CN102091350A - Method for preparing polypeptide chain segment crosslinked hybrid coating - Google Patents

Abstract

The invention provides a method for preparing a polypeptide chain segment crosslinked hybrid coating. By virtue of the polypeptide chain segment crosslinked hybrid coating on the surface of a Ti-based implant, the rapid bone bonding between hard tissues and permanent implant materials is realized, and the polypeptide chain segment crosslinked hybrid coating on the surface of the Ti-based implant is favorable for the long-term stability of the implant materials. According to the method provided by the invention, as RGD (Arg-Gly-Asp) is introduced, the hybrid crosslinked coating is realized, and through introducing disulfide bonds, the biodegradability of a final polyelectrolyte composite membrane has in-vivo responsiveness. According to the invention, the constructed RGD crosslinked hybrid coating is suitable for the surface processing of various in-vivo implanted Ti-based implants, can promote the adhesive proliferation differentiation of somatic cells before bone formation, and can promote the early development of the bone formation around the implants, and bone bonding can be realized soon.

Description

The preparation method of the hybrid coating that a kind of polypeptide segment is crosslinked

Technical field

The invention belongs to the technical field that medical material is made, relate to the crosslinked collagen of a kind of titanio implant surface RGD, the preparation method of nano hydroxylapatite hybridized coating, this coating can realize bone integration fast between sclerous tissues and the permanent implanted material, also helps the long-time stability of planting material.

Background technology

Implantation of tooth is to grow up on the basis of synosteosis theory, be implantation body directly with the ability of osseous tissue formation chemical bonding, this bonding makes material and tissue produce firm synostosis (Bone-binding), rather than isolated by fibrous tissue, just integrate in implantation body and osseous tissue generation bone.Studies show that it is the successful key factor of influence plantation that bone is integrated, get rid of the influence of these two kinds of factors of surgical procedures technology and patient individual difference (situation of plantation position bone amount and sclerotin thereof etc.), implant surface pattern and chemical property have decisive influence for the implant osseointegration performance.

It is found that the implantation body of (HA) coating that has hydroxyapatite and the fast and secular success rate of speed of osseous tissue biological fixation are significantly higher than the implantation body that does not have the HA coating.This is because HA can induce the dystopy crystal growth of bone matrix, forms chemical and connects; The local regulation and control of the Ca2+ of HA oligodynamical are divided into the mineralising that osteocyte promotes bone matrix; Specific absorption extracellular matrix protein of HA and bone matrix protein accelerate that the branch voltinism is osteoblastic sticks and grow; The rough surface of HA also can increase fibrinous adhering to.But, have the HA coating of good combination and peeling off of titanium metal implants body with osseous tissue clinically, be the very serious problem that the titanium implant of this HA of having coating faces.

In recent years, a lot of scholars are devoted to interactional Mechanism Study between extracellular matrix protein and the cell-membrane receptor, thereby have found the bioactive molecule that a lot of regulating cells stick and grow.RGD(Arg-Gly-Asp, arginine-glycine-aspartic acid) be modal basic structure in the various extracellular matrixs, also be the ultimate unit that extensively is present in the cell recognition system.The RGD group can promote osteoblastic growth, suppresses between the osteoclast, sticking between osteoclast and the substrate, stops propagation, migration and the differentiation of osteoclast, thereby promotes skeletonization.Researcher finds that also the RGD small peptide of synthetic also has same function.So people's RGD small peptide of trying is incorporated into implant surface.Physisorphtion is the simplest a kind of, is exactly directly implantation body to be immersed in the RGD small peptide solution.This method also has certain effect really, has promoted the formation of bone regeneration around implant bone.But RGD skewness, thickness that physical absorption is introduced differ, inefficiency, poor stability, poor repeatability and adhesion is little is wiped in implantation body's implantation process easily.Then, there is the scholar that sight has been turned to the method for chemical coupling and introduces RGD.With special method implant surface is activated exactly, make it to produce can with the covalently bound functional group of rgd peptide, as-OH ,-COOH and-NH2 etc.Chemical coupling method makes that RGD can combine with material firmly, stablizes, favorable repeatability.Yet chemical crosslinking changes the best conformation that RGD works easily, has damaged the activity of RGD.As seen seeking a kind of suitable method, is necessary with even, stable, firm being attached to implant surface and not changing the original surface topography of implantation body simultaneously of RGD small peptide.

Layer-by-layer is a kind of effective surface modifying method.Its principle is to utilize electrostatic force, assembles the polyelectrolyte of oppositely charged successively in solid support surface, obtains the multilayer complex films (PEM) from the molecular scale to the submicron-scale at last.It has simple to operate, and the preparation condition gentleness is convenient controlled, characteristics such as energy continuous production, and, all can obtain the poly-electric composite membrane of physicochemical properties homogeneous at last regardless of the solid support pattern.This poly-electric composite membrane has very high stability in dry environment, discover that polyelectrolyte with charge property and nano inoganic particle etc. can utilize the LbL technology to be introduced in support surface.

But the polyelectrolyte composite membrane is not very stable (degraded fully in 4～7 days) under physiological condition.Biological world is very marvellous, and disulfide bond is all arranged in a lot of biomacromolecules, and (existence S-S-) relies on three grades or quarternary structure that disulfide bond is kept biomacromolecule (particularly protein).Studies show that disulfide bond can be interrupted in the effect of glutathion and be reduced into sulfydryl (SH), and sulfydryl is easy to be oxidized to disulfide bond.These reaction conditions are very gentle, and are little to the activity damage of biomacromolecule.This has given us very big enlightenment, so this experiment the present invention discloses a kind of preparation method of the collagen that contains RGD/nano hydroxylapatite hybridized coating of LbL technology preparation, and in introducing RGD, introduce sulfydryl, by the crosslinking hybrid coating that is converted mutually of sulfydryl and disulfide bond, hybrid coating is more stable not to destroy the original biological activity of RGD in the hybrid coating simultaneously as far as possible thereby make.

Summary of the invention

The preparation method that the purpose of this invention is to provide the crosslinked hybrid coating of a kind of polypeptide segment.This coating is crosslinked by titanio implant surface polypeptide segment (RGD), can realize bone integration fast between sclerous tissues and the permanent implanted material, also helps the long-time stability of planting material, and preparation method of the present invention realizes by following steps:

(1) the grafted polyelectrolyte of preparation RGD: synthetic GRGDSPC (S-S) CPSDGRG polypeptide segment (by SciLight Biotechnology, LLC company is synthetic), this polypeptide segment is a water soluble polypeptide.At first polypeptide segment and polyelectrolyte are dissolved in the aqueous solution, be configured to mixed solution, add then carbodiimides [1-ethyl-3-(3-dimethyl ami-nopropyl) carbodiimide, EDC]/N-hydroxy-succinamide (N-hydroxysuccinimide, NHS) catalyst system and catalyzing, reaction is 4 hours under the room temperature, realize the modification of polyelectrolyte, then with 1 week of dialysis in the resulting product pure water, directly add the dissolving of stachyose or glutathion in dialysis solution, room temperature keeps being no less than 4 hours down, then 1 week of dialysis in the pure water, last lyophilizing obtains the grafted polyelectrolyte of RGD.This electrolyte is a kind of polypeptide segment of having modified GRGDSPC, and has the polyelectrolyte of free active sulfydryl.

Wherein:

Described polyelectrolyte is selected a kind of in hyaluronic acid, the molten gelatin of alkali, polyglutamic acid, poly-aspartate, the chondroitin sulfate for use, and the product that obtains is the grafted poly-cloudy electrolyte of RGD.Poly-cloudy electrolytical molecular weight is 100-5000000D.

(2) configuration polyelectrolyte solution: the grafted poly-negative electricity of any RGD of step (1) acquisition is separated plasmogamy be set to solution, as polyanion electrolyte solution; Ι Collagen Type VI (pig source, cattle source or Mus source) is configured to solution, then hydroxyapatite nano particle is scattered in the collagen solution, as poly-positive electrolyte solution;

(3) the crosslinked polyelectrolyte coating of preparation RGD: utilize the static self-assembling technique, at first at the poly-positive electrolyte of titanio implant surface deposition, deposition is gathered cloudy dielectric substrate again, that is: with the poly-positive electrolyte solution in the titanio implantation body immersion step (2), behind the deposition certain hour, fully washing is immersed in the grafted poly-cloudy electrolyte solution of any RGD that step (2) disposes 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, and fully washing obtains the crosslinked hybrid coating of RGD.

In the step (1), the concentration of 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; The consumption of described 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.

The concentration range of polyelectrolyte solution 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.

The particle diameter of nanometer hydroxyapatite is 20nm ~ 100nm in the step (2).

The used sedimentation time of polyelectrolyte layer is that 5min is to 20min in the step (3), the deposition cycle of polyelectrolyte composite bed is 0.5 to 50 time, determine 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, the concentration of toluene-sodium-sulfonchloramide is 0.5-10 mM, 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 for use through acid-treated pure titanium of oxidisability or titanium alloy surface, acid-treated pure titanium of non-oxide type or titanium alloy implant surface, sandblast/acid-treated surface, pure titanium that various alkali full-boiled process obtain or titanium alloy implant surface, the various surfaces that electrochemistry obtains, the pure titanium of various hydroxyapatite coating layers or titanium alloy implant surface, the pure titanium of titanium or hydroxyapatite gunite surface or titanium alloy implant surface, titanium whitewashing/acid-treated pure titanium or titanium alloy surface, ion implantation various pure titanium or titanium alloy implant surface, or the various pure titanium of laser treatment or titanium alloy implant surface.

The crosslinked hybrid coating of RGD that the inventive method is constructed, when introducing RGD, realized the crosslinked of hybrid coating, and by introducing cystine linkage, make the biodegradability of last polyelectrolyte composite membrane have response in the body, this hybrid coating that is rich in RGD is being fit to the titanio implant surface processing that various bodies are implanted into; This hybrid coating can promote the proliferation and differentiation that sticks of skeletonization precursor, can promote the early stage generation of bone regeneration around implant skeletonization, can realize the bone integration early.

Description of drawings

Fig. 1 is the degradation curve of two kinds of coatings.

Fig. 2 detects cell sticking with multiplication capacity on two kinds of coatings.

Fig. 3 is that the alkali phosphatase ALP of osteoblast on two groups of coatings detects.

Fig. 4 is the detection of Bone Gla protein OC.

Fig. 5 is the expression of Colla1mRNA on two groups of coatings.

Fig. 6 is the expression of AKP-2mRNA on two groups of coatings.

Fig. 7 is the expression of OsxmRNA on two groups of coatings.

Fig. 8 is the expression of Runx-2 on two groups of coatings.

The specific embodiment

The present invention is further described in conjunction with the accompanying drawings and embodiments.Because the present invention can permanent implanted implant surface make up, thereby can realize that the quick bone between implant and sclerous tissues integrates and plantation success rate at a specified future date in all bodies.Just as an illustration, still, use the constructed this coating of the present invention in other bodies on the permanent implanted implant surface all within protection scope of the present invention among the embodiment with a kind of sandblast/acid treatment surface.Embodiments of the invention just illustrate feature of the present invention for better, and not exclusively comprise the content of protecting of this patent.

Embodiment 1: the preparation of (collagen+hydroxyapatite)/hyaluronic acid hybrid coating that RGD is crosslinked

Get a certain amount of hyaluronic acid and be dissolved in the water, add the GRGDSPCS-SCPSDGRG dissolving, add the EDC/NHS dissolving at last, reacted 4 hours, 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.

Grafted hyaluronate sodium of this RGD and collagen are dissolved in respectively in the NaCl solution of 0.1mol/L, the two concentration all is 1mg/ml, then hydroxyapatite nano particle is scattered in the collagen solution, wherein the ultimate density of nanometer hydroxyapatite is 0.5mg/ml; 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 collagen hydroxyapatite solution, deionized water flush away physical absorption collagen and hydroxyapatite; Then, be soaked in 10min in the crosslinked hyaluronic acid solution of RGD again, the hyaluronate sodium of deionized water flush away physical absorption.Repeat above operation, obtain 6 (collagen+hydroxyapatite)/hyaluronic acid polyelectrolyte composite beds at last.The implantation body of this hydridization 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 crosslinked (collagen+hydroxyapatite)/hyaluronic acid hybrid coating of RGD.

Embodiment 2: the preparation of the molten gelatin hybrid coating of (collagen+hydroxyapatite)/alkali that RGD is crosslinked

Method is with embodiment one, and difference is to change hyaluronic acid into the molten gelatin of alkali (pig source, Niu Yuan, Mus source).

Embodiment 3: the preparation of (collagen+hydroxyapatite)/polyglutamic acid hybrid coating that RGD is crosslinked

Method is with embodiment one, and difference is to change hyaluronic acid into polyglutamic acid.

Embodiment 4: the preparation of (collagen+hydroxyapatite)/poly-aspartate hybrid coating that RGD is crosslinked

Method is with embodiment one, and difference is to change hyaluronic acid into poly-aspartate.

Embodiment 5: the preparation of (collagen+hydroxyapatite)/chondroitin sulfate hybrid coating that RGD is crosslinked

Method is with embodiment one, and difference is to change hyaluronic acid into chondroitin sulfate.

Embodiment 6: the external degradation test

In pH is 7.2 PBS solution, study the stability of crosslinked (collagen+hydroxyapatite)/hyaluronic acid hybrid coating (preparation method is seen embodiment one) of (collagen+hydroxyapatite)/hyaluronic acid hybrid coating and RGD respectively, as shown in Figure 1, crosslinking hybrid polyelectrolyte coating (is not degraded in six days fully in (Col+Ha)/HA) PBS solution, and crosslinked hydridization polyelectrolyte coating can be degraded in 14 days fully, and the stability of crosslinked hybrid coating (RGD crosslinked hybrid coating) is greatly improved.

Embodiment 7: the extracorporeal biology evaluation

Use mice skeletonization precursor cell line MC3T3-E1(Chinese Academy of Sciences cell bank) evaluate and test the extracorporeal biology performance of this coating.Matched group is that ((Col+Ha)/HA), test group is crosslinked (collagen+hydroxyapatite)/hyaluronic acid hybrid coating (preparation method is seen embodiment one) the titanium sheet group (RGD crosslinked hybrid coating) of RGD to (collagen+hydroxyapatite)/hyaluronic acid hybrid coating titanium sheet group.

A. osteoblast sticks the detection of (attachment), propagation (proliferation) ability on the titanium sheet, referring to Fig. 2;

B. the detection of skeletonization precursor differentiation capability on the titanium sheet

The detection of alkaline phosphatase activities (ALP) is referring to Fig. 3;

The detection of BGP content (OC) is referring to Fig. 4 in the cell culture medium;

C. the detection of skeletonization related gene expression

This experiment adopts the method for real-time quantitative RT-PCR to be parsed into the variation of bone Expression of Related Genes.Detect I Collagen Type VI (Colla1 mRNA) (ginseng Fig. 5) respectively, alkali phosphatase (AKP-2 mRNA) (Fig. 6), Osx mRNA(joins Fig. 7) and Runx2 mRNA(ginseng Fig. 8) expression.

1) extraction of cell total rna

This experiment adopt the dedicated kit can extract high quality RNA (RNeasy Mini kit, Qiagen, German).This test kit comprises following a few part:

a.?RNeasy?Mini?Spin?Columns?(pink) 50

b.?Collection?Tubes?(1.5ml) 50

c.?Collection?Tubes?(2ml) 50

d.?Buffer?RLT 45ml

e.?Buffer?RW1 45ml

f.?Buffer?RPE(5×) 11ml

g.?RNase－Free?Water 10ml

h.?Handbook 1

In order to remove minim DNA residual among total RNA, can use test kit (RNase-free DNase Set), comprise Buffer RDD and DNase I stock solution two parts.

Preparation before RNA extracts:

1. preferably add 10 μ l mercaprols (β-ME) before every milliliter of Buffer RLT uses

2. the Buffer RPE of the 1 volume anhydrous alcohol that adds 4 volumes is made into working solution

The concrete operations step:

1. collecting cell is no more than 10*7 cell.At the official hour point, the Tissue Culture Plate that the titanium sheet is housed is taken out from incubator, outwell culture medium, the titanium sheet is washed in PBS 2 times, and transfer in the new Tissue Culture Plate.Follow every titanium plate surface and add Buffer RLT 200 μ l (parallel samples 3 totally 600 μ l), shaking table was handled several minutes, collected in the RNA-free EP pipe (can-80 degree long preservation).

2. 70% ethanol (DEPC water is joined) that adds a volume, mixing, not centrifugal.

3. get 700 μ l and place pillar, cover, the centrifugal 15s of 10000rpm outwells effluent.

4. get 350 μ l Buffer RW1 and place pillar, the centrifugal 15s of 10000rpm outwells effluent.

10 μ l Dnase stock solutions+70 μ l Buffer RDD totally 80 μ l be added on the pellosil in the pillar, room temperature leaves standstill 15min.

6. get 350 μ l Buffer RW1 and place pillar, the centrifugal 15s of 10000rpm outwells effluent.

7. get 500 μ l Buffer RPE and place pillar, the centrifugal 15s of 10000rpm outwells effluent.

8. get 500 μ l Buffer RPE and place pillar, the centrifugal 2min of 10000rpm, pipe abandon.

9. pillar is positioned in the new pipe of 1.5ml RNA-free EP, 30 μ l RNA-free water are added on the pellosil in the pillar, cover, the centrifugal 1min of 10000rpm is with eluted rna.

Just the total RNA that extracts places on ice at once, and then row RNA purity and concentration detect (using ultraviolet spectrophotometer).When the A260/A280 of RNA between 1.8～2.0, illustrate that the RNA quality of extracting is good, can be used for next step experiment.

2) total RNA reverse transcription becomes cDNA

Used kit: reverse transcription test kit (PrimeScriptTM RT reagent Kit, Code DRR037A, TAKARA, the precious biological engineering company limited in Dalian).It is 10 μ l that this test kit is recommended a reaction system.

Concrete step is:

5×?primescript?buffer 2μl

Primescript?TR?Emyne?Mix?I 0.5μl

Random?6?mer(100um) 0.5μl

Oligo?dT?prime(50um) 0.5μl

Total?RNA 500ng

+ Rnase freeH2O to 10 μ l

37 ° of water-bath 15min

85 ° of water-bath 10S

CDNA-20 ° preservation

3) primer design and synthetic

Realtime RT-PCR the primer is synthesized by the design of the precious Takara of giving birth in Dalian company, specifically sees Table 1.

Table 1: the upstream and downstream primer of genes of interest

3）Realtime?PCR

Reagent: SYBR dye method test kit (SYBRR Premix Ex TaqTM, Perfect Real Time, Takara, Code:DRR041A)

Instrument: quantitative real time PCR Instrument (C1000TM Thermal Cycler, Bio-rad, USA)

What this experiment was adopted is 20 μ l systems, specifically adds quadrat method referring to table 2:

The preparation of table 2:PCR reactant liquor

This tests employed PCR pipe, and (PCRR STRIP TUBES, AXYGEN USA), are added on (operation on ice, attention speed) behind 8 pipes with sample, cover special-purpose lid, and centrifugal to remove the bubble of liquid internal, the machine of going up at last increases for 8 special-purpose pipes.Concrete reaction condition following (preliminary experiment has confirmed that each primer can carry out amplification in vitro efficiently, and product is single, and specificity is good):

Pre-degeneration: 95 ℃, 10s, 1 circulation;

The PCR reaction: 95 ℃, 5s; 60 ℃, 34s, totally 40 circulations.

At last, each reaction system has a Ct value, is confidential reference items with GAPDH expression of gene amount, adopts 2 ^{-△ △ CT}Method comes the relative expression of each gene of computational analysis to change.

Studies show that test group has all significantly improved the sticking of cell, propagation, differentiation capability, and can significantly raise Expression of Related Genes.

The sequence that the present invention relates to

＜110〉Zhejiang University

＜120〉preparation method of the crosslinked hybrid coating of a kind of polypeptide segment

<160>10

 

<210>?1

<211>?21

<212>?DNA

＜213〉artificial sequence

<220>

＜223〉forward primer of mice AKP-2

<440>1

tgc?cta?ctt?gtg?tgg?cgt?gaa

 

<210>?2

<211>?23

<212>?DNA

＜213〉artificial sequence

<220>

＜223〉downstream primer of mice AKP-2

<440>2

tca?ccc?gag?tgg?tag?tca?caa?tg

 

<210>?3

<211>?19

<212>?DNA

＜213〉artificial sequence

<220>

＜223〉forward primer of mice Collal

<440>3

atg?ccg?cga?cct?caa?gat?g

 

<210>?4

<211>?21

<212>?DNA

＜213〉artificial sequence

<220>

＜223〉downstream primer of mice Collal

<440>4

tga?ggc?aca?gac?ggc?tga?gta

 

<210>?5

<211>?19

<212>?DNA

＜213〉artificial sequence

<220>

＜223〉forward primer of mice Runx2

<440>5

cac?tgg?cgg?tgc?aac?aag?a

 

<210>6

<211>?23

<212>?DNA

＜213〉artificial sequence

<220>

＜223〉downstream primer of mice Runx2

<440>6

ttt?cat?aac?agc?gga?ggc?att?tc

 

<210>7

<211>?19

<212>?DNA

＜213〉artificial sequence

<220>

＜223〉forward primer of mice Osx

<440>7

atg?gcg?tcc?tct?ctg?ctt?g

 

210>?8

<211>?22

<212>?DNA

＜213〉artificial sequence

<220>

＜223〉downstream primer of mice Osx

<440>8

gta?tgg?ctt?ctt?tgt?gcc?tcc?t

 

<210>?9

<211>?20

<212>?DNA

＜213〉artificial sequence

<220>

＜223〉forward primer of mice GAPDH

<440>9

tgt?gtc?cgt?cgt?gga?tct?ga

 

<210>10

<211>?21

<212>?DNA

＜213〉artificial sequence

<220>

＜223〉downstream primer of mice GAPDH

<440>10

ttg?ctg?ttg?aag?tcg?cag?gag

 

Claims (7)

1. the preparation method of the crosslinked hybrid coating of a polypeptide segment is characterized in that realizing by following steps:

(1) preparation polypeptide segment grafted polyelectrolyte: GRGDSPC (S-S) CPSDGRG polypeptide segment and polyelectrolyte are dissolved in are configured to mixed solution in the aqueous solution earlier, add carbodiimides/N-hydroxy-succinamide catalyst system and catalyzing then, reaction is 4 hours under the room temperature, then resulting product was dialysed for 1 week in pure water, in dialysis solution, directly add the dissolving of stachyose or glutathion, room temperature kept 4 hours down, in pure water, dialysed for 1 week then, lyophilizing obtains the grafted polyelectrolyte of polypeptide segment;

Wherein: described polyelectrolyte is selected a kind of in hyaluronic acid, the molten gelatin of alkali, polyglutamic acid, poly-aspartate, the chondroitin sulfate for use, and the product that obtains is the grafted poly-cloudy electrolyte of polypeptide segment, and poly-cloudy electrolytical molecular weight is 100-5000000D;

(2) configuration polyelectrolyte solution: the grafted poly-negative electricity of polypeptide segment of step (1) acquisition is separated plasmogamy be set to solution, as polyanion electrolyte solution; The Ι Collagen Type VI is configured to solution, then hydroxyapatite nano particle is scattered in the collagen solution, as poly-positive electrolyte solution;

(3) the crosslinked polyelectrolyte coating of preparation polypeptide segment: with the poly-positive electrolyte solution in the titanio implantation body immersion step (2), deposition, fully washing is immersed in the grafted poly-cloudy electrolyte solution of polypeptide segment that step (2) disposes then, deposition, fully washing; More than operation is designated as the primary depositing circulation, repeats above cycling as required, obtains the polyelectrolyte composite bed; Then, this polyelectrolyte composite bed soaks in toluene-sodium-sulfonchloramide solution or hydrogen peroxide solution, takes out, and fully washing obtains the crosslinked hybrid coating of polypeptide segment.

2. the preparation method of the hybrid coating that a kind of polypeptide segment according to claim 1 is crosslinked, it is characterized in that, in the step (1), the concentration of 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, the consumption of carbodiimides/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 used stachyose or the concentration of glutathion and carbodiimides/N-hydroxy-succinamide system.

3. the preparation method of the hybrid coating that a kind of polypeptide segment according to claim 1 is crosslinked, it is characterized in that, the concentration range of polyelectrolyte solution is 0.1mg/ml-10mg/ml in the step (2), and wherein NaCl concentration is 0.1-0.2mol/L in the polyelectrolyte solution.

4. the preparation method of the hybrid coating that a kind of polypeptide segment according to claim 1 is crosslinked is characterized in that, the particle diameter of nanometer hydroxyapatite is 20 ~ 100nm in the step (2).

5. the preparation method of the hybrid coating that a kind of polypeptide segment according to claim 1 is crosslinked is characterized in that, the used sedimentation time of polyelectrolyte layer is 5-20min in the step (3), and the deposition cycle of polyelectrolyte composite bed is 0.5 to 50 time.

6. the preparation method of the hybrid coating that a kind of polypeptide segment according to claim 1 is crosslinked, it is characterized in that, the soak time of polyelectrolyte composite bed in toluene-sodium-sulfonchloramide is 5-180 second in the step (3), the concentration of toluene-sodium-sulfonchloramide is 0.5-10 mM, soak time is 5-60 minute in hydrogen peroxide, and the concentration of hydrogen peroxide is 1-50 mM.

7. the preparation method of the hybrid coating that a kind of polypeptide segment according to claim 1 is crosslinked, it is characterized in that, used implant surface is permanent embedded type implant surface in the step (3), selects for use through acid-treated pure titanium of oxidisability or titanium alloy surface, acid-treated pure titanium of non-oxide type or titanium alloy implant surface, sandblast/acid-treated surface, pure titanium that the alkali full-boiled process obtains or titanium alloy implant surface, the surface that electrochemistry obtains, the pure titanium of hydroxyapatite coating layer or titanium alloy implant surface, the pure titanium of titanium or hydroxyapatite gunite surface or titanium alloy implant surface, titanium whitewashing/acid-treated pure titanium or titanium alloy surface, ion implantation pure titanium or titanium alloy implant surface, the pure titanium of laser treatment or titanium alloy implant surface.

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