CN113967287B - Antibacterial low-toxicity tooth/bone repair active material and preparation method thereof - Google Patents
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Abstract
The invention discloses an antibacterial low-toxicity tooth/bone repair active material and a preparation method thereof. Dissolving alcohol ammonium and an alkylating agent in n-hexane for reaction, washing with diethyl ether after the reaction, and concentrating to obtain a liquid; dissolving the liquid in water, adding anion exchange resin for exchange, removing water, washing with ethyl acetate and diethyl ether in sequence, and drying to obtain dianion ionic liquid; adding nano calcium phosphate and cationic polymer into the dianionic liquid, uniformly stirring to form sol, and standing to form soft gel; then adding the anionic polymer while heating and stirring at low temperature, pouring the mixture into a mould and standing; washing the gel with ethanol water solution to remove unreacted ionic liquid; and re-dissolving the obtained material in CSP and P11-4 dipeptide aqueous solution to form gel, and then carrying out vacuum freeze drying to obtain the antibacterial low-toxicity tooth/bone repair active material. The invention utilizes a double-anion ionic liquid system combining choline and dicarboxylate ions, can dissolve nano calcium phosphate particles, and has low toxicity.
Description
The technical field is as follows:
the invention belongs to the technical field of biomedical materials, and relates to an antibacterial low-toxicity tooth/bone repair active material and a preparation method thereof.
Background art:
oral diseases are the most common non-infectious diseases and can cause pain, discomfort, disfigurement and even death. According to 2016 global disease burden research, half of the population (35.8 hundred million people) all over the world is estimated to have oral diseases, wherein decayed teeth (decayed teeth) are the most common problems, filling restoration is the most common and effective method for clinically treating decayed teeth, such as composite resin which is widely applied at present, has excellent aesthetic property and biocompatibility, and becomes an important material for treating dental defects, but secondary decayed teeth caused by bacterial infection are one of important reasons for causing the restoration failure of the composite resin. Solving the biocompatibility of the restorative material with the dental pulp-dentin complex and preventing secondary caries is a development trend of the dental restorative material in the next 20 years. Therefore, for the treatment of the odontogenic infection, the key problem is how to induce tooth regeneration and resist bacteria and defend the occurrence of secondary caries on the basis of 'preserving the existing enamel and dentin' and 'preserving the pulp vitality'. Therefore, the development of anti-infection dental biomaterials with antibacterial defense and tooth regeneration promoting capabilities is necessary, and the anti-infection dental biomaterials have important clinical significance and wide market application prospects.
The invention content is as follows:
the invention aims to provide an antibacterial low-toxicity tooth/bone repair active material and a preparation method thereof, which can induce tooth regeneration, can resist bacteria and prevent secondary caries, and can be widely applied to other bone injury parts.
In the present invention we have designed a non-toxic and well biocompatible dianionic liquid based on a combination of choline and dicarboxylate ions. The calcium phosphate particles are dissolved by means of the dianion ionic liquid, and the amide reaction and the anion-cation polyelectrolyte electrostatic complexation are completed without a carboxyl activator, so that the nano calcium phosphate and the active dipeptide are entrapped, and a novel and safe odontogenic antibacterial dental substitute is developed.
In order to achieve the purpose, the invention adopts the following scheme:
a. dissolving alcohol ammonium and an alkylating agent in n-hexane for reaction, washing with diethyl ether after the reaction, and concentrating to obtain a liquid;
b. dissolving the obtained a) liquid in water, adding anion exchange resin for exchange, adding dicarboxylic acid for reaction after completely exchanging hydroxide, removing water, washing with ethyl acetate and diethyl ether in sequence, and finally performing vacuum drying to obtain dianion ionic liquid;
c. adding nano calcium phosphate and cationic polymer into the dianionic liquid, uniformly stirring to form sol, and standing to form soft gel; then adding the anionic polymer under the heating condition of 45-50 ℃, stirring, pouring into a mould, and standing to form gel; washing the gel with an ethanol water solution to remove unreacted ionic liquid; and re-dissolving the obtained material in the CSP and the P11-4 dipeptide aqueous solution to form gel, and then carrying out vacuum freeze drying to obtain the antibacterial low-toxicity tooth/bone repair active material.
Preferably, the reaction of step a is carried out at 60-90 ℃ for 1-4 days with stirring.
Preferably, the reaction of step b is stirred at room temperature for 2-5 hours.
Preferably, the alcohol ammonium is an amino diol compound, more preferably 2,2' -dihydroxydiethylamine, N-methyldiethanolamine or N-ethyldiethanolamine; the concentration of the ammonium alkoxide is preferably 10 to 100mmol/ml.
Preferably, the alkylating agent is: 1-chlorobutane, 1-bromobutane, 2-bromoethyl ether; the mol dosage of the alcohol ammonium is 1.1 to 4.0 times of that of the alkylating agent.
Preferably, the dicarboxylic acid is malonic acid, adipic acid, glutaric acid, succinic acid, fumaric acid; the concentration of the dicarboxylic acid is 0.5 to 1.0 times of the amount concentration of the ammonium alkoxide.
Preferably, the nano calcium phosphate is nano hydroxyapatite, nano alpha-tricalcium phosphate, nano beta-tricalcium phosphate or amorphous tricalcium phosphate; the concentration of the added nano calcium phosphate is 0.01 to 1g/ml, preferably 0.01 to 0.1g/ml.
Preferably, the cationic polymer can be chitosan or polylysine; the anionic polymer is chondroitin sulfate, sodium alginate or polyglutamic acid; the concentration range of the cationic liquid polymer ionic liquid is as follows: 0.03g/ml to 0.25g/ml; the mass concentration ratio of the anionic liquid polymer to the cationic liquid polymer is as follows: 1:1 to 4.
Preferably, the dipeptide is: potent stimulatory peptide (CSP) is a quorum sensing peptide with 21 amino acids, the odontogenic P11-4 peptide: p11-4 (Ace-QQRFEWEFEQQ-NH 2) is a self-assembling peptide that enhances remineralization of early caries lesions. The range of dipeptide concentrations was: 0.01mg/ml to 10mg/ml. The preferred dosage ratio is mass ratio 1:1.
Compared with the prior art, the invention has the following beneficial effects:
1) The antibacterial low-toxicity tooth/bone repair active material provided by the invention utilizes a double-anion ionic liquid system combining choline and dicarboxylic acid radical ions, can dissolve nano calcium phosphate particles, and has low toxicity.
2) According to the antibacterial low-toxicity tooth/bone repair active material provided by the invention, under the condition of no carboxyl activating agent, a cationic polymer and carboxyl in an ionic liquid are subjected to an amide reaction, then an electrostatic complexation effect is generated between the cationic polymer and a polyanion electrolyte to form a gel, and nano calcium phosphate particles are wrapped in the gel, so that calcium phosphate can be gradually separated out in a crystal form in an aqueous solvent to play a bone repair effect.
3) The antibacterial low-toxicity tooth/bone repair active material provided by the invention innovatively enables the composite material to induce tooth/bone regeneration on the basis of 'preserving the existing enamel and dentin' and 'preserving pulp vitality' by virtue of the electrostatic adsorption capacity of stimulating peptide (antibacterial action) and P11-4 odontogenic peptide (promoting tooth/bone regeneration), and can also resist bacteria and prevent the occurrence of secondary caries.
Detailed description of the invention
The technical solutions of the embodiments of the present invention will be described clearly and completely below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without any inventive step, are within the scope of the present invention.
Example 1:
1. 100mmol of N-methyldiethanolamine and 400mmol of 1-chlorobutane are mixed and dissolved in N-hexane, and the mixture is placed in a 100mL pressure reaction vessel and stirred for reaction at 60-70 ℃ for 4 days. Washing with 2 times of diethyl ether, and rotary evaporating under reduced pressure to constant volume to obtain liquid.
2. Dissolving the obtained 1) liquid in distilled water, eluting by using strongly basic quaternary ammonium I type anion exchange resin, slowly adding 0.5 times of propanedicarboxylic acid relative to the molar concentration of N-methyldiethanolamine after the solution obtained by AgNO3-HNO3 detection is completely exchanged to form hydroxide, and stirring for 2 hours at room temperature. Rotating under reduced pressure to remove a large amount of water, washing with 2 times volume of ethyl acetate and 1 time volume of diethyl ether in sequence, and finally vacuum drying to obtain the dianion ionic liquid.
3. Taking 10ml of dianion liquid, sequentially adding 1.0g of nano calcium phosphate and 2.5g of cationic polymer chitosan (the deacetylation degree is more than or equal to 95 percent), stirring uniformly at room temperature to form sol, and standing to form soft gel; then adding 1.25g of anionic polymer polyglutamic acid under the heating condition of 45-50 ℃, stirring for 3 hours, pouring into a mould, and standing to form gel; and washing the gel with a 70% ethanol solution by volume fraction, and removing unreacted ionic liquid.
4. And re-dissolving the obtained material in 10ml of CSP (20 mg) and P11-4 (20 mg) dipeptide aqueous solution to form gel, and then carrying out vacuum freeze drying to obtain the antibacterial low-toxicity bone repair active material.
The antibacterial effect of the antibacterial low-toxicity bone repair active material of the embodiment is tested by an antibacterial experiment, and the specific method is as follows: the test bacterial strain cultured for 24 hours is picked by an inoculating loop, looped in 5mL of sterile physiological saline, and subjected to gradient dilution to be configured into a concentration of 10 3 cfu/mL bacterial liquid. Weighing 10mg of antibacterial low-toxicity bone repair active material, re-dissolving in 10ml of sterile water to form gel, and adding into 10ml of 10-concentration 10 3 After the cfu/mL bacterial solution is subjected to constant temperature oscillation cultivation at 27 ℃ for 2 days, 1mL of bacterial solution is taken for gradient dilution, each gradient is subjected to a plate coating experiment in parallel with three groups, the number of plate colonies is counted after the constant temperature cultivation for 1 day, a plate which is only used for the test bacteria and is not treated by materials is taken as a control, and the bacteriostasis rate is calculated by the bacteriostasis rate = (the number of control colonies-the number of treated colonies)/the number of control colonies multiplied by 100 percent. Experiments prove that the antibacterial low-toxicity bone repair active material of the embodiment has the antibacterial rate of 99.3 percent on staphylococcus aureus and 98.4 percent on escherichia coli under the concentration of 0.2mg/ml, and shows good antibacterial activity.
The antibacterial low-toxicity bone repair of the present example was evaluated by the following experimentThe active material has cytotoxicity to periodontal ligament, and the specific method is as follows: 10mg of the Co60 sterilized antibacterial low-toxicity bone repair active material is placed in a hole of a 48-hole plate, and is redissolved to the maximum extent by using a sterile PBS solution, and the residual PBS solution is removed. Add 300. Mu.L DMEM medium to each well, soak overnight, and blot dry. Selecting 3-5 generation periodontal ligament cells, and formulating with DMEM medium containing 10% FBS at a concentration of 5X 10 4 Cell suspension/mL. The cell suspension was inoculated at 300. Mu.L/well into a well plate containing the active material, incubated in a standard cell incubator for 9d (change of medium every 3 d), stained for live/dead cells as indicated in the kit, and observed under a fluorescent microscope. The number of live/dead cells was softly counted using Image J, and the cell viability = [ (number of live cells)/(number of live cells + number of dead cells) =wascounted]×100％。
The antibacterial low-toxicity bone repair active material in the embodiment has the activity of 124 +/-10% on periodontal ligament cells, and has good biocompatibility.
Example 2:
1. 10mmol of 2-dimethylaminoethanolammonium and 11mmol of 1-bromobutane were mixed and dissolved in n-hexane, and placed in a 100mL pressure reaction vessel, and the reaction was stirred at 80-90 ℃ for 2 days. Washing with 2 times of diethyl ether, and rotary evaporating under reduced pressure to constant volume to obtain liquid.
2. Dissolving the obtained 1) liquid in distilled water, eluting with strongly basic quaternary ammonium I type anion exchange resin (i.e. adding anion exchange resin), slowly adding 1.0 times of adipic acid relative to the molar concentration of 2-dimethylaminoethanol ammonium after completely exchanging into hydroxide, and stirring at room temperature for 3 hours. Rotating under reduced pressure to remove a large amount of water, then washing with 2 times volume of ethyl acetate and 1 time volume of diethyl ether in sequence, and finally drying in vacuum to obtain the dianion ionic liquid.
3. Taking 10ml of dianion liquid, sequentially adding 0.1g of nano calcium phosphate and 0.3g of polylysine (MV 2000-5000), stirring uniformly at room temperature to form sol, and standing to form soft gel; then adding 0.3g chondroitin sulfate under the heating condition of 45-50 ℃, stirring for 1 hour, pouring into a mould, and standing to form gel; the gel was washed with 70% ethanol aqueous solution by volume fraction to remove unreacted ionic liquid.
4. And re-dissolving the obtained material in 10ml of CSP (1 mg) and P11-4 (1 mg) dipeptide aqueous solution to form gel, and then carrying out vacuum freeze drying to obtain the antibacterial low-toxicity bone repair active material.
An antibacterial test and a cell viability evaluation test were performed on antibacterial low-toxic tooth/bone active materials with reference to example 1.
Experiments prove that the antibacterial low-toxicity bone repair active material of the embodiment has the antibacterial rate of 97.3 percent on staphylococcus aureus and the antibacterial rate of 90.4 percent on escherichia coli under the concentration of 0.2mg/ml, and shows good antibacterial activity.
Experiments prove that: the antibacterial low-toxicity bone repair active material in the embodiment has 115 +/-6% of activity on periodontal ligament cells, and has good biocompatibility.
Example 3:
1. 50mmol of 2-dimethylaminoethanolammonium chloride and 100ml of 2-bromoethyl ether are mixed and dissolved in n-hexane, and the mixture is placed in a 100mL pressure reaction vessel and stirred for reaction for 1 day at 70-80 ℃. Washing with diethyl ether of 2 times volume, and rotary evaporating at low temperature under reduced pressure until the volume is constant to obtain liquid.
2. Dissolving the obtained 1) liquid in distilled water, eluting by using strongly basic quaternary ammonium I type anion exchange resin, slowly adding succinic acid which is 0.8 times of the molar concentration of 2-dimethylamino ethanol ammonium after the solution obtained by AgNO3-HNO3 inspection is completely exchanged to form hydroxide, and stirring for 5 hours at room temperature. Rotating under reduced pressure to remove a large amount of water, washing with 2 times volume of ethyl acetate and 1 time volume of diethyl ether in sequence, and finally vacuum drying to obtain the dianion ionic liquid.
3. Taking 10ml of dianion liquid, sequentially adding 0.5g of nano calcium phosphate and 0.8g of carboxymethyl chitosan, stirring uniformly at room temperature to form sol, and standing to form soft gel; then adding 0.2g of sodium alginate under the heating condition of 45-50 ℃, stirring for 2 hours, pouring into a mold, and standing to form gel; the gel was washed with 70% aqueous ethanol to remove unreacted ionic liquid.
4. The obtained material is re-dissolved in 10ml of CSP (0.01 mg) and P11-4 (0.01 mg) dipeptide aqueous solution, and after gel is formed, the gel is frozen and dried in vacuum, thus obtaining the antibacterial low-toxicity bone repair active material.
An antibacterial test and a cell viability evaluation test were performed on antibacterial low-toxic tooth/bone active materials with reference to example 1.
Experiments prove that the antibacterial low-toxicity bone repair active material of the embodiment has a bacteriostasis rate of 89.7% to staphylococcus aureus and a bacteriostasis rate of 86.8% to escherichia coli under the concentration of 0.2mg/ml, and shows good antibacterial activity.
The antibacterial low-toxicity bone repair active material in the embodiment has the activity of 97 +/-14% on periodontal ligament cells, and has good biocompatibility.
Claims (10)
1. The preparation method of the antibacterial low-toxicity tooth/bone repair active material is characterized by comprising the following steps of:
a. dissolving alcohol ammonium and an alkylating agent in n-hexane for reaction, washing with diethyl ether after the reaction, and concentrating to obtain a liquid;
b. dissolving the obtained a) liquid in water, adding anion exchange resin for exchange, adding dicarboxylic acid for reaction after completely exchanging hydroxide, removing water, washing with ethyl acetate and diethyl ether in sequence, and finally performing vacuum drying to obtain dianion ionic liquid;
c. adding nano calcium phosphate and cationic polymer into the dianionic liquid, uniformly stirring to form sol, and standing to form soft gel; then adding the anionic polymer under the heating condition of 45-50 ℃, stirring, pouring into a mould, and standing to form gel; washing the gel with an ethanol water solution to remove unreacted ionic liquid; re-dissolving the obtained material in CSP and P11-4 dipeptide aqueous solution, forming gel, and vacuum freeze-drying to obtain antibacterial low-toxicity tooth/bone repairing active material;
the alkylating agent is: 1-chlorobutane, 1-bromobutane or 2-bromoethyl ether.
2. The method of claim 1, wherein the reaction of step a is carried out at 60-90 ℃ for 1-4 days with stirring.
3. The method of claim 1, wherein the reaction of step b is carried out at room temperature for 2 to 5 hours with stirring.
4. The method of claim 1, wherein the alcohol ammonium is 2,2' -dihydroxydiethylamine, N-methyldiethanolamine or N-ethyldiethanolamine; the concentration of the alcohol ammonium is 10-100 mmol/ml.
5. The process according to claim 1 or 4, wherein the molar amount of ammonium alkoxide used is 1.1 to 4.0 times that of the alkylating agent.
6. The process according to claim 1, wherein the dicarboxylic acid is selected from the group consisting of malonic acid, adipic acid, glutaric acid, succinic acid, fumaric acid; the concentration of the dicarboxylic acid is 0.5 to 1.0 times of the amount concentration of the ammonium alkoxide.
7. The preparation method of claim 1, wherein the nano calcium phosphate is nano hydroxyapatite, nano α -tricalcium phosphate, nano β -tricalcium phosphate or amorphous tricalcium phosphate; the concentration of the added nano calcium phosphate is 0.01g/ml to 1g/ml.
8. The method according to claim 7, wherein the nano calcium phosphate is added at a concentration of 0.01 to 0.1g/ml.
9. The method of claim 1, wherein the cationic polymer is chitosan or polylysine; the anionic polymer is chondroitin sulfate, sodium alginate or polyglutamic acid; the concentration range of the cationic liquid polymer ionic liquid is as follows: 0.03g/ml to 0.25g/ml; the mass concentration ratio of the anionic liquid polymer to the cationic liquid polymer is as follows: 1:1 to 4; the concentration ranges of the dipeptides in the CSP and the aqueous solution of the P11-4 dipeptides are respectively as follows: 0.01mg/ml to 10mg/ml, and the dosage ratio is 1:1.
10. An antibacterial low toxicity dental/bone repair active material prepared according to the process of claim 1.
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| EP1979514A1 (en) * | 2006-01-24 | 2008-10-15 | Basf Se | Polymer backbone for producing artificial tissue |
| WO2009073068A2 (en) * | 2007-11-30 | 2009-06-11 | The Govt. Of U.S.A. As Represented By The Secretary Of The Department Of Health And Human Services | Bone scaffolds, injectable bone repair materials and methods for bone repair |
| CN105315164A (en) * | 2014-08-01 | 2016-02-10 | 中国科学院大连化学物理研究所 | Environment-friendly choline ionic liquid and preparation method therefor |
| AU2020311591A1 (en) * | 2019-07-08 | 2022-02-03 | Theravet Sa | A method for the preparation of a gel-forming composition |
| CN110180022B (en) * | 2019-07-15 | 2021-11-05 | 吉林大学 | Shear force response supramolecular bionic articular cartilage material with dynamic lubrication self-repair capacity and preparation method thereof |
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