Preparation method of BMP-2 sustained-release material and application of BMP-2 sustained-release material in bone defect repair

Abstract

The invention discloses a preparation method of a BMP-2 sustained-release material and application of the BMP-2 sustained-release material in bone defect repair, and belongs to the technical field of drug sustained release. Boric acid, urea and PMMA microspheres are used for preparing a boron nitride porous material, and after the boron nitride porous material is modified through O / Ar mixed gas treatment, the BMP-2 sustained-release material is prepared. The modified boron nitride porous material is soaked in an SBF solution and a BMP-2 solution, the prepared BMP-2 sustained-release material serves as a sustained-release material, the degradation speed of the BMP-2 sustained-release material is low, in-vivo slow release of BMP-2 can be achieved, and cell ALP activity is high.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug sustained release, and specifically relates to a preparation method of a BMP-2 sustained release material and its application in bone defect repair. Background Art

[0002] There are four commonly used bone grafting methods, namely: autologous bone grafting, allogeneic bone grafting, xenogeneic bone grafting, and bone substitute grafting. The materials for autologous bone grafting are usually taken from the patient himself. Although the effect is the best, there are problems of secondary trauma and limited sources in extracting bone tissue from the patient's body. In addition, allogeneic bone grafting and xenogeneic bone grafting have the problem of immune rejection.

[0003] Delivering the drug bone morphogenetic protein-2 (BMP-2) through a sustained release material carrier is currently the best treatment method for replacing autologous bone grafting. Bone morphogenetic protein-2 is a growth factor with obvious osteogenic effects. However, the clinical effect of BMP-2 is not very ideal. BMP-2 will rapidly degrade and inactivate under physiological conditions, resulting in a shortened half-life. The osteogenic effect of too low a dose of BMP-2 is not ideal. In addition, allergic doses of BMP-2 can cause a series of side effects such as heterotopic bone formation, abnormal inflammatory reactions, nerve damage, and cancer. Summary of the Invention

[0004] In order to overcome the problems existing in the prior art, the present invention provides a preparation method of a BMP-2 sustained release material and its application in bone defect repair.

[0005] To achieve the above object, the present invention is realized through the following technical solutions: The first aspect of the present invention provides a preparation method of a BMP-2 sustained release material, comprising the following steps: S1, Take boric acid and urea and dissolve them in deionized water according to a molar ratio of 1:(3-4), then add polyethylene glycol and stir until completely dissolved to obtain a mixed solution; S2, Add PMMA microspheres with a particle size of 300-500 microns to the mixed solution in step S1, assist in impregnation at -0.1 MPa for 2-3 h, and then freeze-dry to remove water to obtain a composite; S3, Keep the composite at 300 °C in an air atmosphere for 2 h. After the heat preservation is completed, transfer it to an ammonia atmosphere, heat it to 1000 °C at a rate of 5 °C / min and keep it for 4 hours, and then sinter it at 1400 °C in nitrogen for 2 h to generate a boron nitride porous material; S4. After cleaning the boron nitride porous material, place it in the cavity of the microwave plasma system and evacuate it to 0.5 - 0.8 Pa. Then, introduce an O₂ / Ar mixed gas at a flow rate of 15 - 25 sccm, stabilize the chamber pressure to 40 - 60 Pa, and then perform plasma excitation pretreatment at a power of 50 w for 2 - 3 min. Then, increase the power to 100 w and process for 10 - 12 min. After turning off the plasma, keep the gas flowing for 5 - 7 min, and slowly cool down to room temperature to obtain the modified boron nitride porous material; S5. Immerse the modified boron nitride porous material in 5×SBF for 24 h; then transfer it to 1×SBF and immerse it for 7 d. During this period, control the pH = 7.2 - 7.6 and change the SBF every 48 h; S6. Wash the immersed boron nitride porous material 3 times with deionized water, vacuum dry it at 37 °C for 24 h, and then transfer it to a 10 mg / ml BMP - 2 solution and immerse it for 2 h to obtain the BMP - 2 sustained - release material.

[0006] Furthermore, in the step S1, the addition amount of polyethylene glycol is 5 - 8 wt% of the boric acid and urea mixed solution.

[0007] Furthermore, in the step S4, the volume ratio of O₂ to Ar is 1:(1 - 4).

[0008] Furthermore, in the step S4, the cleaning method of the boron nitride porous material is as follows: ultrasonically clean it with acetone, ethanol, and deionized water for 15 minutes each in sequence to remove surface organic substances and dust, and dry it at 80 °C for standby.

[0009] The second aspect of the present invention provides a sustained - release material prepared by the method described in the first aspect.

[0010] The third aspect of the present invention provides the application of the sustained - release material prepared by the method described in the first aspect in the preparation of a drug delivery carrier for bone defect repair.

[0011] Through the above - mentioned technical solutions, the present invention can at least achieve the following beneficial effects: The present invention uses boric acid, urea, and PMMA microspheres to prepare a boron nitride porous material. After modifying the boron nitride porous material by treatment with an O₂ / Ar mixed gas, the modified boron nitride porous material is immersed in an SBF solution and a BMP - 2 solution. The prepared BMP - 2 sustained - release material, as a sustained - release material, has a slow self - degradation rate and can achieve the slow release of BMP - 2 in vivo, and has a high cell ALP activity. Description of the Drawings

[0012] Figure 1 is a scanning electron micrograph of the sustained - release material prepared in Example 1 of the present invention; Figure 2It is the experimental result diagram of drug release of the sustained-release material in the present invention; Figure 3 It is the experimental result diagram of degradation of the sustained-release material in the present invention; Figure 4 It is the detection result diagram of alkaline phosphatase (ALP) in the present invention. Specific Embodiments

[0013] Unless otherwise specified, the materials and reagents used in the present invention are commercially available.

[0014] The PMMA microspheres in the present invention, namely polymethyl methacrylate microspheres, are commercially available products; The SBF in the present invention is simulated body fluid and is a commercially available product.

[0015] Example 1: A preparation method of a BMP-2 sustained-release material, comprising the following steps: S1, Take boric acid and urea and dissolve them in deionized water according to a molar ratio of 1:3.5, then add 7 wt% of polyethylene glycol and stir until completely dissolved to obtain a mixed solution; S2, Add PMMA microspheres with a particle size of 400 microns to the mixed solution in step S1, assist in impregnation at -0.1 MPa for 2.5 h, and then freeze-dry to remove moisture to obtain a composite; S3, Keep the composite at 300 °C in an air atmosphere for 2 h. After the heat preservation ends, transfer it to an ammonia atmosphere, heat it to 1000 °C at a rate of 5 °C / min and keep it for 4 hours, and then sinter it at 1400 °C in nitrogen for 2 h to generate a boron nitride porous material; S4, Ultrasonically clean the boron nitride porous material with acetone, ethanol, and deionized water for 15 minutes each to remove surface organic substances and dust. After drying at 80 °C, place it in the cavity of a microwave plasma system, evacuate to 0.6 Pa, then introduce an O2 / Ar mixed gas at a flow rate of 20 sccm, stabilize the chamber pressure to 50 Pa, then perform plasma excitation pretreatment at a power of 50 w for 2.5 min, then increase the power to 100 w and process for 11 min. After turning off the plasma, keep the gas flowing for 6 min and slowly cool to room temperature to obtain a modified boron nitride porous material; wherein, the volume ratio of O2 to Ar is 1:3; S5, Immerse the modified boron nitride porous material in 5×SBF for 24 h; then transfer it to 1×SBF and soak for 7 d. During this period: control the pH = 7.2 - 7.6 and change the SBF every 48 h; S6, Wash the soaked boron nitride porous material 3 times with deionized water, vacuum-dry it at 37 °C for 24 h, and then transfer it to a 10 mg / ml BMP-2 solution and soak for 2 h to obtain the BMP-2 sustained-release material.

[0016] Example 2: A preparation method of a BMP-2 sustained-release material, comprising the following steps: S1, Take boric acid and urea and dissolve them in deionized water according to a molar ratio of 1:3, then add 5 wt% polyethylene glycol and stir until completely dissolved to obtain a mixed solution; S2, Add PMMA microspheres with a particle size of 300 microns to the mixed solution in step S1, assist in impregnation at -0.1 MPa for 2 h, and then freeze-dry to remove moisture to obtain a composite; S3, Keep the composite at 300 °C in an air atmosphere for 2 h. After the heat preservation is completed, transfer it to an ammonia atmosphere, heat it to 1000 °C at a rate of 5 °C / min and hold for 4 hours, and then sinter it at 1400 °C in nitrogen for 2 h to generate a boron nitride porous material; S4, Ultrasonically clean the boron nitride porous material with acetone, ethanol, and deionized water for 15 minutes each in turn to remove surface organic matter and dust. After drying at 80 °C, place it in the cavity of a microwave plasma system, evacuate to 0.5 Pa, and then introduce an O2 / Ar mixed gas at a flow rate of 15 sccm. Stabilize the chamber pressure to 40 Pa, and then pre-treat it with plasma excitation at a power of 50 w for 2 min, and then increase the power to 100 w and treat for 10 min. After turning off the plasma, keep the gas flowing for 5 min and slowly cool to room temperature to obtain a modified boron nitride porous material; wherein, the volume ratio of O2 to Ar is 1:1; S5, Immerse the modified boron nitride porous material in 5×SBF for 24 h; then transfer it to 1×SBF and immerse it for 7 d. During this period: control the pH = 7.2 - 7.6 and change the SBF every 48 h; S6, Wash the immersed boron nitride porous material 3 times with deionized water, vacuum-dry it at 37 °C for 24 h, and then transfer it to a 10 mg / ml BMP-2 solution and immerse it for 2 h to obtain the BMP-2 sustained-release material.

[0017] Example 3: A preparation method of a BMP-2 sustained-release material, comprising the following steps: S1, Take boric acid and urea and dissolve them in deionized water according to a molar ratio of 1:4, then add 8 wt% polyethylene glycol and stir until completely dissolved to obtain a mixed solution; S2, Add PMMA microspheres with a particle size of 500 microns to the mixed solution in step S1, assist in impregnation at -0.1 MPa for 3 h, and then freeze-dry to remove moisture to obtain a composite; S3, Keep the composite at 300 °C in an air atmosphere for 2 h. After the heat preservation is completed, transfer it to an ammonia atmosphere, heat it to 1000 °C at a rate of 5 °C / min and hold for 4 hours, and then sinter it at 1400 °C in nitrogen for 2 h to generate a boron nitride porous material; S4. The boron nitride porous material was ultrasonically cleaned with acetone, ethanol, and deionized water for 15 minutes each to remove surface organic substances and dust. After drying at 80 °C, it was placed in the cavity of a microwave plasma system and evacuated to 0.8 Pa. Then, an O2 / Ar mixed gas was introduced at a flow rate of 25 sccm, and the chamber pressure was stabilized to 60 Pa. Subsequently, it was pretreated by plasma excitation at a power of 50 w for 3 min, and then the power was increased to 100 w for 12 min. After turning off the plasma, the gas flow was maintained for 7 min, and it was slowly cooled to room temperature to obtain the modified boron nitride porous material; wherein, the volume ratio of O2 to Ar is 1:4; S5. The modified boron nitride porous material was immersed in 5×SBF for 24 h; then it was transferred to 1×SBF and immersed for 7 d. During this period, the pH was controlled at 7.2 - 7.6 and the SBF was replaced every 48 h; S6. The immersed boron nitride porous material was washed 3 times with deionized water, vacuum dried at 37 °C for 24 h, and then transferred to a 10 mg / ml BMP-2 solution and immersed for 2 h to obtain the BMP-2 sustained-release material.

[0018] Comparative Example 1:

[0019] The difference between Comparative Example 1 and Example 1 is that in step S4, after the boron nitride porous material was ultrasonically cleaned with acetone, ethanol, and deionized water for 15 minutes each to remove surface organic substances and dust and dried at 80 °C, steps S5 and S6 were carried out immediately.

[0020] Comparative Example 2:

[0021] The difference between Comparative Example 2 and Example 1 is that in step S5, the operation of "immersing the modified boron nitride porous material in 5×SBF for 24 h" was cancelled.

[0022] Experimental Example:

[0023] 1. Drug release experiment of the sustained-release material Take the BMP-2 sustained-release materials prepared in Examples 1 to 3 and Comparative Examples 1 and 2, weigh 50 mg each, and immerse them in 4 mL of PBS solution (pH = 7.4) (using centrifuge tubes as containers). Then, place the centrifuge tubes in a thermostat and oscillator at 37 °C for incubation. At selected times, 100 μL of the supernatant was taken out, and an equal volume of PBS solution was added to the centrifuge tubes. The taken-out supernatant was stored in a -80 °C refrigerator for further use. Finally, according to the steps of the human bone morphogenetic protein-2 (rhBMP-2) ELISA kit, the content of total BMP-2 in the supernatant was calculated, and the results are as shown in the appendix Figure 2 as shown. There are three parallel samples in each group.

[0024] From Figure 2It can be seen that there is little difference in the drug release of the sustained-release materials in Examples 1 to 3 from that in Comparative Example 1 and Comparative Example 2.

[0025] 2. Degradation experiment of the sustained-release material For the degradation experiment of the sustained-release material, first weigh the total mass W0 of the composite material. Immerse the sustained-release material in 10 ml of phosphate buffer solution (PBS), and incubate the solution on an oscillator at 50 rpm at 37 °C for 30 days; then collect the sustained-release material every 2 days, air-dry the remaining sustained-release material at 25 °C for 6 h, and the weighed mass is W1. The results of the degradation test of the sustained-release material are as shown in the appendix Figure 3 . Retention rate (%) = (W1 / W0) * 100%, and 3 parallel experiments are carried out for each sample.

[0026] From the appendix Figure 3 It can be seen that the 30-day retention rate of the sustained-release materials in Examples 1 to 3 is higher than that in Comparative Example 1 and Comparative Example 2, indicating that its degradation rate is slow.

[0027] 3. Detection of alkaline phosphatase (ALP) Alkaline phosphatase (ALP) is a standard feature of the osteogenic differentiation of stem cells, and can also be used to reflect the activity and osteogenic function of osteoblasts.

[0028] The content of alkaline phosphatase is determined using an alkaline phosphatase assay kit. Its basic principle is that p-nitrophenyl phosphate disodium can undergo a chemical reaction with alkaline phosphatase to generate light yellow p-nitrophenol, and p-nitrophenol has a specific absorption peak at a wavelength of 405 nm: the content of alkaline phosphatase in the sample is detected by measuring the absorption intensity at 405 nm.

[0029] After the prepared sustained-release material of the example is aseptically treated, it is placed in a 48-well plate, and 2x10 5Cells were used, and the fresh adipose mesenchymal stem cell basal culture medium (BCM) was replaced every 2 days. On the 3rd and 15th days of culture, the BCM was aspirated, and the above-mentioned sustained-release materials were repeatedly washed with PBS to remove the residual BCM. Then, according to the kit instructions, 600 μL of lysis buffer was added to each well sample (it is necessary to ensure that the sample is completely immersed in the lysis buffer). After lysis on ice for 1 h, strong vibration was carried out to ensure complete cell breakage, and then centrifuged at 1000 rpm at low temperature for 5 min. Detection of total enzyme activity: Take 20 μL of the sample, add 200 μL of p-nitrophenyl phosphate disodium solution and incubate at room temperature for 15 min, then add 100 μL of 0.1 M sodium hydroxide solution. After mixing, use a multifunctional microplate reader to read the absorbance (OD405) at 405 nm. After calculation, the total enzyme activity of the sample can be calculated. Detection of total protein content: Take another 20 μL of the same sample, add 160 μL of protein test detection solution respectively, and incubate at room temperature for 15 min. Use a multifunctional microplate reader to read the absorbance (OD450) at 450 nm. After calculation, the total protein content of the sample can be calculated.

[0030] Using the formula: alkaline phosphatase activity = (total enzyme activity / total protein content), the alkaline phosphatase (ALP) content in the sample can be calculated.

[0031] The results are as follows Figure 4 shown.

[0032] It can be seen from Figure 4 that the cell ALP activity of the sustained-release materials in Examples 1 to 3 on the 15th day was significantly higher than that on the 3rd day, and the cell ALP activity of the sustained-release materials was significantly higher than that in Comparative Example 1 and Comparative Example 3.

[0033] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not restrictive. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A preparation method of a BMP-2 sustained-release material, characterized in that: It includes the following steps: S1. Take boric acid and urea and dissolve them in deionized water according to a molar ratio of 1:(3 - 4), then add polyethylene glycol and stir until completely dissolved to obtain a mixed solution; S2. Add PMMA microspheres with a particle size of 300 - 500 microns to the mixed solution in step S1, assist in impregnation at -0.1 MPa for 2 - 3 h, and then freeze-dry to remove moisture to obtain a composite; S3. Keep the composite at 300 °C in an air atmosphere for 2 h. After the heat preservation ends, transfer it to an ammonia atmosphere, heat it to 1000 °C at a rate of 5 °C / min and hold for 4 h, and then sinter it at 1400 °C in nitrogen for 2 h to generate boron nitride porous material; S4. After cleaning the boron nitride porous material, place it in the cavity of a microwave plasma system, evacuate to 0.5 - 0.8 Pa, then introduce an O2 / Ar mixed gas at a flow rate of 15 - 25 sccm, stabilize the chamber pressure to 40 - 60 Pa, then pre-treat it by plasma excitation at a power of 50 w for 2 - 3 min, then increase the power to 100 w and treat it for 10 - 12 min. After turning off the plasma, keep the gas flowing for 5 - 7 min and slowly cool to room temperature to obtain the modified boron nitride porous material; S5. Immerse the modified boron nitride porous material in 5×SBF for 24 h; then transfer it to 1×SBF and immerse it for 7 d. During this period, control the pH = 7.2 - 7.6 and change the SBF every 48 h; S6. Wash the immersed boron nitride porous material 3 times with deionized water, vacuum-dry it at 37 °C for 24 h, and then transfer it to a 10 mg / ml BMP-2 solution and immerse it for 2 h to obtain the BMP-2 sustained-release material.

2. The preparation method of a BMP-2 sustained-release material according to claim 1, characterized in that: In step S1, the addition amount of polyethylene glycol is 5 - 8 wt% of the boric acid and urea mixed solution.

3. The preparation method of a BMP-2 sustained-release material according to claim 1, characterized in that: In step S4, the volume ratio of O2 to Ar is 1:(1 - 4).

4. The preparation method of a BMP-2 sustained-release material according to claim 1, characterized in that: In step S4, the cleaning method of the boron nitride porous material is: ultrasonically clean it with acetone, ethanol, and deionized water for 15 minutes each in turn to remove surface organic substances and dust, and dry it at 80 °C for standby.

5. A sustained-release material prepared by the method according to any one of claims 1 - 4.

6. Use of the sustained-release material prepared by the method according to any one of claims 1 - 4 in the preparation of a drug delivery carrier for bone defect repair.

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