CN104353109B - Method for modifying titanium with antibacterial property and effect of promoting growth of osteoblasts - Google Patents
Method for modifying titanium with antibacterial property and effect of promoting growth of osteoblasts Download PDFInfo
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- CN104353109B CN104353109B CN201410522585.3A CN201410522585A CN104353109B CN 104353109 B CN104353109 B CN 104353109B CN 201410522585 A CN201410522585 A CN 201410522585A CN 104353109 B CN104353109 B CN 104353109B
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Abstract
The invention discloses a method for modifying titanium with antibacterial property and an effect of promoting growth of osteoblasts. The method comprises the following steps: firstly, preprocessing titanium and preparing mixed liquid which can be adhered to the titanium surface and has a certain cross-linked space structure; secondly, adding the osteoblasts and preprocessed titanium, and drying in vacuum to obtain titanium with the osteoblasts in a coating; finally, soaking in a pancreatic enzyme solution for 15-45 seconds, ultrasonically cleaning, and drying in vacuum to obtain medical modified titanium capable of promoting the growth of the osteoblasts. The method is simple, the preparation conditions are mild, no harmful substances are generated in a preparation process, the obtained product is capable of effectively identifying the osteoblasts and promoting quick growth of the osteoblasts on the titanium surface, and the fixed growth of the osteoblasts on the titanium surface is facilitated after the obtained product serves as an implant to be implanted into a body.
Description
Technical field
The present invention relates to the field of surface modification of bio-medical material, specially one kind have antibiotic property concurrently and promote skeletonization thin
The titanium method of modifying of intracellular growth.
Background technology
With medical skill development, medical titanium is extensively applied as planting materials such as tooth implant and bone planting bodies
In disease treatments such as defect of dentition, bone losses, in terms of saving the life of people, play important function.But such operation
Infection rate is very high, may result in a large amount of antibiotic therapies, removes the serious consequence such as implantation body or even death.The related sense of implantation body
Contaminating main cause occurred frequently has at 2 points：Bacterial plaque is assembled in implant surface and Osseointegrated implants interface resistivity is low.Cause
This, need to improve the anti-microbial property of titanium and promote Oesteoblast growth function.
The concept of molecular engram is proposed in 1975 first by Southern, and molecular imprinting development in recent years is extremely fast
Speed.Multiple action point can be formed with polymer monomer when template molecule (microsphere) is contacted, by this work of polymerization process
With being memorized, after template molecule removes, it is the formation of in polymer matching with template molecule steric configuration
There is the hole of multiple action point, such hole will have selection evident characteristics to template molecule and the like.The present invention
Using the technology of molecular engram, introduce, in surface of metal titanium, the application point matching with osteoblast, to improving titanium
Facilitate bone cell growth ability.
Content of the invention
It is an object of the invention to provide a kind of titanium method of modifying having antibiotic property concurrently and promoting Oesteoblast growth, warp
Cross the stability that the titanium after modification improves coating, strengthen adhesiving effect, pass through to add antibacterial material simultaneously, improving
The antibacterial ability facilitating enhancing titanium while bone cell growth ability of titanium.
The present invention provide technical scheme be：
A kind of titanium method of modifying having antibiotic property and promotion Oesteoblast growth concurrently is it is characterised in that include following
Step：
Step one, titanium pretreatment, by titanium in 10-25ml 0.5-2mol/L acetone soln supersound process 2-3
Secondary, each 15-45 minute, it is then placed in soaking 20-30 hour in 2,3- glycidyl propyl trimethoxy silicane ethanol solution,
Vacuum drying, obtains pre-processing titanium metal；
Step 2, coating preparation, take cross-linking agent solution, lysozyme soln and silver nitrate solution mixing to carry out cross-linking reaction,
Obtain the mixed liquor containing certain crosslinking space structure that may adhere to surface of metal titanium, add skeletonization thin in described mixed liquor
Born of the same parents and pre-processing titanium metal, after soaking 5-10 minute, take out pre-processing titanium metal, vacuum drying obtains being coated with skeletonization in coating
The titanium of cell；
Step 3, osteoblastic remove, be coated with the immersion of osteoblastic titanium in the coating that step 2 is obtained
The 30-50 second in trypsin solution, then immerse distilled water ultrasonic 5-10 minute, make described osteoblast in described crosslinking space structure
Remove, and make pre-processing titanium metal surface form the coating with the space structure matching with osteoblast, obtain final product described energy
Enough promote the medical titanium of modification of Oesteoblast growth.
Preferably, in the described titanium method of modifying having antibiotic property and promotion Oesteoblast growth concurrently, step one
In 2,3- glycidyl propyl trimethoxy silicane ethanol solution consumption be 5-10ml, volume fraction be 5-10%.
Preferably, in the described titanium method of modifying having antibiotic property and promotion Oesteoblast growth concurrently, step 2
Described in the collocation method of cross-linking agent solution and lysozyme soln be respectively：Take 0.1-0.5g cross-linking agent and 3-8ml 75-85%
Ethanol solution is mixed to get cross-linking agent solution；1-2g lysozyme and 30-100ml water is taken to be mixed to prepare lysozyme soln；Take 0.01-
0.05g silver nitrate and 1-5ml distilled water are mixed to prepare silver nitrate solution.
Preferably, in the described titanium method of modifying having antibiotic property and promotion Oesteoblast growth concurrently, step 2
Described in cross-linking agent can be epoxy resin or epoxychloropropane.
Preferably, in the described titanium method of modifying having antibiotic property and promotion Oesteoblast growth concurrently, step 2
Described in osteoblastic consumption be 1-5ml, described osteoblast density be 2-10 × 104/ml.
Preferably, in the described titanium method of modifying having antibiotic property and promotion Oesteoblast growth concurrently, step 3
Described in trypsin solution for consumption be 2-10ml, described trypsin solution concentration be 0.25-1%；Described arrangement water consumption is 30-
50ml.
Preferably, in the described titanium method of modifying having antibiotic property and promotion Oesteoblast growth concurrently, described true
Empty drying condition is pressure 0.1-0.3MPa, and temperature is 80-120 DEG C, and drying time is 10-30 minute.
The beneficial effect that the present invention brings is：The method adopting molecular engram in the present invention, first by the amido in lysozyme
Produce cross-linking reaction with the epoxy radicals in cross-linking agent, form a crosslinked space structure, be re-introduced into osteoblast simultaneously, adding
Under heat condition, osteoblast is coated in crosslinked space structure, simultaneously the ring in 2,3- glycidyl propyl trimethoxy silicane
Epoxide also assists in cross-linking reaction, generates covalent bond, consolidates with titanium and is connected, and stable sticks to surface of metal titanium, increases
The stability in surface of metal titanium for the coating；In the presence of pancreatin, osteoblast is removed, surface of metal titanium stays skeletonization thin
" trace " of born of the same parents, the hole as matching with osteoblast, there is the function to the effectively identification of osteoblast molecule, work as titanium
When the hole of metal surface is contacted with osteoblast, osteoblast is in surface of metal titanium fixed growth；Have in lysozyme itself simultaneously
There are antibacterial, antiinflammatory, belong to contact sterilization, and the silver ion in silver nitrate also has bactericidal action, sterilizes for diffusion,
Both synergism, substantially increase the antibiotic property of gained titanium.Before titanium is modified, first pass through acetone molten
Liquid carries out oil removal treatment, removes the oxide-film of surface of metal titanium, effectively prevents coating shedding, promotes the stability of coating, simultaneously
The lysozyme using derives from human body, has good biocompatibility, so that the implant that titanium is made is had very with human body
Good biocompatibility, the preparation method reducing the harmful effect present invention that rejection causes is simple, and mild condition is no harmful
By-product generates.
Brief description
Fig. 1 is the titanium method of modifying flow process signal having antibiotic property concurrently and promoting Oesteoblast growth of the present invention
Figure；
Fig. 2 carries out the result schematic diagram of antibacterial ability test for products obtained therefrom of the present invention；
Fig. 3 is the result schematic diagram that products obtained therefrom of the present invention and common titanium sheet carry out cytoactive detection；
Fig. 4 is the result schematic diagram that products obtained therefrom of the present invention and common titanium sheet carry out cytotoxicity analysis.
Specific embodiment
With reference to Figure of description, the present invention is described in further detail, to make those skilled in the art's reference say
Bright book word can be implemented according to this.
As shown in figure 1, a kind of titanium method of modifying Preparation Method having antibiotic property concurrently and promoting Oesteoblast growth, its
In, comprise the following steps：
Step one, titanium pretreatment, by titanium in 10-25ml 0.5-2mol/L acetone soln supersound process 2-3
Secondary, each 15-45 minute, it is then placed in 2, the 3- glycidyl propyl trimethoxy silicane that 5-10ml volume fraction is 5-10%
Soak 20-30 hour in ethanol solution, be vacuum dried 10-30 minute in 0.1-0.3MPa temperature 80-120 DEG C, obtain pretreatment
Titanium；
Step 2, coating preparation, take 0.1-0.5g cross-linking agent and 3-8ml 85% ethanol solution to be mixed to get cross-linking agent molten
Liquid；1-2g lysozyme and 30-100ml water is taken to be mixed to prepare lysozyme soln；Take 0.01-0.05g silver nitrate and 1-5ml distilled water
It is mixed to prepare silver nitrate solution；Then described three kinds of solution mixing is carried out cross-linking reaction, obtain may adhere to surface of metal titanium
Containing certain crosslinking space structure mixed liquor, simultaneously the amido in lysozyme the silver ion in silver nitrate is had absorption make
With then adding 1-5ml density to be 2-10 × 10 in described mixed liquor4The osteoblast of/ml, after stirring, adds
Area is 1 × 1cm2Pre-processing titanium metal, soak 5-10 minute after, take out pre-processing titanium metal, in 0.2-0.3MPa temperature
80-120 DEG C of vacuum drying 10-30 minute, obtains being coated with osteoblastic titanium in coating；
Step 3, osteoblastic remove, be coated with the immersion of osteoblastic titanium in the coating that step 2 is obtained
2-10ml concentration stands the 30-50 second in the trypsin solution for 0.25-1%, takes out the immersion ultrasonic 5-10 of 30-50ml distilled water and divides
Clock, is vacuum dried 10-30 minute in 0.1-0.3MPa temperature 80-120 DEG C, makes described osteoblast from described crosslinking space structure
Inside remove, and make pre-processing titanium metal surface form the coating with the space structure matching with osteoblast, obtain final product described
The medical titanium of modification of Oesteoblast growth can be promoted.
In the described titanium method of modifying having antibiotic property and promotion Oesteoblast growth concurrently, crosslinked described in step 2
The collocation method of agent solution and lysozyme soln is respectively：0.1-0.5g cross-linking agent is taken to mix with 3-8ml 75-85% ethanol solution
Conjunction obtains cross-linking agent solution；1-2g lysozyme and 30-100ml water is taken to be mixed to prepare lysozyme soln；Take 0.01-0.05g nitric acid
Silver and 1-5ml distilled water are mixed to prepare silver nitrate solution.
In the described titanium method of modifying having antibiotic property and promotion Oesteoblast growth concurrently, the friendship described in step 2
Connection agent can be epoxy resin or epoxychloropropane.
Embodiment 1
Step one, titanium pretreatment, supersound process 3 times in 10ml 2mol/L acetone soln by titanium, every time 30
Minute, it is then placed in 2, the 3- glycidyl propyl trimethoxy silicane ethanol solution that 10ml volume fraction is 5% soaking 25
Hour, in 0.3MPa temperature, 80 DEG C are vacuum dried 30 minutes, obtain pre-processing titanium metal；
Step 2, coating preparation, take 0.13g cross-linking agent and 5ml 85% ethanol solution to be mixed to get cross-linking agent solution；Take
1.5g lysozyme and 50ml water are mixed to prepare lysozyme soln；0.01g silver nitrate and 1ml distilled water is taken to be mixed to prepare silver nitrate molten
Liquid；Then described three kinds of solution mixing is carried out cross-linking reaction, obtain may adhere to the empty containing certain crosslinking of surface of metal titanium
Between structure mixed liquor, simultaneously the amido in lysozyme have adsorption to the silver ion in silver nitrate, then to described mixing
1-5ml density is added to be 2-10 × 10 in liquid4The osteoblast of/ml, after stirring, adding area is 1 × 1cm2Pre- place
Reason titanium, after soaking 5-10 minute, takes out pre-processing titanium metal, and in 0.3MPa temperature, 100 DEG C are vacuum dried 30 minutes, obtain
It is coated with osteoblastic titanium in coating；
Step 3, osteoblastic remove, be coated with the immersion of osteoblastic titanium in the coating that step 2 is obtained
10ml concentration be 0.25% trypsin solution in stand 50 seconds, take out ultrasonic 10 minutes of 50ml distilled water of immersion, in 0.3MPa temperature
100 DEG C of degree is vacuum dried 30 minutes, so that described osteoblast is removed in described crosslinking space structure, and makes pre-processing titanium metal
Surface is formed has the coating of the space structure matching with osteoblast, obtains final product and described can promote changing of Oesteoblast growth
The medical titanium of property.
1st, antibacterial ability experiment
Will be black to golden yellow staphylococcus, colon bacillus, candida albicans, bacillus subtilises for products therefrom of the present invention
Complexion changed kind carries out bacteriostatic test.Each specimen surface inoculation 1ml concentration is 105Bacterium solution 37 DEG C culture 1d after, sample PBS
Gently rinse 3 times to remove nonadherent antibacterial, then on sample, the antibacterial of adhesion is eluted to sonic oscillation (40W) 5min
In 1ml distilled water, eluent is used for detecting the viable count in specimen surface Adherent bacteria.With doubling dilution and spread plate method inspection
Survey viable count.Result of the test is as shown in Figure 2.
2nd, cytoactive detection
Common titanium sheet and product of the present invention are respectively placed in 24 orifice plates (every group sets three parallel holes).1ml density be 2 ×
104The cell suspension inoculation of/ml, in specimen surface, then cultivates 1,4 and 7d.To after predetermined point of time, sample is with pH=7.4's
Phosphate buffer is transferred in 24 new orifice plates after softly rinsing three times.Every hole add 3- that 200 μ l concentration are 5mg/ml (4,
5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromide and the no phenol red DMEM culture medium of 800 μ l serum-frees.37 DEG C of incubation 4h
Inhale afterwards and abandon supernatant, add the crystal that 1ml dmso solution generates, every hole takes 200 μ l lysates to go to 96 well culture plates,
Survey its OD value with spectrophotometer at 490nm.Result of the test is as shown in Figure 3.
3rd, cytotoxicity analysis
(1) test principle：Product of the present invention is assessed as cytotoxicity index using the activity of lactic acid dehydrogenase (LDH)
Cytotoxicity size, its principle is a kind of stable protein for lactic acid dehydrogenase (LDH), is present in the kytoplasm of normal cell
In, once damaged membrane, LDH is released to extracellular；LDH catalysis lactic acid forms pyruvate, and tetrazolium salt (INT)
The reaction empurpled crystalline material of shape, can be detected by 500nm microplate reader.
(2) test procedure：Common titanium sheet and product of the present invention are respectively put in 24 orifice plates, then by 1 × 10 respectively4Individual thin
The osteoblast of born of the same parents is inoculated in 24 orifice plates, and cultivates 1 day.Collect culture fluid respectively, after centrifugation, take supernatant to be used for LDH activity inspection
Survey.Result of the test is as shown in Figure 4.
4th, result of the test
From figure 2 it can be seen that being wished to staphylococcus aureuses (ATCC 6538), large intestine angstrom by products obtained therefrom of the present invention
Salmonella (ATCC 25922), candida albicans (ATCC 10231), Bacillus subtilis endophyticus (ATCC 9372) all have
Very strong bactericidal properties, its sterilizing rate reaches more than 99%.
As can be seen from Figure 3 the OD value of common titanium sheet and product of the present invention increases with the increase of incubation time, but
It is to can be seen that common titanium sheet increaseing slowly with the increase OD value of incubation time, by contrast, the OD value of product of the present invention
In the increase rapid growth with incubation time, when cultivating 7 days, product of the present invention becomes multiplication compared to the OD value of common titanium sheet
Long.Therefore deduce that, product of the present invention has obvious cell-proliferation activity effect.
It is known that product of the present invention is compared with common titanium sheet from Fig. 4, LDH activity (U/L) value of common titanium sheet is
236, and LDH activity (U/L) value of product of the present invention is 223, less than the LDH value of common titanium sheet, shows that product of the present invention is no thin
Cellular toxicity.
Although embodiment of the present invention is disclosed as above, it is not restricted to listed in description and embodiment
With, it can be applied to various suitable the field of the invention completely, for those skilled in the art, can be easily
Realize other modification, therefore under the general concept being limited without departing substantially from claim and equivalency range, the present invention does not limit
In specific details with shown here as the legend with description.
Claims (6)
1. a kind of have antibiotic property concurrently and facilitate the titanium method of modifying of bone cell growth it is characterised in that comprising the following steps：
Step one, titanium pretreatment, supersound process 2-3 time in 10-25ml 0.5-2mol/L acetone soln by titanium,
15-45 minute every time, is then placed in 2,3- glycidyl propyl trimethoxy silicane ethanol solution soaking 20-30 hour, very
Empty baking temperature is 80-120 DEG C, and drying time is 10-30 minute, obtains pre-processing titanium metal；
Step 2, coating preparation, take cross-linking agent solution, lysozyme soln and silver nitrate solution mixing to carry out cross-linking reaction, obtain
May adhere to surface of metal titanium containing certain crosslinking space structure mixed liquor, in described mixed liquor add osteoblast and
Pre-processing titanium metal, after soaking 5-10 minute, takes out pre-processing titanium metal, vacuum drying obtains being coated with osteoblast in coating
Titanium, described vacuum drying temperature be 80-120 DEG C, drying time be 10-30 minute；
Step 3, osteoblastic remove, be coated with osteoblastic titanium immersion pancreatin in the coating that step 2 is obtained
The 30-50 second in solution, then immerse distilled water ultrasonic 5-10 minute, so that described osteoblast is moved in described crosslinking space structure
Remove, and so that pre-processing titanium metal surface is formed and be there is the coating of the space structure being matched with osteoblast, obtain final product described in have concurrently
Antibiotic property and the titanium facilitating bone cell growth.
2. have antibiotic property and the titanium method of modifying of facilitating bone cell growth as claimed in claim 1 concurrently it is characterised in that
In step one, the consumption of 2,3- glycidyl propyl trimethoxy silicane ethanol solution is 5-10ml, and volume fraction is 5-10%.
3. have antibiotic property and the titanium method of modifying of facilitating bone cell growth as claimed in claim 1 concurrently it is characterised in that
The collocation method carrying out the various solution of cross-linking reaction in step 2 is respectively：Take 0.1-0.5g cross-linking agent and 3-8ml 75-
85% ethanol solution is mixed to get described cross-linking agent solution；1-2g lysozyme and 30-100ml water is taken to be mixed to prepare described lysozyme
Solution；0.01-0.05g silver nitrate and 1-5ml distilled water is taken to be mixed to prepare described silver nitrate solution.
4. have antibiotic property and the titanium method of modifying of facilitating bone cell growth as claimed in claim 2 concurrently it is characterised in that
Cross-linking agent described in step 2 can be epoxy resin or epoxychloropropane.
5. have antibiotic property and the titanium method of modifying of facilitating bone cell growth as claimed in claim 1 concurrently it is characterised in that
Osteoblastic consumption described in step 2 is 1-5ml, and described osteoblast density is 2-10 × 104/ml.
6. have antibiotic property and the titanium method of modifying of facilitating bone cell growth as claimed in claim 1 concurrently it is characterised in that
Trypsin solution described in step 3 is 2-10m1 for consumption, and described trypsin solution concentration is 0.25-1%；Described distillation water consumption
For 30-50ml.
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| CN105288753B (en) * | 2015-11-17 | 2018-06-22 | 广西中医药大学 | Titanium metal material of curcumin derivate modification and its preparation method and application |
| CN108144112A (en) * | 2018-03-05 | 2018-06-12 | 湖北大学 | A kind of preparation method in composite coating of the surface of metal titanium with synergetic antibacterial effect |
| CN111035803B (en) * | 2019-11-07 | 2021-07-06 | 浙江大学 | Titanium implant material with anti-infection and osseointegration promoting functions and preparation method thereof |
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| KR101013999B1 (en) * | 2004-03-19 | 2011-02-14 | 재단법인서울대학교산학협력재단 | Membrane and implant immobilized osteogenic enhancing peptides on the surface |
| JP2009525124A (en) * | 2006-02-02 | 2009-07-09 | メドロジックス・ディバイス・コーポレーション | Bioactive material delivery system comprising a sol-gel composition |
| CN101596329A (en) * | 2009-07-13 | 2009-12-09 | 浙江理工大学 | A kind of method of titanium material surface coating and the application of coating composite material thereof |
| US20110262486A1 (en) * | 2010-04-22 | 2011-10-27 | Taipei Medical University | Bone implant and manufacturing method thereof |
| CN102886071A (en) * | 2011-07-20 | 2013-01-23 | 上海纳米技术及应用国家工程研究中心有限公司 | Medicinal silver-loaded metal bioactive coating as well as preparation method and application thereof |
| KR101612510B1 (en) * | 2013-01-10 | 2016-04-26 | 한양대학교 에리카산학협력단 | Manufacturing method for antibacterial titanium implant and antibacterial titanium implant by thesame |
| CN103463675B (en) * | 2013-08-23 | 2014-10-08 | 广州军区广州总医院 | Antibacterial and antitumor orthopaedic implantation material and preparation method thereof |
| 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 |
| CN104005016A (en) * | 2014-06-06 | 2014-08-27 | 重庆大学 | Medical titanium alloy with antibacterial and osteocyte-facilitating functions and preparation method thereof |
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