Remove celliferous drop then and fill each sample well, and then cultivate the propagation of carrying out 1,3 and 5 day under the same conditions with the DMEM culture medium.Clean sample 3 times with PBS, in formaldehyde, solidify 10min, after 24,72 and 120 hours, in PBS, clean 3 times more respectively.Then with fluorescence microscopy and DAPI dyestuff pair cell counting.And obtain the cellular morphology image.Experiment is carried out in triplicate and each sample repeats twice (each average data point is six samples altogether).Check to determine difference between the matrix with student t-.

Result from protein determination shows synthetic the increasing of all coated portions albumen after 21 days.Silicon for applying approximately increases by 400%, and the UHMWPE for applying approximately increases by 1300%, and for the PTFE that applies, approximately increases by 800%.In these are measured, measure total protein.The propagation that increased at 7,14 and 21 days shown in Figure 11.

Embodiment 9: the osteoblastic proliferation of the increase on silicon, PTFE and UHMWPE

In this embodiment, by IPD technology three kinds of matrixes of Ti 6-4 coating with 200nm.The average nanoparticle size of coating is 30 to 40 nanometers and confirms by sem analysis.

Osteoblast available from ATCC (CRL-11372) and in DMEM with 10%FBS and 1%P/S incubation growth until polymerization.The silicon, PTFE and the UHMWPE sample that apply as the use that provided.With razor uncoated sample is repaired so that adhesive surface is smooth.Before cell experiment, in 70% ethanol, carry out supersound process and high temperature sterilize or under ultraviolet light theirradiation 20 minutes.

With 3500 cells/cm²Osteoblast is seeded on each matrix.Sample is placed 12-hole and 24-porocyte culture dish.Will be in culture medium the celliferous drop of 175 μ l place on the hole and at 37 ℃, 5%CO₂Cultivated 4 hours under the atmosphere.Remove celliferous drop then and fill each sample and cultivation under the same conditions once more, bred 1,3 and 5 day with the DMEM culture medium.Clean sample 3 times with PBS then, in formaldehyde, solidify 10min, after 24,72 and 120 hours, in PBS, clean 3 times more respectively.With fluorescence microscopy and DAPI dyestuff pair cell counting.And obtain the cellular morphology image.Experiment is carried out in triplicate and each sample repeats twice (each average data point is six samples altogether).Analyse (student t-check) with the canonical statistics credit and determine the difference between the matrix.

1,3 and 5 days test results show, compare with its uncoated counterpart, and osteoblastic proliferation increases on all coated substrates.Shown in Figure 12 A, compare the cell proliferation after 1 day on the matrix that applies with uncoated matrix; Be the cell proliferation after 3 days among Figure 12 B and cell proliferation after 5 days has been shown in Figure 12 C.Figure 13 be Ti that apply with uncoated PTFE on, the photo of the fluoroscopic image of the 1st, 3 and 5 day DAPI staining cell on that apply and uncoated PTFE.As far back as first day, compare with uncoated matrix and just to have tangible cell osteoblastic proliferation.Data rows is in table 3.

Table 3

Matrix	1day	3days	5days
				Silicon
	25％	10％	25％
				UHMWPE
	100％	100％	50％
				PTFE
	400％	1000％	400％

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Claims (27)

Translated fromChinese

1.一种用于制备生物涂覆的基体的方法，包括：1. A method for preparing a biocoated substrate comprising:将金属离子等离子体沉积在基体上以形成颗粒的纳米结构表面，所述颗粒的密度为至少约10³颗粒/cm²金属涂层并且尺寸范围为约1微米至约100微米；以及plasma depositing metal ions on the substrate to form a nanostructured surface of particles having a density of at least about¹⁰³ particles/^cm2 of metal coating and a size ranging from about 1 micron to about 100 microns; and使所述沉积的涂层与一种或多种细胞接触一段时间，足以使所述一种或多种细胞附着至所述涂层表面；contacting the deposited coating with one or more cells for a period of time sufficient to allow the one or more cells to attach to the coating surface;其中，附着至所述沉积涂层的所述一种或多种细胞形成了保留所述附着细胞的生物学性能的生物涂覆基体。Wherein, the one or more cells attached to the deposited coating form a biocoated matrix that retains the biological properties of the attached cells.2.根据权利要求1所述的方法，其中，附着至所述涂覆基体的所述一种或多种细胞是单核细胞。2. The method of claim 1, wherein the one or more cells attached to the coated substrate are monocytes.3.根据权利要求2所述的方法，所述一种或多种细胞选自由内皮细胞、成纤维细胞、成骨细胞、软骨细胞、肌细胞以及它们的混合物组成的组。3. The method of claim 2, wherein the one or more cells are selected from the group consisting of endothelial cells, fibroblasts, osteoblasts, chondrocytes, myocytes, and mixtures thereof.4.根据权利要求1所述的方法，其中，所述沉积是通过离子等离子体沉积(IPD)而进行的。4. The method of claim 1, wherein the depositing is by ion plasma deposition (IPD).5.根据权利要求4所述的方法，其中，所述IPD沉积的金属是在约1至约100nm的尺寸范围内的纳米颗粒。5. The method of claim 4, wherein the IPD deposited metals are nanoparticles in the size range of about 1 to about 100 nm.6.根据权利要求1所述的方法，其中，所述基体选自由UHMWPE、EPTFE、PTFE、PEEK、聚丙烯、聚氨酯、聚酰亚胺、聚酯、尼龙、钛、钢、铬、锆、镍、镍钛合金、它们的合金及组合组成的组。6. The method of claim 1, wherein the substrate is selected from the group consisting of UHMWPE, EPTFE, PTFE, PEEK, polypropylene, polyurethane, polyimide, polyester, nylon, titanium, steel, chromium, zirconium, nickel , nickel-titanium alloys, their alloys and combinations thereof.7.根据权利要求4所述的方法，其中，所述离子等离子体包括镍钛合金、CoCrMo、金、铂、铜、钽、钛、锆、铪、锌或它们的组合。7. The method of claim 4, wherein the ion plasma comprises Nitinol, CoCrMo, gold, platinum, copper, tantalum, titanium, zirconium, hafnium, zinc, or combinations thereof.8.根据权利要求7所述的方法，其中，所述离子等离子体是镍钛合金、金或钛。8. The method of claim 7, wherein the ion plasma is nitinol, gold or titanium.10.根据权利要求1所述的方法，其中，所述基体包括选自由导管、瓣、支架以及植入物组成的组的金属或聚合物材料装置。10. The method of claim 1, wherein the substrate comprises a metal or polymer material device selected from the group consisting of a catheter, a valve, a stent, and an implant.11.根据权利要求1所述的方法，其中，所述纳米结构金属涂层具有约10³颗粒/cm²至约10⁴颗粒/cm²的纳米颗粒密度。11. The method of claim 1, wherein the nanostructured metal coating has a nanoparticle density of about¹⁰³ particles/^cm2 to about¹⁰⁴ particles/^cm2 .12.根据权利要求11所述的方法，其中，所述金属涂层具有约1微米至约3微米的厚度。12. The method of claim 11, wherein the metal coating has a thickness of about 1 micron to about 3 microns.13.一种组合物，包括一种或多种附着至纳米结构金属膜的生物可存活细胞，其特征在于，通过离子等离子体沉积的金属颗粒的尺寸达到约1微米，以约10³至10⁴/cm²的密度分布。13. A composition comprising one or more bioviable cells attached to a nanostructured metal film, characterized in that the metal particles deposited by ion plasma have a size of about 1 micron, in the order of about¹⁰³ to 10⁴ /cm² density distribution.14.根据权利要求13所述的组合物，其中，所述离子等离子体沉积的金属颗粒包括Ag、Au、Ti、CoCrMo、或它们的混合物。14. The composition of claim 13, wherein the ion plasma deposited metal particles comprise Ag, Au, Ti, CoCrMo, or mixtures thereof.15.根据权利要求13所述的组合物，其中，所述生物可存活细胞为单核细胞。15. The composition of claim 13, wherein the biologically viable cells are monocytes.16.根据权利要求15所述的组合物，其中，所述单核细胞为成纤维细胞、成骨细胞、软骨细胞、肌细胞、或内皮细胞。16. The composition of claim 15, wherein the monocytes are fibroblasts, osteoblasts, chondrocytes, myocytes, or endothelial cells.17.根据权利要求13所述的组合物，其中，一种或多种细胞为附着至沉积在UHMWPE上的纳米结构钛表面的成骨细胞。17. The composition of claim 13, wherein the one or more cells are osteoblasts attached to a nanostructured titanium surface deposited on UHMWPE.18.根据权利要求13所述的组合物，其中，所述一种或多种细胞为附着至沉积在UHMWPE或PTFE上的纳米结构钛的内皮细胞。18. The composition of claim 13, wherein the one or more cells are endothelial cells attached to nanostructured titanium deposited on UHMWPE or PTFE.19.一种在基体上的纳米结构的金属表面涂层，其中，所述表面包括尺寸达到1微米、以约10³至约10⁴/cm²的密度分布在基体表面上的颗粒，并具有约10微米至约100微米的厚度。19. A nanostructured metal surface coating on a substrate, wherein said surface comprises particles up to 1 micron in size distributed on the surface of the substrate at a density of from about¹⁰³ to about¹⁰⁴ /^cm2 , and having A thickness of about 10 microns to about 100 microns.20.根据权利要求19所述的纳米结构表面，其中，所述金属表面是金、钛或镍钛合金。20. The nanostructured surface of claim 19, wherein the metal surface is gold, titanium or nitinol.21.一种纳米结构的表面涂层，其向金属或聚合物基体渗透达到约10纳米至约100纳米的深度，其中，所述涂层是包括约1nm至约5nm的纳米颗粒的离子等离子体沉积的金属，表面密度为约10³至10⁴/cm²，而厚度为约500nm至约100微米。21. A nanostructured surface coating that penetrates a metal or polymer substrate to a depth of from about 10 nanometers to about 100 nanometers, wherein said coating is an ionic plasma comprising nanoparticles of from about 1 nm to about 5 nm The deposited metal has a surface density of about 10³ to 10⁴ /cm² and a thickness of about 500 nm to about 100 microns.22.根据权利要求21所述的纳米结构的表面涂层，进一步包括附着的组织细胞。22. The nanostructured surface coating of claim 21, further comprising attached tissue cells.23.根据权利要求22所述的纳米结构的表面涂层，其中，所述组织细胞是内皮细胞、成纤维细胞、软骨细胞、成骨细胞、肌细胞或它们的混合物。23. The nanostructured surface coating of claim 22, wherein the tissue cells are endothelial cells, fibroblasts, chondrocytes, osteoblasts, muscle cells, or mixtures thereof.24.根据权利要求21所述的纳米结构的表面涂层，其中，所述涂层是Au、Ag、Ti、CoCrMo或它们的混合物。24. The nanostructured surface coating of claim 21, wherein the coating is Au, Ag, Ti, CoCrMo or mixtures thereof.25.根据权利要求21所述的纳米结构的表面涂层，其中，所述基体是钢、镍钛合金或氧化铝。25. The nanostructured surface coating of claim 21, wherein the substrate is steel, nitinol, or aluminum oxide.26.根据权利要求21所述的纳米结构的表面涂层，其中，所述基体是超高分子量聚乙烯(UHMWPE)、膨体聚四氟乙烯(EPTFE)、聚四氟乙烯(PTFE)、聚醚醚酮(PEEK)、聚丙烯、聚氨酯、聚酰亚胺、聚酯、尼龙、钛、钢、铬、锆、镍、镍钛合金、它们的合金及组合。26. The nanostructured surface coating of claim 21, wherein the substrate is ultra-high molecular weight polyethylene (UHMWPE), expanded polytetrafluoroethylene (EPTFE), polytetrafluoroethylene (PTFE), poly Ether ether ketone (PEEK), polypropylene, polyurethane, polyimide, polyester, nylon, titanium, steel, chromium, zirconium, nickel, nitinol, alloys and combinations thereof.27.根据权利要求21所述的纳米结构的表面涂层，其中，所述离子等离子体沉积的金属是以约300Hz的转换速率由速度受控的等离子体电弧靶而被沉积的。27. The nanostructured surface coating of claim 21, wherein the ion plasma deposited metal is deposited from a velocity controlled plasma arc target at a switching rate of about 300 Hz.28.根据权利要求21所述的纳米结构的表面涂层，其中，所述涂层包括可植入性医疗装置。28. The nanostructured surface coating of claim 21, wherein the coating comprises an implantable medical device.

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