CN101884807A - A kind of preparation method and application of borate glass antibacterial coating with biological activity - Google Patents

Abstract

The invention belongs to the technical field of biological materials, and in particular relates to a preparation method and application of a borate glass antibacterial coating with biological activity. The preparation steps are as follows: prepare borate glass containing sterilizing ions, crush and sieve the glass; uniformly coat the above-mentioned glass powder on the surface of the implant device, and then undergo heat treatment to make the glass powder viscous and form a covering on the implant. antimicrobial coating on the surface of the device. The process equipment is simple, easy to operate and low in cost. The coating of the invention has antibacterial property and biological activity, can be firmly coated on the surface of implant equipment, and can effectively improve the biological activity and antibacterial performance of existing implant materials.

Description

A kind of preparation method and application of borate glass antibacterial coating with biological activity

technical field

The invention belongs to the technical field of biological materials, and in particular relates to a preparation method and application of a borate glass antibacterial coating with biological activity.

Background technique

Titanium alloy (Ti6A14V) is widely used as a load-bearing implant material, such as bone plate, due to its excellent mechanical properties and biocompatibility. The disadvantage of this kind of material is that it has no direct bond with human bone, and due to the presence of vanadium element, it has certain toxicity. At the same time, taking the bone plate as an example, the main method for treating fractures at present is to implant the bone plate to perform fracture internal fixation. However, there are a large number of postoperative infections in internal fixation of fractures, and according to literature reports, the infection rate is about 5% to 20%. Wherein nail track infection can cause serious consequences such as screw loosening more, bring great misery to the patient.

It can be seen that, taking the current bone plate as an example, there are obvious defects in many types of implant equipment. They are usually not antibacterial and cannot be fully adapted to the requirements of surgery, and their improvement is urgently needed.

Contents of the invention

The object of the present invention is to overcome above-mentioned deficiency, provide a kind of preparation method of the borate glass coating that can have biological activity and antibacterial performance.

The preparation method of the borate glass coating with biological activity and antibacterial properties proposed by the present invention, the specific steps are as follows:

(1) Add salts containing sterilizing ions to borate glass, grind and mix evenly, and then use high-temperature melting method to obtain glass blocks; crush and sieve the obtained glass blocks containing sterilizing ions to obtain uniform particle size and average particle size. Glass powder with a diameter of less than 50um; the sterilizing ion is a heavy metal ion, which is any one of silver ion, zinc ion or copper ion; the amount of the sterilizing ion is 0.1wt%-15wt% of the mass of the borate glass; The borate glass network former is B ₂ O ₃ , containing no or a small amount of SiO ₂ /or containing P ₂ O ₅ , wherein the content of B ₂ O ₃ is greater than the content of SiO ₂ /P ₂ O ₅ , and the content of the network former is The total molecular ratio is 30-90mol%; the outer network body contains CaO, and also contains Na ₂ O, K ₂ O alkali metal oxides and MgO, SrO alkaline earth metal oxides; the total molecular ratio of the network outer body ion oxide is 5 ～80mol%; wherein the molecular proportions of CaO and SrO are 5～60mol% respectively;

(2) Uniformly disperse the glass powder obtained in step (1) in the solvent by ultrasonic vibration, and then as the solvent volatilizes, the glass powder is deposited on the surface of the implant device; Spray on the surface of the implanted device; or use the pulling method to adhere the adhesive containing glass powder to the surface of the implanted device;

(3) Put the implanted device that has deposited glass powder or adhered glass powder in step (2) into a muffle furnace, burns the binder at a temperature of 350-400°C, and keeps it warm at 600-1000°C for 2 -30min, the desired antibacterial coating is obtained.

The application of the borate glass coating with biological activity and antibacterial properties on bone repair implants is obtained by using the method of the invention.

The application of the borate glass coating with biological activity and antibacterial performance in stimulating the growth of bone cells is obtained by using the method of the invention.

The application of the borate glass coating with biological activity and antibacterial performance in animal body obtained by using the method of the invention.

The present invention can adjust the thickness of the obtained coating through the amount of glass powder, thereby adjusting the content of sterilizing ions in the coating. The bonding strength between the obtained coating and the implanted device is adjusted by the temperature and time of the heat treatment. The release rate of sterilizing ions in the obtained coating is adjusted by the doping amount of sterilizing ions or the composition of the glass carrier, thereby adjusting the biological activity and antibacterial effect of the coating. Through the composition of the glass powder, the ratio of the various oxides can adjust the bioactivity and biodegradability of the glass coating, as well as the stimulating effect on bone cell growth.

The invention adopts the method of ion sterilization, which combines the excellent mechanical properties of traditional implant equipment with the good biological activity and special structure of borate glass, and sterilizing ions, such as silver ions, which have high antibacterial efficiency and little harm to the human body , The advantages of broad antibacterial spectrum, simple process equipment, easy operation, low cost, can effectively reduce the disease caused by bacteria in the operation, and at the same time applied to bone repair implants, it can obviously stimulate the growth of bone cells.

Description of drawings

Figure 1. Cytocompatibility test of bone plate containing silver borate glass coating, the results of measuring its effect on osteoblasts by MTT colorimetry, indicating that the glass coating has good biocompatibility.

Figure 2 The test that the cells of the bone plate containing silver borate glass coating stimulate the growth of bone cells, and the number of cell proliferation is measured by the alkaline phosphatase (ALP) method, indicating that the glass coating has excellent biological activity and can stimulate bone cell growth.

Fig. 3 The phase analysis of the formation on the surface of the bone plate after the silver-borate glass-coated bone plate was embedded near the rabbit tibia for 10 weeks, suggesting that the inorganic mineral composition of bone tissue has formed on the bone plate, hydroxyphosphorus gray stone. It shows that the bone plate has the function of stimulating the growth of bone cells.

Detailed ways

The present invention is further illustrated below by way of examples.

Embodiment 1 Preparation of silver-containing borate glass coating

(1) Preparation of borate glass with 1.0% silver content

Weigh 2.926g anhydrous sodium carbonate, 7.107g anhydrous potassium carbonate, 4.995g basic magnesium carbonate, 10.294g calcium carbonate, 5.694g strontium carbonate, 6.951g silicon dioxide, 29.473g boric acid, 3.419g sodium dihydrogen phosphate , 0.529g silver phosphate. After grinding, mix well to get the raw ingredients.

After putting the ingredients into a platinum crucible, put it in a temperature of 1150°C for 120 minutes, and then pour the obtained clear glass liquid on a preheated steel plate to obtain a glass block.

The glass block is crushed and sieved to obtain glass particles with an average particle size of about 2-50um.

(2) The preparation of the coating adopts any of the following methods:

(1) After washing and drying the glass particles obtained in step (1), weigh 0.15 g and put it into a 100 ml small beaker.

Put the same cleaned bone plate at the bottom of the beaker, then measure 10ml of absolute ethanol and pour it into the small beaker.

The glass powder was evenly dispersed in ethanol by ultrasonic vibration for 15 minutes, and then the beaker was placed in an oven at 80°C for overnight drying to obtain a bone plate with a uniform layer of glass powder deposited on the surface.

Put the above-mentioned bone plate into a muffle furnace at 600°C, keep it warm for 20 minutes, and then take it out to obtain a uniformly distributed glass coating.

(2) Wash and dry the glass particles obtained in step (1), weigh 4.0 g and put them into a 100 ml small beaker. Measure 20ml of ethanol and weigh 1.2g of ethyl cellulose. After stirring evenly, pour them into a ball mill jar and mill them for 30 minutes to make a slurry.

Then the cleaned bone plate was immersed in the slurry, pulled out from the slurry at a speed of 1 cm/min, and after air drying, a bone plate with a layer of glass powder evenly adhered to the surface was obtained.

Put the above-mentioned bone plate into the muffle furnace, raise the temperature with the furnace to 460°C at a rate of 2.5°C/min, keep it for 1h, then raise the temperature to 660°C at a rate of 10°C/min, keep it for 15min, take it out of the furnace, and get the distribution Uniform glass coating.

(3) Wash and dry the glass particles obtained in step (1) for later use. Mix ethanol and water at a ratio of 1:3, then add glass powder into the solution at a ratio of 0.1g/ml, put them together in an atomization box for atomization, and then be carried by the carrier gas through the nozzle and spray evenly onto the bone After the surface of the plate is air-dried, a bone plate with a layer of glass powder evenly deposited on the surface is obtained.

Put the above-mentioned bone plate into a muffle furnace at 600°C, keep it warm for 20 minutes, and then take it out to obtain a uniformly distributed glass coating.

Example 2: Antimicrobial properties of coatings containing silver borate glass coatings

According to the method of Example 1, 0.265g, 0.529g, 0.794g, and 1.058g of silver phosphate were added to the formula of the glass to prepare four kinds of glass blocks containing AgO of 0.5, 1.0, 1.5 and 2.0wt%, respectively. According to the method of Example 2, four kinds of leaching solutions of glass coating blocks were obtained. Separately place Escherichia coli ATCC259 and Staphylococcus aureus ATCC25923 on the blood agar plate before the test, incubate at 35°C for 18 hours, then pick 2 to 3 colonies of Escherichia coli and Staphylococcus aureus on the blood plate and place them in the broth , 35 ℃ egg incubation 2h. Use a micro-sampler to take a certain amount of bacterial liquid and add it to the extracts arranged from low concentration to high concentration in sequence. The results show that the lowest concentration of the extract without visible growth is its antibacterial performance (MIC ). A certain amount of bacterial solution diluted in a certain proportion was inoculated on two blood plates, and incubated at 35°C for 18 hours. Count the number of colonies on the blood plate, and take the average to calculate the initial inoculum. The minimum concentration of the extract solution at which the number of colonies is lower than 0.1% of the initial inoculum is its bactericidal (killing) ability (MBC) to the bacteria.

The results show that the antibacterial performance of the antibacterial borate glass coating is stronger than that of the basic glass, and it has no antibacterial ability if it is not mixed with silver or contains less silver. The bacteriostasis and bactericidal effect of the coating containing 2% silver on E. The killing effect of Staphylococcus aureus is just worse (see Table 1). With the increase of silver content, the sterilization effect increased significantly.

The sterilization effect comparison when the silver content of table 1 is 1.5wt%

Embodiment 3: Bacteriostatic properties of copper-containing and zinc-containing borate glass coatings

According to the method described in Example 1, in the glass formulation, silver phosphate is replaced by copper sulfate, and 6.818g, 13.636g, and 20.454g CuSO are added respectively to make three kinds of borate glasses with different copper contents, and then Copper-containing borate glass coatings with 5%, 10%, and 15% copper ion mass percentages were prepared. In the same way, 2.172g, 4.344g, and 6.516g of zinc oxide were added to the glass batch material respectively, and three kinds of zinc-containing borate glasses with 5%, 10%, and 15% zinc ion mass percentages were prepared in the same way. coating.

Add sterilized common nutrient agar medium into a sterile petri dish to cover the entire bottom of the petri dish with a thickness of about 2.5 mm, and then evenly coat the Escherichia coli (Escherichia coli) bacterial solution with a concentration of 10 ⁷ cfu/ml. Formation of bacterial fluid film. Cut out six 10*10*0.5mm ³ osteosynthetic plates, the upper surfaces of which have been coated with the above-mentioned six glass coatings, place them flat in a dish, and incubate them at 37°C for 24 hours to observe the growth of bacteria around the coated samples situation, measure the zone of inhibition.

The results showed that with the increase of copper content, the inhibition zone expanded and the antibacterial performance was strengthened. The antibacterial performance of the zinc-containing borate glass coating is also strengthened with the increase of the zinc content, and the law is similar to that of copper ions, but its antibacterial performance is slightly lower than that of the coating obtained by the same mass percentage of copper ions (see Table 2 ).

Table 2

Antibacterial ion Amount added (mass percentage%) Diameter of antibacterial zone (mm) Cu ²⁺ 5 twenty three Cu ²⁺ 10 32 Cu ²⁺ 15 39 Zn ²⁺ 5 18 Zn ²⁺ 10 27 Zn ²⁺ 15 35

Example 4: Biocompatibility of Antimicrobial Borate Glass Coatings

According to the method of Example 1, in the formula, add 0.397g, 0.529g, and 1.058g of silver phosphate respectively to make three kinds of glass coating pieces containing AgO that are 0.75, 1.0 and 2.0wt%, respectively, and the gained three kinds of different silver The glass-coated pieces of the content were soaked in ethanol for 24 hours, and then washed 3 times with deionized water. Then soak the glass in DMEM cell culture medium or α-MEM cell culture medium according to the ratio of 1.25 cm ² surface area to 1 ml solution. After 24 hours, the glass block was removed to obtain three kinds of glass extracts, which were supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin in volume ratio.

Then, MC3T3E1 osteoblasts were introduced into the extract, and the cell lines were planted in a 96-well culture dish at a density of 0.2 ml/well. Then the osteoblasts were cultured in α-MEM medium, which was supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 ug/ml streptomycin. The culture medium without glass extract was used as the control group. All cells were cultured at 37°C in a 5% _CO2 environment. After 24 hours of culture, the growth of cells was evaluated by MTT colorimetry.

The results showed that with the increase of the silver content in the borate glass, the damage to the cells was greater, while the borate glass extracts containing 1% and 0.75% of the silver content were less toxic to the cells, and the boric acid containing 2% silver was less toxic to the cells. Salt glass extracts are also within acceptable limits (see Figure 1).

Example 5: Osteocyte Growth Stimulating Properties of Antimicrobial Borate Glass Coatings

Three borate glasses with different silver contents were prepared according to Example 4, and three corresponding leaching solutions of glass-coated pieces were obtained. MC3T3-E1 osteoblasts were seeded in 24-well culture dishes and cultured in blank medium or medium supplemented with silver borate glass extract. After 48 hours of culture, the cells were washed twice with PBS, and then placed in 500ul of 1% Triton X-100 phosphate buffer, and the cells were lysed by freeze-thaw cycles (-80/37°C). Aliquots of the lysate were packed in 96-well petri dishes and their ALP activity was measured by spectrophotometry.

The results showed that the addition of α-MEM and blank α-MEM medium with silver content of 0%, 0.75%, 1.0%, and 2.0% borate glass leaching solutions had different effects on the performance of osteoblasts. The effect is not much different (see Figure 2). The results showed that the three silver-containing glass-coated blocks all had excellent biological activity and could stimulate the proliferation of bone cells.

Example 6: Effect of Antimicrobial Borate Glass Coating in Animals:

According to embodiment 1, get and be coated with the borate glass coating osteosynthesis plate (20x2x0.5mm) that contains silver oxide 1wt% by sedimentation method, be embedded in the surface of the tibia of 10 rabbits, inject near the tibia of rabbit then Contain 0.1 ml of Staphylococcus aureus ATCC25923 emulsified solution to make animal models containing bacteria. During the experiment with bone plate implantation in rabbits, no signs of inflammation were found in 10 rabbits. After 10 weeks, the embedded bone plate was taken out from the rabbit body, and no signs of bone tissue necrosis were found, indicating that the bone plate coating had an antibacterial function; the surface formations of the bone plate were separated, and XRD surface analysis was performed (see Figure 3), and the prompt showed The surface formation contains inorganic minerals in bone tissue, hydroxyapatite, indicating that the bone plate in the rabbit has a certain stimulating effect on the growth of bone cells.

Claims (4)

1. A preparation method of a borate glass coating with biological activity and antibacterial properties, characterized in that the specific steps are as follows: (1) Add salts containing sterilizing ions to borate glass, grind and mix evenly, and then use high-temperature melting method to obtain glass blocks; crush and sieve the obtained glass blocks containing sterilizing ions to obtain uniform particle size and average particle size. Glass powder with a diameter of less than 50um; the sterilizing ion is a heavy metal ion, which is any one of silver ion, zinc ion or copper ion; the amount of the sterilizing ion is 0.1wt%-15wt% of the mass of the borate glass; The borate glass network former is B ₂ O ₃ , containing no or a small amount of SiO ₂ /or containing P ₂ O ₅ , wherein the content of B ₂ O ₃ is greater than the content of SiO ₂ /P ₂ O ₅ , and the content of the network former is The total molecular ratio is 30-90mol%; the outer network body contains CaO, and also contains Na ₂ O, K ₂ O alkali metal oxides and MgO, SrO alkaline earth metal oxides; the total molecular ratio of the network outer body ion oxide is 5 ～80mol%; wherein the molecular proportions of CaO and SrO are 5～60mol% respectively; (2) Uniformly disperse the glass powder obtained in step (1) in the solvent by ultrasonic vibration, and then as the solvent volatilizes, the glass powder is deposited on the surface of the implant device; Spray on the surface of the implanted device; or use the pulling method to adhere the adhesive containing glass powder to the surface of the implanted device; (3) Put the implanted device that has deposited glass powder or adhered glass powder in step (2) into a muffle furnace, burns the binder at a temperature of 350-400°C, and keeps it warm at 600-1000°C for 2 -30min, the desired antibacterial coating is obtained. 2. An application of the borate glass coating having biological activity and antibacterial properties prepared by the method according to claim 1 on the bone repair implant. 3. The application of the borate glass coating with biological activity and antibacterial properties prepared by the method as claimed in claim 1 in stimulating bone cell growth. 4. The application of the borate glass coating with biological activity and antibacterial properties prepared by the method according to claim 1 in animals.

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