A composition for cartilage repair and its preparation method

Abstract

The present invention relates to the field of biotechnology and medicine, and particularly relates to a composition for cartilage repair and a preparation method thereof. The concentrations of the components of the composition in phosphate buffer are as follows: carboxymethyl chitosan 5-10 mg / mL, modified β-cyclodextrin 1-3 mg / mL, soy protein 1-5 mg / mL, and carperitide 20-30 μg / mL; the preparation process of the modified β-cyclodextrin is as follows: take β-cyclodextrin and add it to water, then add sodium periodate, and after reaction, aldehyde group β-cyclodextrin is obtained; take aldehyde group β-cyclodextrin and add it to water, add 3-aminophenylboronic acid and stem cell exosomes, and stir and react to obtain a mixed solution; add the mixed solution to a coagulating solution, and after post-treatment, modified β-cyclodextrin is obtained. In the present invention, the combined use of modified β-cyclodextrin and carperitide can improve the effect of cartilage repair, and the interaction between modified β-cyclodextrin and carboxymethyl chitosan can improve the biological stability of the material.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology and medicine, and particularly relates 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 undertakes the functions of buffering and dispersing pressure. However, due to various reasons such as trauma, disease, or aging, cartilage tissue often suffers from damage or degeneration, leading to joint pain, stiffness, and loss of function. The treatment of cartilage damage has always been a difficult problem in clinical practice because cartilage tissue itself has a low regenerative ability. Traditional treatment methods such as arthroscopic debridement and drilling microfracture can only relieve symptoms temporarily, but 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, growth factors, etc. 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 parent cells. However, exosomes are easily inactivated in the in vitro environment and are difficult to effectively deliver to the target tissue, which limits their application in cartilage repair. In addition, the current cartilage repair effect is poor. Therefore, developing a carrier material that can effectively encapsulate, stabilize exosomes and improve their activity is of great significance for improving the cartilage repair effect.

[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, so as to improve the cartilage repair effect. Summary of the Invention

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

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

[0007] The object of the present invention is achieved by the following technical solutions:

[0008] A composition for cartilage repair, the concentrations of each component in phosphate buffer are as follows: carboxymethyl chitosan 5 - 10 mg / mL, modified β - cyclodextrin 1 - 3 mg / mL, soy protein 1 - 5 mg / mL, carperitide 20 - 30 μg / mL;

[0009] The preparation process of the modified β - cyclodextrin is as follows:

[0010] (1) Take β-cyclodextrin, add it to water, and then add sodium periodate. After the reaction, aldehyde group β-cyclodextrin is obtained;

[0011] (2) Take the aldehyde group β-cyclodextrin obtained in step (1), add it to water, add 3-aminophenylboronic acid and stem cell exosomes, and stir and react to obtain a mixed solution;

[0012] (3) Add the mixed solution obtained in step (2) to the coagulating liquid, and obtain modified β-cyclodextrin after post-treatment.

[0013] Further, the molar ratio of the β-cyclodextrin to sodium periodate is 1:(1 - 4).

[0014] Further, in step (1), the temperature of the reaction is 35 - 40 °C, and the time of the reaction is 4 - 6 h.

[0015] Further, in step (2), the mass ratio of the 3-aminophenylboronic acid, aldehyde group 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 - 8 μg / mL.

[0016] Further, in step (2), the time of the stirring reaction is 1 - 3 h.

[0017] Further, in step (3), the coagulating liquid is a calcium chloride solution or a potassium chloride solution.

[0018] Further, the concentration of the calcium chloride solution or the potassium chloride solution is both 3 - 8 wt%.

[0019] Further, the stem cell exosomes are bone marrow mesenchymal stem cell exosomes.

[0020] A preparation method of the above composition for cartilage repair includes the following steps:

[0021] Add carboxymethyl chitosan, modified β-cyclodextrin, soy protein, and carperitide to phosphate buffer solution, and stir and mix evenly to obtain.

[0022] Further, the concentration of the phosphate buffer solution is 0.01 - 0.02 mmol / L, and the pH is 6.5.

[0023] The present invention has the following effects compared with the prior art:

[0024] 1. The aldehyde group in the aldehyde group β-cyclodextrin of the present invention can react with the amino group in 3-aminophenylboronic acid to achieve the inclusion of mesenchymal stem cell exosomes, improving the stability and activity of the exosomes. And the combined use of the modified β-cyclodextrin and carperitide can improve the effect of cartilage repair. Among them, the aldehyde group in the modified β-cyclodextrin also has an interaction 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.

[0025] 2. The present invention provides a preparation method for a composition for cartilage repair. The preparation steps of this method are relatively simple and easy to operate. Brief Description of the Drawings

[0026] Figure 1 It is a morphological diagram of passage 3 mesenchymal stem cells obtained by culturing in the present invention;

[0027] Figure 2 It is a diagram of the cytotoxicity test results of the cartilage repair compositions of Examples 1-3 and Comparative Examples 1-2 of the present invention;

[0028] Figure 3 It is a diagram of the effect of the cartilage repair compositions of Examples 1-3 and Comparative Examples 1-2 of the present invention on repairing rat cartilage tissue;

[0029] Figure 4 It is the scoring result of the cartilage repair composition of the present invention. Detailed Description of the Invention

[0030] The present invention will be further described in detail below with reference to examples, but the implementation manners 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 technical field. The test methods without specific experimental conditions 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 through commercial channels.

[0031] Preparation Example 1:

[0032] The preparation method of mesenchymal stem cell exosomes is as follows:

[0033] (1)Isolation and primary culture of bone marrow mesenchymal stem cells: Healthy 2-week-old mice were sacrificed by cervical dislocation, thoroughly disinfected, and their limbs were fixed on the operating table. In a laminar flow hood, the bilateral femurs and tibias of the mice were removed, cleaned, and the muscles and soft tissues were stripped; 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 3 times with PBS; then, under sterile conditions, the bilateral femurs and tibias of the mice were cut off to expose the bone marrow cavity, the bone marrow cavity was punctured with a 5 mL syringe, and then the bone marrow cavity was rinsed with DMEM / F12 medium containing heparin to obtain a bone marrow cell suspension. The suspension was slowly added above an equal volume of Ficoll separation solution, placed in a centrifuge tube and centrifuged (3000 rpm, 20 min). The bone marrow mononuclear cells at the interface of the white membrane layer were aspirated, suspended in DMEM / F12 medium, centrifuged at 1500 rpm for 10 minutes at room temperature, the supernatant was discarded, and the cells were washed 2 times repeatedly with PBS, 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 inoculated into a T25 cell culture flask at a density of 1×10 6 cells / mL. After culturing in an incubator at 37 °C, 5% CO2 and saturated humidity for 24 h, the culture medium was changed, and then the medium was changed every 2 days until the cell confluence reached 85%. The medium was removed, and the cells were washed 2 times repeatedly with PBS. Digested with 0.25% trypsin at 37 °C for 2 min, the action of trypsin was terminated with an equal volume of DMEM / F12 medium, then centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were washed 2 times with PBS. Then the bone marrow mesenchymal stem cells were resuspended in DMEM / F12 medium containing 50 ng / mL basic fibroblast growth factor and 20 ng / mL transferrin, gently blown into a single cell suspension, and subcultured at a ratio of 1:3. The P3 generation of bone marrow mesenchymal stem cells obtained from subculture was collected; the cell morphology and growth status were observed under an inverted microscope, and the results were as Figure 1 shown.

[0034] (2)The P3 generation of bone marrow mesenchymal stem cells obtained from the above culture was inoculated into α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at a density of 1×10 5 cells / mL, placed in an incubator at 37 °C and 5% CO2, and cultured until the confluence reached 80%. Then it was changed to serum-free α-MEM medium and cultured for 48 h. Then it was centrifuged at 1000 g for 30 min at 4 °C to remove cell debris. The supernatant was filtered through a 0.22 µm filter membrane, and the supernatant was centrifuged at 100000 g for 120 min. The supernatant was discarded, and the precipitate was retained; PBS was added and mixed evenly, and then centrifuged at 100000 g for 60 min again. The supernatant was discarded, and the precipitate was retained to obtain bone marrow mesenchymal stem cell exosomes.

[0035] Example 1:

[0036] A composition for cartilage repair, the concentrations of each component in phosphate buffer are as follows: carboxymethyl chitosan 8 mg / mL, modified β-cyclodextrin 2 mg / mL, soy protein 3 mg / mL, carperitide 25 μg / mL;

[0037] The preparation process of the modified β-cyclodextrin is as follows:

[0038] (1) Take β-cyclodextrin and add it to water, then add sodium periodate. Among them, the molar ratio of β-cyclodextrin to sodium periodate is 1:3, and the dosage relationship between β-cyclodextrin and water is 15 g:100 mL. React at 37°C under dark conditions for 5 h, then filter with a filter membrane (220 nm), and then precipitate the filtrate with 95% anhydrous ethanol to obtain aldehyde group β-cyclodextrin;

[0039] (2) Take the aldehyde group β-cyclodextrin obtained in step (1), add it to water, add 3-aminophenylboronic acid, and stir and react with the mesenchymal stem cell exosomes prepared in Preparation Example 1 for 2 h to obtain a mixed solution; among them, the mass ratio of 3-aminophenylboronic acid, aldehyde group β-cyclodextrin and stem cell exosomes is 1:6:0.03, and the concentration of mesenchymal stem cell exosomes in the mixed solution is 6 μg / mL.

[0040] (3) Add the mixed solution in step (2) to 5 wt% calcium chloride solution, filter and freeze-dry to obtain the modified β-cyclodextrin.

[0041] A preparation method of a composition for cartilage repair, comprising the following steps:

[0042] Add the above-mentioned carboxymethyl chitosan, modified β-cyclodextrin, soy protein, and carperitide to phosphate buffer (0.01 mmol / L, pH 6.5), stir and mix evenly to obtain.

[0043] Example 2:

[0044] A composition for cartilage repair, the concentrations of each component in phosphate buffer are as follows: carboxymethyl chitosan 5 mg / mL, modified β-cyclodextrin 1 mg / mL, soy protein 1 mg / mL, carperitide 20 μg / mL;

[0045] The preparation process of the modified β-cyclodextrin is as follows:

[0046] (1) Take β-cyclodextrin and add it to water, then add sodium periodate. Among them, the molar ratio of β-cyclodextrin to sodium periodate is 1:1, and the dosage relationship between β-cyclodextrin and water is 10 g:100 mL. React at 40 °C in the dark for 4 h, filter with a filter membrane (220 nm), and then precipitate the filtrate with 95% absolute ethanol to obtain aldehyde group β-cyclodextrin;

[0047] (2) Take the aldehyde group β-cyclodextrin obtained in step (1), add it to water, add 3-aminophenylboronic acid, and the bone marrow mesenchymal stem cell exosomes prepared in Preparation Example 1, and stir and react for 1 h to obtain a mixed solution; among them, the mass ratio of 3-aminophenylboronic acid, aldehyde group 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.

[0048] (3) Add the mixed solution in step (2) to a 3 wt% calcium chloride solution, filter and freeze-dry to obtain modified β-cyclodextrin.

[0049] A preparation method of a composition for cartilage repair, comprising the following steps:

[0050] Add the above-mentioned carboxymethyl chitosan, modified β-cyclodextrin, soy protein, and carperitide to phosphate buffer (0.015 mmol / L, pH 6.5), and stir and mix evenly to obtain.

[0051] Example 3:

[0052] A composition for cartilage repair, the concentrations of each component in phosphate buffer are as follows: carboxymethyl chitosan 10 mg / mL, modified β-cyclodextrin 3 mg / mL, soy protein 5 mg / mL, carperitide 30 μg / mL;

[0053] The preparation process of the modified β-cyclodextrin is as follows:

[0054] (1) Take β-cyclodextrin and add it to water, then add sodium periodate. Among them, the molar ratio of β-cyclodextrin to sodium periodate is 1:4, and the dosage relationship between β-cyclodextrin and water is 20 g:100 mL. React at 35 °C in the dark for 6 h, filter with a filter membrane (220 nm), and then precipitate the filtrate with 95% absolute ethanol to obtain aldehyde group β-cyclodextrin;

[0055] (2) Take the aldehyde group β-cyclodextrin obtained in step (1), add it to water, add 3-aminophenylboronic acid, and the bone marrow mesenchymal stem cell exosomes prepared in Preparation Example 1, and stir and react for 3 h to obtain a mixed solution; among them, the mass ratio of 3-aminophenylboronic acid, aldehyde group 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.

[0056] (3) Add the mixed solution from step (2) to an 8 wt% calcium chloride solution, filter, and freeze-dry to obtain modified β-cyclodextrin.

[0057] A method for preparing a composition for cartilage repair, comprising the following steps:

[0058] Add the above carboxymethyl chitosan, modified β-cyclodextrin, soy protein, and carperitide to phosphate buffer (0.02 mmol / L, pH 6.5), and stir to mix evenly to obtain the product.

[0059] Comparative Example 1:

[0060] This Comparative Example 1 is basically the same as Example 1, and the only difference is that in the composition for cartilage repair, the modified β-cyclodextrin is replaced with aldehyde group β-cyclodextrin and mesenchymal stem cell exosomes, and the dosages of aldehyde group β-cyclodextrin and mesenchymal stem cell exosomes are the same as those in Example 1.

[0061] Comparative Example 2:

[0062] This Comparative Example 2 is basically the same as Example 1, and the only difference is that carperitide is omitted in the composition for cartilage repair.

[0063] Test Example 1:

[0064] Cytotoxicity test:

[0065] In this invention, L929 mouse fibroblasts are selected for the cytotoxicity experiment. The specific experimental steps are as follows: Select L929 mouse fibroblasts and adjust their concentration to 1×10 5cells / mL, used as the cell suspension for the experiment. The above cell suspension was inoculated into a 96-well culture plate, 100 μL was inoculated into each well to ensure uniform distribution of the cells. The specific treatment methods for the groups of Examples 1-3 were as follows: the corresponding cartilage repair composition prepared was mixed evenly with the above cell suspension at a volume ratio of 2:1. For example, in the group of Example 1: the cartilage repair composition of Example 1 was mixed with the above cell suspension at a ratio of 2:1, and then inoculated into the culture plate, and 3 replicate wells were set in each group. Negative control group: 100 μL of cell suspension was inoculated, and 3 replicate wells were also set. All culture plates were cultured in an incubator at 37 °C and 5% CO2 for 24 h. The original culture medium was discarded, and 20 μL of MTT solution was added to each well. Incubation was continued at 37 °C for 4 h. After the incubation was completed, the supernatant was aspirated, and 150 μL of dimethyl sulfoxide was added to each well. Oscillate for 10 min. The light absorption value (A492) of each well was measured using a microplate reader at a wavelength of 492 nm, and each well was measured 3 times and the average value was taken as the final result. The calculation formula for the relative growth 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 cell cytotoxicity grading are shown in Table 1:

[0066] Table 1 Evaluation criteria for cell cytotoxicity grading:

[0067] ;

[0068] According to the magnitude of 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.

[0069] From Figure 2 the experimental results, it can be seen that the relative growth rates of the cells of the present invention are all above 100%, and the cell cytotoxicity grade is 0, indicating 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.

[0070] Test Example 2:

[0071] (1) To investigate the effect of the cartilage repair composition of the present invention on the repair of cartilage defects in rats, an SD rat articular cartilage defect model was established using the following steps: 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 rats was shaved and disinfected, and then the forelimbs of the rats were fixed. After the lower limbs were disinfected with iodophor, a medial anteromedial knee incision was made medial to the patellar tendon, the medial retinaculum of the patellar tendon was cut (the patellar tendon was kept intact), the joint capsule was incised and the subcutaneous tissue was dissected, the knee joint was extended and the patella was dislocated to expose the articular surface. A Kirschner wire drill was used to drill a hole at the center of the cartilage articular surface to form a cylindrical cartilage defect with a diameter of 1 mm and a depth of 1 mm. In the Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group and Comparative Example 2 group, the corresponding prepared cartilage repair compositions were filled into the defect to ensure no voids. Subsequently, the joint cavity was rinsed with sterile normal saline and sutured. In addition, a blank control group was set up, which was directly sutured without any treatment. There were 10 SD rats in each group above. Each group of rats was injected with the corresponding cartilage repair composition once every 7 days after surgery. After 12 weeks of surgery, the above-mentioned rats in each group were euthanized, and the knee joints were collected for gross observation. The experimental results are shown in Figure 3 , where Figure 3 A - F in

[0072] respectively correspond to the Example 1 - 3 groups, blank control group, Comparative Example 1 - 2 groups. Figure 3 It can be seen from

[0073] that compared with the blank control group, the cartilage repair compositions in the Example 1 - 3 groups of the present invention have been covered by newly formed cartilage tissue at the cartilage defect, and the joint surface is relatively smooth, and the color is similar to that of the surrounding tissues. The cartilage defect has been completely repaired, indicating that the cartilage repair composition of the present invention has a good cartilage repair effect.

[0074] (2)The experimental results of the above groups were scored using the International Cartilage Repair Society (ICRS) scoring criteria to quantify and compare the repair effects. The specific scoring criteria are shown in Table 2. The scoring results of Examples 1-3, Comparative Examples 1-2, and the blank control group are shown in Figure 4 , the higher the score, the better the repair.

[0075] Table 2 Repair effect scoring criteria:

[0076] ;

[0077] From Figure 4 the experimental results, it can be seen that compared with the blank control group, the repair scores of Examples 1-3 are the highest and significantly higher than those of Comparative Examples 1 and 2, indicating that the cartilage repair ability of the compositions in Examples 1-3 is the best.

[0078] The above embodiments are only the 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 those skilled in the art based on the present invention 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) adding the mixed solution of step (2) into the coagulation liquid, and obtaining modified β-cyclodextrin through post-treatment; The stem cell exosomes are bone marrow mesenchymal stem cell exosomes; and the coagulation liquid is a calcium chloride solution.

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 concentration of the calcium chloride solution is 3-8wt%.

7. 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.

8. The method for preparing the composition for cartilage repair according to claim 7, 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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