CN102327645A - Preparation method of bionic coating carrying bioactive factors - Google Patents

Preparation method of bionic coating carrying bioactive factors Download PDF

Info

Publication number
CN102327645A
CN102327645A CN201110288240A CN201110288240A CN102327645A CN 102327645 A CN102327645 A CN 102327645A CN 201110288240 A CN201110288240 A CN 201110288240A CN 201110288240 A CN201110288240 A CN 201110288240A CN 102327645 A CN102327645 A CN 102327645A
Authority
CN
China
Prior art keywords
rgd
polyelectrolyte
solution
grafted
acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201110288240A
Other languages
Chinese (zh)
Inventor
李晓东
黄颖
李晓军
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang University ZJU
Original Assignee
Zhejiang University ZJU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang University ZJU filed Critical Zhejiang University ZJU
Priority to CN201110288240A priority Critical patent/CN102327645A/en
Publication of CN102327645A publication Critical patent/CN102327645A/en
Pending legal-status Critical Current

Links

Images

Landscapes

  • Materials For Medical Uses (AREA)

Abstract

The invention provides a preparation method of bionic coating carrying bioactive factors, which is characterized in that: under a catalyzing system of carbodiimide and N-hydroxy-succinamide, Arg-Gly-Asp (RGD) polypeptide chain containing a disulfide bond is grafted onto polyelectrolyte and then is prepared into RGD grafted polyanion electrolyte solution and RGD grafted bioactive-factor-loaded polycation electrolyte solution, the polyanion electrolyte solution is combined with the polycation electrolyte solution carrying the bioactive factors to establish an organic composite coating carrying the bioactive factors on the surface of planting body through a static self-assembling technology. The composite coating solves the bottleneck problem of bioactive factors used surrounding the planting body, can promote the early growth of osteogenesis surrounding the planting body so as to realize the early osseointegration, and facilitates the long-term stability of the planting body. The method is simple to operate, has moderate preparation conditions, is convenient to control and can realize the continuous production.

Description

A kind of bionical coating production of load bioactie agent
Technical field
The invention belongs to medical material manufacturing technology field; The bionical coating production that relates to a kind of load bioactie agent; Relating in particular to a kind of bionical coating production of biologically active factor slow release function, is the processing method of dentistry titanium implant surface bioactivation modification.Simulated the type of organization of natural extracellular matrix through this method in the bionical coating that implant surface makes up; Existing biomacromolecule constitute its agent structure again load can regulate the bioactie agent of cell behavior; The slowly releasing effect of one or more bioactie agents of realizing through this coating can promote the quick bone between implantation body and the osseous tissue to integrate, and also helps the long-time stability of implantation body.
Background technology
Bone integration (Osteointegration) theory that is proposed by professor Branemark is the core theory in tooth-planting field.Implantation body and surrounding bone tissue realize that synostosis is one of major criterion of estimating the plantation success or not.In order to reach this standard, many scholars are doing a large amount of work aspect planting material and the surface treatment.The enforcement of these implant surface processing methods, its ultimate principle all are through the process of intervention bone regeneration around implant tissue repair healing, accelerate the formation speed of bone regeneration around implant osseous tissue, thereby shorten the treatment cycle of implant denture.Implantation body implants to and realizes that the synostosis with the surrounding bone tissue generally can experience raising, adhere to, breed and breaking up of skeletonization source sexual cell, is the deposition and the sophisticated process of sclerotin mineralising of calcium salt at last.Implantation body contact with blood albumin layer that the back forms in its surface adsorption to skeletonization source sexual cell to the identification of implant surface be stained with important function; And the raising of skeletonization source sexual cell; Propagation and differentiation and calcium deposition process subsequently receive the influence of the local skeletonization microenvironment of bone regeneration around implant, comprise kind and the concentration of local bioactie agent etc.Based on the intensification that bone regeneration around implant tissue repair agglutination is understood; And for cell at the adhesion of substrate surface mechanism and bioactive molecule in the up-to-date understanding of regulating effect aspect cell differentiation and the tissue reconstruction, organic molecule is introduced implant surface becomes the hot issue during the implant surface method of modifying is studied in recent years.On the one hand, these organic molecules have been simulated the main component of extracellular matrix, for skeletonization source sexual cell provides advantageous environment in the implant surface growth; On the other hand, optionally use the bioactie agent of some known functions, particularly have the somatomedin of remarkable promotion cell and osseous tissue response function, can more effectively regulate and control the organization healing process of bone regeneration around implant.
The method of organic molecule being introduced implant surface also is referred to as surperficial biochemical modification.The organic molecule that relates in the biochemical modification of implant surface comprises several types: one type is the composition of macromolecule, mainly is extracellular matrix composition such as collagen and chondroitin sulfate etc.; One type is that the short adhesion polypeptide of small-molecular weight is as containing the peptide sequence of RGD (Arg-Gly-Asp, arginine-glycine-aspartic acid); Also having one type is to have very the somatomedin constituents such as BMP (the bone morphogenetic proteins of strong biological activity; BMPs), and fibroblast growth factor (fibroblast growth factors, FGFs); Insulin like growth factor (insulin-like growth factors; IGFs), and transforming growth factor (transform growth factors, TGFs) etc.The research of existing vivo and vitro confirms, the application of these organic molecules all has positive effect to the cell and the tissue reaction of implant surface.The particularly application of perhaps uniting other organic molecules separately of somatomedin constituents can provide extra clue for the cell growth of implant surface, thereby increase the osteanagenesis of bone regeneration around implant.The bottleneck problem that exists in the biochemical modification of implant surface now is to lack effective means these organic molecules are incorporated into implant surface, especially for the somatomedin constituents with high bioactivity.The ideal technological means that organic molecule is introduced implant surface should have such characteristic: the organic molecule coating that makes up in implant surface not only keeps its BA, and its degraded or the dynamic process that discharges and the process of organization healing are complementary.The requirement of obvious existing technological means as the mode of physical absorption and chemical coupling has satisfied not.Coating skewness, inefficiency, poor stability, poor repeatability and the adhesion that the former introduces is little to be wiped in implantation body's implantation process easily.And the latter causes the conformation of bioactie agent easily and influence its BA.
At enzymolysis, under the effect of hydrolysis and reducing substances, the degraded of above-mentioned polyelectrolyte composite coating under physiological condition still compare very fast (the about 4-7 of life period days).Regulate its degradation rate and realize that also the slow releasing function that loads on the bioactie agent in the coating depends on the further regulation and control to this polyelectrolyte composite coating stability.Research shows, disulfide bond (S-S-) in keeping particularly proteinic native conformation of biomacromolecule and stability, bringing into play important effect, and disulfide bond can be opened under reducing condition and forms sulfydryl (SH), and sulfydryl is easy to be oxidized to disulfide bond.The two reaction condition that conversion each other takes place is gentle relatively, and is less to the activity damage of biomacromolecule.
Summary of the invention
The method for preparing that the purpose of this invention is to provide a kind of bionical coating of load bioactie agent, realize through following steps:
(1) preparation of the grafted polyelectrolyte of RGD:
It at first is the synthetic rgd peptide segment that contains cystine linkage: glycine-arginine-glycine-aspartic acid-serine-proline-cysteine-s-s-cysteine-proline-aspartic acid-glycine-arginine-glycine (GRGDSPC (S-S) CPSDGRG); By SciLight Biotechnology; LLC company is synthetic; Then at carbodiimides [1-ethyl-3-(3-dimethyl ami-nopropyl) carbodiimide; EDC]: N-hydroxy-succinamide (N-hydroxysuccinimide, under the condition that NHS) catalyst system and catalyzing exists, this polypeptide segment is grafted on the polyelectrolyte; Behind resulting product dialysis purification; Add stachyose or glutathion and react with it, the cystine linkage that polypeptide chain is intersegmental is opened and is obtained the grafted polyelectrolyte of glycine-arginine-glycine-aspartic acid-serine-proline-cysteine-SH (GRGDSPC-SH), thereby realizes the modification of polyelectrolyte.Polyelectrolyte is polycation polyelectrolyte and polyanion polyelectrolyte.
RGD of the present invention is a kind of linearity seven peptide structures that contain arginine-glycine-aspartic acid, and its peptide chain structure is glycine-arginine-glycine-aspartic acid-serine-proline-cysteine-SH (GRGDSPC-SH).
The grafted polyelectrolyte of RGD according to the invention all refers to has been modified glycine-arginine-glycine-aspartic acid-serine-proline-cysteine sequence, and has the polyelectrolyte of free active sulfydryl.
Describedly gather cloudy electrolyte and select for use hyaluronic acid, alkali to dissolve in gelatin, polyglutamic acid, the poly-aspartate one or several, the product that obtains is the grafted polyanion electrolyte of RGD.The molecular weight of polyanion electrolyte is from 100-5000000D.
Saidly gather positive electrolyte and select for use Ι Collagen Type VI, chitosan, acid to dissolve in gelatin, poly arginine, polylysine, the polyhistidyl one or several, the product that obtains is the grafted polycation electrolyte of RGD.The molecular weight of polycation electrolyte is from 100-2000000D.
The concentration of said polyelectrolyte is 0.1-50mg/ml, and wherein optium concentration is 5mg/ml; The molar ratio of carboxyl-content is 1:10000-1:1 in segmental amount of described polypeptide and the polyelectrolyte, and wherein the optium concentration ratio is 1:20-50; Described carbodiimides: the consumption of N-hydroxy-succinamide (EDC:NHS) is 1:1-20:1, and wherein the optium concentration ratio is 10-5:1; The molar ratio of the consumption of EDC and the segmental consumption of polypeptide is 1:1-10:1, and wherein the optium concentration ratio is 2-5:1; The molar ratio of cystine linkage is 1:1-50:1 in the used stachyose or the concentration of glutathion and the EDC:NHS system, and wherein the optimum molar concentration ratio is 5:1.
(2) preparation of polyelectrolyte solution:
Be added with the preparation of the polycation electrolyte solution of bioactie agent (BMP-2 or bFGF): will be mixed with solution through grafted polycation electrolyte of RGD or the unmodified polycation electrolyte that step (1) obtains; And add bioactie agent BMP-2 or bFGF therein, be mixed with polycation electrolyte solution with certain bioactie agent concentration.Bioactie agent (bmp-2 or bFGF) only is added in the polycation electrolyte solution, because both are positively charged about 4.0 at pH value.
The preparation of polyanion electrolyte solution: will be mixed with solution through grafted polyanion polyelectrolyte of RGD or the unmodified polyanion electrolyte that step (1) obtains.
The concentration range of various polyelectrolyte solutions is 0.1mg/ml-10mg/ml in the step (2), and wherein optimum concentration range is 0.5-1mg/ml, and wherein NaCl concentration is 0.1-0.2mol/L in the polyelectrolyte solution, and pH value is 4.0.The concentration of BMP-2 is 0.5-1000 μ g/ml, and wherein optimum concentration range is 20-200 μ g/ml, and bFGF concentration is 20-1000 μ g/ml, and wherein optium concentration is 50-500 μ g/ml.
(3) preparation of the bionical coating of the grafted load bioactie agent of RGD:
The solution of step (2) is done following combination: the grafted polycation polyelectrolyte solution of RGD and the unmodified polyanion electrolyte solution that (a) add bioactie agent (BMP-2 or bFGF) can be used as one group of solution; (b) the grafted polyanion polyelectrolyte solution of RGD can be used as one group of solution with the unmodified polycation electrolyte solution that adds bioactie agent (BMP-2 or bFGF); (c) the grafted polycation polyelectrolyte solution of RGD and the grafted polyanion polyelectrolyte solution of RGD of interpolation bioactie agent (BMP-2 or bFGF) can be used as one group of solution.
With (a)-(c) arbitrary group of solution, utilize the static self-assembling technique, at first at titanio implant surface deposition polycation electrolyte and bioactie agent; Behind the deposition certain hour, fully washing deposits the polyanion electrolyte layer then; Behind the deposition certain hour, fully washing; More than operation is designated as the primary depositing circulation, repeats above cycling as required, obtains the polyelectrolyte composite bed that needs at last; Then, this polyelectrolyte composite bed soaks a period of time in toluene-sodium-sulfonchloramide solution or hydrogen peroxide solution, takes out rapidly, fully washes, and obtains the bionical coating of load bioactie agent.It promptly is the bionical coating of polyelectrolyte of the crosslinked biologically active factor slow release function of a kind of RGD.
Wherein depositing the used sedimentation time of single one deck polyanion electrolyte layer or polycation electrolyte layer is 5min-20min; Deposition one deck polycation electrolyte layer and one deck polyanion electrolyte layer are called a deposition cycle; Making up the required deposition cycle of polyelectrolyte composite coating is 0.5-50 time; Confirm last deposition cycle number according to the actual clinical purposes, wherein the oral cavity is 4-10 time with the surface deposition optimum cycle of tooth implant, and outermost layer is the polyelectrolyte layer that RGD modifies.
The soak time of polyelectrolyte composite bed in toluene-sodium-sulfonchloramide is 5-180 second in the step (3); Best Times is 30-90 second, and the concentration of toluene-sodium-sulfonchloramide is 0.5-10 mM, and wherein optium concentration is 2mM; Soak time is 5-60 minute in hydrogen peroxide; Best Times is 10-20 minute, and the concentration of hydrogen peroxide is 1-50 mM, and wherein optium concentration is 10-15mM.
Used implant surface is various permanent embedded type implant surface in the step (3), can select pure titanium or titanium alloy implant surface, titanium whitewashing/acid-treated pure titanium or the titanium alloy surface of pure titanium or titanium alloy implant surface, titanium or the hydroxyapatite gunite surface of the pure titanium that obtains through the acid-treated pure titanium of oxidisability or titanium alloy surface, the acid-treated pure titanium of non-oxide type or titanium alloy implant surface, sandblast/acid-treated surface, various alkali full-boiled process or titanium alloy implant surface, the various surfaces that electrochemistry obtains, various hydroxyapatite coating layers, ion implantation various pure titanium or the various pure titanium or the titanium alloy implant surface of titanium alloy implant surface or laser treatment for use.
The present invention makes up the organic coating that load has bioactie agent through layer-by-layer (layer by layer technique, LBL technology) in implant surface.The LBL technology is proposed by French scientist G.Decher the earliest, and this method has simple to operate, and preparation condition is gentle, and is convenient controlled, characteristics such as ability continuous production.Its ultimate principle is under electrostatic force, have the polyelectrolyte of opposite charges can alternating deposit in solid support surface, obtain the polyelectrolyte composite membrane of physicochemical properties homogeneous.Discover; Polyelectrolyte and polyampholyte such as protein, polypeptide, DNA with charge property etc. can utilize the LBL technology to be introduced in support surface; Bioactive macromolecule activity in multilayer complex films has almost obtained whole reservations simultaneously, and has the ability of stronger antagonism environmental change.Polyanion electrolyte that the present invention selected for use and polycation electrolyte are natural or through the biomacromolecule of RGD sequence graft modification.Somatomedin such as BMP-2 or bFGF is added in the polycation electrolyte solution, in polycation electrolyte deposits to the process of implant surface, is loaded into simultaneously in the polyelectrolyte composite coating.
The RGD sequential design that is used for the grafting biomacromolecule among the present invention becomes end to have sulfydryl; Under the condition of toluene-sodium-sulfonchloramide/anhydrous morpholino b acid or hydrogen peroxide existence; Can realize the crosslinked of polyelectrolyte composite coating through being converted each other of sulfydryl and disulfide bond, thereby increase the stability of coating and realize the slow release of bioactie agent.Simultaneously, realize regulation and control through the number of plies of change assembling and the time of cross-linking agent effect to the coating degradation time.And, because the existence of cystine linkage is given the polyelectrolyte composite coating again to biodegradable response in the body.
The polyelectrolyte composite bed of the load bioactie agent that the constructed RGD of the inventive method is crosslinked has been simulated the main composition of extracellular matrix on composition, the cell that is introduced as of rgd peptide provides more clue to the identification of substrate.Another effect of more wanting that functional r GD peptide sequence is introduced is the gentleness crosslinked load bioactie agent carrier of specificity effectively, has solved and has limited the bottleneck problem that bioactie agent is used in bone regeneration around implant for a long time.Expectation can promote the early stage generation of bone regeneration around implant skeletonization, can realize the bone integration early.
The present invention is a kind of method that makes up the imitative extracellular matrix organic coating of load bioactie agent at titanium implant surface.The preparation of implant surface polyelectrolyte composite coating is through layer-by-layer (layer by layer technique; LBL technology) realize, and the somatomedin constituents (BMP-2 or bFGF etc.) with high bioactivity is carried in the coating along with the assembling process of coating.It is the RGD sequence graft modification that has sulfydryl through end that the polyelectrolyte that makes up coating has at least a kind of.Utilize sulfydryl to increase the stability of polyelectrolyte coating through the reaction of oxidation generation cystine linkage; And the cystine linkage in the coating of crosslinked back has determined the degraded of coating to have biological responding to the response of reducing substances under the physiological environment, also makes the release of bioactie agent have slowly releasing effect.The precondition of the introducing bioactie agent ability steady load of rgd peptide sequence plays synergism with bioactie agent aspect the biological effect again simultaneously.The preparation of this coating can realize the early stage bone integration of implantation body, and helps the long-time stability of implantation body.
Description of drawings
Fig. 1 is the slow release behavior of the BMP-2 of load in the polyelectrolyte coating at phosphate buffer.
Fig. 2 is the influence that the coating of the crosslinked load BMP-2 of RGD is expressed MC3T3 cell Bone Gla protein (OC).
Fig. 3 is the influence that the coating of the crosslinked load BMP-2 of RGD is expressed MC3T3 cell Runx2 mRNA.
Fig. 4 is the influence that the coating of the crosslinked load BMP-2 of RGD is expressed MC3T3 cell AKP-2 mRNA.
Fig. 5 is in the different tissues healing cycle, the torque magnitude of the implantation body of experimental group and matched group.
Fig. 6 is the slow release behavior of the bFGF of load in the polyelectrolyte coating at phosphate buffer.
Fig. 7 is the influence of the coating of the crosslinked load bFGF of RGD to MC3T3 cell adhesion, propagation behavior.
Fig. 8 is the influence that the coating of the crosslinked load bFGF of RGD is expressed MC3T3 cell Bone Gla protein (OC).
Fig. 9 is the influence that the coating of the crosslinked load bFGF of RGD is expressed MC3T3 cell Runx2 mRNA.
Figure 10 is the influence that the coating of the crosslinked load bFGF of RGD is expressed MC3T3 cell AKP-2 mRNA.
Figure 11 is in the different tissues healing cycle, the torque magnitude of the implantation body of experimental group and matched group.
The specific embodiment
The present invention combines accompanying drawing and embodiment to be further described, and owing to the present invention can permanent implanted implant surface make up in all bodies, thereby can realize the quick bone integration and plantation success rate at a specified future date between implant and sclerous tissues.Among the embodiment just with a kind of sandblast/acid treatment surface as explaining, still, in other bodies on the permanent implanted implant surface with the constructed this coating of the present invention all within protection scope of the present invention.Embodiments of the invention are just explained characteristic of the present invention for better, and not exclusively comprise the application's the content of protecting.
Embodiment one: the preparation of the collagen of the load bioactie agent that RGD is crosslinked/hyaluronic acid polyelectrolyte composite bed
Polypeptide segment GRGDSPC (S-S) CPSDGRG and a certain amount of hyaluronic acid that will contain RGD are soluble in water, add EDC/NHS subsequently, react 4 hours, and product was dialysed for 1 week, lyophilizing; Product directly is dissolved in the water, and adds stachyose, reacts 4 hours, dialyses a week, and lyophilizing obtains having the grafted hyaluronate sodium of RGD of free sulfhydryl group.
In the NaCl of 0.1mol/L solution, both concentration all is 1mg/ml with the grafted hyaluronic acid of RGD and collagenolysis.In the collagen solution for preparing, add BMP-2, making its ultimate density in solution is 20 μ g/ml (as if what add are bFGF, and then final concentration is 0.1 mg/ml).The pH value of the above-mentioned solution that preparation is accomplished adjusts to 4.0.Implant surface is through bulky grain sandblast, H 2SO 4Handled 1 minute with the HCl mixed acid solution, lot of pure is washed clean implant surface, then this implantation body is soaked in (10min in the solution of collagen+BMP-2), the physical absorption of deionized water flush away (collagen+BMP-2); Then, be soaked in 10min in the hyaluronic acid solution that RGD modifies again, the hyaluronate sodium of deionized water flush away physical absorption.Repeat above operation, obtain 6 collagens/hyaluronic acid polyelectrolyte composite bed at last.The implantation body of this polyelectrolyte composite bed coating is soaked in 30 seconds in the toluene-sodium-sulfonchloramide, takes out immediately, massive laundering, flush away toluene-sodium-sulfonchloramide.Obtain the implantation body of the collagen/hyaluronic acid polyelectrolyte composite bed coating of the crosslinked load bioactie agent of RGD.
Embodiment two: the preparation of gelatin/hyaluronic acid polyelectrolyte composite bed is dissolved in the acid of the load bioactie agent that RGD is crosslinked
Method is with embodiment one, and difference is that collagen is replaced by acid dissolves gelatin (its source is pig source, cattle source or Mus source).
Embodiment three: the preparation of the chitosan of the load bioactie agent that RGD is crosslinked/hyaluronic acid polyelectrolyte composite bed
Method is with embodiment one, and difference is collagen is replaced by chitosan.
Embodiment four: the preparation of the poly arginine of the load bioactie agent that RGD is crosslinked/hyaluronic acid polyelectrolyte composite bed
Method is with embodiment one, and difference is collagen is replaced by poly arginine.
Embodiment five: the preparation of the polylysine of the load bioactie agent that RGD is crosslinked/hyaluronic acid polyelectrolyte composite bed
Method is with embodiment one, and difference is collagen is replaced by polylysine.
Embodiment six: the preparation of the polyhistidyl of the load bioactie agent that RGD is crosslinked/hyaluronic acid polyelectrolyte composite bed
Method is with embodiment one, and difference is collagen is replaced by polyhistidyl.
Embodiment seven: the collagen/alkali of the load bioactie agent that RGD is crosslinked dissolves the preparation of gelatin polyelectrolyte composite bed
Polypeptide segment GRGDSPC (S-S) CPSDGRG and the molten gelatin of a certain amount of alkali that will contain RGD are soluble in water, add EDC/NHS subsequently, react 4 hours, and product was dialysed for 1 week, lyophilizing; Product directly is dissolved in the water, and adds stachyose, react 4 hours, dialyses a week, and lyophilizing obtains having the molten gelatin of the grafted alkali of RGD of free sulfhydryl group.
The grafted alkali of RGD is dissolved gelatin and collagenolysis in the NaCl of 0.1mol/L solution, and both concentration all is 1mg/ml.In the collagen solution for preparing, add BMP-2, making its ultimate density in solution is 5 μ g/ml (as if what add are bFGF, and then final concentration is 0.1 mg/ml).The pH value of the above-mentioned solution that preparation is accomplished adjusts to 4.0.Implant surface is through bulky grain sandblast, H 2SO 4Handled 1 minute with the HCl mixed acid solution, lot of pure is washed clean implant surface, then this implantation body is soaked in (10min in the solution of collagen+BMP-2), the physical absorption of deionized water flush away (collagen+BMP-2); Then, the alkali that is soaked in the RGD modification again dissolves 10min in the gelatin solution, and the alkali of deionized water flush away physical absorption dissolves gelatin.Repeat above operation, obtain 6 collagen/alkali at last and dissolve gelatin polyelectrolyte composite bed.The implantation body of this polyelectrolyte composite bed coating is soaked in 30 seconds in the toluene-sodium-sulfonchloramide, takes out immediately, massive laundering, flush away toluene-sodium-sulfonchloramide.Collagen/the alkali that obtains the crosslinked load bioactie agent of RGD dissolves the implantation body of gelatin polyelectrolyte composite bed coating.
Embodiment eight: the preparation that gelatin/alkali dissolves gelatin polyelectrolyte composite bed is dissolved in the acid of the load bioactie agent that RGD is crosslinked
Method is with embodiment seven, and difference is that collagen is replaced by acid dissolves gelatin (its source is pig source, cattle source or Mus source).
Embodiment nine: the chitosan/alkali of the load bioactie agent that RGD is crosslinked dissolves the preparation of gelatin polyelectrolyte composite bed
Method is with embodiment seven, and difference is collagen is replaced by chitosan.
Embodiment ten: the poly arginine/alkali of the load bioactie agent that RGD is crosslinked dissolves the preparation of gelatin polyelectrolyte composite bed
Method is with embodiment seven, and difference is collagen is replaced by poly arginine.
Embodiment 11: the polylysine/alkali of the load bioactie agent that RGD is crosslinked dissolves the preparation of gelatin polyelectrolyte composite bed
Method is with embodiment seven, and difference is collagen is replaced by polylysine.
Embodiment 12: the polyhistidyl/alkali of the load bioactie agent that RGD is crosslinked dissolves the preparation of gelatin polyelectrolyte composite bed
Method is with embodiment seven, and difference is collagen is replaced by polyhistidyl.
Embodiment 13: the preparation of collagen/polyglutamic acid polyelectrolyte composite bed that RGD is crosslinked
Polypeptide segment GRGDSPC (S-S) CPSDGRG and a certain amount of polyglutamic acid that will contain RGD are soluble in water, add EDC/NHS subsequently, react 4 hours, and product was dialysed for 1 week, lyophilizing; Product directly is dissolved in the water, and adds stachyose, reacts 4 hours, dialyses a week, and lyophilizing obtains having the grafted polyglutamic acid of RGD of free sulfhydryl group.
In the NaCl of 0.1mol/L solution, both concentration all is 1mg/ml with the grafted polyglutamic acid of RGD and collagenolysis.In the collagen solution for preparing, add BMP-2, making its ultimate density in solution is 5 μ g/ml (as if what add are bFGF, and then final concentration is 0.1 mg/ml).The pH value of the above-mentioned solution that preparation is accomplished adjusts to 4.0.Implant surface is through bulky grain sandblast, H 2SO 4Handled 1 minute with the HCl mixed acid solution, lot of pure is washed clean implant surface, then this implantation body is soaked in (10min in the solution of collagen+BMP-2), the physical absorption of deionized water flush away (collagen+BMP-2); Then, be soaked in 10min in the polyglutamic acid solution that RGD modifies again, the polyglutamic acid of deionized water flush away physical absorption.Repeat above operation, obtain 6 collagens/polyglutamic acid polyelectrolyte composite bed at last.The implantation body of this polyelectrolyte composite bed coating is soaked in 30 seconds in the toluene-sodium-sulfonchloramide, takes out immediately, massive laundering, flush away toluene-sodium-sulfonchloramide.Obtain the implantation body of the collagen/polyglutamic acid polyelectrolyte composite bed coating of the crosslinked load bioactie agent of RGD.
Embodiment 14: the preparation of gelatin/polyglutamic acid polyelectrolyte composite bed is dissolved in the acid of the load bioactie agent that RGD is crosslinked
Method is with embodiment 13, and difference is that collagen is replaced by acid dissolves gelatin (its source is pig source, cattle source or Mus source).
Embodiment 15: the preparation of the chitosan of the load bioactie agent that RGD is crosslinked/polyglutamic acid polyelectrolyte composite bed
Method is with embodiment 13, and difference is collagen is replaced by chitosan.
Embodiment 16: the preparation of the poly arginine of the load bioactie agent that RGD is crosslinked/polyglutamic acid polyelectrolyte composite bed
Method is with embodiment 13, and difference is collagen is replaced by poly arginine.
Embodiment 17: the preparation of the polylysine of the load bioactie agent that RGD is crosslinked/polyglutamic acid polyelectrolyte composite bed
Method is with embodiment 13, and difference is collagen is replaced by polylysine.
Embodiment 18: the preparation of the polyhistidyl of the load bioactie agent that RGD is crosslinked/polyglutamic acid polyelectrolyte composite bed
Method is with embodiment 13, and difference is collagen is replaced by polyhistidyl.
Embodiment 19: the preparation of the collagen of the load bioactie agent that RGD is crosslinked/poly-aspartate polyelectrolyte composite bed
Polypeptide segment GRGDSPC (S-S) CPSDGRG and a certain amount of poly-aspartate that will contain RGD are soluble in water, add EDC/NHS subsequently, react 4 hours, and product was dialysed for 1 week, lyophilizing; Product directly is dissolved in the water, and adds stachyose, reacts 4 hours, dialyses a week, and lyophilizing obtains having the grafted poly-aspartate of RGD of free sulfhydryl group.
In the NaCl of 0.1mol/L solution, both concentration all is 1mg/ml with the grafted poly-aspartate of RGD and collagenolysis.In the collagen solution for preparing, add BMP-2, making its ultimate density in solution is 5 μ g/ml (as if what add are bFGF, and then final concentration is 0.1 mg/ml).The pH value of the above-mentioned solution that preparation is accomplished adjusts to 4.0.Implant surface is through bulky grain sandblast, H 2SO 4Handled 1 minute with the HCl mixed acid solution, lot of pure is washed clean implant surface, then this implantation body is soaked in (10min in the solution of collagen+BMP-2), the physical absorption of deionized water flush away (collagen+BMP-2); Then, be soaked in 10min in the poly-aspartate solution that RGD modifies again, the poly-aspartate of deionized water flush away physical absorption.Repeat above operation, obtain 6 collagens/poly-aspartate polyelectrolyte composite bed at last.The implantation body of this polyelectrolyte composite bed coating is soaked in 30 seconds in the toluene-sodium-sulfonchloramide, takes out immediately, massive laundering, flush away toluene-sodium-sulfonchloramide.Obtain the implantation body of the collagen/poly-aspartate polyelectrolyte composite bed coating of the crosslinked load bioactie agent of RGD.
Embodiment 20: the preparation of gelatin/poly-aspartate polyelectrolyte composite bed is dissolved in the acid of the load bioactie agent that RGD is crosslinked
Method is with embodiment 19, and difference is that collagen is replaced by acid dissolves gelatin (its source is pig source, cattle source or Mus source).
Embodiment 21: the preparation of the chitosan of the load bioactie agent that RGD is crosslinked/poly-aspartate polyelectrolyte composite bed
Method is with embodiment 19, and difference is collagen is replaced by chitosan.
Embodiment 22: the preparation of the poly arginine of the load bioactie agent that RGD is crosslinked/poly-aspartate polyelectrolyte composite bed
Method is with embodiment 19, and difference is collagen is replaced by poly arginine.
Embodiment 23: the preparation of the polylysine of the load bioactie agent that RGD is crosslinked/poly-aspartate polyelectrolyte composite bed
Method is with embodiment 19, and difference is collagen is replaced by polylysine.
Embodiment 24: the preparation of the polyhistidyl of the load bioactie agent that RGD is crosslinked/poly-aspartate polyelectrolyte composite bed
Method is with embodiment 19, and difference is collagen is replaced by polyhistidyl.
Embodiment 25: the preparation of the collagen of the load bioactie agent that RGD is crosslinked/hyaluronic acid polyelectrolyte composite bed
Polypeptide segment GRGDSPC (S-S) CPSDGRG and a certain amount of collagen that will contain RGD are soluble in water, add EDC/NHS subsequently, react 4 hours, and product was dialysed for 1 week, lyophilizing; Product directly is dissolved in the water, and adds stachyose, reacts 4 hours, dialyses a week, and lyophilizing obtains having the grafted collagen of RGD of free sulfhydryl group.
Grafted collagen of this RGD and hyaluronic acid are dissolved in the NaCl solution of 0.1mol/L, and both concentration all is 1mg/ml.In the collagen solution for preparing, add BMP-2, making its ultimate density in solution is 5 μ g/ml (as if what add are bFGF, and then final concentration is 0.1 mg/ml).The pH value of the above-mentioned solution that preparation is accomplished adjusts to 4.0.Implant surface is through bulky grain sandblast, H 2SO 4Handled 1 minute with the HCl mixed acid solution, lot of pure is washed clean implant surface, then this implantation body is soaked in (10min in the solution of the grafted collagen of RGD+BMP-2), the physical absorption of deionized water flush away (the grafted collagen of RGD+BMP-2); Then, be soaked in 10min in the hyaluronic acid solution again, the hyaluronic acid of deionized water flush away physical absorption.Repeat above operation, obtain 6 collagens/hyaluronic acid polyelectrolyte composite bed at last.The implantation body of this polyelectrolyte composite bed coating is soaked in 30 seconds in the toluene-sodium-sulfonchloramide, takes out immediately, massive laundering, flush away toluene-sodium-sulfonchloramide.Obtain the implantation body of the collagen/hyaluronic acid polyelectrolyte composite bed coating of the crosslinked load bioactie agent of RGD.
Embodiment 26: the collagen/alkali of the load bioactie agent that RGD is crosslinked dissolves the preparation of gelatin polyelectrolyte composite bed
Method is with embodiment 25, and difference is that hyaluronic acid is replaced by alkali dissolves gelatin (its source is pig source, cattle source or Mus source).
Embodiment 27: the preparation of the collagen of the load bioactie agent that RGD is crosslinked/polyglutamic acid polyelectrolyte composite bed
Method is with embodiment 25, and difference is hyaluronic acid is replaced by polyglutamic acid.
Embodiment 28: the preparation of the poly arginine of the load bioactie agent that RGD is crosslinked/poly-aspartate polyelectrolyte composite bed
Method is with embodiment 25, and difference is hyaluronic acid is replaced by poly-aspartate.
Embodiment 29: the preparation of the chitosan of the load bioactie agent that RGD is crosslinked/hyaluronic acid polyelectrolyte composite bed
Polypeptide segment GRGDSPC (S-S) CPSDGRG and the certain amount of chitosan that will contain RGD are soluble in water, add EDC/NHS subsequently, react 4 hours, and product was dialysed for 1 week, lyophilizing; Product directly is dissolved in the water, and adds stachyose, reacts 4 hours, dialyses a week, and lyophilizing obtains having the grafted chitosan of RGD of free sulfhydryl group.
Grafted chitosan of this RGD and hyaluronic acid are dissolved in the NaCl solution of 0.1mol/L, and both concentration all is 1mg/ml.In the chitosan solution for preparing, add BMP-2, making its ultimate density in solution is 5 μ g/ml (as if what add are bFGF, and then final concentration is 0.1 mg/ml).The pH value of the above-mentioned solution that preparation is accomplished adjusts to 4.0.Implant surface is through bulky grain sandblast, H 2SO 4Handled 1 minute with the HCl mixed acid solution, lot of pure is washed clean implant surface, then this implantation body is soaked in (10min in the solution of the grafted chitosan of RGD+BMP-2), the physical absorption of deionized water flush away (the grafted chitosan of RGD+BMP-2); Then, be soaked in 10min in the hyaluronic acid solution again, the hyaluronic acid of deionized water flush away physical absorption.Repeat above operation, obtain 6 chitosans/hyaluronic acid polyelectrolyte composite bed at last.The implantation body of this polyelectrolyte composite bed coating is soaked in 30 seconds in the toluene-sodium-sulfonchloramide, takes out immediately, massive laundering, flush away toluene-sodium-sulfonchloramide.Obtain the implantation body of the chitosan/hyaluronic acid polyelectrolyte composite bed coating of the crosslinked load bioactie agent of RGD.
Embodiment 30: the chitosan/alkali of the load bioactie agent that RGD is crosslinked dissolves the preparation of gelatin polyelectrolyte composite bed
Method is with embodiment 25, and difference is that hyaluronic acid is replaced by alkali dissolves gelatin (its source is pig source, cattle source or Mus source).
The preparation of the chitosan of the load bioactie agent that embodiment hentriaconta-: RGD is crosslinked/polyglutamic acid polyelectrolyte composite bed
Method is with embodiment 25, and difference is hyaluronic acid is replaced by polyglutamic acid.
Embodiment 32: the preparation of the chitosan of the load bioactie agent that RGD is crosslinked/poly-aspartate polyelectrolyte composite bed
Method is with embodiment 25, and difference is hyaluronic acid is replaced by poly-aspartate.
Embodiment 33: the preparation of gelatin/hyaluronic acid polyelectrolyte composite bed is dissolved in the acid of the load bioactie agent that RGD is crosslinked
Polypeptide segment GRGDSPC (S-S) CPSDGRG and the molten gelatin of a certain amount of acid that will contain RGD are soluble in water, add EDC/NHS subsequently, react 4 hours, and product was dialysed for 1 week, lyophilizing; Product directly is dissolved in the water, and adds stachyose, react 4 hours, dialyses a week, and lyophilizing obtains having the molten gelatin of the grafted acid of RGD of free sulfhydryl group.
Molten gelatin of the grafted acid of this RGD and hyaluronic acid are dissolved in the NaCl solution of 0.1mol/L, and both concentration all is 1mg/ml.In the molten gelatin solution of the acid for preparing, add BMP-2, making its ultimate density in solution is 5 μ g/ml (as if what add are bFGF, and then final concentration is 0.1 mg/ml).The pH value of the above-mentioned solution that preparation is accomplished adjusts to 4.0.Implant surface is through bulky grain sandblast, H 2SO 4Handled 1 minute with the HCl mixed acid solution, lot of pure is washed clean implant surface, then this implantation body is soaked in (10min in the solution of gelatin+BMP-2) is dissolved in the grafted acid of RGD, the physical absorption of deionized water flush away (gelatin+BMP-2) is dissolved in the grafted acid of RGD; Then, be soaked in 10min in the hyaluronic acid solution again, the hyaluronic acid of deionized water flush away physical absorption.Repeat above operation, obtain 6 acid at last and dissolve gelatin/hyaluronic acid polyelectrolyte composite bed.The implantation body of this polyelectrolyte composite bed coating is soaked in 30 seconds in the toluene-sodium-sulfonchloramide, takes out immediately, massive laundering, flush away toluene-sodium-sulfonchloramide.The implantation body of gelatin/hyaluronic acid polyelectrolyte composite bed coating is dissolved in the acid that obtains the crosslinked load bioactie agent of RGD.
Embodiment 34: the preparation that gelatin/alkali dissolves gelatin polyelectrolyte composite bed is dissolved in the acid of the load bioactie agent that RGD is crosslinked
Method is with embodiment 33, and difference is that hyaluronic acid is replaced by alkali dissolves gelatin (its source is pig source, cattle source or Mus source).
Embodiment 35: the preparation of gelatin/polyglutamic acid polyelectrolyte composite bed is dissolved in the acid of the load bioactie agent that RGD is crosslinked
Method is with embodiment 33, and difference is hyaluronic acid is replaced by polyglutamic acid.
Embodiment 36: the preparation of gelatin/poly-aspartate polyelectrolyte composite bed is dissolved in the acid of the load bioactie agent that RGD is crosslinked
Method is with embodiment 33, and difference is hyaluronic acid is replaced by poly-aspartate.
Embodiment 37: the preparation of the poly arginine of the load bioactie agent that RGD is crosslinked/hyaluronic acid polyelectrolyte composite bed
Polypeptide segment GRGDSPC (S-S) CPSDGRG and a certain amount of poly arginine that will contain RGD are soluble in water, add EDC/NHS subsequently, react 4 hours, and product was dialysed for 1 week, lyophilizing; Product directly is dissolved in the water, and adds stachyose, reacts 4 hours, dialyses a week, and lyophilizing obtains having the grafted poly arginine of RGD of free sulfhydryl group.
Grafted poly arginine of this RGD and hyaluronic acid are dissolved in the NaCl solution of 0.1mol/L, and both concentration all is 1mg/ml.In the poly arginine solution for preparing, add BMP-2, making its ultimate density in solution is 5 μ g/ml (as if what add are bFGF, and then final concentration is 0.1 mg/ml).The pH value of the above-mentioned solution that preparation is accomplished adjusts to 4.0.Implant surface is through bulky grain sandblast, H 2SO 4Handled 1 minute with the HCl mixed acid solution, lot of pure is washed clean implant surface, then this implantation body is soaked in (10min in the solution of the grafted poly arginine of RGD+BMP-2), the physical absorption of deionized water flush away (the grafted poly arginine of RGD+BMP-2); Then, be soaked in 10min in the hyaluronic acid solution again, the hyaluronic acid of deionized water flush away physical absorption.Repeat above operation, obtain 6 poly arginines/hyaluronic acid polyelectrolyte composite bed at last.The implantation body of this polyelectrolyte composite bed coating is soaked in 30 seconds in the toluene-sodium-sulfonchloramide, takes out immediately, massive laundering, flush away toluene-sodium-sulfonchloramide.Obtain the implantation body of the poly arginine/hyaluronic acid polyelectrolyte composite bed coating of the crosslinked load bioactie agent of RGD.
Embodiment 38: the poly arginine/alkali of the load bioactie agent that RGD is crosslinked dissolves the preparation of gelatin polyelectrolyte composite bed
Method is with embodiment 37, and difference is that hyaluronic acid is replaced by alkali dissolves gelatin (its source is pig source, cattle source or Mus source).
Embodiment 39: the preparation of the poly arginine of the load bioactie agent that RGD is crosslinked/polyglutamic acid polyelectrolyte composite bed
Method is with embodiment 37, and difference is hyaluronic acid is replaced by polyglutamic acid.
Embodiment 40: the preparation of the poly arginine of the load bioactie agent that RGD is crosslinked/poly-aspartate polyelectrolyte composite bed
Method is with embodiment 37, and difference is hyaluronic acid is replaced by poly-aspartate.
Embodiment 41: the preparation of the polylysine of the load bioactie agent that RGD is crosslinked/hyaluronic acid polyelectrolyte composite bed
Polypeptide segment GRGDSPC (S-S) CPSDGRG and a certain amount of polylysine that will contain RGD are soluble in water, add EDC/NHS subsequently, react 4 hours, and product was dialysed for 1 week, lyophilizing; Product directly is dissolved in the water, and adds stachyose, reacts 4 hours, dialyses a week, and lyophilizing obtains having the grafted polylysine of RGD of free sulfhydryl group.
Grafted polylysine acid of this RGD and hyaluronic acid are dissolved in the NaCl solution of 0.1mol/L, and both concentration all is 1mg/ml.In the polylysine solution for preparing, add BMP-2, making its ultimate density in solution is 5 μ g/ml (as if what add are bFGF, and then final concentration is 0.1 mg/ml).The pH value of the above-mentioned solution that preparation is accomplished adjusts to 4.0.Implant surface is through bulky grain sandblast, H 2SO 4Handled 1 minute with the HCl mixed acid solution, lot of pure is washed clean implant surface, then this implantation body is soaked in (10min in the solution of the grafted polylysine of RGD+BMP-2), the physical absorption of deionized water flush away (the grafted polylysine of RGD+BMP-2); Then, be soaked in 10min in the hyaluronic acid solution again, the hyaluronic acid of deionized water flush away physical absorption.Repeat above operation, obtain 6 polylysines/hyaluronic acid polyelectrolyte composite bed at last.The implantation body of this polyelectrolyte composite bed coating is soaked in 30 seconds in the toluene-sodium-sulfonchloramide, takes out immediately, massive laundering, flush away toluene-sodium-sulfonchloramide.Obtain the implantation body of the polylysine/hyaluronic acid polyelectrolyte composite bed coating of the crosslinked load bioactie agent of RGD.
Embodiment 42: the polylysine/alkali of the load bioactie agent that RGD is crosslinked dissolves the preparation of gelatin polyelectrolyte composite bed
Method is with embodiment 41, and difference is that hyaluronic acid is replaced by alkali dissolves gelatin (its source is pig source, cattle source or Mus source).
Embodiment 43: the preparation of the polylysine of the load bioactie agent that RGD is crosslinked/polyglutamic acid polyelectrolyte composite bed
Method is with embodiment 41, and difference is hyaluronic acid is replaced by polyglutamic acid.
Embodiment 44: the preparation of the polylysine of the load bioactie agent that RGD is crosslinked/poly-aspartate polyelectrolyte composite bed
Method is with embodiment 41, and difference is hyaluronic acid is replaced by poly-aspartate.
Embodiment 45: the preparation of the polyhistidyl of the load bioactie agent that RGD is crosslinked/hyaluronic acid polyelectrolyte composite bed
Polypeptide segment GRGDSPC (S-S) CPSDGRG and a certain amount of polyhistidyl that will contain RGD are soluble in water, add EDC/NHS subsequently, react 4 hours, and product was dialysed for 1 week, lyophilizing; Product directly is dissolved in the water, and adds stachyose, reacts 4 hours, dialyses a week, and lyophilizing obtains having the grafted polyhistidyl of RGD of free sulfhydryl group.
Grafted polyhistidyl of this RGD and hyaluronic acid are dissolved in the NaCl solution of 0.1mol/L, and both concentration all is 1mg/ml.In the polyhistidyl solution for preparing, add BMP-2, making its ultimate density in solution is 5 μ g/ml (as if what add are bFGF, and then final concentration is 0.1 mg/ml).The pH value of the above-mentioned solution that preparation is accomplished adjusts to 4.0.Implant surface is through bulky grain sandblast, H 2SO 4Handled 1 minute with the HCl mixed acid solution, lot of pure is washed clean implant surface, then this implantation body is soaked in (10min in the solution of the grafted polyhistidyl of RGD+BMP-2), the physical absorption of deionized water flush away (the grafted polyhistidyl of RGD+BMP-2); Then, be soaked in 10min in the hyaluronic acid solution again, the hyaluronic acid of deionized water flush away physical absorption.Repeat above operation, obtain 6 polyhistidyls/hyaluronic acid polyelectrolyte composite bed at last.The implantation body of this polyelectrolyte composite bed coating is soaked in 30 seconds in the toluene-sodium-sulfonchloramide, takes out immediately, massive laundering, flush away toluene-sodium-sulfonchloramide.Obtain the implantation body of the polyhistidyl/hyaluronic acid polyelectrolyte composite bed coating of the crosslinked load bioactie agent of RGD.
Embodiment 46: the polyhistidyl/alkali of the load bioactie agent that RGD is crosslinked dissolves the preparation of gelatin polyelectrolyte composite bed
Method is with embodiment 45, and difference is that hyaluronic acid is replaced by alkali dissolves gelatin (its source is pig source, cattle source or Mus source).
Embodiment 47: the preparation of the polyhistidyl of the load bioactie agent that RGD is crosslinked/polyglutamic acid polyelectrolyte composite bed
Method is with embodiment 45, and difference is hyaluronic acid is replaced by polyglutamic acid.
Embodiment 48: the preparation of the polyhistidyl of the load bioactie agent that RGD is crosslinked/poly-aspartate polyelectrolyte composite bed
Method is with embodiment 45, and difference is hyaluronic acid is replaced by poly-aspartate.
Embodiment 49: the preparation of the collagen of the load bioactie agent that RGD is crosslinked/hyaluronic acid polyelectrolyte composite bed
Polypeptide segment GRGDSPC (S-S) CPSDGRG that will contain RGD is soluble in water with a certain amount of collagen and hyaluronic acid respectively, adds EDC/NHS subsequently, reacts 4 hours, and product was dialysed for 1 week, lyophilizing; Product directly is dissolved in the water, and adds stachyose, reacts 4 hours, dialyses a week lyophilizing, the grafted hyaluronic acid of RGD that obtains having the grafted collagen of RGD of free sulfhydryl group and have free sulfhydryl group.
Grafted collagen of RGD and the grafted hyaluronic acid of RGD are dissolved in the NaCl solution of 0.1mol/L, and both concentration all is 1mg/ml.In the collagen solution for preparing, add BMP-2, making its ultimate density in solution is 5 μ g/ml (as if what add are bFGF, and then final concentration is 0.1 mg/ml).The pH value of the above-mentioned solution that preparation is accomplished adjusts to 4.0.Implant surface is through bulky grain sandblast, H 2SO 4Handled 1 minute with the HCl mixed acid solution, lot of pure is washed clean implant surface, then this implantation body is soaked in (10min in the solution of the grafted collagen of RGD+BMP-2), the physical absorption of deionized water flush away (the grafted collagen of RGD+BMP-2); Then, be soaked in 10min in the hyaluronic acid solution again, the hyaluronic acid of deionized water flush away physical absorption.Repeat above operation, obtain 6 collagens/hyaluronic acid polyelectrolyte composite bed at last.The implantation body of this polyelectrolyte composite bed coating is soaked in 30 seconds in the toluene-sodium-sulfonchloramide, takes out immediately, massive laundering, flush away toluene-sodium-sulfonchloramide.Obtain the implantation body of the collagen/hyaluronic acid polyelectrolyte composite bed coating of the crosslinked load bioactie agent of RGD.
Embodiment 50: the collagen/alkali of the load bioactie agent that RGD is crosslinked dissolves the preparation of gelatin polyelectrolyte composite bed
Method is with embodiment 49, and difference is that hyaluronic acid is replaced by alkali dissolves gelatin (its source is pig source, cattle source or Mus source).
Embodiment 51: the preparation of the collagen of the load bioactie agent that RGD is crosslinked/polyglutamic acid polyelectrolyte composite bed
Method is with embodiment 49, and difference is hyaluronic acid is replaced by polyglutamic acid.
Embodiment 52: the preparation of the poly arginine of the load bioactie agent that RGD is crosslinked/poly-aspartate polyelectrolyte composite bed
Method is with embodiment 49, and difference is hyaluronic acid is replaced by poly-aspartate.
Embodiment 53: the preparation of the chitosan of the load bioactie agent that RGD is crosslinked/hyaluronic acid polyelectrolyte composite bed
Polypeptide segment GRGDSPC (S-S) CPSDGRG that will contain RGD is soluble in water with certain amount of chitosan and hyaluronic acid respectively, adds EDC/NHS subsequently, reacts 4 hours, and product was dialysed for 1 week, lyophilizing; Product directly is dissolved in the water, and adds stachyose, reacts 4 hours, dialyses a week, and lyophilizing obtains having the grafted chitosan of RGD and the grafted hyaluronic acid of RGD of free sulfhydryl group respectively.
Grafted chitosan of this RGD and the grafted hyaluronic acid of RGD are dissolved in the NaCl solution of 0.1mol/L, and both concentration all is 1mg/ml.In the chitosan solution for preparing, add BMP-2, making its ultimate density in solution is 5 μ g/ml (as if what add are bFGF, and then final concentration is 0.1 mg/ml).The pH value of the above-mentioned solution that preparation is accomplished adjusts to 4.0.Implant surface is through bulky grain sandblast, H 2SO 4Handled 1 minute with the HCl mixed acid solution, lot of pure is washed clean implant surface, then this implantation body is soaked in (10min in the solution of the grafted chitosan of RGD+BMP-2), the physical absorption of deionized water flush away (the grafted chitosan of RGD+BMP-2); Then, be soaked in 10min in the grafted hyaluronic acid solution of RGD again, the grafted hyaluronic acid of RGD of deionized water flush away physical absorption.Repeat above operation, obtain 6 chitosans/hyaluronic acid polyelectrolyte composite bed at last.The implantation body of this polyelectrolyte composite bed coating is soaked in 30 seconds in the toluene-sodium-sulfonchloramide, takes out immediately, massive laundering, flush away toluene-sodium-sulfonchloramide.Obtain the implantation body of the chitosan/hyaluronic acid polyelectrolyte composite bed coating of the crosslinked load bioactie agent of RGD.
Embodiment 54: the chitosan/alkali of the load bioactie agent that RGD is crosslinked dissolves the preparation of gelatin polyelectrolyte composite bed
Method is with embodiment 53, and difference is that hyaluronic acid is replaced by alkali dissolves gelatin (its source is pig source, cattle source or Mus source).
Embodiment 55: the preparation of the chitosan of the load bioactie agent that RGD is crosslinked/polyglutamic acid polyelectrolyte composite bed
Method is with embodiment 53, and difference is hyaluronic acid is replaced by polyglutamic acid.
Embodiment 56: the preparation of the chitosan of the load bioactie agent that RGD is crosslinked/poly-aspartate polyelectrolyte composite bed
Method is with embodiment 53, and difference is hyaluronic acid is replaced by poly-aspartate.
Embodiment 57: the preparation of gelatin/hyaluronic acid polyelectrolyte composite bed is dissolved in the acid of the load bioactie agent that RGD is crosslinked
Polypeptide segment GRGDSPC (S-S) CPSDGRG that will contain RGD is soluble in water with molten gelatin of a certain amount of acid and hyaluronic acid respectively, adds EDC/NHS subsequently, reacts 4 hours, and product was dialysed for 1 week, lyophilizing; Product directly is dissolved in the water, and adds stachyose, react 4 hours, dialyses a week, and lyophilizing obtains having the molten gelatin of the grafted acid of RGD and the grafted hyaluronic acid of RGD of free sulfhydryl group.
Molten gelatin of the grafted acid of this RGD and the grafted hyaluronic acid of RGD are dissolved in the NaCl solution of 0.1mol/L, and both concentration all is 1mg/ml.In the molten gelatin solution of the acid for preparing, add BMP-2, making its ultimate density in solution is 5 μ g/ml (as if what add are bFGF, and then final concentration is 0.1 mg/ml).The pH value of the above-mentioned solution that preparation is accomplished adjusts to 4.0.Implant surface is through bulky grain sandblast, H 2SO 4Handled 1 minute with the HCl mixed acid solution, lot of pure is washed clean implant surface, then this implantation body is soaked in (10min in the solution of gelatin+BMP-2) is dissolved in the grafted acid of RGD, the physical absorption of deionized water flush away (gelatin+BMP-2) is dissolved in the grafted acid of RGD; Then, be soaked in 10min in the grafted hyaluronic acid solution of RGD again, the hyaluronic acid of deionized water flush away physical absorption.Repeat above operation, obtain 6 acid at last and dissolve gelatin/hyaluronic acid polyelectrolyte composite bed.The implantation body of this polyelectrolyte composite bed coating is soaked in 30 seconds in the toluene-sodium-sulfonchloramide, takes out immediately, massive laundering, flush away toluene-sodium-sulfonchloramide.The implantation body of gelatin/hyaluronic acid polyelectrolyte composite bed coating is dissolved in the acid that obtains the crosslinked load bioactie agent of RGD.
Embodiment 58: the preparation that gelatin/alkali dissolves gelatin polyelectrolyte composite bed is dissolved in the acid of the load bioactie agent that RGD is crosslinked
Method is with embodiment 57, and difference is that hyaluronic acid is replaced by alkali dissolves gelatin (its source is pig source, cattle source or Mus source).
Embodiment 59: the preparation of gelatin/polyglutamic acid polyelectrolyte composite bed is dissolved in the acid of the load bioactie agent that RGD is crosslinked
Method is with embodiment 57, and difference is hyaluronic acid is replaced by polyglutamic acid.
Embodiment 60: the preparation of gelatin/poly-aspartate polyelectrolyte composite bed is dissolved in the acid of the load bioactie agent that RGD is crosslinked
Method is with embodiment 57, and difference is hyaluronic acid is replaced by poly-aspartate.
Embodiment 61: the preparation of the poly arginine of the load bioactie agent that RGD is crosslinked/hyaluronic acid polyelectrolyte composite bed
Polypeptide segment GRGDSPC (S-S) CPSDGRG that will contain RGD is soluble in water with a certain amount of poly arginine and hyaluronic acid respectively, adds EDC/NHS subsequently, reacts 4 hours, and product was dialysed for 1 week, lyophilizing; Product directly is dissolved in the water, and adds stachyose, reacts 4 hours, dialyses a week, and lyophilizing obtains having the grafted poly arginine of RGD and the grafted hyaluronic acid of RGD of free sulfhydryl group.
Grafted poly arginine of this RGD and the grafted hyaluronic acid of RGD are dissolved in the NaCl solution of 0.1mol/L, and both concentration all is 1mg/ml.In the poly arginine solution for preparing, add BMP-2, making its ultimate density in solution is 5 μ g/ml (as if what add are bFGF, and then final concentration is 0.1 mg/ml).The pH value of the above-mentioned solution that preparation is accomplished adjusts to 4.0.Implant surface is through bulky grain sandblast, H 2SO 4Handled 1 minute with the HCl mixed acid solution, lot of pure is washed clean implant surface, then this implantation body is soaked in (10min in the solution of the grafted poly arginine of RGD+BMP-2), the physical absorption of deionized water flush away (the grafted poly arginine of RGD+BMP-2); Then, be soaked in 10min in the grafted hyaluronic acid solution of RGD again, the grafted hyaluronic acid of RGD of deionized water flush away physical absorption.Repeat above operation, obtain 6 poly arginines/hyaluronic acid polyelectrolyte composite bed at last.The implantation body of this polyelectrolyte composite bed coating is soaked in 30 seconds in the toluene-sodium-sulfonchloramide, takes out immediately, massive laundering, flush away toluene-sodium-sulfonchloramide.Obtain the implantation body of the poly arginine/hyaluronic acid polyelectrolyte composite bed coating of the crosslinked load bioactie agent of RGD.
Embodiment 62: the poly arginine/alkali of the load bioactie agent that RGD is crosslinked dissolves the preparation of gelatin polyelectrolyte composite bed
Method is with embodiment 61, and difference is that hyaluronic acid is replaced by alkali dissolves gelatin (its source is pig source, cattle source or Mus source).
Embodiment 63: the preparation of the poly arginine of the load bioactie agent that RGD is crosslinked/polyglutamic acid polyelectrolyte composite bed
Method is with embodiment 61, and difference is hyaluronic acid is replaced by polyglutamic acid.
Embodiment 64: the preparation of the poly arginine of the load bioactie agent that RGD is crosslinked/poly-aspartate polyelectrolyte composite bed
Method is with embodiment 61, and difference is hyaluronic acid is replaced by poly-aspartate.
Embodiment 65: the preparation of the polylysine of the load bioactie agent that RGD is crosslinked/hyaluronic acid polyelectrolyte composite bed
Polypeptide segment GRGDSPC (S-S) CPSDGRG that will contain RGD is soluble in water with a certain amount of polylysine and hyaluronic acid respectively, adds EDC/NHS subsequently, reacts 4 hours, and product was dialysed for 1 week, lyophilizing; Product directly is dissolved in the water, and adds stachyose, reacts 4 hours, dialyses a week, and lyophilizing obtains having the grafted polylysine of RGD and the grafted hyaluronic acid of RGD of free sulfhydryl group.
Grafted polylysine acid of this RGD and the grafted hyaluronic acid of RGD are dissolved in the NaCl solution of 0.1mol/L, and both concentration all is 1mg/ml.In the polylysine solution for preparing, add BMP-2, making its ultimate density in solution is 5 μ g/ml (as if what add are bFGF, and then final concentration is 0.1 mg/ml).The pH value of the above-mentioned solution that preparation is accomplished adjusts to 4.0.Implant surface is through bulky grain sandblast, H 2SO 4Handled 1 minute with the HCl mixed acid solution, lot of pure is washed clean implant surface, then this implantation body is soaked in (10min in the solution of the grafted polylysine of RGD+BMP-2), the physical absorption of deionized water flush away (the grafted polylysine of RGD+BMP-2); Then, be soaked in 10min in the grafted hyaluronic acid solution of RGD again, the grafted hyaluronic acid of RGD of deionized water flush away physical absorption.Repeat above operation, obtain 6 polylysines/hyaluronic acid polyelectrolyte composite bed at last.The implantation body of this polyelectrolyte composite bed coating is soaked in 30 seconds in the toluene-sodium-sulfonchloramide, takes out immediately, massive laundering, flush away toluene-sodium-sulfonchloramide.Obtain the implantation body of the polylysine/hyaluronic acid polyelectrolyte composite bed coating of the crosslinked load bioactie agent of RGD.
Embodiment 66: the polylysine/alkali of the load bioactie agent that RGD is crosslinked dissolves the preparation of gelatin polyelectrolyte composite bed
Method is with embodiment 65, and difference is that hyaluronic acid is replaced by alkali dissolves gelatin (its source is pig source, cattle source or Mus source).
Embodiment 67: the preparation of the polylysine of the load bioactie agent that RGD is crosslinked/polyglutamic acid polyelectrolyte composite bed
Method is with embodiment 65, and difference is hyaluronic acid is replaced by polyglutamic acid.
Embodiment 68: the preparation of the polylysine of the load bioactie agent that RGD is crosslinked/poly-aspartate polyelectrolyte composite bed
Method is with embodiment 65, and difference is hyaluronic acid is replaced by poly-aspartate.
Embodiment 69: the preparation of the polyhistidyl of the load bioactie agent that RGD is crosslinked/hyaluronic acid polyelectrolyte composite bed
Polypeptide segment GRGDSPC (S-S) CPSDGRG that will contain RGD is soluble in water with a certain amount of polyhistidyl and hyaluronic acid respectively, adds EDC/NHS subsequently, reacts 4 hours, and product was dialysed for 1 week, lyophilizing; Product directly is dissolved in the water, and adds stachyose, reacts 4 hours, dialyses a week, and lyophilizing obtains having the grafted polyhistidyl of RGD and the grafted hyaluronic acid of RGD of free sulfhydryl group respectively.
Grafted polyhistidyl of this RGD and the grafted hyaluronic acid of RGD are dissolved in the NaCl solution of 0.1mol/L, and both concentration all is 1mg/ml.In the polyhistidyl solution for preparing, add BMP-2, making its ultimate density in solution is 5 μ g/ml (as if what add are bFGF, and then final concentration is 0.1 mg/ml).The pH value of the above-mentioned solution that preparation is accomplished adjusts to 4.0.Implant surface is through bulky grain sandblast, H 2SO 4Handled 1 minute with the HCl mixed acid solution, lot of pure is washed clean implant surface, then this implantation body is soaked in (10min in the solution of the grafted polyhistidyl of RGD+BMP-2), the physical absorption of deionized water flush away (the grafted polyhistidyl of RGD+BMP-2); Then, be soaked in 10min in the grafted hyaluronic acid solution of RGD again, the grafted hyaluronic acid of RGD of deionized water flush away physical absorption.Repeat above operation, obtain 6 polyhistidyls/hyaluronic acid polyelectrolyte composite bed at last.The implantation body of this polyelectrolyte composite bed coating is soaked in 30 seconds in the toluene-sodium-sulfonchloramide, takes out immediately, massive laundering, flush away toluene-sodium-sulfonchloramide.Obtain the implantation body of the polyhistidyl/hyaluronic acid polyelectrolyte composite bed coating of the crosslinked load bioactie agent of RGD.
Embodiment 70: the polyhistidyl/alkali of the load bioactie agent that RGD is crosslinked dissolves the preparation of gelatin polyelectrolyte composite bed
Method is with embodiment 69, and difference is that hyaluronic acid is replaced by alkali dissolves gelatin (its source is pig source, cattle source or Mus source).
Embodiment 71: the preparation of the polyhistidyl of the load bioactie agent that RGD is crosslinked/polyglutamic acid polyelectrolyte composite bed
Method is with embodiment 69, and difference is hyaluronic acid is replaced by polyglutamic acid.
Embodiment 72: the preparation of the polyhistidyl of the load bioactie agent that RGD is crosslinked/poly-aspartate polyelectrolyte composite bed
Method is with embodiment 69, and difference is hyaluronic acid is replaced by poly-aspartate.
Embodiment 73: the BMP-2 of load is in the slow release behavior of phosphate buffer in the polyelectrolyte coating
Use that (collagen+BMP-2) and the hyaluronic acid with the terminal RGD graft modification of sulfydryl prepare 6 bilayers on the titanium sheet the crosslinked load of RGD has the polyelectrolyte coating (BEC-CL-TI group) (crosslinked group) of BMP-2.Use that (collagen+BMP-2) and the hyaluronic acid of the RGD graft modification of end of tape sulfydryl not prepare 6 bilayers on the titanium sheet load has the polyelectrolyte coating (BEC-TI group) (uncrosslinked group) of BMP-2.The titanium sheet that covers two kinds of different coating is placed phosphate buffer (PBS solution) respectively, and the method for using enzyme linked immunological to adsorb detects the release behavior of BMP-2 in these two kinds of coatings.The result has shown that referring to Fig. 1 the release of the uncrosslinked group of BMP-2 in the coating has only continued 3 days, and first day burst size is very high, is explosive and discharges, and ensuing burst size just sharply descends.And the release of crosslinked group of BMP-2 slowly and lasting, to also having a spot of BMP-2 to be released out on the tenth day.
Embodiment 74: the influence that the coating of the load BMP-2 that RGD is crosslinked is expressed MC3T3 cell Bone Gla protein (OC)
Use collagen and hyaluronic acid on the titanium sheet, to prepare the polyelectrolyte coating (CHC-TI group) (matched group) of 6 bilayers.The hyaluronic acid of use collagen and RGD modification prepares the polyelectrolyte coating (CHRC-TI group) (experimental group) of 6 bilayers on the titanium sheet.Use that (collagen+BMP-2) and the hyaluronic acid with the terminal RGD graft modification of sulfydryl prepare 6 bilayers on the titanium sheet the crosslinked load of RGD has the polyelectrolyte coating (BEC-CL-TI group) (experimental group) of BMP-2.Mice skeletonization precursor cell line MC3T3-E1 (Chinese Academy of Sciences's cell bank) is used for estimating the influence of the pair cell differentiation behavior of coating.The expression of Bone Gla protein is one of important indicator of skeletonization precursor differentiation.During 2 weeks of measuring between four groups the result of cell Bone Gla protein expression show (referring to Fig. 2), the facilitation that the group of rgd peptide sequence modification (CHRC-TI group) pair cell breaks up will be higher than does not have RGD modification group (CHC-TI group).BMP-2 is as the somatomedin with induced strong bone formation ability; The facilitation of its pair cell differentiation is very tangible; Therefore show the highest OC expression in the BEC-CL-TI group, embodied the advantage of the slowly releasing effect of the bioactie agent that crosslinked action brings.(" * " represents P, and < 0.05, the difference between experimental group and contrast has statistical significance; " # " represent P 0.05, the difference between experimental group and experimental group has statistical significance).
Embodiment 75: the coating of the load BMP-2 that RGD is crosslinked is to the influence of MC3T3 cell skeletonization related gene expression
The cell line of experiment grouping and use is with embodiment 74.Adopt the method for real-time quantitative RT-PCR to analyze skeletonization Expression of Related Genes variation between different groups.Detected the expression of alkali phosphatase (AKP-2 mRNA) and Runx2 mRNA.The expression of Runx2 is to start the important transcription factor of skeletonization precursor to osteoblast differentiation, and alkali phosphatase (ALP) is the sign of the early stage differentiation of osteoblast.The result shows, early stage at cell culture, the rise of BEC-CL-TI group Runx2 and ALP mRNA expression just apparently higher than other two groups.And when 7 days and 14 days, the advantage of BEC-CL-TI group is just more obvious.(" * " represents P, and < 0.05, the difference between experimental group and contrast has statistical significance; " # " represent P 0.05, the difference between experimental group and experimental group has statistical significance).Referring to Fig. 3, Fig. 4.
Embodiment 76: in vivo test
New zealand rabbit is as laboratory animal, implants biological effect in the body that implantation body is used to estimate the implantation body after coating modified at the bilateral femur distal end of bull rabbit.Matched group (Control) is the implantation body (SLA) that handles through the surface treatment of sandblast acid etching.Experimental group (Test) is the coating modified SLA implantation body of polyelectrolyte that the crosslinked load of RGD of 6 bilayers has BMP-2.Respectively 2,4 and 8 weeks after implantation body implants, adopt portable digital torsional test appearance to analyze the adhesion of implantation body and osseous tissue.The torque value size is represented the adhesion size.Experimental result shows; In 2 weeks after implantation body implants, implantation body's synosteosis power of experimental group is greater than matched group, and to 4 and 8 weeks; The advantage of experimental group is then more obvious, has confirmed also that coating in implant surface preparation has to promote the bonded ability of osseous tissue-implantation body in early days.(" * " represent P 0.05, experimental group and the contrast between difference have statistical significance).Referring to Fig. 5.
Embodiment 77: the bFGF of load is in the slow release behavior of phosphate buffer in the polyelectrolyte coating
Use that (collagen+bFGF) and the hyaluronic acid with the terminal RGD graft modification of sulfydryl prepare 6 bilayers on the titanium sheet the crosslinked load of RGD has the polyelectrolyte coating (Crossliked) (crosslinked group) of bioactie agent.Use that (collagen+bFGF) and the hyaluronic acid of the RGD graft modification of end of tape sulfydryl not prepare 6 bilayers on the titanium sheet load has the polyelectrolyte coating (Ti-[(Col+bFGF)/HA-RGD]) (uncrosslinked group) of bioactie agent.The titanium sheet that covers two kinds of different coating is placed phosphate buffer (PBS solution) respectively, and the method for using enzyme linked immunological to adsorb detects the release behavior of bioactie agent (bFGF) in these two kinds of coatings.The result shows, the bFGF pro-in uncrosslinked group of coating just discharged and finishes in 4 days, and the speed that discharges is very fast, and is very big in the amount that a few days ago discharges.And the slowly releasing effect of crosslinked group of bFGF can continue about 14 days.Referring to Fig. 6.
Embodiment 78: the coating of the load bFGF that RGD is crosslinked is to the influence of MC3T3 cell adhesion, propagation behavior
Use collagen and hyaluronic acid on the titanium sheet, to prepare the polyelectrolyte coating (CHC-Ti) (matched group) of 6 bilayers.Use the collagen and the hyaluronic acid of the RGD modification of terminal band sulfydryl on the titanium sheet, to prepare the crosslinked polyelectrolyte coating (CHC-RGD-Ti) (experimental group) of RGD of 6 bilayers.Use that (collagen+bFGF) and the hyaluronic acid with the terminal RGD graft modification of sulfydryl prepare 6 bilayers on the titanium sheet the crosslinked load of RGD has the polyelectrolyte coating (CC-Ti) (experimental group) of bFGF.Mice skeletonization precursor cell line MC3T3-E1 is used for estimating the influence of the on cell proliferation behavior of coating.Dna content how much represented what of cell number.To organize adherent cell concentration be maximum for CHC-RGD-Ti and CC-Ti when 1h, shown the adherent facilitation of rgd peptide pair cell.Because bFGF has the effect that promotes cell mitogen, when 3d, it is significantly to increase that the cell of CC-Ti group compares to other two groups.(" * " represents P, and < 0.05, the difference between experimental group and contrast has statistical significance; " # " represent P 0.05, the difference between experimental group and experimental group has statistical significance).Referring to Fig. 7.
Embodiment 79: the influence that the coating of the load bFGF that RGD is crosslinked is expressed MC3T3 cell Bone Gla protein (OC)
The cell line of testing grouping and being used to detect the differentiation behavior is with embodiment 78.During 2 weeks of measuring between four groups the result of cell Bone Gla protein expression show that the facilitation that the group of rgd peptide sequence modification (CHC-RGD-Ti group) pair cell breaks up will be higher than does not have RGD modification group (CHC-TI group).The highest the showing of OC amount that the last cultured cells of CC-Ti is expressed, the facilitation of bFGF pair cell differentiation also is very tangible.(" * " represents P, and < 0.05, the difference between experimental group and contrast has statistical significance; " # " represent P 0.05, the difference between experimental group and experimental group has statistical significance).Referring to Fig. 8.
Embodiment 80: the coating of the load bFGF that RGD is crosslinked is to the influence of MC3T3 cell skeletonization related gene expression
The cell line of testing grouping and being used to detect gene expression is with embodiment 78.Adopt the method for real-time quantitative RT-PCR to analyze skeletonization Expression of Related Genes variation between different groups.Detected the expression of alkali phosphatase (AKP-2 mRNA) and Runx2 mRNA.The expression of Runx2 is to start the important transcription factor of skeletonization precursor to osteoblast differentiation, and alkali phosphatase is the sign of the early stage differentiation of osteoblast.The biological effect of every kind of somatomedin is discrepant.Visibly differently with the effect of the intensive promotion osteoblast differentiation of BMP-2 be that the rise that bFGF expresses Runx2 and AKP-2mRNA shows remarkable advantages and comes when acting on 2W.(" * " represents P, and < 0.05, the difference between experimental group and contrast has statistical significance; " # " represent P 0.05, the difference between experimental group and experimental group has statistical significance).Referring to Fig. 9, Figure 10.
Embodiment 81: in vivo test
New zealand rabbit is as laboratory animal, implants biological effect in the body that implantation body is used to estimate the implantation body after coating modified at the bilateral femur distal end of bull rabbit.Matched group (Control) is the implantation body (SLA) that handles through the surface treatment of sandblast acid etching.Experimental group (Test) is that the crosslinked load of RGD of 6 bilayers has the not coating modified SLA implantation body of polyelectrolyte of FGF.Respectively 2,4 and 8 weeks after implantation body implants, adopt portable digital torsional test appearance to analyze the adhesion of implantation body and osseous tissue.The torque value size is represented the adhesion size.Experimental result shows that in 2 and 4 weeks after implantation body implants, implantation body's synosteosis power of experimental group is greater than matched group.To 8 whens week, both torque values all reduce before slightly, and both values are more approaching." * " represent P 0.05, experimental group and the contrast between difference have statistical significance).Referring to Figure 11.

Claims (4)

1. the method for preparing of the bionical coating of a load bioactie agent, realize through following steps:
(1) preparation of the grafted polyelectrolyte of RGD:
Rgd peptide segment GRGDSPC (S-S) CPSDGRG that will contain cystine linkage is at carbodiimides: under the condition that the N-hydroxy-succinamide catalyst system and catalyzing exists; Be grafted on the polyelectrolyte; Behind resulting product dialysis purification; Add stachyose or glutathion and react with it, obtain the grafted polyelectrolyte of glycine-arginine-glycine-aspartic acid-serine-proline-cysteine-SH, polyelectrolyte is respectively polycation polyelectrolyte and polyanion polyelectrolyte;
Described RGD is the linearity seven peptide structures that contain arginine-glycine-aspartic acid, and its peptide chain structure is glycine-arginine-glycine-aspartic acid-serine-proline-cysteine-SH;
The concentration of said polyelectrolyte is 0.1-50mg/ml; The mol ratio of carboxyl-content is 1:10000-1:1 in segmental amount of polypeptide and the polyelectrolyte; Carbodiimides: the consumption of N-hydroxy-succinamide is 1:1-20:1; The mol ratio of the consumption of carbodiimides and the segmental consumption of polypeptide is 1:1-10:1, and the mol ratio of cystine linkage is 1:1-50:1 in the concentration of stachyose or glutathion and the catalyst system and catalyzing;
(2) preparation of polyelectrolyte solution:
The polycation electrolyte solution allocation: grafted polycation polyelectrolyte of RGD or unmodified polyelectrolyte cation that step (1) is obtained are mixed with solution, and add bioactie agent BMP-2 or bFGF therein;
The preparation of polyanion electrolyte solution: grafted polyanion polyelectrolyte of RGD or unmodified polyanion electrolyte that step (1) is obtained are mixed with solution;
Wherein the concentration range of various polyelectrolyte solutions is 0.1mg/ml-10mg/ml, and NaCl concentration is 0.1-0.2mol/L in the polyelectrolyte solution, and pH value is 4.0, and the concentration of BMP-2 is 0.5-1000 μ g/ml, and bFGF concentration is 20-1000 μ g/ml;
(3) preparation of bionical coating:
The solution of step (2) configuration is done following combination: the grafted polycation polyelectrolyte solution of RGD and the unmodified polyanion electrolyte solution that (a) add bioactie agent BMP-2 or bFGF; (b) grafted polyanion polyelectrolyte solution of RGD and the unmodified polycation electrolyte solution that adds bioactie agent BMP-2 or bFGF; (c) grafted polycation polyelectrolyte solution of RGD and the grafted polyanion polyelectrolyte solution of RGD of interpolation bioactie agent BMP-2 or bFGF;
With (a)-(c) arbitrary group of solution, at first at titanio implant surface deposition polycation electrolyte and bioactie agent, behind the deposition certain hour; Fully washing deposits the polyanion electrolyte layer then, behind the deposition certain hour; Fully washing, more than operation is designated as the primary depositing circulation, repeats above cycling as required; Obtaining the polyelectrolyte composite bed that needs at last, then, is to soak in the toluene-sodium-sulfonchloramide solution of 0.5-10 mM or the hydrogen peroxide solution that concentration is 1-50 mM in concentration; Take out, fully wash, obtain the bionical coating of load bioactie agent; Soak time in toluene-sodium-sulfonchloramide is 5-180 second, and soak time is 5-60 minute in hydrogen peroxide.
2. the method for preparing of the bionical coating of a kind of load bioactie agent according to claim 1; It is characterized in that; Said described in the step (1) gathers positive electrolyte and selects for use Ι Collagen Type VI, chitosan, acid to dissolve in gelatin, poly arginine, polylysine, the polyhistidyl one or several; The product that obtains is the grafted polycation electrolyte of RGD, and the molecular weight of polycation electrolyte is 100-2000000D; Gather cloudy electrolyte and select for use hyaluronic acid, alkali to dissolve in gelatin, polyglutamic acid, the poly-aspartate one or several, the product that obtains is the grafted polyanion electrolyte of RGD, and the molecular weight of polyanion electrolyte is 100-5000000D.
3. the method for preparing of the bionical coating of a kind of load bioactie agent according to claim 1; It is characterized in that; One deck polyanion electrolyte layer or the used sedimentation time of polycation electrolyte layer are 5min-20min in the step (3), and required deposition cycle is 0.5-50 time.
4. the method for preparing of the bionical coating of a kind of load bioactie agent according to claim 1; It is characterized in that; Used implant surface is various permanent embedded type implant surface in the step (3), selects pure titanium or titanium alloy implant surface, titanium whitewashing/acid-treated pure titanium or the titanium alloy surface of pure titanium or titanium alloy implant surface, titanium or the hydroxyapatite gunite surface of the pure titanium that obtains through the acid-treated pure titanium of oxidisability or titanium alloy surface, the acid-treated pure titanium of non-oxide type or titanium alloy implant surface, sandblast/acid-treated surface, alkali full-boiled process or titanium alloy implant surface, surface that electrochemistry obtains, hydroxyapatite coating layer, ion implantation pure titanium or the pure titanium or the titanium alloy implant surface of titanium alloy implant surface or laser treatment for use.
CN201110288240A 2011-09-26 2011-09-26 Preparation method of bionic coating carrying bioactive factors Pending CN102327645A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201110288240A CN102327645A (en) 2011-09-26 2011-09-26 Preparation method of bionic coating carrying bioactive factors

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201110288240A CN102327645A (en) 2011-09-26 2011-09-26 Preparation method of bionic coating carrying bioactive factors

Publications (1)

Publication Number Publication Date
CN102327645A true CN102327645A (en) 2012-01-25

Family

ID=45479768

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201110288240A Pending CN102327645A (en) 2011-09-26 2011-09-26 Preparation method of bionic coating carrying bioactive factors

Country Status (1)

Country Link
CN (1) CN102327645A (en)

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103893826A (en) * 2014-03-03 2014-07-02 重庆大学 Titanium alloy surface modification method for regulating and controlling stem cell differentiation and promoting in-vivo bone formation
CN105126165A (en) * 2015-01-21 2015-12-09 中国人民解放军第三〇七医院 Titanium dental implant material and preparing method and application of titanium dental implant material
CN106491225A (en) * 2016-12-23 2017-03-15 大连三生科技发展有限公司 Implant system based on titanium-zirconium alloy
CN106491226A (en) * 2016-12-23 2017-03-15 大连三生科技发展有限公司 A kind of implant system with straight base station
CN106539627A (en) * 2016-12-23 2017-03-29 大连三生科技发展有限公司 A kind of tooth implant of the bioactivity surface containing RGD
CN106618765A (en) * 2016-12-23 2017-05-10 大连三生科技发展有限公司 Antibacterial peptide layer for dental implants
CN106620853A (en) * 2016-12-23 2017-05-10 大连三生科技发展有限公司 Preparation method of implant active surface with high-content and high-stability BMP (bone morphogenetic protein) coating
CN106693051A (en) * 2016-12-23 2017-05-24 大连三生科技发展有限公司 Preparation method of implant active surface with chitosan-RGD composite coating layer
CN106729995A (en) * 2016-12-23 2017-05-31 大连三生科技发展有限公司 A kind of construction method of the planting body active surface with high content BMP/RGD coatings
CN106725931A (en) * 2016-12-23 2017-05-31 大连三生科技发展有限公司 A kind of tooth implant for the bioactivity surface containing antibacterial peptide
CN106725937A (en) * 2016-12-23 2017-05-31 大连三生科技发展有限公司 A kind of dentistry implant for being coated with antibacterial peptide layer
CN106729992A (en) * 2016-12-23 2017-05-31 大连三生科技发展有限公司 Planting body active surface with high content, high stability BMP coatings
CN106725935A (en) * 2016-12-23 2017-05-31 大连三生科技发展有限公司 Planting body with high content, high stability BMP coatings
CN106729994A (en) * 2016-12-23 2017-05-31 大连三生科技发展有限公司 A kind of planting body active surface with shitosan RGD composite coatings
CN107117962A (en) * 2017-05-04 2017-09-01 郑州汉东科技有限公司 A kind of preparation method of the dental all-ceramics repair materials based on electrostatic self-assembled technology
CN107854724A (en) * 2017-12-25 2018-03-30 大连三生科技发展有限公司 A kind of construction method of planting body active surface
CN109316622A (en) * 2017-07-31 2019-02-12 华东理工大学 Chitosan hydrochloride anti-biotic material and its preparation method and application
CN110214176A (en) * 2016-12-15 2019-09-06 格勒诺布尔理工学院 The robotic method of porous plate is coated by polyelectrolyte multilayer film
CN110403736A (en) * 2019-08-09 2019-11-05 常州市第二人民医院 A kind of super porous titanium alloy of 3D printing facilitates bone surface method of modifying
CN113304317A (en) * 2021-06-01 2021-08-27 嘉兴学院 Preparation method of functional coating material for biological tissue stent or catheter

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1515324A (en) * 2003-08-25 2004-07-28 浙江大学 Method for layer-layer self-assembling and fixing biological active factor on tissue engineering material
CN101006944A (en) * 2007-01-31 2007-08-01 浙江大学 A method for assembling RGD on the surface of porous pure titanium dental implant

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1515324A (en) * 2003-08-25 2004-07-28 浙江大学 Method for layer-layer self-assembling and fixing biological active factor on tissue engineering material
CN101006944A (en) * 2007-01-31 2007-08-01 浙江大学 A method for assembling RGD on the surface of porous pure titanium dental implant

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
张锋: "钛基种植体表面仿细胞外基质活性涂层的设计、构建及其生物学评价", 《中国博士学位论文全文数据库 医药卫生科技辑》 *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103893826A (en) * 2014-03-03 2014-07-02 重庆大学 Titanium alloy surface modification method for regulating and controlling stem cell differentiation and promoting in-vivo bone formation
CN105126165A (en) * 2015-01-21 2015-12-09 中国人民解放军第三〇七医院 Titanium dental implant material and preparing method and application of titanium dental implant material
CN110214176B (en) * 2016-12-15 2023-08-08 格勒诺布尔理工学院 Robotic method for coating multiwell plates by polyelectrolyte multilayer films
CN110214176A (en) * 2016-12-15 2019-09-06 格勒诺布尔理工学院 The robotic method of porous plate is coated by polyelectrolyte multilayer film
CN106725937A (en) * 2016-12-23 2017-05-31 大连三生科技发展有限公司 A kind of dentistry implant for being coated with antibacterial peptide layer
CN106729994A (en) * 2016-12-23 2017-05-31 大连三生科技发展有限公司 A kind of planting body active surface with shitosan RGD composite coatings
CN106620853A (en) * 2016-12-23 2017-05-10 大连三生科技发展有限公司 Preparation method of implant active surface with high-content and high-stability BMP (bone morphogenetic protein) coating
CN106693051A (en) * 2016-12-23 2017-05-24 大连三生科技发展有限公司 Preparation method of implant active surface with chitosan-RGD composite coating layer
CN106729995A (en) * 2016-12-23 2017-05-31 大连三生科技发展有限公司 A kind of construction method of the planting body active surface with high content BMP/RGD coatings
CN106725931A (en) * 2016-12-23 2017-05-31 大连三生科技发展有限公司 A kind of tooth implant for the bioactivity surface containing antibacterial peptide
CN106539627A (en) * 2016-12-23 2017-03-29 大连三生科技发展有限公司 A kind of tooth implant of the bioactivity surface containing RGD
CN106729992A (en) * 2016-12-23 2017-05-31 大连三生科技发展有限公司 Planting body active surface with high content, high stability BMP coatings
CN106725935A (en) * 2016-12-23 2017-05-31 大连三生科技发展有限公司 Planting body with high content, high stability BMP coatings
CN106618765A (en) * 2016-12-23 2017-05-10 大连三生科技发展有限公司 Antibacterial peptide layer for dental implants
CN106491225A (en) * 2016-12-23 2017-03-15 大连三生科技发展有限公司 Implant system based on titanium-zirconium alloy
CN106491226A (en) * 2016-12-23 2017-03-15 大连三生科技发展有限公司 A kind of implant system with straight base station
CN106539627B (en) * 2016-12-23 2019-06-11 大连三生科技发展有限公司 A kind of dental implant of the bioactivity surface containing RGD
CN107117962B (en) * 2017-05-04 2019-08-16 中山市瑞丰医疗器械有限公司 A kind of preparation method of the dental all-ceramics repair materials based on electrostatic self-assembled technology
CN107117962A (en) * 2017-05-04 2017-09-01 郑州汉东科技有限公司 A kind of preparation method of the dental all-ceramics repair materials based on electrostatic self-assembled technology
CN109316622A (en) * 2017-07-31 2019-02-12 华东理工大学 Chitosan hydrochloride anti-biotic material and its preparation method and application
CN107854724A (en) * 2017-12-25 2018-03-30 大连三生科技发展有限公司 A kind of construction method of planting body active surface
CN110403736A (en) * 2019-08-09 2019-11-05 常州市第二人民医院 A kind of super porous titanium alloy of 3D printing facilitates bone surface method of modifying
CN113304317A (en) * 2021-06-01 2021-08-27 嘉兴学院 Preparation method of functional coating material for biological tissue stent or catheter

Similar Documents

Publication Publication Date Title
CN102327645A (en) Preparation method of bionic coating carrying bioactive factors
Hanawa A comprehensive review of techniques for biofunctionalization of titanium
Abbass et al. Hydrogels and dentin–pulp complex regeneration: from the benchtop to clinical translation
US9623146B2 (en) Bone implant materials comprising cross-linked bioactive hydrogel matrices
Almine et al. Elastin-based materials
CN103200971B (en) Complex layered materials, its manufacture method and application thereof
Vasita et al. Growth factor-delivery systems for tissue engineering: a materials perspective
CN102950102B (en) Preparation method of multi-growth-factor slow release coating of titanium and titanium alloy surface
US10383981B2 (en) Structural lattice and method of making same
Taylor Biological matrices and bionanotechnology
JP2007532211A (en) Bone morphogenetic matrix composite, process for its production and tissue engineering implant and skeleton with coating of bone morphogenetic composite matrix
Yang et al. Biofunctionalization of zirconia with cell-adhesion peptides via polydopamine crosslinking for soft tissue engineering: effects on the biological behaviors of human gingival fibroblasts and oral bacteria
JP2011160817A (en) Implantation support material
Santin et al. Biomimetic, bioresponsive, and bioactive materials: An introduction to integrating materials with tissues
Hofmann et al. Bioactive scaffolds for the controlled formation of complex skeletal tissues
Scarritt et al. Biologic scaffolds composed of extracellular matrix for regenerative medicine
Wei et al. Aptamer/Hydroxyapatite-Functionalized Titanium substrate promotes Implant Osseointegration via recruiting mesenchymal stem cells
Kim et al. Potential of hydrogels based on poly (ethylene glycol) and sebacic acid as orthopedic tissue engineering scaffolds
CN102977391B (en) Method for quantitatively loading growth factors on tissue engineering scaffold, and applications thereof
Li et al. Cathelicidin LL37 promotes osteogenic differentiation in vitro and bone regeneration in vivo
Chen et al. Biofunctional coatings for dental implants
CN102940909B (en) Method for modifying tissue engineering scaffold
CN101961508A (en) Method for preparing polyelectrolyte composite coating
Zhu et al. Biomimetic hydrogels as scaffolds for tissue-engineering applications
EP2173396B1 (en) Bioactive coating for an implantable device or bioprosthesis

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C02 Deemed withdrawal of patent application after publication (patent law 2001)
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20120125