A bone repair membrane and its preparation method

Abstract

The present invention belongs to the field of biomedical materials, and discloses a bone repair membrane and a preparation method thereof. The preparation method includes: successively treating poultry eggshells with phosphoric acid and acetic acid, dissolving the eggshell membrane with hexafluoroisopropanol, adding polylysine, and electrospinning to form a nanofiber membrane; adding the phosphoric acid treatment solution to hexamine, putting the nanofiber membrane into it for hydrothermal reaction to obtain a nanofiber membrane deposited with hydroxyapatite nanowires; grafting lactic acid activated by EDC / NHS to the membrane, and finally obtaining the bone repair membrane. The bone repair membrane promotes the healing of bone cracks through the release of an appropriate amount of lactic acid, and then the exposed polylysine groups contribute to the adhesion of osteoblasts, and the surface hydroxyapatite can also stimulate osteoblast differentiation to promote the repair of defective tissues.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and in particular relates to a bone repair membrane and a preparation method thereof. Background Art

[0002] Bone repair materials usually refer to devices and materials that are surgically implanted into the human body to repair bone defects (bone defects refer to the destruction of the structural integrity of bones. A series of causes such as tumors, trauma, necrosis, congenital malformations, etc. often lead to large bone defects). The interaction between seed cells and materials is an important link in bone tissue engineering research. The adhesion, migration, differentiation and proliferation of osteoblasts on the surface of materials and bone remodeling are key steps in bone healing and are regulated by multiple molecular mechanisms. At present, existing bone repair materials can only be designed for one of the key points, such as bone replacement, recruitment of osteoblasts, etc., and there is a lack of molecular regulation methods. Therefore, the treatment of bone defects has not been ideal. What's more, bone repair materials made of polylactic acid materials will release a large amount of lactic acid during use, causing chronic inflammation, which is not conducive to bone defect repair. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a bone repair membrane and a preparation method thereof, which promotes bone crack healing by releasing an appropriate amount of lactic acid. The exposed polylysine groups thereafter help the adhesion of osteoblasts, and the hydroxyapatite on the surface can also stimulate osteoblast differentiation to promote the repair of defective tissue.

[0004] In order to solve the above technical problems, the present invention provides a bone repair membrane and a preparation method thereof, comprising the following steps:

[0005] S1. The eggshells are treated with phosphoric acid solution for 4-6 hours, filtered, and the filtrate is recovered, that is, the phosphoric acid treatment solution. The filtered residue is treated with acetic acid solution for 8-12 hours, and then filtered. The residue is washed with water and dried to obtain the eggshell membrane;

[0006] S2. dissolving the eggshell membrane obtained in step S1 with hexafluoroisopropanol, adding poly-lysine, and electrospinning into a nanofiber membrane;

[0007] S3. After adding urotropine to the phosphoric acid treatment solution obtained in step S1, the nanofiber membrane obtained in step S2 is placed therein for hydrothermal reaction to obtain a nanofiber membrane with hydroxyapatite nanowires deposited thereon;

[0008] S4. Add lactic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to 0.1 mol / L 2-morpholineethanesulfonic acid buffer at pH 5.5, mix thoroughly for pre-reaction, add the nanofiber membrane deposited with hydroxyapatite nanowires prepared in step S3, immerse in reaction, take out and freeze-dry to finally obtain a bone repair membrane.

[0009] Preferably, in step S1, the feeding ratio of the phosphoric acid solution, acetic acid solution and poultry eggshells is (100 - 120) ml : (100 - 200) ml : 10 g.

[0010] Preferably, in step S1, the concentration of the phosphoric acid solution is 0.6 - 1.0% v / v, and the concentration of the acetic acid solution is 3 - 5% v / v.

[0011] Preferably, in step S2, the feeding ratio of the eggshell membrane, hexafluoroisopropanol and polylysine is (800 - 1200) mg : 10 ml : (200 - 300) mg.

[0012] Preferably, in step S2, the electrospinning conditions are a voltage of 20 - 26 kV, a receiving distance of 10 - 14 cm, and an injection rate of 0.5 - 0.7 ml / h.

[0013] Preferably, in step S3, the feeding ratio of the phosphoric acid treatment solution, hexamethylenetetramine, and nanofiber membrane is (24 - 30) ml : (1.0 - 1.7) g : 20 mg.

[0014] Preferably, in step S3, the hydrothermal reaction temperature is 180 - 200 °C, and the time is 4 - 8 h.

[0015] Preferably, in step S4, the dosage ratio of lactic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, 0.1 mol / L 2-morpholinoethanesulfonic acid buffer solution at pH 5.5, and the nanofiber membrane deposited with hydroxyapatite nanowires is (25 - 40) μl : (100 - 150) mg : (50 - 100) mg : 10 ml : (5 - 10) mg.

[0016] Preferably, in step S4, the pre-reaction temperature is 0 - 8 °C, and the time is 10 - 15 min; the temperature of the impregnation reaction is 15 - 20 °C, and the time is 2 - 4 h.

[0017] The present invention also provides a bone repair membrane prepared by the above preparation method.

[0018] Compared with the prior art, the present invention utilizes the natural calcium component of eggshells. After being dissolved by phosphoric acid, hydroxyapatite nanowires are formed on the surface of the reconstructed eggshell membrane protein nanofiber membrane through hydrothermal synthesis to stimulate osteoblast differentiation and promote the repair of defective tissues (hydroxyapatite can promote osteoblast differentiation and has a stimulating or inducing effect on bone hyperplasia. The nanowire structure has a larger specific surface area and can enrich more precursor cells of osteoblasts, namely pluripotent mesenchymal stem cells). By incorporating polylysine into the nanofiber membrane and grafting lactylation on its amino group, it can slowly release lactic acid. Through intracellular lactic acid accumulation, the lactylation modification process of histones is activated, promoting cell differentiation and tissue repair. The use of amidation to achieve lactylation has more controllable amide bond hydrolysis than ester hydrolysis of polylactic acid when releasing lactic acid, avoiding the inflammatory reaction caused by a large amount of lactic acid release. After the gradual release of lactic acid, polylysine can also promote the adhesion of osteoblasts on the membrane, realizing multi-point radial bone reconstruction of osteoblasts on the membrane and greatly shortening the bone repair time. Detailed implementation manners

[0019] To further understand the present invention, the preferred implementation schemes of the present invention are described below in conjunction with embodiments. However, it should be understood that these descriptions are only for further explaining the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0020] Example 1

[0021] A bone repair membrane and its preparation method, comprising the following steps:

[0022] 1. 10 g of poultry eggshells are sequentially treated with 110 ml of a phosphoric acid solution with a concentration of 0.8% v / v for 5 h, then filtered, and the filtrate is recovered as the phosphoric acid treatment solution. The filtered residue is treated with 140 ml of an acetic acid solution with a concentration of 4% v / v for 10 h, then filtered again, and the filtered residue is washed with water and dried to obtain an eggshell membrane;

[0023] 2. 1100 mg of the eggshell membrane obtained in step 1 is dissolved in 10 ml of hexafluoroisopropanol. After adding 240 mg of polylysine, electrospinning is carried out at a voltage of 23 kV, a receiving distance of 12 cm, and an injection rate of 0.6 ml / h to form a nanofiber membrane;

[0024] 3. 26 ml of the phosphoric acid treatment solution obtained in step 1 is added with 1.5 g of hexamine, and then 20 mg of the nanofiber membrane prepared in step 2 is put in for hydrothermal reaction at a temperature of 185 °C for 6 h to obtain a nanofiber membrane with hydroxyapatite nanowires deposited thereon;

[0025] 4. Add 37 μl of lactic acid, 120 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 60 mg of N-hydroxysuccinimide to 10 ml of 0.1 mol / L 2-morpholinoethanesulfonic acid buffer solution with a pH of 5.5. Mix well and pre-react at 5 °C for 12 min. Then add 8 mg of the nanofiber membrane deposited with hydroxyapatite nanowires prepared in step 3, and immerse and react at 18 °C for 3 h. Take out and freeze-dry to finally obtain the bone repair membrane.

[0026] Example 2

[0027] A bone repair membrane and its preparation method, including the following steps:

[0028] 1. Treat 10 g of poultry eggshells with 100 ml of phosphoric acid solution with a concentration of 0.6% v / v for 6 h in sequence, then filter, and recover the filtrate, namely the phosphoric acid treatment solution. Treat the filtered residue with 100 ml of acetic acid solution with a concentration of 3% v / v for 12 h, then filter again. Wash the filtered residue with water and dry to obtain the eggshell membrane.

[0029] 2. Dissolve 800 mg of the eggshell membrane obtained in step 1 in 10 ml of hexafluoroisopropanol. After adding 200 mg of polylysine, electrospin into a nanofiber membrane at a voltage of 20 kV, a receiving distance of 10 cm, and an injection rate of 0.5 ml / h.

[0030] 3. Add 24 ml of the phosphoric acid treatment solution obtained in step 1 to 1 g of hexamine, then put 20 mg of the nanofiber membrane prepared in step 2 for hydrothermal reaction at a temperature of 180 °C for 8 h to obtain the nanofiber membrane deposited with hydroxyapatite nanowires.

[0031] 4. Add 25 μl of lactic acid, 100 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 50 mg of N-hydroxysuccinimide to 10 ml of 0.1 mol / L 2-morpholinoethanesulfonic acid buffer solution with a pH of 5.5. Mix well and pre-react at 0 °C for 15 min. Then add 5 mg of the nanofiber membrane deposited with hydroxyapatite nanowires prepared in step 3, and immerse and react at 15 °C for 4 h. Take out and freeze-dry to finally obtain the bone repair membrane.

[0032] Example 3

[0033] A bone repair membrane and its preparation method, including the following steps:

[0034] 1. Treat 10 g of poultry eggshells with 120 ml of phosphoric acid solution with a concentration of 1.0% v / v for 4 h in sequence, then filter, and recover the filtrate, namely the phosphoric acid treatment solution. Treat the filtered residue with 200 ml of acetic acid solution with a concentration of 5% v / v for 8 h, then filter again. Wash the filtered residue with water and dry to obtain the eggshell membrane.

[0035] 2. Dissolve 1200 mg of the eggshell membrane obtained in step 1 in 10 ml of hexafluoroisopropanol. After adding 300 mg of polylysine, electrospin it into a nanofiber membrane at a voltage of 26 kV, a receiving distance of 14 cm, and an injection rate of 0.7 ml / h.

[0036] 3. Add 1.7 g of hexamine to 30 ml of the phosphoric acid treatment solution obtained in step 1, then put 20 mg of the nanofiber membrane prepared in step 2 for hydrothermal reaction at a temperature of 200 °C for 4 h to obtain a nanofiber membrane with hydroxyapatite nanowires deposited thereon.

[0037] 4. Add 40 μl of lactic acid, 150 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 100 mg of N-hydroxysuccinimide to 10 ml of 0.1 mol / L 2-(N-morpholino)ethanesulfonic acid buffer solution with a pH of 5.5. After fully mixing and pre-reacting at 8 °C for 10 min, add 10 mg of the nanofiber membrane with hydroxyapatite nanowires deposited thereon prepared in step 3, and carry out an impregnation reaction at 20 °C for 2 h. Take it out and freeze-dry to finally obtain a bone repair membrane.

[0038] Comparative Example 1 (Changing the lactic acid modification to a polylactic acid membrane)

[0039] A bone repair membrane and its preparation method, comprising the following steps:

[0040] 1. Treat 10 g of poultry eggshells with 110 ml of a phosphoric acid solution with a concentration of 0.8% v / v for 5 h, then filter to recover the filtrate, which is the phosphoric acid treatment solution. Treat the filtered residue with 140 ml of an acetic acid solution with a concentration of 4% v / v for 10 h, then filter again. Wash the filtered residue with water and dry it to obtain an eggshell membrane.

[0041] 2. Dissolve 1100 mg of the eggshell membrane obtained in step 1 in 10 ml of hexafluoroisopropanol. After adding 240 mg of polylysine, electrospin it into a nanofiber membrane at a voltage of 23 kV, a receiving distance of 12 cm, and an injection rate of 0.6 ml / h.

[0042] 3. Add 1.5 g of hexamine to 26 ml of the phosphoric acid treatment solution obtained in step 1, then put 20 mg of the nanofiber membrane prepared in step 2 for hydrothermal reaction at a temperature of 185 °C for 6 h to obtain a nanofiber membrane with hydroxyapatite nanowires deposited thereon.

[0043] 4. Thermocompression bond the nanofiber membrane with hydroxyapatite nanowires deposited thereon prepared in step 3 and a polylactic acid nanofiber membrane at 105 °C and 0.2 MPa to finally obtain a bone repair membrane.

[0044] Comparative Example 2 (Changing the hydrothermal reaction to a room temperature reaction)

[0045] A bone repair membrane and its preparation method, comprising the following steps:

[0046] 1. After 10 g of poultry eggshells are treated with 110 ml of a phosphoric acid solution with a concentration of 0.8% v / v for 5 h in sequence, filtration is carried out, and the filtrate is recovered as the phosphoric acid treatment solution. The filter residue after filtration is treated with 140 ml of an acetic acid solution with a concentration of 4% v / v for 10 h, and then filtered again. The filter residue after filtration is washed with water and dried to obtain eggshell membrane;

[0047] 2. Dissolve 1100 mg of the eggshell membrane obtained in step 1 with 10 ml of hexafluoroisopropanol. After adding 240 mg of polylysine, electrospinning is carried out at a voltage of 23 kV, a receiving distance of 12 cm, and an injection rate of 0.6 ml / h to form a nanofiber membrane;

[0048] 3. Add 26 ml of the phosphoric acid treatment solution obtained in step 1 to 1.5 g of hexamine, and then put 20 mg of the nanofiber membrane prepared in step 2 into it and react at room temperature for 6 h to obtain a nanofiber membrane with hydroxyapatite deposition;

[0049] 4. Add 37 μl of lactic acid, 120 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, and 60 mg of N-hydroxysuccinimide to 10 ml of 0.1 mol / L 2-(N-morpholino)ethanesulfonic acid buffer solution with a pH of 5.5. After fully mixing and pre-reacting at 5°C for 12 min, add 8 mg of the nanofiber membrane with hydroxyapatite deposition prepared in step 3, and carry out an impregnation reaction at 18°C for 3 h. Take it out and freeze-dry to finally obtain a bone repair membrane.

[0050] Comparative Example 3 (without polylysine)

[0051] A bone repair membrane and its preparation method, comprising the following steps:

[0052] 1. After 10 g of poultry eggshells are treated with 110 ml of a phosphoric acid solution with a concentration of 0.8% v / v for 5 h in sequence, filtration is carried out, and the filtrate is recovered as the phosphoric acid treatment solution. The filter residue after filtration is treated with 140 ml of an acetic acid solution with a concentration of 4% v / v for 10 h, and then filtered again. The filter residue after filtration is washed with water and dried to obtain eggshell membrane;

[0053] 2. Dissolve 1100 mg of the eggshell membrane obtained in step 1 with 10 ml of hexafluoroisopropanol, and electrospin it into a nanofiber membrane at a voltage of 23 kV, a receiving distance of 12 cm, and an injection rate of 0.6 ml / h;

[0054] 3. Add 26 ml of the phosphoric acid treatment solution obtained in step 1 to 1.5 g of hexamine, and then put 20 mg of the nanofiber membrane prepared in step 2 into it for hydrothermal reaction. The temperature is 185°C and the time is 6 h to obtain a nanofiber membrane with hydroxyapatite nanowire deposition;

[0055] 4. 37 μl of lactic acid, 120 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 60 mg of N-hydroxysuccinimide were added to 10 ml of 0.1 mol / L 2-morpholinoethanesulfonic acid buffer solution with a pH of 5.5. After thorough mixing and pre-reacting at 5°C for 12 min, 8 mg of the nanofiber membrane deposited with hydroxyapatite nanowires prepared in step 3 was added, and the impregnation reaction was carried out at 18°C for 3 h. After taking it out and freeze-drying, a bone repair membrane was finally obtained.

[0056] Femoral defect healing experiment in New Zealand white rabbits

[0057] A femoral defect model was constructed using 6-month-old healthy New Zealand white rabbits, that is, a segmental bone and periosteal defect with a length of 1.5 cm in the middle of the left femur. The bone repair membranes of Examples 1-3 and Comparative Examples 1-3 were used for treatment, with 5 rabbits in each group, and both sides of the defect were fixed. The anteroposterior X-ray films of the left femur were taken every day after the operation to observe the healing of the bone defect, and the healing time was calculated. The results are shown in Table 1.

[0058] Table 1 Healing of femoral defects

[0059]

[0060] As can be seen from Table 1, the healing rate of the femoral defects treated with the bone repair membranes of Examples 1-3 was significantly higher than that of Comparative Examples 1-3. The bone repair membrane of the example promoted the healing of bone cracks through the release of an appropriate amount of lactic acid. Subsequently, the exposed polylysine groups contributed to the adhesion of osteoblasts, and the hydroxyapatite on the surface could also stimulate the differentiation of osteoblasts to promote the repair of the defect tissue. In the bone repair membrane of Comparative Example 1, the lactic acid graft modification was replaced with a polylactic acid membrane, and the esterase hydrolysis rate was relatively large, resulting in a large amount of lactic acid release, leading to chronic inflammation, which greatly affected the proliferation and differentiation of osteoblasts and bone remodeling. In the preparation process of the bone repair membrane of Comparative Example 2, the hydrothermal synthesis reaction was not used. Under high temperature and high pressure conditions, the solubility curve of hydroxyapatite changed, and a nanowire structure could be formed on the membrane surface, which greatly promoted the adhesion and differentiation of osteoblasts. This process was missing in Comparative Example 2. In the bone repair membrane of Comparative Example 3, no polylysine was added, the adhesion of osteoblasts was weakened, and lactic acid could only be grafted on the nanofiber membrane of eggshell membrane protein. There were not enough amino groups for grafting, the lactic acid grafting amount was low, the lactic acid release amount was small, and it could not reach the level of lactylation modification of histone, resulting in a reduction in tissue repair ability.

[0061] Osteoblast adhesion experiment

[0062] Take osteoblasts from the skulls of 3-day-old SD rats and perform primary culture. Inoculate them onto the bone repair membranes of Examples 1-3 and Comparative Examples 1-3 respectively and continue culturing for 4 days. Wash with PBS to remove surface free cells, add digestive solution to wash off the adherent cells, count with a cell counting plate, and calculate the cell adhesion rate with the untreated bone repair membrane as the control. The results are shown in Table 2.

[0063] Table 2 Osteoblast adhesion rate

[0064]

[0065] As can be seen from Table 2, the bone repair membranes of Examples 1-3 have a higher osteoblast adhesion rate, while the bone repair membranes of Comparative Examples 1-3 have a poorer cell adhesion rate. The reason is that the bone repair membrane of Comparative Example 1 releases more lactic acid, and the high lactic acid level induces inflammation, which is extremely unfavorable for cell adhesion; the bone repair membrane of Comparative Example 2 lacks hydroxyapatite nanowires and cannot induce the adhesion and differentiation of osteoblasts; the bone repair membrane of Comparative Example 3 lacks polylysine and has poor cell adhesion.

[0066] The present invention provides an idea and method for a bone repair membrane and its preparation method. There are many specific methods and ways to implement this technical solution. The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be realized by existing technologies.

Claims

1. A preparation method of a bone repair membrane, characterized in that, It includes the following steps: S1. The poultry eggshell is treated with a phosphoric acid solution for 4 - 6 h in sequence, then filtered, and the filtrate is recovered. The filter residue after filtration is treated with an acetic acid solution for 8 - 12 h, then filtered again, and the filter residue is washed with water and dried to obtain an eggshell membrane; S2. The eggshell membrane obtained in step S1 is dissolved in hexafluoroisopropanol, and after adding polylysine, it is electrospun into a nanofiber membrane; S3. Urotropine and the nanofiber membrane are added to the filtrate, and a hydrothermal reaction is carried out to obtain a nanofiber membrane with hydroxyapatite nanowires deposited thereon; S4. Lactic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide are added to a 0.1 mol / L 2-morpholinoethanesulfonic acid buffer solution with a pH of 5.

5. After thorough mixing and pre-reaction, the nanofiber membrane with hydroxyapatite nanowires deposited thereon prepared in step S3 is added, and an impregnation reaction is carried out. Then it is taken out and freeze-dried to obtain a bone repair membrane.

2. The preparation method according to claim 1, characterized in that, In step S1, the feeding ratio of the phosphoric acid solution, acetic acid solution, and poultry eggshell is (100 - 120) mL : (100 - 200) mL : 10 g.

3. The preparation method according to claim 1, characterized in that In step S1, the concentration of the phosphoric acid solution is 0.6 - 1.0% v / v, and the concentration of the acetic acid solution is 3 - 5% v / v.

4. The preparation method according to claim 1, wherein, In step S2, the feeding ratio of the eggshell membrane, hexafluoroisopropanol, and polylysine is (800 - 1200) mg : 10 mL : (200 - 300) mg.

5. The preparation method according to claim 1, wherein In step S2, the electrospinning conditions are a voltage of 20 - 26 kV, a receiving distance of 10 - 14 cm, and an injection rate of 0.5 - 0.7 mL / h.

6. The preparation method according to claim 1, characterized in that, In step S3, the feeding ratio of the phosphoric acid treatment solution, urotropine, and nanofiber membrane is (24 - 30) mL : (1.0 - 1.7) g : 20 mg.

7. The preparation method according to claim 1, characterized in that, In step S3, the hydrothermal reaction temperature is 180 - 200 °C, and the time is 4 - 8 h.

8. The preparation method according to claim 1, characterized in that, In step S4, the dosage ratio of lactic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, the 0.1 mol / L 2-morpholinoethanesulfonic acid buffer solution with a pH of 5.5, and the nanofiber membrane with hydroxyapatite nanowires deposited thereon is (25 - 40) μL : (100 - 150) mg : (50 - 100) mg : 10 mL : (5 - 10) mg.

9. The preparation method according to claim 1, characterized in that, In step S4, the pre-reaction temperature is 0 - 8 °C, and the time is 10 - 15 min; the temperature of the impregnation reaction is 15 - 20 °C, and the time is 2 - 4 h.

10. The bone repair membrane is prepared by the preparation method according to any one of claims 1 - 9.

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