Antibacterial nickel-titanium alloy bone tissue engineering scaffold as well as preparation method and application thereof
By employing a layer-by-layer self-assembly technique that forms a chitosan quaternary ammonium salt coating on the surface of nickel-titanium alloys, the problem of easy infection in titanium alloy implants has been solved, the antibacterial and osteogenic activities of nickel-titanium alloys have been improved, and the safety of implants has been enhanced.
Patent Information
- Application Number
- CN202511014638.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-23
- Publication Date
- 2025-11-07
AI Technical Summary
Titanium alloy implants are prone to bacterial infections. Existing surface-modified antibacterial coating processes are complex and have poor adhesion, resulting in poor long-term antibacterial performance and making it difficult to effectively solve the problem of bacterial infection in titanium alloy implants.
A chitosan quaternary ammonium salt coating was formed on the surface of a nickel-titanium alloy using a layer-by-layer self-assembly technique. An antibacterial nickel-titanium alloy bone tissue engineering scaffold was prepared by polishing, acid etching, glutaraldehyde crosslinking, and chitosan coating.
It improves the biocompatibility of nickel-titanium alloys, exhibits good antibacterial activity, enhances osteogenic activity of osteoblasts, and reduces the risk of bacterial infection.
Smart Images
Figure CN120899993A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of bone tissue engineering, and particularly relates to an antibacterial nickel-titanium alloy bone tissue engineering scaffold and a preparation method and application thereof. BACKGROUND
[0002] Titanium alloy has good biocompatibility, comprehensive mechanical properties, mechanical properties, processing properties and corrosion resistance in a biological environment, is the most widely used metal implant material at present, is used to replace human hard tissues that have lost physiological functions and perform functions thereof, and has a wide application prospect in the medical field.
[0003] However, titanium alloy is a kind of biologically inert material and has no biological antibacterial property itself, and after being implanted into the human body, foreign body rejection occurs, inflammation is easily induced, and harmful bacteria are possibly brought in, thereby causing infection. The spread of diseases and harmful bacteria poses a major threat to human health and survival, and is one of the important social problems faced by countries all over the world. The incidence of bacterial infection caused by titanium alloy implants is very high, bacterial infection affects wound healing and the use effect of implants, and the infection is difficult to control and recurs repeatedly, which has been a thorny problem that has plagued medical staff for a long time. In addition, titanium alloy is also commonly used in some kitchen utensils, household equipment, medical and health utensils and catering equipment, and is easy to adhere to bacteria, thereby bringing harm to human health and safety. At present, methods such as magnetron sputtering, electrochemical deposition, ion implantation and micro-arc oxidation are usually used to load inorganic or organic antibacterial agents on the surface of medical titanium alloy, so as to endow the titanium alloy with antibacterial property. However, the antibacterial coating layer has the following disadvantages although its advantages are obvious, for example, the coating manufacturing process is relatively complex, the production cost is increased, the combination between the antibacterial coating layer and the substrate is poor, the coating layer is easy to wear, and long-term antibacterial property is poor.
[0004] Therefore, it is urgent to develop a new type of antibacterial titanium alloy bone tissue engineering scaffold, so as to solve the problem of frequent bacterial infection of implant titanium alloy, and to be applied to dental implants and bone replacement materials in dentistry and orthopedics. Through the antibacterial effect of the titanium alloy, the probability of bacterial infection is effectively reduced, the success rate of implantation of the implant material is improved, and the health of patients is better protected. SUMMARY
[0005] Based on the problem of frequent bacterial infection of implant titanium alloy in the prior art, a first object of the present application is to provide a preparation method of a new type of antibacterial nickel-titanium alloy bone tissue engineering scaffold, a second object of the present application is to provide an antibacterial nickel-titanium alloy bone tissue engineering scaffold prepared by the preparation method, and a third object of the present application is to provide application of the new type of antibacterial nickel-titanium alloy bone tissue engineering scaffold in dental implants and bone replacement materials in orthopedics.
[0006] The objects of the present application are achieved by the following technical solutions.
[0007] In one aspect, the application provides a preparation method of an antibacterial nickel-titanium alloy bone tissue engineering scaffold, comprising the following steps:
[0008] Step one, polishing treatment of the nickel-titanium alloy sheet;
[0009] Step two, acid etching treatment of the polished nickel-titanium alloy sheet;
[0010] Step three, then glutaraldehyde cross-linking is used to introduce a chitosan coating;
[0011] Step four, then a sodium alginate coating and a chitosan quaternary ammonium salt coating are introduced to prepare the antibacterial nickel-titanium alloy bone tissue engineering scaffold.
[0012] In the above preparation method, preferably, the specific method of step one comprises:
[0013] First, the nickel-titanium alloy sheet is polished by using progressively finer grits of emery paper to remove surface dirt;
[0014] Then, ultrasonic cleaning is performed; finally, the polished nickel-titanium alloy sheet is obtained by nitrogen drying treatment.
[0015] In the above preparation method, preferably, the nickel-titanium alloy sheet is polished by using progressively finer grits of emery paper, i.e., 400#, 600#, 800#, and 1200#.
[0016] In the above preparation method, preferably, acetone, anhydrous ethanol, and ultrapure water are used for ultrasonic cleaning in sequence.
[0017] In the above preparation method, preferably, the frequency of ultrasonic treatment is 100 HZ; the ultrasonic cleaning time is 15 minutes, and the cleaning is repeated 3 times.
[0018] In the above preparation method, preferably, the specific method of step two comprises:
[0019] First, the polished nickel-titanium alloy sheet is placed in a mixed solution of sulfuric acid and hydrogen peroxide for etching reaction for 1 h; after the reaction is completed, the sheet is cleaned with deionized water and dried with nitrogen blowing;
[0020] Then, the sheet is placed in an amino-terminated polyether solution for reaction for 1 h; after being taken out, the sheet is repeatedly cleaned with acetone and dried with nitrogen blowing to obtain the acid-etched nickel-titanium alloy sheet.
[0021] In the above preparation method, preferably, the mixed solution of sulfuric acid and hydrogen peroxide is prepared by mixing 98% sulfuric acid and 30% hydrogen peroxide in a volume ratio of 3:1.
[0022] In the preparation method, preferably, the specific method of step three comprises:
[0023] Firstly, the acid-etched nickel-titanium alloy sheet is put into glutaraldehyde solution for cross-linking reaction under magnetic stirring, and then the reaction product is washed with deionized water and dried by nitrogen blowing.
[0024] Then, the sample is taken out after being soaked in chitosan solution for 8 hours, and then washed repeatedly with deionized water and dried by nitrogen blowing, thereby obtaining a nickel-titanium alloy sheet with a glutaraldehyde-cross-linked chitosan layer.
[0025] In the preparation method, preferably, the concentration of the glutaraldehyde solution is 4% v / v.
[0026] In the preparation method, preferably, the concentration of the chitosan solution is 0.2% w / v.
[0027] In the preparation method, preferably, the specific method of step four comprises:
[0028] Firstly, the nickel-titanium alloy sheet with a glutaraldehyde-cross-linked chitosan layer is put into sodium alginate solution for 30 min, and then put into calcium chloride solution for 10 min, and finally put into chitosan quaternary ammonium salt solution for 30 min; after the soaking, the sample is taken out, washed with deionized water and dried by nitrogen blowing, thereby obtaining an antibacterial nickel-titanium alloy bone tissue engineering scaffold.
[0029] In the preparation method, preferably, the concentration of the sodium alginate solution is 2% m / v.
[0030] In the preparation method, preferably, the concentration of the calcium chloride solution is 5 mM.
[0031] In the preparation method, preferably, the concentration of the chitosan quaternary ammonium salt solution is 2% m / v.
[0032] In another aspect, the present application also provides an antibacterial nickel-titanium alloy bone tissue engineering scaffold, which is prepared by the above preparation method.
[0033] In still another aspect, the present application also provides the application of the above antibacterial nickel-titanium alloy bone tissue engineering scaffold in dental implants and bone replacement materials in orthopedics.
[0034] The present application has the following beneficial effects:
[0035] The antibacterial nickel-titanium alloy bone tissue engineering scaffold of the present application forms a chitosan quaternary ammonium salt coating on the surface of the nickel-titanium alloy through layer-by-layer self-assembly, which can effectively improve the biocompatibility of the nickel-titanium alloy, has better antibacterial activity, and can enhance the osteogenic activity of osteoblasts, thereby being beneficial to the proliferation of osteoblasts.
[0036] The above description is only a summary of the technical solutions of the present application. In order to make the technical means of the present application clearer and to enable the implementation according to the content of the description, the preferred embodiments of the present application are described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS
[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.
[0038] Figure 1 The scanning electron microscope image of the untreated nickel-titanium alloy sheet.
[0039] Figure 2 The scanning electron microscope image of the nickel-titanium alloy sheet after acid etching treatment prepared in Comparative Example 1.
[0040] Figure 3 The scanning electron microscope image of the nickel-titanium alloy sheet with glutaraldehyde cross-linked chitosan layer prepared in Comparative Example 2.
[0041] Figure 4 The scanning electron microscope image of the antibacterial nickel-titanium alloy bone tissue engineering scaffold prepared in Example 1.
[0042] Figure 5 The comparison chart of the change of the hydrophilic angle of the nickel-titanium alloy and the layer-by-layer treated nickel-titanium alloy.
[0043] Figure 6 The statistical chart of the change of the hydrophilic angle of the nickel-titanium alloy and the layer-by-layer treated nickel-titanium alloy. Figure 7 The chart of the change of the alkaline phosphatase activity of the osteoblasts. DETAILED DESCRIPTION
[0044] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. The process, conditions, reagents, experimental methods, etc. for implementing the present application are the general knowledge and common sense in the art, and the present application does not have special limitations.
[0045] The related solutions used in the following embodiments are prepared as follows:
[0046] Preparation of chitosan solution (2% m / v): chitosan powder 100 mg was weighed by electronic balance and sterilized by CO60 irradiation with a radiation dose of 8000 Gy for 12 h. 0.3 ml of anhydrous acetic acid was measured by a measuring cylinder and poured into a 30 ml sterile beaker. 29.7 ml of sterile deionized water was added to the beaker, and after stirring thoroughly, a 1% anhydrous ethanol solution was obtained. 0.04 mg of chitosan was weighed by electronic balance and dissolved in 20 ml of the ethanol solution (1% v / v). After stirring thoroughly until the chitosan powder was completely dissolved, the solution was transferred to a sterile glass bottle and stored at low temperature after sealing.
[0047] Preparation of chitosan quaternary ammonium salt (2% m / v): chitosan quaternary ammonium salt (degree of substitution 26%) 6 mg was weighed by electronic balance and sterilized by CO60 irradiation with a radiation dose of 8000 Gy for 12 h. It was dissolved in 30 ml of sterile ultrapure water, and after stirring thoroughly, a chitosan quaternary ammonium salt solution with a concentration of 2 mg / ml was obtained. A sodium alginate solution with a concentration of 2 mg / ml can be obtained according to the above method.
[0048] Example 1:
[0049] The present embodiment provides an antibacterial nickel-titanium alloy bone tissue engineering scaffold and a preparation method thereof. The specific preparation method comprises:
[0050] (1) Polishing treatment of nickel-titanium alloy sheet: the nickel-titanium alloy sheet is sequentially ground and polished by 400#, 600#, 800# and 1200# diamond sandpaper to remove surface dirt; then it is cleaned by ultrasonic wave (frequency 100 Hz) in acetone, anhydrous ethanol and ultrapure water respectively, the cleaning time is 15 minutes, and the cleaning is repeated 3 times, and the sample is dried by nitrogen.
[0051] (2) Acid etching treatment of nickel-titanium alloy sheet: the polished nickel-titanium alloy sheet is placed in a mixed solution (98% sulfuric acid + 30% hydrogen peroxide; volume ratio 3:1) for 1 hour, then it is repeatedly washed with deionized water for 3 times and dried by nitrogen. Then it is placed in an amino-terminated polyether solution for 1 hour, and then the sample is taken out with tweezers and the surface of the sample is repeatedly cleaned with acetone for 5 minutes (repeated 5 times). The cleaned sample is dried by nitrogen to obtain the acid-etched nickel-titanium alloy sheet.
[0052] (3) Glutaraldehyde crosslinking introduction of chitosan coating: a 30 ml glutaraldehyde solution (4% v / v) was measured in a measuring cylinder and poured into a round-bottom flask, and the nickel-titanium alloy sheet after acid etching treatment was placed therein and continuously stirred on a magnetic stirrer for 8 hours. The stirring speed was adjusted from small to large to avoid splashing of the solution. After 8 hours, the sample was fished out and repeatedly washed with deionized water for three times and then dried with nitrogen. Then the sample was immersed in a chitosan solution (0.2% w / v) for 8 hours, and then fished out and repeatedly washed with deionized water for three times and then dried with nitrogen, to obtain a nickel-titanium alloy sheet with a glutaraldehyde crosslinked chitosan layer.
[0053] (4) Preparation of antibacterial nickel-titanium alloy bone tissue engineering scaffold: the nickel-titanium alloy sheet with a glutaraldehyde crosslinked chitosan layer was placed in a 0.2% sodium alginate solution for 30 min, then placed in a 5 mM CaCl2 solution for 10 min, and finally placed in a chitosan quaternary ammonium salt solution (2% m / v) for 30 min. After soaking, the sample was fished out, washed with deionized water and dried with nitrogen, to obtain an antibacterial nickel-titanium alloy bone tissue engineering scaffold.
[0054] Comparative Example 1
[0055] This comparative example provides a nickel-titanium alloy sheet after acid etching treatment and a preparation method thereof. The specific preparation method comprises:
[0056] (1) Polishing treatment of nickel-titanium alloy sheet: the nickel-titanium alloy sheet was sequentially ground and polished by 400#, 600#, 800# and 1200# diamond sandpaper to remove surface dirt; then ultrasonic cleaning (frequency 100 Hz) was performed in acetone, anhydrous ethanol and ultrapure water, respectively, with a cleaning time of 15 minutes, and the cleaning was repeated 3 times, and the sample was dried with nitrogen.
[0057] (2) Acid etching treatment of nickel-titanium alloy sheet: the polished nickel-titanium alloy sheet was placed in a mixed solution (98% sulfuric acid + 30% hydrogen peroxide; volume ratio 3:1) for 1 hour, and then repeatedly washed with deionized water for 3 times and dried with nitrogen. Then it was placed in an ATPES solution for 1 hour, and the sample was fished out with tweezers and the surface of the sample was repeatedly cleaned with acetone, and the unreacted silane was cleaned for 5 min (repeated 5 times). The cleaned sample was dried with nitrogen, to obtain a nickel-titanium alloy sheet after acid etching treatment.
[0058] Comparative Example 2
[0059] This comparative example provides a nickel-titanium alloy sheet with a glutaraldehyde crosslinked chitosan layer and a preparation method thereof. The specific preparation method comprises:
[0060] (1) Polishing treatment of the nickel-titanium alloy sheet: The nickel-titanium alloy sheet was polished by using 400#, 600#, 800# and 1200# emery paper in sequence to remove the dirt on the surface; then the nickel-titanium alloy sheet was cleaned in acetone, anhydrous ethanol and ultrapure water by using ultrasonic cleaning (frequency 100 Hz) for 15 minutes, and the cleaning was repeated for 3 times, and the nickel-titanium alloy sheet was dried by using nitrogen.
[0061] (2) Acid etching treatment of the nickel-titanium alloy sheet: The polished nickel-titanium alloy sheet was put into a mixed solution (98% sulfuric acid + 30% hydrogen peroxide; volume ratio 3:1) for reaction for 1 hour, and then the nickel-titanium alloy sheet was repeatedly washed with deionized water for 3 times and dried by using nitrogen. Then the nickel-titanium alloy sheet was put into an ATPES solution for 1 hour, and then the nickel-titanium alloy sheet was taken out by using tweezers and repeatedly cleaned with acetone, and the unreacted silane was cleaned for 5 minutes (repeated for 5 times), and the cleaned nickel-titanium alloy sheet was dried by using nitrogen, thereby obtaining the nickel-titanium alloy sheet treated by acid etching.
[0062] (3) Cross-linking of glutaraldehyde to introduce a chitosan coating: 30 ml of a glutaraldehyde solution (4% v / v) was measured by using a measuring cylinder, and then the glutaraldehyde solution was poured into a round-bottom flask, and then the nickel-titanium alloy sheet treated by acid etching was put into the round-bottom flask, and then the round-bottom flask was placed on a magnetic stirrer for continuous stirring for 8 hours, and the stirring speed was adjusted from small to large to avoid splashing of the solution, and then the nickel-titanium alloy sheet was taken out after 8 hours, and then the nickel-titanium alloy sheet was repeatedly washed with deionized water for 3 times and dried by using nitrogen; then the nickel-titanium alloy sheet was put into a chitosan solution (0.2% w / v) for immersion for 8 hours, and then the nickel-titanium alloy sheet was taken out, repeatedly washed with deionized water for 3 times and dried by using nitrogen, thereby obtaining the nickel-titanium alloy sheet with a glutaraldehyde cross-linked chitosan layer.
[0063] Comparative Example 3
[0064] The embodiment provides an antibacterial nickel-titanium alloy bone tissue engineering scaffold and a preparation method thereof, and the specific preparation method comprises the following steps:
[0065] (1) Polishing treatment of the nickel-titanium alloy sheet: The nickel-titanium alloy sheet was polished by using 400#, 600#, 800# and 1200# emery paper in sequence to remove the dirt on the surface; then the nickel-titanium alloy sheet was cleaned in acetone, anhydrous ethanol and ultrapure water by using ultrasonic cleaning (frequency 100 Hz) for 15 minutes, and the cleaning was repeated for 3 times, and the nickel-titanium alloy sheet was dried by using nitrogen.
[0066] (2) Acid etching treatment of the nickel-titanium alloy sheet: The polished nickel-titanium alloy sheet was put into a mixed solution (98% sulfuric acid + 30% hydrogen peroxide; volume ratio 3:1) for reaction for 1 hour, and then the nickel-titanium alloy sheet was repeatedly washed with deionized water for 3 times and dried by using nitrogen. Then the nickel-titanium alloy sheet was put into an ATPES solution for 1 hour, and then the nickel-titanium alloy sheet was taken out by using tweezers and repeatedly cleaned with acetone, and the unreacted silane was cleaned for 5 minutes (repeated for 5 times), and the cleaned nickel-titanium alloy sheet was dried by using nitrogen, thereby obtaining the nickel-titanium alloy sheet treated by acid etching.
[0067] (3) Glutaraldehyde crosslinking to introduce chitosan coating: 30 ml of glutaraldehyde solution (4% v / v) was measured with a graduated cylinder and poured into a round-bottom flask. The acid-etched nickel-titanium alloy sheet was placed in the flask and stirred continuously on a magnetic stirrer for 8 hours. When adjusting the speed, it should be increased from small to large to avoid splashing of the solution. After 8 hours, the sample was taken out and rinsed three times with deionized water and then dried with nitrogen. Then the sample was immersed in chitosan solution (0.2% w / v) for 8 hours. After that, the sample was taken out and rinsed three times with deionized water and then dried with nitrogen to obtain a nickel-titanium alloy sheet with glutaraldehyde crosslinked chitosan layer.
[0068] (4) Preparation of antibacterial nickel-titanium alloy bone tissue engineering scaffold: The nickel-titanium alloy sheet with glutaraldehyde cross-linked chitosan layer was placed in 0.2% sodium alginate solution for 30 min. After soaking, the sample was taken out, washed with deionized water and dried with nitrogen to obtain the antibacterial nickel-titanium alloy bone tissue engineering scaffold.
[0069] Example 2 Scanning Electron Microscopy Experiment
[0070] The morphology of the antibacterial nickel-titanium alloy bone tissue engineering scaffold prepared in Example 1, the acid-etched nickel-titanium alloy sheet prepared in Comparative Example 1, the nickel-titanium alloy sheet with a glutaraldehyde cross-linked chitosan layer prepared in Comparative Example 2, and the untreated nickel-titanium alloy sheet (blank control group) were observed by scanning electron microscopy. The experimental results are as follows: Figures 1-4 As shown. Figure 1 A scanning electron microscope image of an untreated nickel-titanium alloy sheet; Figure 2 The image shows a scanning electron microscope (SEM) image of the nickel-titanium alloy sheet prepared in Comparative Example 1 after acid etching. Figure 3 A scanning electron microscope image of a nickel-titanium alloy sheet with a glutaraldehyde cross-linked chitosan layer prepared in Comparative Example 2; Figure 4 The image shows a scanning electron microscope (SEM) image of the antibacterial nickel-titanium alloy bone tissue engineering scaffold prepared in Example 1.
[0071] Depend on Figure 4 It can be seen that the antibacterial nickel-titanium alloy bone tissue engineering scaffold prepared by this invention has a relatively smooth surface compared to... Figure 2 Comparative Example 1 and Figure 3 In Comparative Example 2, the uneven surface of the nickel-titanium alloy was basically covered, suggesting that the surface of the nickel-titanium alloy was covered with multiple layers of coating material.
[0072] Example 3: Hydrophilic Angle Experiment
[0073] The hydrophilic angle detector was used to detect the hydrophilic angle of the nickel-titanium alloy sheet after each layer treatment during the preparation of the material, i.e. the nickel-titanium alloy sheet without any treatment (blank control group), the nickel-titanium alloy sheet with glutaraldehyde cross-linked chitosan layer (comparative example 2), the nickel-titanium alloy sheet after sodium alginate coating treatment (comparative example 3), and the nickel-titanium alloy sheet after chitosan quaternary ammonium salt coating treatment (example 1). The experimental results are shown in FIGS. Figure 5 and Figure 6 .
[0074] Figure 5 FIG. 4 is a comparison chart of the hydrophilic angle of the nickel-titanium alloy and the nickel-titanium alloy after each layer treatment. Figure 6 FIG. 5 is a statistical chart of the change in the hydrophilic angle of the nickel-titanium alloy and the nickel-titanium alloy after each layer treatment (* indicates that the hydrophilic angle has a statistical difference compared with the nickel-titanium alloy group, P < 0.05). Figure 5 FIGS. 1, 2, 3 and 4 in FIG. 6 are the hydrophilic angles of the blank control group, comparative example 2, comparative example 3 and example 1, respectively, and it can be seen that the hydrophilic angle of the nickel-titanium alloy decreases after each layer treatment. Figure 6 FIG. 5 is a statistical chart of the change in the hydrophilic angle of the nickel-titanium alloy and the nickel-titanium alloy after each layer treatment (* indicates that the hydrophilic angle has a statistical difference compared with the nickel-titanium alloy group, P < 0.05).
[0075] Example 4: Bone cell osteogenic activity experiment
[0076] Experimental grouping: Group A: nickel-titanium alloy sheet without any treatment; Group B: example 1; Group C: blank control group (no material added, only osteoblasts).
[0077] (1) Cell inoculation: The prepared and sterilized material samples were sequentially placed in the 24-well plate in the clean bench, and the osteoblast suspension with a concentration of 1 × 10 5 / ml was prepared according to the experimental content in the second part and inoculated into the 24-well plate. After 2 days, 4 days and 6 days, the AKP alkaline phosphatase kit was used for detection, and the measurement was repeated 3 times.
[0078] (2) Protein extraction: At the set time point, the 24-well plate was taken out of the cell incubator, the culture medium was removed, and the plate was washed with PBS solution. The residual liquid in the well was aspirated with a pipette gun, and 100 ul of prepared cell lysis solution was added to the well. The plate was placed on ice for 30 minutes, and the cells were fully lysed by shaking the plate from time to time. Then the cell lysis solution on the surface of the nickel-titanium alloy and in the 24-well plate was scraped off with a cell scraper, and the cell lysis solution was transferred to a 1.5 ml centrifuge tube with a pipette gun. The tube was placed in a 4°C centrifuge for 20 minutes (12000 rpm). The supernatant was removed and transferred to another 1.5 ml centrifuge tube, and the precipitate in the centrifuge tube was discarded.
[0079] (3) BCA kit for determining protein concentration: According to the kit instructions, standard protein was configured into a protein solution with a concentration gradient, and 20 ul of the protein solution was added to each well of a 96-well plate. Then the reagents in the BCA kit were added in sequence according to the instructions. After incubation in a 37°C water bath for 30 minutes, the absorbance was measured on an enzyme marker. The standard curve was drawn according to the absorbance and protein concentration. The extracted protein solution was transferred to a 96-well plate, and the reagents in the BCA kit were added. The absorbance was measured on an enzyme marker (wavelength 562 nm). The absorbance of the sample was calculated according to the standard curve.
[0080] (4) Determination of osteogenic activity: According to the AKP alkaline phosphatase kit instructions, the protein extract of the sample was transferred to a 96-well plate. The reagents in the kit were added in sequence according to the instructions, and after incubation in a 37°C water bath for 15 minutes, a specific color developing agent was added to measure the absorbance on an enzyme marker (wavelength set to 520 nm). The alkaline phosphatase activity was calculated according to the formula.
[0081] The calculation formula is: AKP activity in cells = (measured OD value - blank OD value) / (standard OD value - blank OD value) x standard concentration (0.1 mg / ml) / protein concentration of sample to be tested
[0082] The experimental results are shown in Figure 7 . Figure 7 The change of alkaline phosphatase activity of osteoblasts in each experimental group is shown in Figure 7 It can be seen that the activity of osteoblasts in each group increases with time. Compared with other groups, the osteogenic activity of group C is higher than that of the other two groups, and the difference in alkaline phosphatase activity is statistically significant (P<0.05). There is no statistically significant difference in alkaline phosphatase activity between group A and group B (P>0.05), which indicates that chitosan quaternary ammonium salt coating can increase the osteogenic activity of nickel-titanium alloy.
[0083] As can be seen from the above, the chitosan quaternary ammonium salt coating is prepared on the surface of the nickel-titanium alloy through the layer-by-layer self-assembly mode, so that the tissue compatibility of the nickel-titanium alloy can be effectively improved, the antibacterial activity is better, and the osteogenic activity of the osteoblasts can be enhanced, thereby being beneficial to the proliferation of the osteoblasts.
[0084] The principles and implementation manners of the present application are described by using specific examples in the present application, and the above example description is only used for helping to understand the method and core idea of the present application; meanwhile, for the general technical personnel in the field, the specific implementation manners and application ranges will be changed according to the idea of the present application, and the above description should not be understood as the limitation of the present application.
Claims
1. A method for preparing an antibacterial nickel-titanium alloy bone tissue engineering scaffold, comprising the following steps: Step one, polishing the nickel-titanium alloy sheet; Step two, acid etching the polished nickel-titanium alloy sheet; Step three, introducing a chitosan coating layer by glutaraldehyde cross-linking; Step four, introducing a sodium alginate coating layer and a chitosan quaternary ammonium salt coating layer to obtain the antibacterial nickel-titanium alloy bone tissue engineering scaffold.
2. The production method according to claim 1, characterized by, The specific method of step one comprises: First, polishing the nickel-titanium alloy sheet by using emery paper in stages to remove surface dirt; Then, ultrasonic cleaning; and finally, drying the polished nickel-titanium alloy sheet by using nitrogen.
3. The preparation method according to claim 2, characterized in that, The nickel-titanium alloy sheet is polished by using emery paper in stages, in the order of 400#, 600#, 800# and 1200#. Preferably, ultrasonic cleaning is performed by using acetone, anhydrous ethanol and ultrapure water in sequence. Preferably, the ultrasonic treatment frequency is 100 HZ, the ultrasonic cleaning time is 15 minutes, and the cleaning is repeated three times.
4. The method of claim 1, wherein: The specific method of step two comprises: First, placing the polished nickel-titanium alloy sheet into a mixed solution of sulfuric acid and hydrogen peroxide for etching reaction for 1 h, then washing with deionized water and drying by nitrogen blowing; Then, placing the sheet into an amino-terminated polyether solution for reaction for 1 h, then repeatedly washing with acetone and drying by nitrogen blowing to obtain the acid-etched nickel-titanium alloy sheet.
5. The method of claim 4, wherein: The mixed solution of sulfuric acid and hydrogen peroxide is prepared by mixing 98% sulfuric acid and 30% hydrogen peroxide in a volume ratio of 3:
1.
6. The method of claim 1, wherein, The specific method of step three comprises: First, placing the acid-etched nickel-titanium alloy sheet into a glutaraldehyde solution for cross-linking reaction by magnetic stirring, then washing with deionized water and drying by nitrogen blowing; Then, placing the sheet into a chitosan solution for immersion for 8 hours, then repeatedly washing with deionized water and drying by nitrogen blowing to obtain the nickel-titanium alloy sheet with a glutaraldehyde cross-linked chitosan layer. Preferably, the concentration of the glutaraldehyde solution is 4% v / v, and the concentration of the chitosan solution is 0.2% w / v.
7. The preparation method according to claim 1, characterized in that, The specific method of step four comprises: First, placing the nickel-titanium alloy sheet with a glutaraldehyde cross-linked chitosan layer into a sodium alginate solution for immersion for 30 min, then into a calcium chloride solution for immersion for 10 min, and finally into a chitosan quaternary ammonium salt solution for immersion for 30 min; then washing the sample with deionized water and drying by nitrogen blowing to obtain the antibacterial nickel-titanium alloy bone tissue engineering scaffold.
8. The method of claim 7, wherein: The concentration of the sodium alginate solution is 2% m / v, the concentration of the calcium chloride solution is 5 mM, and the concentration of the chitosan quaternary ammonium salt solution is 2% m / v. 9.An antibacterial nickel-titanium alloy bone tissue engineering scaffold prepared by the method of any one of claims 1-8. 10.The antibacterial nickel-titanium alloy bone tissue engineering scaffold of claim 9 is used in dental implants and bone replacement materials in orthopedics.