Composition for cartilage repair and preparation method thereof

Abstract

The invention relates to the technical field of biology and medicine, in particular to a composition for cartilage repair and a preparation method thereof. The concentration of each component of the composition in a phosphate buffer solution is as follows: 5-10 mg / mL of carboxymethyl chitosan, 1-3 mg / mL of modified beta-cyclodextrin, 1-5 mg / mL of soybean protein and 20-30 [mu] g / mL of carperitide. A preparation process of the modified beta-cyclodextrin comprises the following steps: adding beta-cyclodextrin into water, then adding sodium periodate, and reacting to obtain aldehyde beta-cyclodextrin; adding aldehyde group beta-cyclodextrin into water, adding 3-aminophenylboronic acid and the stem cell exosome, and stirring for reaction to obtain a mixed solution; and adding the mixed solution into the solidification liquid, and carrying out post-treatment to obtain the modified beta-cyclodextrin. According to the invention, the modified beta-cyclodextrin and the carperitide are matched for use, so that the cartilage repair effect can be improved, and the modified beta-cyclodextrin interacts with carboxymethyl chitosan, so that the biological stability of the material can be improved.

Description

Technical Field

[0001] The present invention relates to the field of biology and medicine technology, and in particular to a composition for cartilage repair and a preparation method thereof. Background Art

[0002] Cartilage tissue is an important component of human joints. It not only provides a smooth contact surface between joints, but also plays the role of buffering and dispersing pressure. However, due to various reasons such as trauma, disease or aging, cartilage tissue often suffers damage or degeneration, leading to joint pain, stiffness and loss of function. The treatment of cartilage damage has always been a clinical problem because cartilage tissue itself has a low regeneration capacity. Although traditional treatments such as arthroscopic cleaning and drilling microfracture can temporarily relieve symptoms, it is often difficult to achieve true cartilage regeneration.

[0003] In recent years, with the rapid development of tissue engineering and regenerative medicine, more and more researchers have begun to explore the use of bioactive materials, cell therapy and growth factors to promote cartilage repair. Among them, stem cell exosomes, as an important medium for intercellular communication, show great potential in cartilage repair because they carry the genetic information and bioactive molecules of the mother cells. However, exosomes are easily inactivated in vitro and difficult to be effectively delivered to the target tissue, which limits their application in cartilage repair. In addition, the current cartilage repair effect is poor. Therefore, the development of a carrier material that can effectively encapsulate, stabilize exosomes and improve their activity is of great significance for improving the effect of cartilage repair.

[0004] Based on this, the present application aims to prepare a cartilage repair composition with good biocompatibility and bioactivity by optimizing the proportion of each component and the preparation process, thereby improving the effect of cartilage repair. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, the primary purpose of the present invention is to provide a composition for cartilage repair.

[0006] Another object of the present invention is to provide a method for preparing the composition for cartilage repair.

[0007] The purpose of the present invention is achieved through the following technical solutions: A composition for cartilage repair, wherein the concentrations of the components in phosphate buffer are as follows: 5-10 mg / mL of carboxymethyl chitosan, 1-3 mg / mL of modified β-cyclodextrin, 1-5 mg / mL of soy protein, and 20-30 μg / mL of caperitide; The preparation process of the modified β-cyclodextrin is as follows: (1) Add β-cyclodextrin to water, and then add sodium periodate to obtain aldehyde β-cyclodextrin after reaction; (2) adding the aldehyde β-cyclodextrin prepared in step (1) into water, adding 3-aminophenylboronic acid and stem cell exosomes, and stirring to react to obtain a mixed solution; (3) The mixed solution of step (2) is added to the coagulation liquid, and modified β-cyclodextrin is obtained through post-treatment.

[0008] Furthermore, the molar ratio of the β-cyclodextrin to sodium periodate is 1:(1-4).

[0009] Furthermore, in step (1), the reaction temperature is 35-40° C., and the reaction time is 4-6 h.

[0010] Furthermore, in step (2), the mass ratio of 3-aminophenylboronic acid, aldehyde β-cyclodextrin and stem cell exosomes is 1:5-7:0.01-0.05, and the concentration of the stem cell exosomes in the mixed solution is 2-8µg / mL.

[0011] Furthermore, the stirring reaction time in step (2) is 1-3 hours.

[0012] Furthermore, the coagulation liquid in step (3) is a calcium chloride solution or a potassium chloride solution.

[0013] Furthermore, the concentration of the calcium chloride solution or the potassium chloride solution is 3-8wt%.

[0014] Furthermore, the stem cell exosomes are bone marrow mesenchymal stem cell exosomes.

[0015] A method for preparing the above-mentioned composition for cartilage repair comprises the following steps: Add carboxymethyl chitosan, modified β-cyclodextrin, soybean protein and caperitide into phosphate buffer, and stir and mix evenly to obtain the product.

[0016] Furthermore, the concentration of the phosphate buffer is 0.01-0.02 mmol / L, and the pH is 6.5.

[0017] The present invention has the following effects compared with the prior art: 1. The aldehyde group in the aldehyde β-cyclodextrin of the present invention can react with the amino group in 3-aminophenylboronic acid to undergo a Schiff base reaction, thereby achieving inclusion of bone marrow mesenchymal stem cell exosomes and improving the stability and activity of the exosomes. In addition, the modified β-cyclodextrin and caperitide can be used together to improve the effect of cartilage repair. The aldehyde group in the modified β-cyclodextrin also interacts with the amino group in carboxymethyl chitosan, which can further improve the biological stability of the material and provide a new solution for cartilage repair.

[0018] 2. The present invention proposes a method for preparing a composition for cartilage repair, which has relatively simple preparation steps and is easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a morphological diagram of P3 bone marrow mesenchymal stem cells cultured in the present invention; Figure 2 The cytotoxicity test results of the cartilage repair compositions of Examples 1-3 and Comparative Examples 1-2 of the present invention are shown; Figure 3 The effect diagram of the cartilage repair composition of Examples 1-3 and Comparative Examples 1-2 on the cartilage tissue repair of rats; Figure 4 This is the scoring result of the cartilage repair composition of the present invention. DETAILED DESCRIPTION

[0020] The present invention will be described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0021] Preparation Example 1: The preparation method of bone marrow mesenchymal stem cell exosomes is as follows: (1) Isolation and primary culture of bone marrow mesenchymal stem cells: Two-week-old healthy mice were killed by cervical dislocation, fully disinfected, and their limbs were fixed on the operating table. In the clean bench, the femurs and tibias of the mice were removed, and the muscles and soft tissues were cleaned and peeled off; a small amount of PBS buffer was added to a sterile culture dish, and the femurs and tibias of the mice were immersed in it and rinsed with PBS three times; then, under sterile conditions, the femurs and tibias of the mice were cut off to expose the bone marrow cavity, and the bone marrow cavity was punctured with a 5mL syringe, and then the bone marrow cavity was rinsed with DMEM / F12 culture medium containing heparin to obtain a bone marrow cell suspension. The suspension was slowly added to an equal volume of Ficoll separation solution, placed in a centrifuge tube for centrifugation (3000rpm, 20min), and the bone marrow mononuclear cells on the interface of the white film layer were aspirated, suspended with DMEM / F12 culture medium, and centrifuged at 1500rpm for 10 minutes at room temperature, the supernatant was discarded, and the cells were repeatedly washed with PBS twice, and the PBS was discarded. The obtained bone marrow mesenchymal stem cells were resuspended in DMEM / F12 medium containing 50 ng / mL basic fibroblast growth factor and 20 ng / mL transferrin, and the culture medium was diluted to 1×10 6 / mL was inoculated into a T25 cell culture flask, and the culture medium was replaced after culturing for 24 hours in a 37°C, 5% CO2, and saturated humidity incubator. The medium was replaced every 2 days thereafter until the cell confluence reached 85%, the culture medium was removed, and the cells were washed twice with PBS. Digested with 0.25% trypsin at 37°C for 2 minutes, and the trypsin effect was terminated with an equal volume of DMEM / F12 culture medium. Then, the cells were centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, and the cells were rinsed twice with PBS. The bone marrow mesenchymal stem cells were then resuspended in DMEM / F12 culture medium containing 50ng / mL basic fibroblast growth factor and 20ng / mL transferrin, and gently blown into a single cell suspension. The cells were subcultured at a ratio of 1:3, and the P3 bone marrow mesenchymal stem cells obtained by subculture were collected. The morphology and growth of the cells were observed under an inverted microscope. The results are as follows Figure 1 shown.

[0022] (2) Take the P3 bone marrow mesenchymal stem cells obtained from the above culture and add 1×10 5 The cells were inoculated at a density of 100 / mL in α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, placed in an incubator at 37°C and 5% CO2, and cultured until the confluence reached 80%. The medium was then replaced with serum-free α-MEM medium and cultured for 48 hours. The cells were then centrifuged at 1000 g for 30 minutes at 4°C to remove cell debris. The supernatant was filtered with a 0.22 µm filter membrane, centrifuged at 100,000 g for 120 minutes, the supernatant was discarded, and the precipitate was retained. PBS was added to mix, and the cells were centrifuged again at 100,000 g for 60 minutes. The supernatant was discarded, and the precipitate was retained to obtain bone marrow mesenchymal stem cell exosomes.

[0023] Embodiment 1: A composition for cartilage repair, wherein the concentrations of the components in phosphate buffer are as follows: 8 mg / mL carboxymethyl chitosan, 2 mg / mL modified β-cyclodextrin, 3 mg / mL soy protein, and 25 μg / mL caperitide; The preparation process of the modified β-cyclodextrin is as follows: (1) Add β-cyclodextrin to water, and then add sodium periodate, wherein the molar ratio of β-cyclodextrin to sodium periodate is 1:3, and the amount of β-cyclodextrin to water is 15 g:100 mL. After reacting at 37°C in the dark for 5 hours, filter with a filter membrane (220 nm), and then precipitate the filtrate with 95% anhydrous ethanol to obtain aldehyde β-cyclodextrin; (2) Take the aldehyde β-cyclodextrin prepared in step (1) and add it into water, then add 3-aminophenylboronic acid and the bone marrow mesenchymal stem cell exosomes prepared in Preparation Example 1, and stir to react for 2 h to obtain a mixed solution; wherein the mass ratio of 3-aminophenylboronic acid, aldehyde β-cyclodextrin and stem cell exosomes is 1:6:0.03, and the concentration of bone marrow mesenchymal stem cell exosomes in the mixed solution is 6 µg / mL.

[0024] (3) The mixed solution of step (2) is added into a 5 wt % calcium chloride solution, filtered and freeze-dried to obtain modified β-cyclodextrin.

[0025] A method for preparing a composition for cartilage repair comprises the following steps: The above-mentioned carboxymethyl chitosan, modified β-cyclodextrin, soybean protein and caperitide are added into phosphate buffer (0.01 mmol / L, pH 6.5), and stirred to mix evenly.

[0026] Embodiment 2: A composition for cartilage repair, wherein the concentrations of the components in phosphate buffer are as follows: 5 mg / mL carboxymethyl chitosan, 1 mg / mL modified β-cyclodextrin, 1 mg / mL soy protein, and 20 μg / mL caperitide; The preparation process of the modified β-cyclodextrin is as follows: (1) β-cyclodextrin is added to water, and then sodium periodate is added, wherein the molar ratio of β-cyclodextrin to sodium periodate is 1:1, and the amount of β-cyclodextrin to water is 10 g:100 mL. After reacting at 40°C in the dark for 4 hours, the mixture is filtered with a filter membrane (220 nm), and then the filtrate is precipitated with 95% anhydrous ethanol to obtain aldehyde β-cyclodextrin; (2) Take the aldehyde β-cyclodextrin prepared in step (1) and add it into water, then add 3-aminophenylboronic acid and the bone marrow mesenchymal stem cell exosomes prepared in Preparation Example 1, stir and react for 1 hour to obtain a mixed solution; wherein the mass ratio of 3-aminophenylboronic acid, aldehyde β-cyclodextrin and stem cell exosomes is 1:5:0.01, and the concentration of bone marrow mesenchymal stem cell exosomes in the mixed solution is 2 µg / mL.

[0027] (3) The mixed solution of step (2) is added into a 3 wt % calcium chloride solution, filtered and freeze-dried to obtain modified β-cyclodextrin.

[0028] A method for preparing a composition for cartilage repair comprises the following steps: The above-mentioned carboxymethyl chitosan, modified β-cyclodextrin, soybean protein and caperitide are added into phosphate buffer (0.015 mmol / L, pH 6.5), and stirred and mixed to obtain a product.

[0029] Embodiment 3: A composition for cartilage repair, wherein the concentrations of the components in phosphate buffer are as follows: 10 mg / mL carboxymethyl chitosan, 3 mg / mL modified β-cyclodextrin, 5 mg / mL soy protein, and 30 μg / mL caperitide; The preparation process of the modified β-cyclodextrin is as follows: (1) Add β-cyclodextrin to water, and then add sodium periodate, wherein the molar ratio of β-cyclodextrin to sodium periodate is 1:4, and the amount of β-cyclodextrin to water is 20 g:100 mL. After reacting at 35°C in the dark for 6 hours, filter with a filter membrane (220 nm), and then precipitate the filtrate with 95% anhydrous ethanol to obtain aldehyde β-cyclodextrin; (2) Take the aldehyde β-cyclodextrin prepared in step (1) and add it into water, then add 3-aminophenylboronic acid and the bone marrow mesenchymal stem cell exosomes prepared in Preparation Example 1, and stir to react for 3 hours to obtain a mixed solution; wherein the mass ratio of 3-aminophenylboronic acid, aldehyde β-cyclodextrin and stem cell exosomes is 1:7:0.05, and the concentration of bone marrow mesenchymal stem cell exosomes in the mixed solution is 8 µg / mL.

[0030] (3) The mixed solution of step (2) is added into 8 wt % calcium chloride solution, filtered and freeze-dried to obtain modified β-cyclodextrin.

[0031] A method for preparing a composition for cartilage repair comprises the following steps: The above carboxymethyl chitosan, modified β-cyclodextrin, soybean protein and caperitide are added into phosphate buffer (0.02 mmol / L, pH 6.5), and stirred to mix evenly.

[0032] Comparative Example 1: This comparative example 1 is basically the same as Example 1, with the only difference being that in the composition for cartilage repair, the modified β-cyclodextrin is replaced with aldehyde β-cyclodextrin and bone marrow mesenchymal stem cell exosomes, and the amounts of aldehyde β-cyclodextrin and bone marrow mesenchymal stem cell exosomes are consistent with those in Example 1.

[0033] Comparative Example 2: This comparative example 2 is substantially the same as example 1, with the only difference being that caperitide is omitted from the composition for cartilage repair.

[0034] Test Example 1: Cytotoxicity assay: The present invention uses L929 mouse fibroblasts to conduct cytotoxicity experiments. The specific experimental steps are as follows: select L929 mouse fibroblasts and adjust their concentration to 1×10 5 / mL, as the cell suspension for the experiment. The above cell suspension was inoculated into a 96-well culture plate, with 100 µL inoculated into each well to ensure uniform distribution of cells. The specific treatment method of Example 1-3 groups is: the corresponding cartilage repair composition prepared is mixed evenly with the above cell suspension in a volume ratio of 2:1. For example, Example 1 group: the cartilage repair composition of Example 1 is mixed with the above cell suspension in a ratio of 2:1, and then inoculated into the culture plate, with 3 replicates set for each group. Negative control group: 100 µL of cell suspension was inoculated, and 3 replicates were also set. All culture plates were placed in an incubator at 37°C and 5% CO2 for 24 hours. Discard the original culture medium and add 20 µL of MTT solution to each well. Continue to incubate at 37°C for 4 hours. After the incubation is completed, the supernatant is aspirated and 150 µL of dimethyl sulfoxide is added to each well. Oscillate for 10 minutes. The light absorbance value (A492) of each well was measured at a wavelength of 492 nm using an ELISA reader. Each well was measured 3 times and the average value was taken as the final result. The formula for calculating the relative proliferation rate (RGR) of cells is as follows: RGR = A / A0 × 100% (where A is the average absorbance of the experimental group and A0 is the average absorbance of the negative control group). The experimental results are shown in Figure 2 ; The evaluation criteria for cytotoxicity grading are shown in Table 1: Table 1 Evaluation criteria for cytotoxicity grading: ; According to the RGR value, it can be judged whether the cartilage repair composition of the present invention has cytotoxicity. If the RGR value is close to or higher than 100%, it indicates that the material is non-toxic to cells or has a promoting effect; if the RGR value is significantly reduced, it indicates that the material has cytotoxicity.

[0035] Depend on Figure 2 The experimental results show that the relative proliferation rate of the cells of the present invention is above 100%, and the cytotoxicity level is level 0, which shows that the cartilage repair composition of the present invention is non-toxic to cells, has good biocompatibility, and can meet the requirements of cartilage repair materials for cytotoxicity.

[0036] Test Example 2: (1) In order to explore the effect of the cartilage repair composition of the present invention on the repair of cartilage defects in rats, a SD rat articular cartilage defect model was established, and the following steps were adopted: First, SD rats were anesthetized with 3% sodium pentobarbital at a dose of 0.15 mL / 100 g. After the anesthesia took effect, the hair in the knee joint area of ​​the rat was shaved and disinfected, and then the forelimbs of the rat were fixed. After the lower limbs were disinfected with iodine, an anterior medial incision of the knee joint was made on the inner side of the patellar tendon, the medial retinaculum of the patellar tendon was cut off (the patellar tendon was kept intact), the joint capsule was cut and the subcutaneous tissue was peeled off, the knee joint was straightened and the patella was dislocated to expose the articular surface. A Kirschner wire drill was used to drill a hole in the middle position of the cartilage articular surface to form a cylindrical cartilage defect with a diameter of 1 mm and a depth of 1 mm. Example 1 Group, Example 2 Group, Example 3 Group, Comparative Example 1 Group and Comparative Example 2 Group respectively filled the corresponding cartilage repair composition prepared into the defect to ensure that there was no gap. The joint cavity was then rinsed with sterile saline and sutured. In addition, a blank control group was set up, which was not treated and directly sutured. There were 10 SD rats in each group. Each group of rats was injected with the corresponding cartilage repair composition every 7 days after surgery. The rats in each group were euthanized 12 weeks after surgery, and the knee joints were collected for gross observation. The experimental results are shown in Figure 3 ,in Figure 3 AF in the figure correspond to Example 1-3 group, blank control group, and Comparative Example 1-2 group, respectively.

[0037] Depend on Figure 3 It can be seen that compared with the blank control group, the cartilage repair composition of Examples 1-3 of the present invention has been covered with new cartilage tissue at the cartilage defect, and the joint surface is relatively smooth, the color is similar to the color of the surrounding tissue, and the cartilage defect has been completely repaired, indicating that the cartilage repair composition of the present invention has a good cartilage repair effect.

[0038] In the comparative example 1 group, there is still white fibrous tissue in the cartilage repair area, and there is a slight depression, indicating that its repair effect 12 weeks after surgery is not ideal. In the comparative example 2 group, the cartilage repair area is improved to a certain extent compared with the comparative example 1, and the depression area is slightly reduced, but the color of the cartilage repair area is still quite different from the surrounding tissue. The above results show that the aldehyde group in the aldehyde β-cyclodextrin of the present invention can react with the amino group in 3-aminophenylboronic acid to undergo Schiff base reaction, realize the inclusion of bone marrow mesenchymal exosomes, and improve the stability and activity of exosomes. And the modified β-cyclodextrin and carpelitide can be used together to improve the effect of cartilage repair. The aldehyde group in the modified β-cyclodextrin also interacts with the amino group in carboxymethyl chitosan, which can further improve the biological stability of the material.

[0039] (2) The experimental results of the above groups were scored using the International Cartilage Repair Society (ICRS) scoring criteria to quantify the repair effects and compare them. The specific scoring criteria are shown in Table 2. The scoring results of Example 1-3, Comparative Example 1-2 and the blank control group are shown in Table 2. Figure 4 , the higher the score, the better the repair.

[0040] Table 2 Restoration effect scoring criteria: ; Depend on Figure 4 The experimental results show that, compared with the blank control group, the repair scores of Examples 1-3 are the highest, and are significantly higher than those of Comparative Examples 1 and 2, indicating that the compositions of Examples 1-3 have the best cartilage repair capabilities.

[0041] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A composition for cartilage repair, characterized in that: The concentrations of the components in phosphate buffer were as follows: carboxymethyl chitosan 5-10 mg / mL, modified β-cyclodextrin 1-3 mg / mL, soy protein 1-5 mg / mL, caperitide 20-30 μg / mL; The preparation process of the modified β-cyclodextrin is as follows: (1) Add β-cyclodextrin to water, and then add sodium periodate to obtain aldehyde β-cyclodextrin after reaction; (2) adding the aldehyde β-cyclodextrin prepared in step (1) into water, adding 3-aminophenylboronic acid and stem cell exosomes, and stirring to react to obtain a mixed solution; (3) The mixed solution of step (2) is added to the coagulation liquid, and modified β-cyclodextrin is obtained through post-treatment.

2. The composition for cartilage repair according to claim 1, characterized in that: The molar ratio of the β-cyclodextrin to sodium periodate is 1:(1-4).

3. The composition for cartilage repair according to claim 1, characterized in that: In step (1), the reaction temperature is 35-40°C and the reaction time is 4-6h.

4. The composition for cartilage repair according to claim 1, characterized in that: In step (2), the mass ratio of 3-aminophenylboronic acid, aldehyde β-cyclodextrin and stem cell exosomes is 1:5-7:0.01-0.05, and the concentration of the stem cell exosomes in the mixed solution is 2-8µg / mL.

5. The composition for cartilage repair according to claim 1, characterized in that: The stirring reaction time in step (2) is 1-3 hours.

6. The composition for cartilage repair according to claim 1, characterized in that: The coagulation liquid in step (3) is a calcium chloride solution or a potassium chloride solution.

7. The composition for cartilage repair according to claim 6, characterized in that: The concentration of the calcium chloride solution or potassium chloride solution is 3-8wt%.

8. The composition for cartilage repair according to claim 1, characterized in that: The stem cell exosomes are bone marrow mesenchymal stem cell exosomes.

9. A method for preparing the composition for cartilage repair according to claim 1, characterized in that: The steps include: Add carboxymethyl chitosan, modified β-cyclodextrin, soybean protein and caperitide into phosphate buffer, and stir and mix evenly to obtain the product.

10. The method for preparing the composition for cartilage repair according to claim 9, characterized in that: The concentration of the phosphate buffer is 0.01-0.02 mmol / L, and the pH is 6.5.

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