CN118059301A - Preparation method of difunctional membrane for periodontal bone tissue repair - Google Patents
Preparation method of difunctional membrane for periodontal bone tissue repair Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/025—Other specific inorganic materials not covered by A61L27/04 - A61L27/12
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/20—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
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Abstract
The invention discloses a preparation method of a difunctional membrane for periodontal bone tissue repair. Firstly, mixing carboxymethyl chitosan, sodium alginate and a composite piezoelectric material into purified water, stirring, performing ultrasonic vibration, then pouring into a mould, standing, casting, drying in a drying box, adding CaCl 2 solution for crosslinking, cutting and forming after crosslinking, pre-freezing in a refrigerator at-80 ℃, freeze-drying in a freeze dryer, and finally performing polarization treatment. The invention aims at realizing periodontal tissue repair and regeneration, and prepares the gel composite membrane with piezoelectric effect from two angles of bacterial control and bone regeneration, and has good biocompatibility, biomechanical property and stable degradation rate.
Description
Technical Field
The invention belongs to the technical field of periodontal bone tissue repair, and particularly relates to a preparation method of a difunctional membrane for periodontal bone tissue repair.
Background
Periodontal disease is a common oral disease that can lead to periodontal soft tissue defects, severe alveolar bone resorption, and is a major factor in tooth loss. The periodontal tissue structure is fine and complex, and once destroyed, it is quite difficult to reconstruct the physiological structure of the periodontal tissue. 80% -90% of adults in China are affected by periodontal diseases, the incidence rate of periodontal diseases is high, the harm is large, the complications are serious, and the life quality of patients is seriously affected. Therefore, reconstructing the anatomical structure and physiological function of periodontal tissue is a problem that researchers have dedicated to solving. Guided Tissue Regeneration (GTR) is currently the most effective method for achieving periodontal regeneration, and GTRM is the key to this technique, GTRM, which is currently in clinical use, achieves its barrier function only by preventing fibroblast growth to the tissue defect site, and has low osteoinductive activity and cannot inhibit the destructive action of bacteria. In order to solve the clinical problems of easy infection of the operation area, poor osteoinductive activity and the like. Researchers have generally increased osteogenic capacity by adding preparations such as bioactive proteins, growth factors, bioactive molecules, and the like, or new strategies for regeneration based on tissue engineering, stem cell therapy, and the like, and although some osteogenic activity has been increased, the side effects of adding active factors are also not negligible. In addition, many researchers have achieved antibacterial effects by loading antibiotics or antibacterial nano ions, etc., but antibiotic resistance and a single improvement GTRM of a certain characteristic have not achieved periodontal tissue regeneration in a true sense.
In order for the piezoelectric material to exhibit its optimal piezoelectric performance, it is necessary to perform polarization treatment. Polarization is a mandatory process for exciting the piezoelectric properties of a piezoelectric material by applying a strong electric field across the material. Under the action of the applied electric field, the dipoles are redirected from random directions to alignment in the solid region according to the direction of the electric field. The polarization treatment is applied manually, that is, a high enough direct current electric field is applied to the piezoelectric ceramic, and the piezoelectric ceramic is kept at a certain temperature and time to force the electric domain to turn or force the spontaneous polarization to be oriented. The polarization treatment of a piezoelectric material refers to the rearrangement of polarized molecules or ions inside the material by applying an electric field, thereby enhancing the piezoelectric effect thereof. During heating, the crystal structure of the ceramic changes, causing the polarized molecules or ions to rearrange. Then, under the action of mechanical stress, polarized molecules or ions are kept in an aligned state for a period of time, thereby realizing polarization improvement in piezoelectricity.
Disclosure of Invention
The invention aims to provide a preparation method of a difunctional membrane for periodontal bone tissue repair.
A preparation method of a difunctional membrane for periodontal bone tissue repair is carried out according to the following method:
(1) Mixing carboxymethyl chitosan, sodium alginate and a composite piezoelectric material in purified water, stirring, and performing ultrasonic vibration for 1-3h to uniformly mix the materials;
(2) Pouring the uniformly mixed materials in the step (1) into a mould, standing for casting, and placing the materials in a drying box at 34-40 ℃ for drying for 1-3h;
(3) Taking out the dried material in the step (2), and adding CaCl 2 solution to crosslink for 4-8h; cutting and forming after crosslinking, pre-freezing in a refrigerator at-80 ℃, and freeze-drying in a freeze dryer for 8-12h;
(4) And (3) carrying out polarization treatment on the material prepared in the step (3) to prepare the difunctional membrane for periodontal bone tissue repair.
The composite piezoelectric material in the step (1) is barium titanate and cysteine according to the mass ratio of 1: 1.
The carboxymethyl chitosan, the sodium alginate and the composite piezoelectric material in the step (1) respectively account for 2-4%, 2-4% and 6-9% of the total mass of the uniformly mixed materials.
The molar concentration of the CaCl 2 solution in the step (3) is 0.5-1.5mol/L.
The voltage of the polarization treatment in the step (4) is 1500-2500V, the polarization temperature is 23-26 ℃, and the polarization time is 0.5-1.5h.
Preferably, the drying temperature in the step (2) is 37 ℃ and the drying time is 2 hours.
Preferably, the crosslinking time in step (3) is 6 hours.
Preferably, the lyophilization time in step (3) is 10 hours.
The invention has the beneficial effects that: the invention aims at realizing periodontal tissue repair and regeneration, and prepares the gel composite membrane with piezoelectric effect from two angles of bacterial control and bone regeneration. The composite membrane is prepared from carboxymethyl chitosan, sodium alginate and composite piezoelectric materials, and has good biocompatibility, biomechanical property and stable degradation rate. Under the polarization voltage, the piezoelectric material has crystal grain electric domain arrangement which tends to be orderly and externally shows polarity. Bound charges are formed near the two surfaces, and free charges are adsorbed on the surfaces to balance the free charges under the action of the bound charges. When the composite piezoelectric material is pressed, the electric dipole moment of the bound charge changes, and the amount of free charge on the surface is inevitably changed through charge and discharge to balance, so that a piezoelectric effect is formed. Therefore, the material after polarization treatment has piezoelectric coefficient which is more matched with the piezoelectric property of human bones, namely, can fully play the osteogenesis role, further, under the stimulation effect of an ultrasonic instrument, the piezoelectric signal is excited to promote the dual functions of antibiosis and bone regeneration, so that the periodontal tissue regeneration is rapidly and efficiently promoted, and a novel technology and means are provided for guiding the periodontal tissue regeneration film.
Drawings
FIG. 1 is a state diagram of the composite membrane material before and after lyophilization.
Fig. 2 is a comparison of piezoelectric coefficients before and after polarization.
FIG. 3 shows the effect of material polarization on BMSCs proliferation before and after material polarization.
Fig. 4 is the effect of the material on the antimicrobial effect before and after polarization.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Example 1
A preparation method of a difunctional membrane for periodontal bone tissue repair is carried out according to the following method:
(1) Mixing carboxymethyl chitosan, sodium alginate and a composite piezoelectric material in purified water, stirring, and performing ultrasonic vibration for 2 hours to uniformly mix the materials; the composite piezoelectric material is prepared from barium titanate and cysteine according to a mass ratio of 1: 1; the carboxymethyl chitosan, sodium alginate and the composite piezoelectric material respectively account for 3%, 3% and 7.5% of the total mass of the uniformly mixed materials;
(2) Pouring the uniformly mixed materials in the step (1) into a mould, standing for casting, and placing the materials in a drying box at 37 ℃ for drying for 2 hours;
(3) Taking out the dried material in the step (2), and adding 1mol/L CaCl 2 solution to crosslink for 6 hours; cutting and forming after crosslinking, pre-freezing in a refrigerator at-80 ℃, and freeze-drying in a freeze dryer for 10 hours;
(4) Polarizing the material prepared in the step (3) to prepare a difunctional membrane for periodontal bone tissue repair; the voltage of the polarization treatment is 2000V, the polarization temperature is 25 ℃, and the polarization time is 1h.
Example 2
A preparation method of a difunctional membrane for periodontal bone tissue repair is carried out according to the following method:
(1) Mixing carboxymethyl chitosan, sodium alginate and a composite piezoelectric material in purified water, stirring, and performing ultrasonic vibration for 1h to uniformly mix the materials; the composite piezoelectric material is prepared from barium titanate and cysteine according to a mass ratio of 1: 1; the carboxymethyl chitosan, sodium alginate and the composite piezoelectric material respectively account for 2%, 2% and 6% of the total mass of the uniformly mixed materials;
(2) Pouring the uniformly mixed materials in the step (1) into a mould, standing for casting, and placing the materials in a 34 ℃ drying box for drying for 1h;
(3) Taking out the dried material in the step (2), and adding 0.5mol/L CaCl 2 solution to crosslink for 4 hours; cutting and forming after crosslinking, pre-freezing in a refrigerator at-80 ℃, and freeze-drying for 8 hours in a freeze dryer;
(4) Polarizing the material prepared in the step (3) to prepare a difunctional membrane for periodontal bone tissue repair; the voltage of the polarization treatment is 1500V, the polarization temperature is 23 ℃, and the polarization time is 1.5h.
Example 3
A preparation method of a difunctional membrane for periodontal bone tissue repair is carried out according to the following method:
(1) Mixing carboxymethyl chitosan, sodium alginate and a composite piezoelectric material in purified water, stirring, and performing ultrasonic vibration for 3 hours to uniformly mix the materials; the composite piezoelectric material is prepared from barium titanate and cysteine according to a mass ratio of 1: 1; the carboxymethyl chitosan, sodium alginate and the composite piezoelectric material respectively account for 4%, 4% and 9% of the total mass of the uniformly mixed materials;
(2) Pouring the uniformly mixed materials in the step (1) into a mould, standing for casting, and placing the materials in a drying box at 40 ℃ for drying for 3 hours;
(3) Taking out the dried material in the step (2), and adding 1.5mol/L CaCl 2 solution to crosslink for 8 hours; cutting and forming after crosslinking, pre-freezing in a refrigerator at-80 ℃, and freeze-drying in a freeze dryer for 12 hours;
(4) Polarizing the material prepared in the step (3) to prepare a difunctional membrane for periodontal bone tissue repair; the voltage of the polarization treatment is 2500V, the polarization temperature is 26 ℃, and the polarization time is 1.5h.
Comparative example 1
A preparation method of a difunctional membrane for periodontal bone tissue repair is carried out according to the following method:
(1) Mixing carboxymethyl chitosan, sodium alginate and barium titanate in purified water, stirring, and performing ultrasonic vibration for 2 hours to uniformly mix the materials; the carboxymethyl chitosan, sodium alginate and barium titanate respectively account for 3%, 3% and 7.5% of the total mass of the uniformly mixed materials;
(2) Pouring the uniformly mixed materials in the step (1) into a mould, standing for casting, and placing the materials in a drying box at 37 ℃ for drying for 2 hours;
(3) Taking out the dried material in the step (2), and adding 1mol/L CaCl 2 solution to crosslink for 6 hours; cutting and forming after crosslinking, pre-freezing in a refrigerator at-80 ℃, and freeze-drying in a freeze dryer for 10 hours;
(4) Polarizing the material prepared in the step (3) to prepare a difunctional membrane for periodontal bone tissue repair; the voltage of the polarization treatment is 2000V, the polarization temperature is 25 ℃, and the polarization time is 1h.
Comparative example 2
A preparation method of a difunctional membrane for periodontal bone tissue repair is carried out according to the following method:
(1) Mixing carboxymethyl chitosan, sodium alginate and cysteine in purified water, stirring, and performing ultrasonic vibration for 2 hours to uniformly mix the materials; the carboxymethyl chitosan, sodium alginate and cysteine respectively account for 3%, 3% and 7.5% of the total mass of the uniformly mixed materials;
(2) Pouring the uniformly mixed materials in the step (1) into a mould, standing for casting, and placing the materials in a drying box at 37 ℃ for drying for 2 hours;
(3) Taking out the dried material in the step (2), and adding 1mol/L CaCl 2 solution to crosslink for 6 hours; cutting and forming after crosslinking, pre-freezing in a refrigerator at-80 ℃, and freeze-drying in a freeze dryer for 10 hours;
(4) Polarizing the material prepared in the step (3) to prepare a difunctional membrane for periodontal bone tissue repair; the voltage of the polarization treatment is 2000V, the polarization temperature is 25 ℃, and the polarization time is 1h.
Experimental example:
The material prepared in example 1 is taken as a detection object, the state of the composite film prepared in example 1 before and after freeze-drying is shown in fig. 1, and after freeze-drying in step (3), the surface of the material is full and glossy. The material is processed into round films with uniform size and thickness before freeze-drying, the color is semitransparent, and the color after freeze-drying is almost pure white.
Piezoelectric coefficient: the piezoelectric coefficient d33 is an important parameter of the piezoelectric material, and is the ratio of the amount of charge that the material should generate under the action of pressure to the pressure exerted on the material. The larger the value of d33, the stronger the piezoelectric effect of the material, with higher piezoelectric performance.
Experimental facilities: RK2671AM pressure-resistant tester
Experimental parameters: polarization conditions: 2KV, normal temperature 25 ℃ and 1h.
The experimental steps are as follows: and 3, preparing a silver paste electrode with the thickness of 3mm, airing, clamping the electrode polarized by adopting a copper needle point, maximally bearing 10KV voltage, carrying out 2KV polarization for 1h at the normal temperature of 25 ℃, ensuring that leakage current is not more than 0.05, and testing the piezoelectric value.
The piezoelectric coefficient of the composite membrane prepared in example 1 before and after the polarization treatment is as shown in fig. 2, the piezoelectric coefficient before polarization is low, and the piezoelectric coefficient after polarization treatment is obviously improved because the piezoelectric coefficient before polarization treatment is used as a piezoelectric material which cannot provide enough piezoelectric performance and further cannot meet the requirement of ideal bone tissue repair and regeneration.
And (3) mechanical detection: standard test pieces of 2cm×2mm were prepared, and the conventional mechanical properties (tensile properties and elastic modulus) of the dual-functional films for periodontal bone tissue repair prepared in examples 1 to 3 and comparative examples 1 to 2 were compared and studied, thereby preparing film materials conforming to the mechanical conditions of barrier films. And (3) after a certain mechanical stimulus is applied to the prepared standard test piece with the length of 2cm multiplied by 2mm, a ZL-11-SYJ-01 electronic universal material testing machine is adopted to carry out a comparative test on tensile property and elastic modulus, and the influence of piezoelectric stimulus on the mechanical property is detected.
3 Parallel experiments are carried out on each group of experiments, average values are obtained, SPSS 24.0 software is adopted for statistical analysis, the measurement data result is expressed by x ̅ +/-s (mean square error), a Kolmogorov-Smirnov test method is adopted for data normalization test, t test is adopted for comparison of average value difference between two groups of data conforming to normal distribution, and P <0.05 is used as difference to have statistical significance.
The measurement results are shown in Table 1:
TABLE 1
Note that: * Representing a comparison of P <0.05 with example 1 group.
The experimental results are shown in table 1, and the mechanical properties of the composite piezoelectric material are obviously higher than those of the single-component piezoelectric material.
Cell proliferation assay: osteoblasts were seeded into 96-well plates according to a suitable number of plated cells (2×10 4), and approximately 100uL of cell suspension per well was seeded onto membranes pre-soaked with culture medium, the material being divided into polarized and unpolarized groups; culturing the pore plate in a 37 ℃ incubator until the cells adhere, adding 10uL of CCK8 after the cells adhere, and measuring the absorbance at 450nm to evaluate the proliferation condition of the cells.
As shown in the experimental result in FIG. 3, the material before polarization has little effect on the proliferation of BMSCs cells, while the BMSCs cells cultured by the material after polarization proliferate in large quantity, and the material shows obvious promotion effect on the cells.
Antibacterial experiment:
1, preparing an LB agar medium, and then autoclaving.
2. Resuscitates bacteria in a ratio of 1:1000, and then places them on a shaker under conditions of 37℃for 120 revolutions overnight.
3. Bacteria were cultured, the material and the bacterial liquid were co-cultured at a concentration of 1X 10 6, and placed on a shaker, and the conditions were set at 120 revolutions and 37 ℃.
4. Plating, the bacterial liquid which is successfully cultured is diluted by 1 ten thousand times and plated on an LB agar medium, and each colony takes 5 ul. Then, the cells were placed in an incubator at 37℃and incubated for 12 hours to take a photograph.
The experimental result is shown in fig. 4, and the polarized piezoelectric material has stronger antibacterial effect.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (8)
1. A method for preparing a difunctional membrane for periodontal bone tissue repair, which is characterized by comprising the following steps:
(1) Mixing carboxymethyl chitosan, sodium alginate and a composite piezoelectric material in purified water, stirring, and performing ultrasonic vibration for 1-3h to uniformly mix the materials;
(2) Pouring the uniformly mixed materials in the step (1) into a mould, standing for casting, and placing the materials in a drying box at 34-40 ℃ for drying for 1-3h;
(3) Taking out the dried material in the step (2), and adding CaCl 2 solution to crosslink for 4-8h; cutting and forming after crosslinking, pre-freezing in a refrigerator at-80 ℃, and freeze-drying in a freeze dryer for 8-12h;
(4) And (3) carrying out polarization treatment on the material prepared in the step (3) to prepare the difunctional membrane for periodontal bone tissue repair.
2. The method for preparing a bifunctional film for periodontal bone tissue repair of claim 1, wherein the composite piezoelectric material in step (1) is barium titanate and cysteine in a mass ratio of 1:1.
3. The preparation method of the difunctional film for periodontal bone tissue repair according to claim 1, wherein the carboxymethyl chitosan, sodium alginate and composite piezoelectric material in the step (1) respectively account for 2-4%, 2-4% and 6-9% of the total mass of the uniformly mixed materials.
4. The method for preparing a bifunctional film for periodontal bone tissue repair of claim 1, wherein the molar concentration of CaCl 2 solution in step (3) is 0.5-1.5mol/L.
5. The method for preparing a bifunctional film for periodontal bone tissue repair of claim 1, wherein the voltage of the polarization treatment in step (4) is 1500-2500V, the polarization temperature is 23-26℃and the polarization time is 0.5-1.5h.
6. The method for preparing a bifunctional film for periodontal bone tissue repair of claim 1, wherein the drying temperature in step (2) is 37℃and the drying time is 2 hours.
7. The method for preparing a bifunctional film for periodontal bone tissue repair of claim 1, wherein the crosslinking time in step (3) is 6h.
8. The method for preparing a bifunctional film for periodontal bone tissue repair of claim 1, wherein the lyophilization time in step (3) is 10 hours.
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