CN104353109A - 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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- CN104353109A CN104353109A CN201410522585.3A CN201410522585A CN104353109A CN 104353109 A CN104353109 A CN 104353109A CN 201410522585 A CN201410522585 A CN 201410522585A CN 104353109 A CN104353109 A CN 104353109A
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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, be specially a kind of titanium method of modifying having antibiotic property concurrently and promote Oesteoblast growth.
Background technology
Along with medical skill development, medical titanium metal is widely used in the disease treatment such as defect of dentition, bone loss by as planting materials such as tooth implant and bone implantation bodys, in the life saving people, played important function.But infection rate of such operation is very high, a large amount of antibiotic therapy may be caused, remove the serious consequences such as implantation body is even dead.Implantation body's infections relating main cause occurred frequently has 2 points: bacterial plaque implant surface assemble and Osseointegrated implants interface resistivity low.Therefore, the anti-microbial property and the promotion Oesteoblast growth function that improve titanium is needed.
First the Objective Concept Southern of molecular engram proposed in 1975, and molecular imprinting development is in recent years very rapid.Multiple action point can be formed when template molecule (microsphere) contacts with polymer monomer, will be memorized by this effect of polymerization process, when after template molecule removing, just define the hole with multiple action point matched with template molecule steric configuration in polymer, such hole will have selective recognition characteristic to template molecule and analog thereof.The present invention utilizes the technology of molecular engram, introduces the application point matched with osteoblast in surface of metal titanium, facilitates bone cell growth ability to what improve titanium.
Summary of the invention
The object of this invention is to provide a kind of titanium method of modifying having antibiotic property concurrently and promote Oesteoblast growth, titanium after modification improves the stability of coating, strengthen adhesiving effect, simultaneously by adding antibacterial material, improve titanium facilitate bone cell growth ability while strengthen the antibacterial ability of titanium.
Technical scheme provided by the invention is:
Have a titanium method of modifying for antibiotic property and promotion Oesteoblast growth concurrently, it is characterized in that, comprise the following steps:
Step one, titanium pretreatment, by titanium supersound process 2-3 time in 10-25ml 0.5-2mol/L acetone soln, each 15-45 minute, then 2 are put into, soak 20-30 hour, vacuum drying in 3-glycidyl propyl trimethoxy silicane alcoholic solution, obtain pre-processing titanium metal;
Prepared by step 2, coating, get the mixing of cross-linking agent solution, lysozyme soln and silver nitrate solution and carry out cross-linking reaction, obtain the mixed liquor containing certain crosslinked space structure that can stick to surface of metal titanium, osteoblast and pre-processing titanium metal is added in described mixed liquor, soak after 5-10 minute, take out pre-processing titanium metal, vacuum drying obtains being coated with osteoblastic titanium in coating;
Step 3, osteoblasticly to remove, be coated with osteoblastic titanium in coating step 2 obtained and immerse in trypsin solution 30-50 second, immerse the ultrasonic 5-10 minute of distilled water again, described osteoblast is removed in described crosslinked space structure, and make pre-processing titanium metal surface form the coating with the space structure matched with osteoblast, obtain the described modification medical titanium metal that can promote Oesteoblast growth.
Preferably, described has concurrently in the titanium method of modifying of antibiotic property and promotion Oesteoblast growth, and in step one, the consumption of 2,3-glycidyl propyl trimethoxy silicane alcoholic solution is 5-10ml, and volume fraction is 5-10%.
Preferably, described have antibiotic property concurrently and promote that in the titanium method of modifying of Oesteoblast growth, the collocation method of cross-linking agent solution described in step 2 and lysozyme soln is respectively: get 0.1-0.5g cross-linking agent and 3-8ml 75-85% alcoholic solution is mixed to get cross-linking agent solution; Get 1-2g lysozyme and mix obtained lysozyme soln with 30-100ml water; Get 0.01-0.05g silver nitrate and mix obtained silver nitrate solution with 1-5ml distilled water.
Preferably, described has concurrently in the titanium method of modifying of antibiotic property and promotion Oesteoblast growth, and the cross-linking agent described in step 2 can be epoxy resin or epoxychloropropane.
Preferably, described has concurrently in the titanium method of modifying of antibiotic property and promotion Oesteoblast growth, and osteoblastic consumption described in step 2 is 1-5ml, and described osteoblast density is 2-10 × 10
4/ ml.
Preferably, described has concurrently in the titanium method of modifying of antibiotic property and promotion Oesteoblast growth, and trypsin solution described in step 3 is consumption is 2-10ml, and described trypsin solution concentration is 0.25-1%; Described arrangement water consumption is 30-50ml.
Preferably, described has concurrently in the titanium method of modifying of antibiotic property and promotion Oesteoblast growth, and described vacuum 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 the amido in lysozyme and the epoxy radicals in cross-linking agent are produced cross-linking reaction, form a crosslinked space structure, introduce osteoblast more simultaneously, in a heated condition osteoblast is coated in crosslinked space structure, simultaneously 2, epoxy radicals in 3-glycidyl propyl trimethoxy silicane also participates in cross-linking reaction, generate covalent bond, firmly be connected with titanium, stable sticks to surface of metal titanium, adds the stability of coating in surface of metal titanium; Under the effect of pancreatin, osteoblast is removed, surface of metal titanium stays osteoblastic " trace ", be the hole matched with osteoblast, there is the function effectively identified osteoblast molecule, when the hole of surface of metal titanium contacts with osteoblast, osteoblast is in surface of metal titanium fixed growth; Have effect that is antibacterial, antiinflammatory at lysozyme itself, belong to contact sterilization, and the silver ion in silver nitrate also has bactericidal action, is diffusion sterilization, both synergism, substantially increase the antibiotic property of gained titanium simultaneously.Titanium is being carried out before modified, first carry out oil removal treatment by acetone soln, the oxide-film of removing surface of metal titanium, effectively prevents coating shedding, promotes the stability of coating, the lysozyme simultaneously used derives from human body, have good biocompatibility, the implant that titanium is made and human body have good biocompatibility, and the harmful effect preparation method of the present invention that minimizing rejection causes is simple, mild condition, generates without harmful side product.
Accompanying drawing explanation
Fig. 1 is the titanium method of modifying Preparation Method schematic flow sheet figure having antibiotic property concurrently and promote Oesteoblast growth of the present invention;
Fig. 2 is the result schematic diagram that products obtained therefrom of the present invention and common titanium sheet carry out cytoactive detection;
Fig. 3 is the result schematic diagram that products obtained therefrom of the present invention and common titanium sheet carry out cytotoxicity analysis.
Fig. 4 is the result schematic diagram that products obtained therefrom of the present invention and common titanium sheet carry out cytotoxicity analysis.
Detailed description of the invention
Below in conjunction with Figure of description, the present invention is described in further detail, can implement according to this with reference to description word to make those skilled in the art.
As shown in Figure 1, a kind of titanium method of modifying Preparation Method having antibiotic property concurrently and promote Oesteoblast growth, wherein, comprises the following steps:
Step one, titanium pretreatment, by titanium supersound process 2-3 time in 10-25ml 0.5-2mol/L acetone soln, each 15-45 minute, then put into that 5-10ml volume fraction is 5-10% 2,20-30 hour is soaked in 3-glycidyl propyl trimethoxy silicane alcoholic solution, at 0.1-0.3MPa temperature 80-120 DEG C of vacuum drying 10-30 minute, obtain pre-processing titanium metal;
The preparation of step 2, coating, gets 0.1-0.5g cross-linking agent and 3-8ml 85% alcoholic solution is mixed to get cross-linking agent solution; Get 1-2g lysozyme and mix obtained lysozyme soln with 30-100ml water; Get 0.01-0.05g silver nitrate and mix obtained silver nitrate solution with 1-5ml distilled water; Then cross-linking reaction is carried out in described three kinds of solution mixing, obtain the mixed liquor containing certain crosslinked space structure that can stick to surface of metal titanium, amido simultaneously in lysozyme has adsorption to the silver ion in silver nitrate, and then in described mixed liquor, adding 1-5ml density is 2-10 × 10
4the osteoblast of/ml, after stirring, then to add area be 1 × 1cm
2pre-processing titanium metal, soak after 5-10 minute, take out pre-processing titanium metal, at 0.2-0.3MPa temperature 80-120 DEG C of vacuum drying 10-30 minute, obtain in coating, being coated with osteoblastic titanium;
Step 3, osteoblasticly to remove, being coated with osteoblastic titanium immersion 2-10ml concentration in coating step 2 obtained is leave standstill 30-50 second in the trypsin solution of 0.25-1%, take out and immerse the ultrasonic 5-10 minute of 30-50ml distilled water, at 0.1-0.3MPa temperature 80-120 DEG C of vacuum drying 10-30 minute, described osteoblast is removed in described crosslinked space structure, and make pre-processing titanium metal surface form the coating with the space structure matched with osteoblast, obtain the described modification medical titanium metal that can promote Oesteoblast growth.
Described have antibiotic property concurrently and promote that in the titanium method of modifying of Oesteoblast growth, the collocation method of cross-linking agent solution described in step 2 and lysozyme soln is respectively: get 0.1-0.5g cross-linking agent and 3-8ml 75-85% alcoholic solution is mixed to get cross-linking agent solution; Get 1-2g lysozyme and mix obtained lysozyme soln with 30-100ml water; Get 0.01-0.05g silver nitrate and mix obtained silver nitrate solution with 1-5ml distilled water.
Described has antibiotic property concurrently and promotes that in the titanium method of modifying of Oesteoblast growth, the cross-linking agent described in step 2 can be epoxy resin or epoxychloropropane.
Embodiment 1
Step one, titanium pretreatment, by titanium supersound process 3 times in 10ml 2mol/L acetone soln, each 30 minutes, then put into that 10ml volume fraction is 5% 2, soak 25 hours in 3-glycidyl propyl trimethoxy silicane alcoholic solution, 0.3MPa temperature 80 DEG C of vacuum dryings 30 minutes, obtain pre-processing titanium metal;
The preparation of step 2, coating, gets 0.13g cross-linking agent and 5ml 85% alcoholic solution is mixed to get cross-linking agent solution; Get 1.5g lysozyme and mix obtained lysozyme soln with 50ml water; Get 0.01g silver nitrate and mix obtained silver nitrate solution with 1ml distilled water; Then cross-linking reaction is carried out in described three kinds of solution mixing, obtain the mixed liquor containing certain crosslinked space structure that can stick to surface of metal titanium, amido simultaneously in lysozyme has adsorption to the silver ion in silver nitrate, and then in described mixed liquor, adding 1-5ml density is 2-10 × 10
4the osteoblast of/ml, after stirring, then to add area be 1 × 1cm
2pre-processing titanium metal, soak after 5-10 minute, take out pre-processing titanium metal, 0.3MPa temperature 100 DEG C of vacuum dryings 30 minutes, obtain in coating, being coated with osteoblastic titanium;
Step 3, osteoblasticly to remove, being coated with osteoblastic titanium immersion 10m1 concentration in coating step 2 obtained is leave standstill 50 seconds in the trypsin solution of 0.25%, take out and immerse 50ml distilled water ultrasonic 10 minutes, 0.3MPa temperature 100 DEG C of vacuum dryings 30 minutes, described osteoblast is removed in described crosslinked space structure, and make pre-processing titanium metal surface form the coating with the space structure matched with osteoblast, obtain the described modification medical titanium metal that can promote Oesteoblast growth.
1, antibacterial ability experiment
Products therefrom of the present invention is carried out bacteriostatic test to golden yellow staphylococcus, colon bacillus, candida albicans, Bacillus subtilis endophyticus.Each specimen surface inoculation 1ml concentration is 10
5bacterium liquid cultivate after 1d at 37 DEG C, sample PBS gently rinsing 3 times to remove the antibacterial do not adhered to, then antibacterial sample adhered to sonic oscillation (40W) 5min is eluted in 1ml distilled water, and eluent is used for the viable count in test samples surface adhesion antibacterial.Viable count is detected with doubling dilution and spread plate method.Result of the test as shown in Figure 2.
2, cytoactive detects
Common titanium sheet and product of the present invention are placed in respectively 24 orifice plates (often group establishes three parallel holes).1ml density is 2 × 10
4the cell suspension inoculation of/ml, in specimen surface, then cultivates 1,4 and 7d.After predetermined point of time, transfer in 24 new orifice plates after the soft rinsing of phosphate buffer of sample pH=7.4 three times.Every hole adds 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide bromine salt and 800 μ l serum-frees that 200 μ l concentration are 5mg/ml without phenol red DMEM culture medium.37 DEG C hatch 4h after inhale abandon supernatant, add 1ml dmso solution generate crystal, every hole is got 200 μ l lysates and is forwarded 96 well culture plates to, with spectrophotometer in 490nm place survey its OD value.Result of the test as shown in Figure 3.
3, cytotoxicity analysis
(1) test principle: the cytotoxicity size assessing product of the present invention using the activity of lactic acid dehydrogenase (LDH) as cytotoxicity index, its principle is lactic acid dehydrogenase (LDH) is a kind of stable protein, be present in Normocellular kytoplasm, once damaged membrane, namely LDH is released to extracellular; LDH catalysis lactic acid forms pyruvate, and tetrazolium salt (INT) reacts the empurpled crystalline material of shape, detects by 500nm microplate reader.
(2) test procedure: common titanium sheet and product of the present invention put into 24 orifice plates, respectively then by 1 × 10 respectively
4the osteoblast of individual cell is inoculated in 24 orifice plates, and cultivates 1 day.Collect culture fluid respectively, get supernatant after centrifugal and detect for LDH activity.Result of the test as shown in Figure 4.
4, result of the test
As can be seen from Figure 2, all have very strong bactericidal properties by products obtained therefrom of the present invention to staphylococcus aureus (ATCC 6538), colon bacillus (ATCC 25922), candida albicans (ATCC 10231), Bacillus subtilis endophyticus (ATCC 9372), 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 along with the increase of incubation time, but can find out, common titanium sheet is along with the increaseing slowly of increase OD value of incubation time, by contrast, the OD value of product of the present invention increases fast in the increase along with incubation time, when cultivation 7 days, product of the present invention was doubled and redoubled compared to the OD value of common titanium sheet.Can draw thus, product of the present invention has obvious cell-proliferation activity effect.
Can know that from Fig. 4 product of the present invention is compared with common titanium sheet, LDH activity (U/L) value of common titanium sheet is 236, and the LDH activity of product of the present invention (U/L) value is 223, lower than the LDH value of common titanium sheet, show product no cytotoxicity of the present invention.
Although embodiment of the present invention are open as above, but it is not restricted to listed in description and embodiment utilization, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other amendment, therefore do not deviating under the general concept that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the legend described.
Claims (7)
1. have antibiotic property concurrently and facilitate the titanium method of modifying of bone cell growth, it is characterized in that, comprise the following steps:
Step one, titanium pretreatment, by titanium supersound process 2-3 time in 10-25ml0.5-2mol/L acetone soln, each 15-45 minute, then 2 are put into, soak 20-30 hour, vacuum drying in 3-glycidyl propyl trimethoxy silicane alcoholic solution, obtain pre-processing titanium metal;
Prepared by step 2, coating, get the mixing of cross-linking agent solution, lysozyme soln and silver nitrate solution and carry out cross-linking reaction, obtain the mixed liquor containing certain crosslinked space structure that can stick to surface of metal titanium, osteoblast and pre-processing titanium metal is added in described mixed liquor, soak after 5-10 minute, take out pre-processing titanium metal, vacuum drying obtains being coated with osteoblastic titanium in coating;
Step 3, osteoblasticly to remove, be coated with osteoblastic titanium in coating step 2 obtained and immerse in trypsin solution 30-50 second, immerse the ultrasonic 5-10 minute of distilled water again, described osteoblast is removed in described crosslinked space structure, and make pre-processing titanium metal surface form the coating with the space structure matched with osteoblast, obtain the described modification medical titanium metal that can promote Oesteoblast growth.
2. have antibiotic property as claimed in claim 1 concurrently and facilitate the titanium method of modifying of bone cell growth, it is characterized in that, in step one, the consumption of 2,3-glycidyl propyl trimethoxy silicane alcoholic solution is 5-10ml, and volume fraction is 5-10%.
3. have antibiotic property as claimed in claim 1 concurrently and facilitate the titanium method of modifying of bone cell growth, it is characterized in that, the collocation method carrying out the various solution of cross-linking reaction in step 2 is respectively: get 0.1-0.5g cross-linking agent and 3-8ml75-85% alcoholic solution is mixed to get described cross-linking agent solution; Get 1-2g lysozyme and mix obtained described lysozyme soln with 30-100ml water; Get 0.01-0.05g silver nitrate and mix obtained described silver nitrate solution with 1-5ml distilled water.
4. have antibiotic property as claimed in claim 2 concurrently and facilitate the titanium method of modifying of bone cell growth, it is characterized in that, the cross-linking agent described in step 2 can be epoxy resin or epoxychloropropane.
5. have antibiotic property as claimed in claim 1 concurrently and facilitate the titanium method of modifying of bone cell growth, it is characterized in that, osteoblastic consumption described in step 2 is 1-5ml, and described osteoblast density is 2-10 × 10
4/ ml.
6. have antibiotic property as claimed in claim 1 concurrently and facilitate the titanium method of modifying of bone cell growth, it is characterized in that, trypsin solution described in step 3 is consumption is 2-10ml, and described trypsin solution concentration is 0.25-1%; Described distilled water consumption is 30-50ml.
7. have antibiotic property as claimed in claim 1 concurrently and facilitate the titanium method of modifying of bone cell growth, it is characterized in that, described vacuum drying condition is pressure 0.1-0.3MPa, and temperature is 80-120 DEG C, and drying time is 10-30 minute.
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