CN114404663A - Bone repair material with inflammation relieving characteristic and preparation method thereof - Google Patents
Bone repair material with inflammation relieving characteristic and preparation method thereof Download PDFInfo
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- CN114404663A CN114404663A CN202210161153.9A CN202210161153A CN114404663A CN 114404663 A CN114404663 A CN 114404663A CN 202210161153 A CN202210161153 A CN 202210161153A CN 114404663 A CN114404663 A CN 114404663A
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- repair material
- bone repair
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- sodium alginate
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Abstract
The invention belongs to the field of bone regeneration materials, and particularly relates to a bone repair material with inflammation relieving characteristics, and a preparation method and application thereof. The bone repair material with inflammation relieving characteristic comprises the components of an osteoinductive growth factor rhBMP-2, PLGA microspheres for wrapping the rhBMP-2 and a basic additive for neutralizing the acidity of PLGA acidic degradation products in situ, and is characterized by also comprising an activity protective agent for maintaining the activity of the rhBMP-2. The material not only has a good BMP activity protection effect, but also has good acid-base regulation capacity, and the inflammatory reaction is greatly slowed down.
Description
Technical Field
The invention belongs to the field of bone regeneration materials, and particularly relates to a bone repair material with inflammation relieving characteristics, and a preparation method and application thereof.
Background
According to statistics, the number of bone defect patients in 300-700 thousands per year in China is increased year by year along with the development of traffic facilities and the acceleration of population aging process. The larger bone defect is difficult to repair by self, and the bone graft is needed to reconstruct the shape and the function of the bone defect part in clinic so as to shorten the treatment period and reduce the disability rate, therefore, the demand of bone repair materials is huge. At present, the bone implant material is endowed with osteoinductive activity mainly by introducing osteoinductive signal molecules into the scaffold material. The recombinant bone morphogenetic protein-2 (rhBMP-2) is a key factor for promoting osteogenesis, belongs to a TGF-alpha family member, and the rhBMP-2 is a main signal molecule for differentiating cells into mineral deposit osteoblasts and plays a role in inducing osteoblast differentiation. The expression is performed during limb growth, endochondral ossification and fracture, plays an important role in bone growth and development and regeneration repair, and has good application prospect in clinical treatment of bone injury. However, rhBMP-2 has a short half-life in vivo and is easily hydrolyzed by enzymes, which does not provide a good therapeutic effect for bones that require a long period of growth and restoration.
The poly (lactic-co-glycolic acid) (PLGA) microspheres are one of the most widely used and studied drug carrier materials due to their good biocompatibility and biodegradability, and have been approved by the US FDA for clinical application. In recent years, in the microspheres studied at home and abroad, polylactic-co-glycolic acid (PLGA) is often used as a framework material, polypeptide and protein drugs are wrapped or dispersed in the PLGA to prepare a microsphere preparation, and after administration, the microsphere preparation can achieve the purpose of protecting the drugs and release the drugs in the modes of diffusion, erosion and the like along with the degradation of the polymer, thereby achieving the purpose of slow release and long acting. For protein and polypeptide drugs which need to be continuously administered for a long time, the PLGA microsphere controlled release system becomes an ideal drug delivery system at present because the drugs are easy to be hydrolyzed by enzyme and have short biological half-life in vivo. Many studies have shown that the inclusion of bioactive cytokines or drugs into PLGA microspheres not only allows for slow release but also better protection against growth factor inactivation. However, the PLGA microspheres can generate acidic monomers lactic acid and glycolic acid during in vivo degradation, and if these acidic products cannot be timely discharged out of the body through normal metabolism, the pH of the tissue microenvironment around the implant can be reduced, and the aggregation of the acidic products can not only affect the growth, differentiation and extracellular matrix synthesis of cells adhered thereon, but also cause local aseptic reaction of tissues around the implant, and seriously affect the tissue repair effect.
In order to improve the problem of acidic degradation products, a simple and feasible method is provided by adopting additives to blend into the existing biodegradable polyester material which is actually used clinically. For example, by introducing certain specific additives, the degradation rate of the polyester material can be regulated, and the acidic degradation product can be neutralized after the additive is released, so that the aim of reducing the influence of the acidic degradation product on the cell function is fulfilled. At present, substances such as lysine, sodium bicarbonate, chitosan, sodium alginate and the like are reported to relieve or eliminate the degree of local acid collection, and different alkaline additives have different regulating effects.
The comb-shaped amphiphilic polysaccharide derivative can form a thermodynamically stable micelle through weak interaction in molecules and among molecules, and is a biopolymer surfactant when the concentration in a solution is lower than the Critical Aggregation Concentration (CAC); when the concentration is higher than CAC, the nano micelle is formed by self-assembly, and hydrophobic drug small molecules are embedded for slow release and targeted positioning of the drug in vivo; or form stable complex nano-micelle with hydrophilic polypeptide and protein macromolecule, and prevent protein molecule from precipitating in water.
Object of the Invention
In order to solve the problem of activity reduction of the rhBMP-2-loaded microspheres in the preparation and in-vivo release processes, a Tween-20 (Tween-20) and beta-cyclodextrin mixture is added into the water phase of the microspheres as an activity protective agent, and then researches show that after an alkaline additive is further added, the acidity of an acidic environment caused by degradation of the PLGA microspheres can be adjusted, the problem of low protective effect of combination of the Tween-20 and the beta-cyclodextrin on the rhBMP-2 is solved, the activity of the rhBMP-2 is further improved, and the long-acting use of the rhBMP-2 in the drug release process is ensured.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the bone repairing material with inflammation relieving characteristic includes bone inducing growth factor rhBMP-2, PLGA microballoon for coating rhBMP-2, alkaline additive for in-situ neutralizing the acidity of PLGA acid degradation product and activity protecting agent for maintaining the activity of rhBMP-2.
Preferably, the alkaline additive is lysine or sodium alginate. The literature reports that lysine in basic amino acid has a good comprehensive adjusting effect on acidity in the PLGA degradation process, and sodium alginate also has a certain adjusting effect but is not obvious.
Preferably, the activity protective agent is Tween-20 or beta-cyclodextrin. Because the Tween-20 can inhibit the aggregation and precipitation of the protein on an oil-water interface, the beta-cyclodextrin can improve the hydrophilicity of the protein and make the protein difficult to enter the oil-water interface, thereby avoiding the aggregation and denaturation of the protein, and the Tween-20 and the protein have different protein protection mechanisms.
Preferably, the alkaline additive is modified sodium alginate, and the activity protective agent is Tween-20 and beta-cyclodextrin.
Preferably, the modified sodium alginate is obtained by adding sodium alginate into a DMSO solution, stirring at 50-60 ℃ until the sodium alginate is completely dissolved, then cooling to room temperature, dissolving cholesterol in chloroform, slowly dropwise adding the dissolved solution into a sodium alginate solution, uniformly stirring, then adding a mixed solution of a coupling agent DCC and a catalyst DMAP, stirring at room temperature for reaction for 20-30 h, precipitating with 4-6 times of volume of absolute ethyl alcohol, centrifuging to remove supernatant, and then drying in vacuum. The cholesterol has good biocompatibility, and the sodium alginate after being grafted and modified by the cholesterol is easier to form stable micelles in an aqueous solution, so that protein precipitation can be prevented.
Preferably, the mass ratio of the modified sodium alginate to the Tween-20 to the beta-cyclodextrin is 2: 1: 1.
further, the present application provides a method for preparing the bone repair material, the method comprising the steps of: (1) preparing a PLGA microsphere which is loaded with rhBMP-2 and contains an active protective agent; (2) preparing the PLGA microspheres and alkaline additive composite bone repair material.
Preferably, the operation process of the step (1) is as follows: dissolving rhBMP-2 freeze-dried powder in an acetic acid buffer solution, adding an active protective agent, fully stirring, adding acetonitrile containing PLGA, stirring and emulsifying, adding a mixed emulsion of liquid paraffin and Tween-80, stirring, standing, alternately washing with petroleum ether and isopropanol, and freeze-drying to obtain the dry microspheres.
Preferably, the operation process of the step (2) is as follows: suspending PLGA microspheres in acetic acid aqueous solution, fully grinding an alkaline additive, adding the ground PLGA microspheres into the PLGA microsphere suspension, uniformly stirring, standing, washing to remove the solvent, and drying to obtain the bone repair material.
Further, the application provides the application of the bone repair material and the preparation method thereof in bone defect regeneration repair.
Because PLGA is insoluble in water, severe chemical and mechanical factors such as organic solvents, ultrasonic emulsification and the like are involved in the process of preparing microspheres by using PLGA, and the factors can directly influence the super structure and the biological activity of protein, so that the protein is denatured, aggregated and degraded, and therefore, the activity loss of the rhBMP-2 in the preparation process is reduced by selecting the activity protective agent. According to the fact that the Tween-20 can inhibit protein from aggregating and precipitating at an oil-water interface, the beta-cyclodextrin can improve the hydrophilicity of the protein so that the protein is difficult to enter the oil-water interface, and therefore the protein aggregation and denaturation are avoided, the applicant of the invention speculates that the two are combined to play a synergistic effect, and the activity of the rhBMP-2 protein can be further protected and improved.
However, in the test process, the applicant finds that the combination of tween-20 and beta-cyclodextrin rather reduces the activity of the protein, wherein when sodium alginate is used as an alkaline additive, the acidity regulation effect is poor, but the activity of the protein is slightly improved; when lysine is used as the alkaline additive, the acidity regulating effect is better, but the activity of the protein is basically unchanged. Therefore, the applicant selects to carry out hydrophobic modification on sodium alginate, and obtains the modified sodium alginate by carrying out esterification reaction with cholesterol, and the result shows that in the material added by the tween-20 and the beta-cyclodextrin composition together with the modified sodium alginate, the activity of rhBMP-2 protein is better than that when tween-20 is used as a protective agent and lysine is used as an alkaline additive, and meanwhile, the acidity regulation effect is close to that of the lysine. Therefore, the activity of the rhBMP-2 protein is greatly improved by adding the Tween-20, the beta-cyclodextrin and the modified sodium alginate, and the prepared bone repair material has good acidity regulation capability and inflammation relieving property.
Drawings
FIG. 1: the pH value changes during the degradation of the bone repair material added with different alkaline additives.
Detailed Description
The technical solution of the present invention is further described below by means of specific examples.
All samples in this application are exemplified by the preparation of the finished product of example 1, differing only in composition.
Example 1 bone repair Material
The main experimental materials: the rhBMP-2 is produced by enterprises by self, the specific production process refers to the production method (expression in escherichia coli, purification by ion exchange chromatography and detection of escherichia coli host residual protein < 0.005%) in the Chinese patent application No. CN201910030649.0 of the applicant, and the antibiotic residue <0.1ppm is detected by a bacteriostatic circle method. Poly (lactic-co-glycolic acid) (PLGA) is manufactured by DURECT, Inc. of USA. Dimethyl sulfoxide (DMSO), N '-Dicyclohexylcarbodiimide (DCC), 4- (N, N' -dimethylamino) pyridine (DMAP), Tween-20, beta-cyclodextrin, sodium alginate and cholesterol were all purchased from commercial products.
The preparation method comprises the following steps:
modified sodium alginate: adding sodium alginate into a DMSO solution, stirring at 50 ℃ until the sodium alginate is completely dissolved, then cooling to room temperature, dissolving cholesterol into chloroform, slowly dropwise adding the solution into a sodium alginate solution, stirring uniformly, then adding a mixed solution of a coupling agent DCC and a catalyst DMAP, stirring at room temperature for reaction for 24 hours, precipitating with 4 times of volume of absolute ethyl alcohol, centrifuging to remove supernatant, and drying in vacuum to obtain the sodium alginate-based composite material.
Bone repair materials: (1) preparing a PLGA microsphere which is loaded with rhBMP-2 and contains an active protective agent: dissolving rhBMP-2 freeze-dried powder in an acetic acid buffer solution, adding an active protective agent, fully stirring, adding acetonitrile containing PLGA, stirring and emulsifying, adding a mixed emulsion of liquid paraffin and Tween-80, stirring, standing, alternately washing with petroleum ether and isopropanol, and freeze-drying to obtain dried microspheres; (2) preparing a PLGA microsphere and alkaline additive composite bone repair material: suspending PLGA microspheres in acetic acid aqueous solution, fully grinding an alkaline additive, adding the ground PLGA microspheres into the PLGA microsphere suspension, uniformly stirring, standing, washing to remove the solvent, and drying to obtain the bone repair material.
EXAMPLE 2 determination of rhBMP-2 protein Activity
The determination method comprises the following steps: in vitro alkaline phosphatase method: according to the fact that rhBMP-2 can induce C2C12 cells to differentiate into osteoblasts, ALP (alkaline phosphatase) in the osteoblasts can catalyze p-nitrophenol disodium phosphate to be converted into p-nitrophenol, and the p-nitrophenol can be used as a color indicator, and the color intensity of the p-nitrophenol is in direct proportion to the concentration. The method adopts the amount of the p-nitrophenol catalytically generated by the rhBMP-2 with unit mass per minute at 37 ℃ to represent the activity of the rhBMP-2, and the unit is U/mg rhBMP-2.
The samples of each group were repeatedly washed with 10% streptomycin-containing PBS buffer, and placed in 6-well plates, 1mL of sample solution was added to each well, 5mL of serum-free alpha-MEM medium was added to each well, and the plates were incubated at 37 ℃ and 5% in a CO2 incubator. Extracting the conditioned medium from the incubated 1d, 5d, 10d and 20d, respectively, centrifuging the extracted conditioned medium at 1000rpm for 10 minutes after extraction, collecting the supernatant, filtering the supernatant with a 0.22 μm filter, respectively labeling the supernatant as alpha-MEM + (1d, 5d, 10d and 20d), adding 10% fetal calf serum and 1% double antibody solution, respectively transferring the mixture into a 50mL sterile centrifuge tube, and storing the tube in a refrigerator at-80 ℃ for later use. Preparation of standard solution: WHO International Standard for biological Activity (NIBSC code: 93/574): 10 μ g/ampoule, 5000 units of biological activity per ampoule. Conversion of biological Activity 5X 105U/mg. Completely using rhBMP-2 international standardThe culture medium is dissolved, diluted to a proper concentration, and subpackaged and stored in a refrigerator at-20 ℃ (the shelf life is 2 weeks). When in use, one part is slowly dissolved in an environment of 4 ℃. In 96-well cell culture plates, 2-fold serial dilutions were made for 8 dilutions, each dilution making 2 wells, and the above procedure was performed under sterile conditions. Preparation of sample solution (test solution): (1) samples No. 1-5 were taken and dissolved completely in about 0.5mg/ml solution by adding 0.5% acetic acid solution, and each sample was divided into 2 parts. One sample solution was assayed for protein content by the Coomassie Brilliant blue method (Bradford method). (2) The other sample solution was stored in a 2-8 ℃ freezer for exchange. The concentration corresponding to the standard (about 1. mu.g/ml) was prepared as an estimate of the specific activity. In 96-well cell culture plates, complete medium was added for 2-fold serial dilutions for a total of 8 dilutions, each dilution making 2 wells, and the above procedure was performed under sterile conditions.
The method for measuring the biological activity of the rhBMP-2 standard comprises the following steps:
(1) cell culture: firstly, use complete culture medium, in the presence of 5% CO2C2C12 cells were cultured in a carbon dioxide incubator at 37 ℃. The cells were cultured at about 2X 104/cm2Was inoculated into a culture flask (3.5 ml of cell sap was added to 25 cm)2In a culture flask). ② when the confluence degree of the cells reaches 70-80%, adding pancreatin digestive juice to digest the cells. Cells were adjusted to 10X 10 with complete medium4The single cell suspension/ml was plated in 96-well plates at 50. mu.l/well and incubated in a carbon dioxide incubator.
(2) Liquid changing: after 24 hours of cell culture, the old medium was poured off, and 100. mu.l of fresh complete medium was added to each well. ② standard solution and sample solution with 8 dilutions are added into each well with 100 mul, each dilution is 2 wells. And a blank was made with fresh medium without rhBMP-2. Thirdly, placing the 96-well plate in a carbon dioxide incubator to continue culturing for 46-52 hours.
(3) Cell lysis treatment: after the end of the culture, the medium was discarded, the cells were washed once with 100. mu.l/well of cold physiological saline, the residual liquid was blotted, and the cells were cooled on ice. Add 25. mu.l of cell lysate to each well.
(4) Alkaline phosphatase determination: and adding 25 mul of detection buffer solution and 50 mul of chromogenic substrate into the detection hole, lightly blowing and uniformly mixing by using a gun head, sealing by using a sealing film, placing in a water bath kettle at 37 ℃ for reaction for 15min, taking out the hole plate, and adding 100 mul of reaction termination solution. The absorbance was measured at a wavelength of 405nm, and the measurement results were recorded. If the color is too dark, the measurement solution may be diluted by an appropriate factor.
And (4) calculating a result:
the test data is processed by a computer program (such as softMax software) or a four-parameter regression calculation method, and the result is calculated according to the following formula:
in the formula, Pr: biological activity of standard substance, U/ml; ds: pre-dilution times of samples; dr: pre-dilution times of standard products; es: the test sample is equivalent to the half-effective dilution multiple of the standard sample; er: half-effective dilution factor of standard.
EXAMPLE 3 detection of rhBMP-2 Activity in bone repair Material with different Activity protecting Agents
Sample group:
TABLE 1 drug-loaded microspheres of different activity protection additives
Note: "-" indicates that no component is present.
TABLE 2 bone repair materials with different active protectants and alkaline protectants
Note: "-" indicates that no component is present.
The following measurements were obtained according to the test method of example 2:
TABLE 3 results of the determination of the biological Activity of rhBMP-2
| Biological Activity (U/ml) | 1d | 5d | 10d | 20d |
| Sample No. 1 | 2.0×105 | 1.95×105 | 1.76×105 | 1.52×105 |
| Sample No. 2 | 1.99×105 | 1.84×105 | 1.67×105 | 1.43×105 |
| Sample No. 3 | 1.89×105 | 1.71×105 | 1.50×105 | 1.33×105 |
| Sample No. 4 | 1.95×105 | 1.78×105 | 1.60×105 | 1.39×105 |
| Sample No. 5 | 1.91×105 | 1.75×105 | 1.54×105 | 1.36×105 |
| Sample No. 6 | 2.01×105 | 1.96×105 | 1.75×105 | 1.53×105 |
| Sample No. 7 | 2.05×105 | 1.96×105 | 1.74×105 | 1.51×105 |
| Sample No. 8 | 2.09×105 | 1.98×105 | 1.77×105 | 1.55×105 |
| Sample No. 9 | 1.94×105 | 1.74×105 | 1.52×105 | 1.30×105 |
| Sample No. 10 | 1.93×105 | 1.76×105 | 1.61×105 | 1.40×105 |
| Sample No. 11 | 2.51×105 | 2.03×105 | 1.96×105 | 1.88×105 |
From the experimental data in table 3, it is found that the rhBMP-2 protein activities of the sample No. 1 and the sample No. 2, in which only Tween-20 or beta-cyclodextrin is added as an activity protective agent, are higher, and the activity protective effects are both greater than those of the sample No. 3-5 in which Tween-20 and beta-cyclodextrin are used together. It is demonstrated that in the case of adding only the activity protecting agent without the alkaline additive, wherein the activity of rhBMP-2 protein is rather decreased when Tween-20 and beta-cyclodextrin are used together as the activity protecting agent, the activity protecting effect is smaller than that when Tween-20 or beta-cyclodextrin is used alone, which is inconsistent with what the applicant speculates.
The applicant selects the Tween-20 with higher protein protection activity as an activity protective agent, and further adds three different alkaline additives, so that the activity of the protein is basically not influenced. Meanwhile, the experimental data of the sample No. 9 and the sample No. 10 show that when tween-20 and beta-cyclodextrin are added in the same proportion, the activity improvement effect on protein is not good when sodium alginate or lysine serving as an alkaline additive is further added. However, the protein activity of the obtained sample No. 11 is greatly improved and is higher than that of the sample No. 1 after the sodium alginate modified by cholesterol esterification is added. The combination of the modified sodium alginate, the Tween-20 and the beta-cyclodextrin is proved to have larger rhBMP-2 activity protection effect.
Example 4 evaluation of in vitro degradation behavior of bone repair materials with different basic additives
TABLE 4 bone repair materials with different basic additives
Note: "-" indicates that no component is present.
And (3) taking a sample 9-13 with the same mass, decontaminating, washing with distilled water, drying, adding 10mL of PBS (pH 7.4) buffer solution for complete infiltration, and putting the sample into a constant-temperature water bath kettle at 37 ℃ for degradation experiments, wherein no intervention measures are taken in the degradation process, so that the degradation system is kept in a normal state. The pH values of the soaking solutions of the 5 samples were measured with a pH meter every week for a fixed period of time, and a pH change curve was plotted.
As can be seen from fig. 1, the bone repair material to which the basic additive is added has an ability to alleviate the degree of acid accumulation during degradation. The pH value adjusting effect of the sample No. 9 on the degradation process is weaker, the adjusting effect of the sample No. 8 is stronger, which shows that the acidity relieving capability of lysine as an alkaline additive is better than that of sodium alginate, and the pH reduction amplitude of the sample No. 10 is close to that of the sample No. 9, which shows that the acidity relieving capability of the sodium alginate modified by esterification is close to that of lysine. In addition, the pH adjusting effect of the sample No. 11 is found to be stronger through the composition of the Tween-20 and the beta-cyclodextrin in different proportions, which shows that the acidity relieving capacity of the bone repairing material prepared by the active protective agent and the alkaline additive in the content ratio is better.
Claims (10)
1. The bone repair material with inflammation relieving characteristic comprises the components of an osteoinductive growth factor rhBMP-2, PLGA microspheres for wrapping the rhBMP-2 and a basic additive for neutralizing the acidity of PLGA acidic degradation products in situ, and is characterized by also comprising an activity protective agent for maintaining the activity of the rhBMP-2.
2. The bone repair material of claim 1, wherein the alkaline additive is lysine, sodium alginate or modified sodium alginate.
3. The bone repair material of claim 1, wherein the active protective agent is Tween-20 or β -cyclodextrin.
4. The bone repair material of claim 1, wherein the alkaline additive is modified sodium alginate and the activity protector is Tween-20 and β -cyclodextrin.
5. The bone repair material of claim 4, wherein the modified sodium alginate is prepared by adding sodium alginate into DMSO solution, stirring at 50-60 ℃ until completely dissolved, then cooling to room temperature, dissolving cholesterol in chloroform, slowly adding dropwise into sodium alginate solution, stirring uniformly, adding a mixed solution of coupling agent DCC and catalyst DMAP, stirring at room temperature for 20-30 h, precipitating with 4-6 times volume of anhydrous ethanol, centrifuging to remove supernatant, and vacuum drying.
6. The bone repair material of claim 4, wherein the mass ratio of the modified sodium alginate to the Tween-20 to the beta-cyclodextrin is 2: 1: 1.
7. a method of preparing the bone repair material of claim 1, comprising the steps of: (1) preparing a PLGA microsphere which is loaded with rhBMP-2 and contains an active protective agent; (2) preparing the PLGA microspheres and alkaline additive composite bone repair material.
8. The method for preparing a bone repair material according to claim 7, wherein the operation process of the step (1) is as follows: dissolving rhBMP-2 freeze-dried powder in an acetic acid buffer solution, adding an active protective agent, fully stirring, adding acetonitrile containing PLGA, stirring and emulsifying, adding a mixed emulsion of liquid paraffin and Tween-80, stirring, standing, alternately washing with petroleum ether and isopropanol, and freeze-drying to obtain the dry microspheres.
9. The method for preparing bone repair material according to claim 7, wherein the operation process of the step (2) is: suspending PLGA microspheres in acetic acid aqueous solution, fully grinding an alkaline additive, adding the ground PLGA microspheres into the PLGA microsphere suspension, uniformly stirring, standing, washing to remove the solvent, and drying to obtain the bone repair material.
10. The bone repair material according to any one of claims 1 to 9 and the use of the method for the preparation thereof for the regenerative repair of bone defects.
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