CN101461965B - Method for preparing chitosan/protein composite micrographics on surface of material - Google Patents
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- CN101461965B CN101461965B CN2009100581282A CN200910058128A CN101461965B CN 101461965 B CN101461965 B CN 101461965B CN 2009100581282 A CN2009100581282 A CN 2009100581282A CN 200910058128 A CN200910058128 A CN 200910058128A CN 101461965 B CN101461965 B CN 101461965B
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Abstract
The invention discloses a method for preparing a chitosan/protein composite micrographic on the surface of a material. The method comprises the following steps that: silicon is selected as a substrate; the silicon is oxidized by dry-oxygen at 1,000 DEG C, so that the surface of the silicon obtains a compact and even oxide film; and the silicon is treated by alkali, and is washed and dried, so that a silicon chip full of hydroxide radical on the surface is obtained; bovine serum albumin is prepared into a solution with certain concentration with phosphate buffer, chitosan and acetic acid respectively; a slot shaped micrographic of the chitosan is prepared on the hydroxylated silicon surface by a microtransfer molding method; a slot shaped micrographic of the protein is crossly prepared on the coating; and finally, a chitosan/protein composite micrographic with regular shape on the surface of the material is obtained.
Description
Affiliated technical field
The present invention relates to a kind of method of preparing chitosan/protein composite micrographics on surface of material.This compound micrographics has antibiotic property, and promotes effects such as absorption of osteoblast location and growth.
Background technology
A lot of materials can instruct the growth of cell, but some specific tissues when being organized in internal regeneration like nerve, bone, blood vessel and cornea etc. the requirement cell obtain the guidance of more special signal.Therefore, desirable material should be able to optionally be participated in interacting special sticking with growth factor receptors of the expression of target cell in the repair tissue.Research shows; Various kinds of cell can respond micro-meter scale; The material surface topological structure of nanoscale even; The form of cell, orientation, growth and differentiation also receive the influence of surface texture, in order to study the interactional process of biomolecule and base material, hope that these molecules can have clear and definite location and appear according to predetermined mode in specific substrate; Then need earlier substrate to be carried out surface treatment or made up little graphics processing at substrate surface, to reach the purpose of absorption of cell guiding location and the growth of control cell directional.
Micrographicsization is meant on the space that each zone or site of confirming contains the chemical substance of known micro-concentrations and structure, and utilizes such micrographics to obtain interactional information between they and analyte and the surrounding." soft etching " technology that the Whitesides of Harvard University professor research group has been invented multinomial micrographics transfer techniques such as comprising micro-contact printing, duplicating molded, micrometastasis moulding, nanometer embossing.The two combines with soft lithographic technique and adsorption technology, can be on material substrate the different group of grafting or different chemical substances, and obtain the substrate of different patternization.This substrate can not only be adjusted the kind and the quantity of adsorbed material flexibly, can also control the absorption deposition process at the surface chemistry material.At present, work out many micro fabrications and realized protein and material surface radical reaction, realized proteinic graphical or array.
Compound micrographics is meant that the molecule with two or more types is combined in the same substrate jointly; Utilize its different chemical property; Produce different chemical actions, and then seek different effect targets in vivo, reach and utilize same block of material can produce the different efficacies purpose.Chitosan is the natural polycation polysaccharide that de-acetyl chitin makes; As the unique alkaline polysaccharide of occurring in nature; Have performances such as favorable biological degradability, recyclability and antibiotic anti-corrosive properties; Its derivant has good anticoagulant property, and chitosan is applied to a plurality of fields such as medicine, food, agricultural, health care widely.Albumin is the rich in protein of content in the blood plasma, and bovine serum albumin has been proved to be has excellent biological compatibility.
In recent years, the existing research of adopting " soft etching " technology that the biomolecule arrays of micrographics pattern is arranged in the material surface preparation.Feng etc. have adopted micro-contact-printing to self-assemble to the glass substrate surface that is full of aldehyde radical to chitosan and bovine serum albumin, obtain the compound micrographics of the two.The deficiency of this method is to be in the process of pre-treatment substrate; Introduced extra aldehyde radical functional group; Increased the genotoxic potential of material, and littlely connect that to separate print process only be the surface that biomolecule solution to be assembled is printed on template, the bearing capacity of template is little; The preparation gained biomolecule on the low side, the biological agent of performance also corresponding a little less than.
Summary of the invention
In view of the above shortcoming of prior art, the purpose of this invention is to provide a kind of method of the compound micrographics at two kinds of materials of material surface preparation, the method that promptly adopts micro-processing technology to combine with surface adsorption prepares the compound micrographics of chitosan/protein.Concrete means are:
A kind of method of preparing chitosan/protein composite micrographics on surface of material is that raw material adopts the micrometastasis method of molding to make CS and two kinds of materials of BSA obtain compound micrographics in the even absorption of silicon face with chitosan CS and bovine serum albumin BSA, comprises the steps:
(1) preparation of silicon chip: adopt dry-oxygen oxidation to handle the pure silicon sheet, even compact oxide layer silicon chip is arranged to obtain the surface; Said surperficial even compact oxide layer silicon chip after alkali treatment, obtains silicon chip that surface be full of hydroxyl after cleaning up drying with distilled water at last through cleaning.
(2) preparation of solution: CS is dissolved in CH
3Among the COOH, be mixed with the CH of 2mg/ml CS
3COOH solution; It is in 6.6 the phosphate buffer that BSA is dissolved in pH value, is mixed with the BSA solution of 2mg/ml.
(3) elastomeric stamp preparation: fully mix polydimethylsiloxane PDMS and silicone rubber firming agent in 10: 1 ratio of mass ratio, the gained mixture casts on the silicon template that the surface has the micron-scale groove structure; After the curing solidfied material is peeled off from the silicon template down in 80 ℃, after hydrophilic improves processing, obtained the elastomeric stamp of groove topography again through distilled water cleaning, drying.
(4) system of opening up of micrographics: CS solution and the BSA solution of using (2) to obtain respectively drip respectively on the elastomeric stamp that groove topography is arranged that obtains two (3); Leave standstill and strike off surperficial excessive solution; Obtain two elastomeric stamps that have CS micrographics and BSA micrographics respectively, promptly accomplish the system of opening up respectively of micrographics;
(5) the elastomeric stamp left-hand thread with CS micrographics that (4) is obtained is on silicon chip surface that (1) step makes; The fully closely contact that keeps the two; After 20 ℃ of following dryings, peel off elastomeric stamp, in the NaOH of 0.0125mol/L, soak 1min again, with in the acidity of CS; The silicon chip surface adsorption stays the groove micrographics of CS.
On the silicon chip of the groove micrographics that the elastomeric stamp left-hand thread with BSA micrographics that (6) (4) is obtained obtains in (5) with CS; On the silicon chip surface that is adsorbed with CS, the BSA micrographics is gone up in absorption across, obtains the compound micrographics of chitosan/protein of material surface regular shape.
Compared with prior art, the inventive method has overcome the shortcoming of prior art, has prepared the big micrometer structure of material bearing capacity at material surface, and need not introduce the chemical substance that other has genotoxic potential in the course of reaction.Its beneficial effect is embodied in:
At first, the present invention has prepared bigger chitosan and the compound micrographics of bovine serum albumin of material bearing capacity at material surface, can be than the antibiotic property of good utilisation chitosan and the good biocompatibility of bovine serum albumin.
Secondly; In substrate and chitosan and the interactional process of bovine serum albumin, when being carried out alkali treatment, substrate introduces the oh group, and do not re-use other organic reagent and handle; Avoid introducing extra chemical substance, and then reduced the genotoxic potential of pair cell.
Once more; The inventive method has realized that chitosan and protein co-absorbed are on the same material surface; Can be simultaneously and utilize antibacterial action and the proteinic short celluar localization absorption and the growth effect of chitosan fully; Thereby on micro-scale, realized chitosan and proteinic evenly compound, helped sticking and growth at the micron order regulating cell.
At last; The size of micrographics and the angle between compound pattern can be carried out suitable adjustment as required, and only the size through the adjustment graphics template just can obtain, thereby preparation is simple; The goods cycle is short, can apply to neatly in the various micron-sized tissues.
Description of drawings is following
Preparation technology's flow process of Fig. 1 embodiment of the invention 1 chitosan/compound micrographics of bovine serum albumin.
The infared spectrum of Fig. 2 embodiment of the invention 1 chitosan/compound micrographics of bovine serum albumin.(a) the compound micrographics of CS/BSA; (b) BSA; (c) CS.
The light microscopic figure of Fig. 3 embodiment of the invention 1 chitosan/compound micrographics of bovine serum albumin.
The AFM figure of Fig. 4 embodiment of the invention 1 chitosan/compound micrographics of bovine serum albumin.
The light microscopic figure of the cellular morphology of Fig. 5 embodiment of the invention 1 chitosan/compound micrographics of bovine serum albumin.
The antibacterial experiment installation drawing of Fig. 6 embodiment of the invention 1 chitosan/compound micrographics of bovine serum albumin.
Fig. 7 embodiment of the invention 1 chitosan/compound micrographics antibiotic rate of bovine serum albumin data.
The specific embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is done further description.
Embodiment 1
(1) get the pure silicon sheet as base material, 1000 ℃ of following sintering 4 hours, obtaining the fine and close oxide layer uniformly of silicon face, ultrasonic through ethanol, water;
(2) get 40g NaOH and be dissolved in the 500ml distilled water, be mixed with the 5mol/L aqueous slkali, the silicon chip that (1) is made alkali treatment 3 hours in 60 ℃ of water-baths cleans up with distilled water, and drying is to obtain surperficial competent hydroxyl;
(3) get 0.4g acetic acid and be dissolved in the 19.6g distilled water, be mixed with mass fraction and be 2% 20ml acetic acid solution, take by weighing 40mg CS again and add in the acetic acid solution, stir, obtain the CS solution of 2mg/ml;
(4) get 0.087g NaH
2PO
4, 0.135g Na
2HPO
4Be dissolved in the 20ml distilled water with 0.085g NaCl, and to regulate pH value with HCl or Tris be 6.6, be mixed with phosphate buffer; Get 40mg BSA and be dissolved in the phosphate buffer, stir, obtain the BSA solution of 2mg/ml;
(5) get 70g polydimethylsiloxane (PDMS), silicone rubber firming agent 7g fully stirs, and leaves standstill to the bubble complete obiteration, casts in mixture on the silicon template that the surface has the micron-scale groove structure, after decompression vacuum pumping removes bubble, solidifies 2 hours down in 80 ℃.Behind the completion of cure PDMS is peeled off from the silicon template, promptly obtain the elastomeric stamp of figure;
(6) earlier the PDMS seal is immersed in 12h in the 1mol/L hydrochloric acid solution, improves its hydrophilic, clean up drying with distilled water.Drip step (3) the CS solution for preparing on PDMS seal surface, leave standstill 10min, strike off surperficial excessive solution; With the elastomeric stamp left-hand thread at substrate surface; Keep the fully closely contact of the two, peel off the PDMS seal behind the dry 2h down, promptly get the groove micrographics sample of silicon face absorption CS in 20 ℃; Sample is soaked 1min in the NaOH of 0.0125mol/L, with in the acidity of CS;
(7) step for preparing (4) BSA drips of solution is added to the PDMS surface, leaves standstill 10min, with the method for step (6), the silicon chip surface being adsorbed with CS prepares the BSA micrographics, across to obtain the compound micrographics of the two.
Can see the typical process flow of the compound micrographics absorption of micrographics of the present invention system of opening up and material surface among Fig. 1.
Fig. 2 is the infared spectrum of the compound micrographics of CS/BSA.(a) the compound micrographics of CS/BSA; (b) BSA; (c) CS.Can know that through comparison CS and BSA characteristic peak the two forms compound micrographics by CS and BSA.
Fig. 3 is the light microscopic figure of the compound micrographics of CS/BSA.Can find out that the two has formed the compound micrographics structure of groove cross-like at silicon face.
Fig. 4 is the AFM figure of the compound micrographics of CS/BSA.
Fig. 5 is the MC3T3-E osteoblast at 1 day light microscopic figure of the compound micrographics superficial growth of CS/BSA: a. * 200; B. * 400.Can observe and learn; The MC3T3-E osteoblast is better at the surface adhesion of the compound micrographics of CA/BSA in the time of 1 day; Can be observed the pseudopodium of cell, can see that obviously cell has produced location absorption on the micrographics surface of groove cross-like, can be along orthogonal micrographics growth; Arrange out corresponding cross-like pattern, explain that micrographics has very strong inductivity for osteoblast in the direction of growth of material surface.From cellular morphology and sprawl situation and can know, the cell well-grown, CS/BSA is compound, and micrographics has better biocompatibility.
Fig. 7 is the antibiotic rate data of the compound micrographics of embodiment of the invention 1CS/BSA, and clump count method working sample is to the bacteriostasis rate (n=3) of escherichia coli, Staphylococcus albus
Antibiotic rate data according among Fig. 7 can find out, at CS/BSA after compound micrographics surface action a period of time, compound micrographics has reached 34.3% to colibacillary bacteriostasis rate, and the staphylococcic bacteriostasis rate of white is reached 30.5% with the bacterium liquid of concentration.The compound micrographics of proof CS/BSA all has sterilizing ability to these two strains, and colibacillary sterilizing ability is higher than Staphylococcus albus.The step of antibacterial experiment:
(1) activation of thalline:
On aseptic workbench, an amount of sterilized tryptone (LB) culture medium to be poured in the flat board, the bacterial classification inoculation of getting 50 μ l then with spreader with antibacterial coating evenly, is put under 37 ℃ of the constant incubators, at constant temperature culture 12h on culture medium.
(2) preparation of bacteria suspension:
Get an amount of step 1 with inoculating loop and cultivate 12 hours (h) postactivated good thalline, put into the test tube that fills 10ml liquid LB culture medium.Then, the test tube that inoculation is good is put into 37 ℃, in the shaking table of 200rmp, cultivates 12h.Adopt the concentration of gradient dilution method test bacterium liquid subsequently, and adjusting concentration is 1 * 10
8Individual/ml.
(3) add sample
With prepare in the step 21 * 10
8The bacterium liquid of individual/ml dilutes 10 respectively
3Doubly.Sample is placed on the microscope slide, is placed in the culture dish, the culture dish bottom adds sterilized water, and is to prevent the evaporation of bacterium liquid, as shown in Figure 6: (1) sterilized water (2) slide (3) sample (4) bacterium liquid.Select the compound micrographics sample of CS/BSA, pure silicon sheet sample totally 2 experimental grouies for use, 3 every group parallel appearance.Get 50 μ L bacterium drops in sample surfaces, behind 37 ℃ of cultivation 12h bacterium liquid is moved in the 500 μ L PBS solution, shake up.
(4) observe counting
Draw the liquid of 50 μ l steps 3 with liquid-transfering gun, coat on the solid medium, put into constant incubator and cultivate 12h, observe the growing state of antibacterial, record, and observed clump count counted.
(5) bacteriostasis rate calculates
The antibacterial action of sample can represent that the computational methods of antibiotic rate are suc as formula shown in 1 through the antibiotic rate of calculation sample.Each experimental group carries out three parallel tests, to obtain antibiotic rate with 3 parallel appearance.Antibiotic rate is calculated by following formula.
Conclusion: can find out with the contrast of pure Si sheet:
(1) the Staphylococcus albus amount of bacteria is 100 μ l, bacterial concentration 1 * 10
8Under the condition of individual/ml, behind the cultivation 12h, the compound micrographics of CS/BSA reaches 30.5% to the staphylococcic bacteriostasis rate of white, has certain germicidal efficacy.
(2) the escherichia coli amount of bacteria is 100 μ l, bacterial concentration 1 * 10
8Under the condition of individual/ml; After cultivating 12h; The compound micrographics of CS/BSA reaches 34.3% to colibacillary bacteriostasis rate, has certain germicidal efficacy, and the compound micrographics of CS/BSA will be superior to the staphylococcic fungistatic effect of white colibacillary fungistatic effect a little.
Comprehensive above experimental result can know that the micrometastasis method of molding is successfully prepared the compound micrographics of CS/BSA of CS and the even absorption of BSA.The compound micrographics of CS/BSA has antibiotic resistance performance and excellent biological compatibility simultaneously.
Change the bovine serum albumin of embodiment 1 into collagen protein, get collagen protein and be dissolved in the phosphate buffer, be mixed with the 2mg/ml collagen solution, other conditions are constant, use experimental technique of the present invention, the compound micrographics of preparation chitosan/collagen protein.
Embodiment 3
Change the pure silicon sheet among the embodiment 1 into pure titanium sheet, with pure titanium sheet with acetone, water ultrasonic after, be placed in the NaOH solution of 5mol/L; In 60 ℃ of following alkali treatment 3h, clean up drying with distilled water; Promptly obtain the sufficient and uniform hydroxyl in titanium surface; Other conditions are constant, use experimental technique of the present invention, the compound micrographics of the chitosan/bovine serum albumin of preparation titanio basal surface.
Embodiment 4
Change the pure silicon sheet among the embodiment 1 into titanium alloy (like Ti6Al4V), with the titanium alloy sheet with acetone, water ultrasonic after, be placed in the NaOH solution of 5mol/L; In 60 ℃ of following alkali treatment 3h, clean up drying with distilled water; Promptly obtain the sufficient and uniform hydroxyl of titanium alloy surface; Other conditions are constant, use experimental technique of the present invention, prepare the compound micrographics of the chitosan/bovine serum albumin of titanium alloy-based basal surface.
The compound micrographics of preparation CS/BSA provided by the invention; In whole process of preparation; Only adopt alkali treatment during the substrate pre-treatment, except introducing oh group, just no longer introduce extra group, and then reduced the genotoxic potential of other chemical group the human body cell effect.Microsoft moves the amount of substance that method of molding can carry and will so can improve the content of preparation sample, strengthen its biological activity more than other micro-processing method in addition.Can adjust micrographics size and the two compound pattern angle as required, thereby applicable surface is wider.Preparation is simple for compound micrographics, and the goods cycle is short, can access the compound micrographics of the antibiotic and good biocompatibility of good combination property.
Claims (2)
1. the method for a preparing chitosan/protein composite micrographics on surface of material is that raw material adopts the micrometastasis method of molding to make CS and two kinds of materials of BSA obtain compound micrographics in the even absorption of silicon face with chitosan CS and bovine serum albumin BSA, comprises the steps:
(1) preparation of silicon chip: adopt dry-oxygen oxidation to handle the pure silicon sheet, the silicon chip of even compact oxide layer is arranged to obtain the surface; The silicon chip of said surperficial even compact oxide layer again through alkali treatment, cleans up with distilled water after cleaning at last, obtains the silicon chip that the surface is full of hydroxyl after the drying;
(2) preparation of solution: CS is dissolved in CH
3Among the COOH, be mixed with the CH of 2mg/ml CS
3COOH solution; It is in 6.6 the phosphate buffer that BSA is dissolved in pH value, is mixed with the BSA solution of 2mg/ml;
(3) elastomeric stamp preparation: fully mix polydimethylsiloxane PDMS and silicone rubber firming agent in 10: 1 ratio of mass ratio, the gained mixture casts on the silicon template that the surface has the micron-scale groove structure; After the curing solidfied material is peeled off from the silicon template down in 80 ℃, after hydrophilic improves processing, obtained the elastomeric stamp of groove topography again through distilled water cleaning, drying, said hydrophilic improves processing and adopts immersion processing in 12 hours in the 1mol/L hydrochloric acid solution;
(4) system of opening up of micrographics: CS solution and the BSA solution of using (2) to obtain respectively drip respectively on the elastomeric stamp that groove topography is arranged that obtains two (3); Leave standstill and strike off surperficial excessive solution; Obtain two elastomeric stamps that have CS micrographics and BSA micrographics respectively, promptly accomplish the system of opening up respectively of micrographics;
(5) the elastomeric stamp left-hand thread with CS micrographics that (4) is obtained is on silicon chip surface that (1) step makes; The fully closely contact that keeps the two; After 20 ℃ of following dryings, peel off elastomeric stamp, in the NaOH of 0.0125mol/L, soak 1min again, with in the acidity of CS; The silicon chip surface adsorption stays the groove micrographics of CS;
On the silicon chip of the groove micrographics that the elastomeric stamp left-hand thread with BSA micrographics that (6) (4) is obtained obtains in (5) with CS; On the silicon chip surface that is adsorbed with CS; The BSA micrographics is gone up in absorption across, obtains the compound micrographics of chitosan/protein of material surface regular shape.
2. the method for a kind of preparing chitosan/protein composite micrographics on surface of material according to claim 1 is characterized in that, said pure silicon sheet adopts dry-oxygen oxidation to handle, and is employed in 1000 ℃ of following sintering 4 hours.
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| CN103951280B (en) * | 2014-04-22 | 2016-05-18 | 西南交通大学 | A kind of method of utilizing titanium oxide seal to prepare fibrinogen figure on target material surface |
| CN104527254B (en) * | 2015-01-04 | 2017-02-01 | 浙江农林大学 | Method for printing double-protein composite micro pattern on surface of material |
| CN105388056B (en) * | 2015-12-23 | 2018-07-06 | 哈尔滨工业大学 | A kind of method for preparing giant phospholipid vesica array using point face Electrode Field based on microcontact printing techniques |
| CN105527139B (en) * | 2015-12-23 | 2018-07-06 | 哈尔滨工业大学 | A kind of method for the giant phospholipid vesica array for preparing phase separation using point face Electrode Field based on micro- contact lift-off technology |
| CN107789666A (en) * | 2016-08-30 | 2018-03-13 | 北京航空航天大学 | A kind of inwall micro-patterning small-caliber artificial blood vessel |
| CN106310366B (en) * | 2016-09-29 | 2019-04-30 | 武汉生物工程学院 | A kind of Guide Periodontal Tissue Regeneration barrier film and the preparation method and application thereof |
| CN107601913A (en) * | 2017-09-29 | 2018-01-19 | 重庆科技学院 | A kind of preparation method of the micro- pattern of material surface |
| CN116477849B (en) * | 2023-04-10 | 2024-04-26 | 之江实验室 | Bismuth ferrite nano-pillar array and preparation method thereof |
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| CN1425920A (en) * | 2002-12-26 | 2003-06-25 | 浙江大学 | Method for fixing biological macro molecule in common pattern on inorganic silicone material surface |
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|---|---|---|---|---|
| CN1425920A (en) * | 2002-12-26 | 2003-06-25 | 浙江大学 | Method for fixing biological macro molecule in common pattern on inorganic silicone material surface |
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