CN106620878B - Collagen-chitosan composite material capable of slowly releasing icariin and preparation method thereof - Google Patents
Collagen-chitosan composite material capable of slowly releasing icariin and preparation method thereof Download PDFInfo
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Abstract
The invention provides a preparation method of a collagen-chitosan composite material capable of slowly releasing icariin, which comprises the following steps: s1.0.336% collagen solution and 2% chitosan solution in a volume ratio of 5: 5-6: 4, mixing, pouring into a bracket mould, freeze-drying, and sequentially soaking in alcohol with different concentrations for gradient deacidification; s2, adding 0.5% genipin for crosslinking for 24 hours, and cleaning to obtain a collagen-chitosan scaffold material; s3, uniformly dripping 0.005-0.5 mg/mL icariin solution into the collagen-chitosan scaffold material, and freeze-drying to obtain the collagen-chitosan composite material capable of slowly releasing icariin; the preparation method of the scaffold material is simple and convenient, the cost of the used material is low, the collagen-chitosan composite material capable of slowly releasing icariin obtained by the method can effectively release icariin for a long time, and the scaffold material has good drug slow release performance and better biological safety.
Description
Technical Field
The invention relates to the technical field of biomedical material application, and in particular relates to a collagen-chitosan composite material capable of slowly releasing icariin, and a preparation method and application thereof.
Background
Periodontitis is a chronic inflammation of periodontal tissues, often causing damage to the periodontal tissues, causing alveolar bone resorption, and finally leading to a common cause of tooth loss. The materials applied to alveolar bone defect repair have some defects and shortcomings, for example, the collagen-chitosan scaffold material has good biocompatibility, is beneficial to cell adhesion and proliferation, but has poor osteogenesis effect. For example, hydroxyapatite or tricalcium phosphate may be combined with collagen-chitosan scaffold to form composite material, with the hydroxyapatite or tricalcium phosphate particles being combined with immune cells such as mononuclearAfter cell contact or phagocytosis, there is a risk of causing aseptic inflammation of local tissues. Limiting the clinical use of these materials. Icariin (C)33H40O15Molecular weight of 676.67) is a very effective Chinese medicinal ingredient for treating osteoporosis. Many studies have proved that it can promote the proliferation of osteoblasts and the expression of genes related to osteogenesis, and at the same time, can inhibit the activity of osteoclasts, thus being beneficial to the healing of bone tissues of patients with osteoporosis. In addition, icariin also has the advantages of simple extraction process, stable chemical property, easy storage and the like. The icariin and the calcium phosphate are mixed to prepare the biological scaffold material, so that the activity of the biological material is improved.
The addition of the traditional Chinese medicine preparation with the effect of promoting osteogenesis into the collagen-chitosan is a relatively safe method, and overcomes potential adverse reactions of the scaffold material. So far, no collagen-chitosan stent slowly releases icariin to form a relatively safer alveolar bone defect repair stent material.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a collagen-chitosan composite material capable of slowly releasing icariin.
The second purpose of the invention is to provide a preparation method of the collagen-chitosan composite material for slowly releasing icariin.
The third purpose of the invention is to provide the application of the collagen-chitosan composite material for slowly releasing the icariin.
The purpose of the invention is realized by the following technical scheme:
a preparation method of a collagen-chitosan composite material capable of slowly releasing icariin comprises the following steps:
s1.0.336% collagen solution and 2% chitosan solution in a volume ratio of 5: 5-6: 4, mixing, pouring into a bracket mould, freeze-drying, and sequentially soaking in alcohol with different concentrations for gradient deacidification;
s2, adding 0.5% genipin for crosslinking for 24h, and cleaning to obtain a collagen-chitosan scaffold material;
s3, uniformly dripping 0.005-0.5 mg/mL icariin solution into the collagen-chitosan scaffold material, and freeze-drying to obtain the collagen-chitosan composite material capable of slowly releasing icariin.
Preferably, the gradient deacidification in S1 is performed by sequentially soaking in 100%, 75%, 50%, 25%, 0% ethanol.
Specifically, the soaking time in 100% alcohol is 2h, the soaking time in 75% alcohol is 1.5h, the soaking time in 50% alcohol is 1h, and the soaking time in 25% alcohol is 30 min.
Preferably, the ratio of the 0.336% collagen solution to the 2% chitosan solution is 5:5 the size distribution of the gaps of the scaffold material obtained by mixing is uniform.
Preferably, the freeze-drying of S1 is carried out overnight at-80 deg.C, the freeze-drying temperature is-30 deg.C, and the freezing time is 48 h.
Preferably, step S3 is repeated before obtaining the collagen-chitosan composite material slowly releasing icariin; the stent material can be made to carry more drug.
The invention also provides a collagen-chitosan composite material capable of slowly releasing icariin, which is obtained by the method.
The collagen-chitosan composite material capable of slowly releasing icariin can be used as a material for repairing alveolar bone defects, so that the collagen-chitosan composite material capable of slowly releasing icariin is protected to be applied as a material for repairing alveolar bone defects.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a preparation method of a collagen-chitosan composite material capable of slowly releasing icariin, which comprises the following steps: s1.0.336% collagen solution and 2% chitosan solution in a volume ratio of 5: 5-6: 4, mixing, pouring into a bracket mould, freeze-drying, and sequentially soaking in alcohol with different concentrations for gradient deacidification; s2, adding 0.5% genipin for crosslinking for 24h, and cleaning to obtain a collagen-chitosan scaffold material; s3, uniformly dripping 0.005-0.5 mg/mL icariin solution into the collagen-chitosan scaffold material, and freeze-drying to obtain the collagen-chitosan composite material capable of slowly releasing icariin; the preparation method of the scaffold material is simple and convenient, the cost of the used material is low, the collagen-chitosan composite material capable of slowly releasing icariin obtained by the method can effectively release icariin for a long time, and the scaffold material has good drug slow release performance and better biological safety.
Drawings
Fig. 1 is a flow chart of the preparation of the sustained-release icariin-collagen-chitosan composite material.
FIG. 2 is a scanning electron microscope image of a collagen-chitosan scaffold material obtained by cross-linking 1% glutaraldehyde; wherein, fig. 2A, 2B, 2C are collagen: the chitosan is 7: the stent material at 3 was individually 100/200/500 times larger; fig. 2D, 2E, 2F are collagen: the chitosan is 6: the stent material at 4 was individually 100/200/500 times larger; FIGS. 2G, 2H, 2I are collagen: the chitosan is 5: the stent material at 5 was individually 100/200/500 times larger.
FIG. 3 is a scanning electron micrograph of different collagen-chitosan scaffold materials cross-linked with 0.5% genipin; wherein, fig. 3A, 3B, 3C are collagen: the chitosan is 7: the stent material at 3 was individually 100/200/500 times larger; fig. 3D, 3E, 3F are collagen: the chitosan is 6: the stent material at 4 was individually 100/200/500 times larger; FIGS. 3G, 3H, and 3I are collagen: the chitosan is 5: the stent material at 5 was individually 100/200/500 times larger.
FIG. 4 is a scanning electron micrograph of the obtained collagen-chitosan scaffold material crosslinked with 0.5% genipin; wherein FIGS. 4A, 4B, 4C are collagen: the chitosan is 8: 2 was 100/200/500 times magnified; fig. 4D, 4E, 4F are collagen: the chitosan is 4: the scaffold materials of 6 were individually amplified 100/200/500 times.
Fig. 5 shows the cell adhesion results of the sustained-release icariin-collagen-chitosan composite scaffold material, wherein fig. 5A shows the collagen-chitosan scaffold without drug loading, and no cell adhesion is observed; FIG. 5B is a collagen-chitosan scaffold loaded with 0.005mg/mL icariin, with no apparent cell adhesion seen; FIG. 5C is a collagen-chitosan scaffold loaded with 0.05mg/mL icariin, showing cell adhesion; FIG. 5D is a collagen-chitosan scaffold loaded with 0.5mg/mL icariin, showing multiple cell adhesion.
Detailed Description
The invention will be further described with reference to the drawings and specific examples, which are not to be construed as limiting the invention. Modifications and substitutions of the methods, procedures, and conditions of the present invention can be made without departing from the spirit and substance of the invention. Unless otherwise indicated, the experimental procedures used in the examples are all conventional procedures and techniques well known to those skilled in the art, and reagents or materials are all commercially available.
EXAMPLE 1 preparation of collagen-chitosan scaffold Material
The method for preparing the collagen-chitosan scaffold material comprises the following steps:
chitosan was purchased from Guangzhou Qiyun biotechnology, and a certain amount of chitosan powder was dissolved in 1% acetic acid solution prepared with ultrapure water, stirred uniformly with a stirrer for 1h at room temperature (20 ℃) to prepare a 2% chitosan solution. Collagen was purchased from sigma and the concentration of collagen swelling solution was 0.336%.
The volume ratio of the 0.336% collagen solution to the 2% chitosan solution is 7: 3, uniformly mixing, pouring into a bracket mould, standing overnight at-80 ℃, freezing for 48 hours at-30 ℃ in a freeze dryer, and soaking the bracket in gradient alcohol of 100%, 75%, 50%, 25% and 0% respectively after freezing for gradient deacidification. The deacidification time is 2h in 100% alcohol, 1.5h in 80% alcohol, 1h in 50% alcohol, 30min in 30% alcohol, and 30min in 0% alcohol. After the deacidification step, the collagen-chitosan scaffold is soaked in a cell culture medium for 24 hours, and the pH value of the cell culture medium is detected after 24 hours. If no effective deacidification occurs, the pH of the cell culture medium is affected, the survival state of the cells is affected, and even cytotoxicity is caused.
Selecting two cross-linking agents, namely adding 1% of glutaraldehyde into the deacidified collagen-chitosan scaffold for cross-linking for 24 hours, and performing ddH2O washing for 3 times, 15min each time. And observing the surface appearance of the support material after the crosslinking of different crosslinking agents by a scanning electron microscope.
EXAMPLE 2 preparation of collagen-Chitosan scaffold Material
The experimental procedure is the same as in example 1, with the only difference that: the volume ratio of the 0.336% collagen solution to the 2% chitosan solution is 6:4, uniformly mixing.
EXAMPLE 3 preparation of collagen-Chitosan scaffold Material
The experimental procedure is the same as in example 1, with the only difference that: the volume ratio of the 0.336% collagen solution to the 2% chitosan solution is respectively 5:5, uniformly mixing.
EXAMPLE 4 preparation of collagen-Chitosan scaffold Material
The experimental procedure is the same as in example 1, with the only difference that: 0.5% genipin was used as the crosslinking agent.
EXAMPLE 5 preparation of collagen-Chitosan scaffold Material
The experimental procedure is the same as in example 1, with the only difference that: the volume ratio of the 0.336% collagen solution to the 2% chitosan solution is 6:4, and 0.5% genipin is used as the cross-linking agent.
EXAMPLE 6 preparation of collagen-chitosan scaffold Material
The experimental procedure is the same as in example 1, with the only difference that: the volume ratio of the 0.336% collagen solution to the 2% chitosan solution is respectively 5:5, and 0.5% genipin is used as the cross-linking agent.
First, the surface morphology of the collagen-chitosan materials obtained in examples 1 to 6 was observed, and the results showed that: the porous structure of the collagen-chitosan scaffold after cross-linking by 1% of glutaraldehyde is not obvious, the scaffold material after cross-linking by 0.5% of genipin is in a porous structure, and in addition, the earlier stage research result shows that: when the proportion of collagen is larger (the proportion of collagen to chitosan is 8: 2), the pores of the stent material are larger, and the stent material has low strength and large brittleness. When the proportion of chitosan is too large (the proportion of collagen to chitosan is 4: 6), the pores of the stent are too small, the strength of the stent material is too large, the stent is not easy to decompose (figure 4), and finally, the proportion of collagen to chitosan is 5: and 5, the pore size distribution of the stent is most uniform, and the shape is good (figure 2 and figure 3).
Example 7 preparation of sustained-release icariin-collagen-chitosan composite scaffold Material
Icariin (ICA) standards (purity > 99.9%) were purchased from drug laboratory of guangzhou city. An amount of ICA was weighed and dissolved in absolute ethanol/DMSO (DMSO <1% vol%) to prepare concentrations of 0.005mg/mL, 0.05mg/mL and 0.5mg/mL, respectively. And carrying out ultrasonic oscillation for 30min after preparation, wherein the ultrasonic oscillation frequency is 20KHz, so that the icariin is fully and uniformly mixed in the solution.
0.005mg/mL icariin solution was added to the collagen-chitosan scaffold material prepared in example 4, and freeze-dried twice. The method comprises the following specific steps: preparing icariin solutions with different concentrations, ultrasonically shaking for 30min to fully mix icariin, sucking the solution with a syringe, and uniformly dripping the solution in the collagen-chitosan scaffold. And then, naturally drying the scaffold material immersed in the icariin in a ventilated place, repeatedly dripping the medicine once after drying, obtaining the icariin-collagen-chitosan three-dimensional scaffold material with different proportions after drying, and then carrying out secondary freeze drying.
Placing the prepared icariin-collagen-chitosan material in a 24-pore plate, adding 1mL of PBS buffer solution, tightly sealing the 24-pore plate by a sealing film, covering the 24-pore plate with tinfoil paper in a dark place, placing in a shaking table at 37 ℃ and shaking at 100rpm, taking out PBS solution for detecting the content of icariin at 12h, 1d, 3d and 6d respectively, and simultaneously adding fresh 1mL of PBS. The collected PBS solution was stored in a refrigerator at 4 ℃ in the dark. And detecting the sustained-release concentration of the icariin by adopting a high performance liquid chromatograph. The method comprises the following specific steps: preparing icariin solutions with concentration gradients of 0.5, 1, 2.5, 5 and 10 μ g/mL by using chromatographic grade methanol, and measuring peak areas of the icariin solutions to prepare an icariin standard curve. And filtering the collected sample, and taking the supernatant for detection. The detection conditions are that the temperature is 25 ℃, the flow rate is 1mL/min, and the water: the acetonitrile is 65: 35, the sample adding amount is 20 μ L, the wavelength is 270nm, and the time is 8 min. And calculating the icariin content according to the standard curve.
Example 8 preparation of sustained-Release icariin-collagen-Chitosan composite scaffold Material
The experimental procedure was the same as in example 7, except that: 0.05mg/mL of icariin solution was added to the collagen-chitosan scaffold material prepared in example 4.
Example 9 preparation of sustained-Release icariin-collagen-Chitosan composite scaffold Material
The experimental procedure was the same as in example 7, except that: 0.5mg/mL icariin solution was added to the collagen-chitosan scaffold material prepared in example 4.
Example 10 preparation of sustained-Release icariin-collagen-Chitosan composite scaffold Material
The experimental procedure was the same as in example 7, except that: 0.005mg/mL of icariin solution was added to the collagen-chitosan scaffold material prepared in example 5.
Example 11 preparation of sustained-Release icariin-collagen-Chitosan composite scaffold Material
The experimental procedure was the same as in example 7, except that: 0.05mg/mL of icariin solution was added to the collagen-chitosan scaffold material prepared in example 5.
Example 12 preparation of sustained-Release icariin-collagen-Chitosan composite scaffold Material
The experimental procedure was the same as in example 7, except that: 0.5mg/mL icariin solution was added to the collagen-chitosan scaffold material prepared in example 5.
Example 13 preparation of sustained-Release icariin-collagen-Chitosan composite scaffold Material
The experimental procedure was the same as in example 7, except that: 0.005mg/mL of icariin solution was added to the collagen-chitosan scaffold material prepared in example 6.
Example 14 preparation of sustained-Release icariin-collagen-Chitosan composite scaffold Material
The experimental procedure was the same as in example 7, except that: 0.05mg/mL of icariin solution was added to the collagen-chitosan scaffold material prepared in example 6.
Example 15 preparation of sustained-Release icariin-collagen-Chitosan composite scaffold Material
The experimental procedure was the same as in example 7, except that: 0.5mg/mL icariin solution was added to the collagen-chitosan scaffold material prepared in example 6.
The sustained-release icariin-collagen-chitosan composite scaffold materials prepared in examples 7 to 15 were examined for their properties, and the results are shown in table 1.
The results show that the collagen-chitosan ratio is 7: 3, 0.005mg/mL, 0.05mg/mL, 0.5mg/mL icariin concentrations, the drug release at 6d was nearly 0. mu.g/mL. The collagen-chitosan ratio was 5:5, the medicine can be continuously released for more than 6 days when the concentration of the icariin is 0.5mg/mL, and the release concentration of the medicine is higher than that when the ratio of the collagen to the chitosan is 6: 4. Therefore, the optimal release formulation is a collagen-chitosan ratio of 5:5, the icariin concentration is 0.5 mg/mL.
Example 16 adhesion of the surface of the Slow-Release icariin-collagen-Chitosan composite scaffold
Primarily culturing mouse bone marrow mesenchymal stem cells, irradiating the slow-release icariin-collagen-chitosan scaffold material prepared in the example 15 under ultraviolet rays for 24 hours for disinfection, and drying. The material was placed face up in a 6-well plate. The cells were digested with 0.25% pancreatin containing 0.1% EDTA, collected, centrifuged at 2X 104Density of/well seeded on scaffold material. After 7 days of cell culture, the composite material was removed, the cells that did not adhere were removed by washing with PBS, and the composite material was fixed with 3% glutaraldehyde at room temperature for 3h, ddH2Cleaning, dehydrating with 50%, 70%, 90%, 100% alcohol for 15min, air drying overnight, observing cell adhesion by scanning electron microscope, and showing that no drug-loaded collagen-chitosan scaffold is adhered to the cells in FIG. 5, FIG. 5A; FIG. 5B is a collagen-chitosan scaffold loaded with 0.005mg/mL icariin, with no apparent cell adhesion seen; FIG. 5C is a collagen-chitosan scaffold loaded with 0.05mg/mL icariin, showing cell adhesion; FIG. 5D is a collagen-chitosan scaffold loaded with 0.5mg/mL icariin, showing multiple cell adhesion.
Claims (7)
1. A preparation method of a collagen-chitosan composite material capable of slowly releasing icariin is characterized by comprising the following steps:
mixing S1.0.336% collagen solution and 2% chitosan solution at a volume ratio of 5:5, pouring into a bracket mould, freeze-drying, and sequentially soaking in alcohol with different concentrations for gradient deacidification;
s2, adding 0.5% genipin for crosslinking for 24 hours, and cleaning to obtain a collagen-chitosan scaffold material;
s3, uniformly dripping 0.5mg/mL icariin solution into the collagen-chitosan scaffold material, and freeze-drying to obtain the collagen-chitosan composite material capable of slowly releasing icariin.
2. The method for preparing a collagen-chitosan composite material capable of slowly releasing icariin according to claim 1, wherein the gradient deacidification in S1 is performed by sequentially soaking in 100% alcohol, 75% alcohol, 50% alcohol, 25% alcohol and water.
3. The method for preparing a collagen-chitosan composite material capable of slowly releasing icariin according to claim 1, wherein the soaking time in 100% alcohol is 2 hours, the soaking time in 75% alcohol is 1.5 hours, the soaking time in 50% alcohol is 1 hour, and the soaking time in 25% alcohol is 30 min.
4. The method for preparing a collagen-chitosan composite material capable of slowly releasing icariin according to claim 1, wherein the collagen-chitosan composite material is placed at-80 ℃ overnight before the freeze-drying in S1, the freeze-drying temperature is-30 ℃, and the freezing time is 48 hours.
5. The method for preparing an icariin-releasing collagen-chitosan composite material according to claim 1, wherein step S3 is repeated before obtaining the icariin-releasing collagen-chitosan composite material.
6. A collagen-chitosan composite material slowly releasing icariin, obtained by the production method of any one of claims 1 to 5.
7. The use of the collagen-chitosan composite material slowly releasing icariin according to claim 6 as a material for alveolar bone defect repair.
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| 骨组织工程诱导性支架材料修复骨缺损;唐俊杰等;《中国组织工程研究》;20150115;第19卷(第3期);第340-346页 * |
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