ASP / BFP-1 sustained-release nanofiber membrane, preparation method and application thereof

Abstract

The application belongs to the technical field of regenerative medicine, and particularly relates to an ASP / BFP-1 sustained-release nanofiber membrane and a preparation method and application thereof. In the initial stage of bone repair, matrix metalloproteinase-2 (MMP-2) secreted by a large number of stem cells can effectively degrade gelatin and polylysine in the nanofiber membrane coating, so as to sequentially release aspirin and bone formation peptide-1 in the drug-loaded coating. The released aspirin can control inflammation, and the bone formation peptide-1 can enhance the osteogenic potential of stem cells and guide bone tissue regeneration.

Description

Technical Field

[0001] This invention belongs to the field of regenerative medicine technology, specifically relating to an ASP / BFP-1 sustained-release nanofiber membrane, its preparation method, and its application. Background Technology

[0002] Periodontal bone defects caused by chronic periodontitis not only lead to functional limitations and aesthetic defects in patients, but also affect their overall health. Therefore, efficient repair of periodontal bone defects has always been a hot and challenging issue in the field of oral medicine. Currently, guided tissue regeneration (GTR) is often used in clinical practice to repair periodontal bone defects, but existing GTR membranes lack biomimetic design and only serve as a simple physical barrier, without anti-inflammatory or bone-promoting effects.

[0003] In the current bone repair industry, the mainstream bone repair methods include bone grafting, non-degradable GTR membranes such as titanium mesh, and degradable GTR membranes. However, these methods all have certain limitations. For example, autologous bone grafting has a high probability of complications, allogeneic bone grafting carries the risk of immune rejection and disease transmission, non-degradable GTR membranes such as titanium mesh require secondary surgery, causing multiple injuries, and existing degradable GTR membranes are merely physical barriers without anti-inflammatory or bone-forming functions. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides an ASP / BFP-1 sustained-release nanofiber membrane, its preparation method, and its application. The ASP / BFP-1 sustained-release nanofiber membrane provided by this invention has anti-inflammatory and bone-forming functions.

[0005] This invention provides an ASP / BFP-1 sustained-release nanofiber membrane, comprising a nanofiber membrane, a first drug loading layer loaded on the surface and pores of the nanofibers, and a second drug loading layer loaded on the first drug loading layer;

[0006] The nanofiber membrane comprises biomimetic nano-hydroxyapatite and polycaprolactone.

[0007] The first drug-loaded layer includes a plurality of first drug-loaded units; each first drug-loaded unit includes alternating layers of a first polylysine-based drug-loaded layer and a first gelatin-based drug-loaded layer; the innermost and outermost layers of the first drug-loaded unit are both first polylysine-based drug-loaded layers; the components of the first polylysine-based drug-loaded layer include bone morphogenetic peptide-1 and polylysine; the components of the first gelatin-based drug-loaded layer include bone morphogenetic peptide-1 and gelatin.

[0008] The second drug-loaded layer includes a plurality of second drug-loaded units; each second drug-loaded unit includes a second polylysine-based drug-loaded layer and a second gelatin-based drug-loaded layer stacked sequentially; the innermost and outermost layers of the second drug-loaded unit are both second polylysine-based drug-loaded layers;

[0009] The second polylysine-based drug-loaded layer includes aspirin and polylysine; the second gelatin-based drug-loaded layer includes aspirin, gelatin, and genipin.

[0010] Preferably, the first polylysine-based drug-carrying layer in the first drug-carrying unit has 16 layers; the first gelatin-based drug-carrying layer has 15 layers.

[0011] Preferably, the second polylysine-based drug-carrying layer in the second drug-carrying unit has 6 layers; the second gelatin-based drug-carrying layer has 5 layers.

[0012] The present invention also provides a method for preparing the ASP / BFP-1 sustained-release nanofiber membrane according to any one of the claims, comprising the following steps:

[0013] A solution of biomimetic nano-hydroxyapatite and polycaprolactone was mixed to obtain an electrospinning solution.

[0014] The electrospinning solution was sequentially electrospinned and dried to obtain a nanofiber membrane.

[0015] The nanofiber membrane was first immersed in a mixed solution of poly-L-lysine and bone morphogenetic peptide-1 to obtain the first composite nanofiber membrane.

[0016] The first composite nanofiber membrane was immersed in a mixed solution of gelatin and bone morphogenetic peptide-1 to obtain the second composite nanofiber membrane.

[0017] The second composite nanofiber membrane was immersed in a mixed solution of poly-L-lysine and bone morphogenetic peptide-1 to obtain the third composite nanofiber membrane.

[0018] The second and third immersions were repeated 15 times in sequence to obtain a nanofiber membrane loaded with the first drug-loaded layer.

[0019] The nanofiber membrane loaded with the first drug-carrying layer was immersed in a mixed solution of poly-L-lysine and aspirin to obtain a fourth composite nanofiber membrane.

[0020] The fourth composite nanofiber membrane was immersed in a mixed solution of gelatin and aspirin to obtain the fifth composite nanofiber membrane.

[0021] The fifth composite nanofiber membrane was immersed in a mixed solution of poly-L-lysine and aspirin to obtain the sixth composite nanofiber membrane.

[0022] The fifth and sixth immersions were repeated five times in sequence to obtain a nanofiber membrane loaded with the second drug-loaded layer.

[0023] The nanofiber membrane loaded with the second drug-carrying layer was mixed with genipin solution and subjected to a cross-linking reaction to obtain the ASP / BFP-1 sustained-release nanofiber membrane.

[0024] Preferably, the solvent in the polycaprolactone solution is hexafluoro-2-propanol; the mass concentration of polycaprolactone in the polycaprolactone solution is 8-15%; and the mass concentration of biomimetic nano-hydroxyapatite in the electrospinning solution is 8-12%.

[0025] Preferably, the mass concentration of polylysine in the mixed solution of polylysine and bone morphogenetic peptide-1 is 0.8-1.5%; and the concentration of bone morphogenetic peptide-1 in the mixed solution of polylysine and bone morphogenetic peptide-1 is 80-120 μg / mL.

[0026] Preferably, the mass concentration of gelatin in the mixed solution of gelatin and bone morphogenetic peptide-1 is 0.8-1.5%; and the concentration of bone morphogenetic peptide-1 in the mixed solution of gelatin and bone morphogenetic peptide-1 is 80-120 μg / mL.

[0027] Preferably, the concentration of aspirin in the mixed solution of polylysine and aspirin is 80-120 μg / mL; the concentration of aspirin in the mixed solution of gelatin and aspirin is 80-120 μg / mL.

[0028] Preferably, the crosslinking reaction is carried out at a temperature of 20–30°C for 6–10 hours.

[0029] The present invention also provides the application of the above-described ASP / BFP-1 sustained-release nanofiber membrane or the ASP / BFP-1 sustained-release nanofiber membrane prepared by the above-described preparation method in the preparation of materials for treating bone defects and repairing them.

[0030] This invention provides an ASP / BFP-1 sustained-release nanofiber membrane, comprising a nanofiber membrane, a first drug-loaded layer loaded on the surface and pores of the nanofibers, and a second drug-loaded layer loaded on the first drug-loaded layer; the nanofiber membrane comprises biomimetic nano-hydroxyapatite and polycaprolactone; the first drug-loaded layer comprises a plurality of first drug-loaded units; each first drug-loaded unit comprises alternating layers of a first polylysine-based drug-loaded layer and a first gelatin-based drug-loaded layer; the innermost and outermost layers of the first drug-loaded unit are both first polylysine-based drug-loaded layers; the first polylysine-based drug-loaded layer... The polylysine-based drug-loaded layer comprises bone morphogenetic peptide-1 and polylysine; the first gelatin-based drug-loaded layer comprises bone morphogenetic peptide-1 and gelatin; the second drug-loaded layer comprises several second drug-loaded units; each second drug-loaded unit comprises a second polylysine-based drug-loaded layer and a second gelatin-based drug-loaded layer stacked sequentially; the innermost and outermost layers of the second drug-loaded unit are both second polylysine-based drug-loaded layers; the second polylysine-based drug-loaded layer comprises aspirin and polylysine; the second gelatin-based drug-loaded layer comprises aspirin, gelatin, and genipin. During the early stages of bone repair, the matrix metalloproteinase 2 (MMP-2) secreted in large quantities by stem cells can effectively degrade the gelatin and polylysine in the nanofiber membrane coating, thereby sequentially releasing aspirin and bone morphogenetic peptide-1 from the drug-loaded coating; the released aspirin can control inflammation, and bone morphogenetic peptide-1 can enhance the osteogenic potential of stem cells and guide bone tissue regeneration. Attached Figure Description

[0031] Figure 1 Scanning electron microscopy images of BMSCs after 6 hours of cell culture in Test Example 1;

[0032] Figure 2 Fluorescent staining images of the BMSCs cytoskeleton after 3 and 6 hours of cell culture in Example 2;

[0033] Figure 3 The CCK8 results for cells cultured in Example 3 at days 1, 4, and 7 are shown in the figure.

[0034] Figure 4 Three-dimensional reconstructed images of skull defects in test case four;

[0035] Figure 5 Semi-quantitative analysis charts of bone volume fraction (BV / TV) and bone mineral density (BMD) for test case four;

[0036] Figure 6 Microscopic observations of different staining methods in test example five. Detailed Implementation

[0037] This invention provides an ASP / BFP-1 sustained-release nanofiber membrane, comprising a nanofiber membrane, a first drug loading layer loaded on the surface and pores of the nanofibers, and a second drug loading layer loaded on the first drug loading layer;

[0038] The nanofiber membrane comprises biomimetic nano-hydroxyapatite and polycaprolactone.

[0039] The first drug-loaded layer includes a plurality of first drug-loaded units; each first drug-loaded unit includes alternating layers of a first polylysine-based drug-loaded layer and a first gelatin-based drug-loaded layer; the innermost and outermost layers of the first drug-loaded unit are both first polylysine-based drug-loaded layers; the components of the first polylysine-based drug-loaded layer include bone morphogenetic peptide-1 and polylysine; the components of the first gelatin-based drug-loaded layer include bone morphogenetic peptide-1 and gelatin.

[0040] The second drug-loaded layer includes a plurality of second drug-loaded units; each second drug-loaded unit includes a second polylysine-based drug-loaded layer and a second gelatin-based drug-loaded layer stacked sequentially; the innermost and outermost layers of the second drug-loaded unit are both second polylysine-based drug-loaded layers;

[0041] The second polylysine-based drug-loaded layer includes aspirin and polylysine; the second gelatin-based drug-loaded layer includes aspirin, gelatin, and genipin.

[0042] In this invention, the ASP / BFP-1 sustained-release nanofiber membrane includes a nanofiber membrane, a first drug loading layer loaded on the surface and pores of the nanofibers, and a second drug loading layer loaded on the first drug loading layer.

[0043] In this invention, the nanofiber membrane comprises biomimetic nano-hydroxyapatite and polycaprolactone.

[0044] In this invention, the drug-carrying layer includes a plurality of first drug-carrying units, each first drug-carrying unit including a first polylysine-based drug-carrying layer and a first gelatin-based drug-carrying layer that are alternately stacked.

[0045] In this invention, the components of the first polylysine-based drug-loaded layer include bone morphogenetic peptide-1 and polylysine.

[0046] In this invention, the components of the first gelatin-based drug-loaded layer include bone-forming peptide-1 and gelatin. Preferably, the first drug-loaded unit comprises 16 layers of a first polylysine-based drug-loaded layer and 15 layers of a first gelatin-based drug-loaded layer.

[0047] In this invention, the innermost and outermost layers of the first drug-loading unit are both first polylysine-based drug-loading layers.

[0048] In this invention, the second drug-loaded layer includes a plurality of second drug-loaded units; each second drug-loaded unit includes a second polylysine-based drug-loaded layer and a second gelatin-based drug-loaded layer stacked sequentially; in this invention, the second polylysine-based drug-loaded layer includes aspirin and polylysine.

[0049] In this invention, the second gelatin-based drug-loaded layer comprises aspirin, gelatin, and genipin.

[0050] In this invention, the second drug-carrying unit preferably comprises 6 layers of second polylysine-based drug-carrying layers and 5 layers of second gelatin-based drug-carrying layers.

[0051] In this invention, the innermost and outermost layers of the second drug-carrying unit are both second polylysine-based drug-carrying layers.

[0052] This invention also provides a method for preparing the above-described ASP / BFP-1 sustained-release nanofiber membrane, comprising the following steps:

[0053] A solution of biomimetic nano-hydroxyapatite and polycaprolactone was mixed to obtain an electrospinning solution.

[0054] The electrospinning solution was sequentially electrospinned and dried to obtain a nanofiber membrane.

[0055] The nanofiber membrane was first immersed in a mixed solution of poly-L-lysine and bone morphogenetic peptide-1 to obtain the first composite nanofiber membrane.

[0056] The first composite nanofiber membrane was immersed in a mixed solution of gelatin and bone morphogenetic peptide-1 to obtain the second composite nanofiber membrane.

[0057] The second composite nanofiber membrane was immersed in a mixed solution of poly-L-lysine and bone morphogenetic peptide-1 to obtain the third composite nanofiber membrane.

[0058] The second and third immersions were repeated 15 times in sequence to obtain a nanofiber membrane loaded with the first drug-loaded layer.

[0059] The nanofiber membrane loaded with the first drug-carrying layer was immersed in a mixed solution of poly-L-lysine and aspirin to obtain a fourth composite nanofiber membrane.

[0060] The fourth composite nanofiber membrane was immersed in a mixed solution of gelatin and aspirin to obtain the fifth composite nanofiber membrane.

[0061] The fifth composite nanofiber membrane was immersed in a mixed solution of poly-L-lysine and aspirin to obtain the sixth composite nanofiber membrane.

[0062] The fifth and sixth immersions were repeated five times in sequence to obtain a nanofiber membrane loaded with the second drug-loaded layer.

[0063] The nanofiber membrane loaded with the second drug-carrying layer was mixed with genipin solution and subjected to a cross-linking reaction to obtain the ASP / BFP-1 sustained-release nanofiber membrane.

[0064] This invention mixes a solution of biomimetic nano-hydroxyapatite and polycaprolactone to obtain an electrospinning solution.

[0065] In this invention, the biomimetic nano-hydroxyapatite is prepared.

[0066] In this invention, the preparation of the biomimetic nano-hydroxyapatite preferably includes the following steps:

[0067] CaCl2, water, and dopamine hydrochloride are mixed to obtain a CaCl2 solution in which dopamine is dissolved.

[0068] An aqueous solution of Na2HPO4·12H2O was added dropwise to a CaCl2 solution containing dopamine to grow apatite, thus obtaining biomimetic nano-hydroxyapatite.

[0069] In this invention, the concentration of dopamine in the CaCl2 solution containing dopamine is preferably 2 mg / mL. In this invention, the concentration of CaCl2 in the CaCl2 solution containing dopamine is preferably 16-20 mg / mL. In this invention, the molar ratio of CaCl2 to Na2HPO4·12H2O is preferably 1.67:1.

[0070] In this invention, during the dropwise addition process, the pH value of the CaCl2 solution containing dopamine is preferably 8.5. In this invention, the pH adjusting agent is preferably a Tris buffer.

[0071] In this invention, the apatite growth is preferably carried out under stirring conditions, the apatite growth temperature is preferably 60°C, and the growth time is preferably 12 hours.

[0072] After the apatite is grown, the present invention further includes aging, centrifuging, washing, and drying the apatite-grown system sequentially. In this invention, the aging temperature is preferably room temperature, and the aging time is preferably 24 hours. In this invention, the washing is preferably a sequential process of washing with deionized water and washing with anhydrous ethanol, and the washing is preferably performed until the supernatant becomes clear. In this invention, the drying temperature is preferably 60°C, and the drying time is preferably ≥48 hours.

[0073] In this invention, the solvent in the polycaprolactone solution is hexafluoro-2-propanol; the mass concentration of polycaprolactone in the polycaprolactone solution is preferably 8-15%, more preferably 10%; the mass concentration of biomimetic nano-hydroxyapatite in the electrospinning solution is preferably 8-12%, more preferably 10%.

[0074] In this invention, the electrospinning conditions preferably include: an applied voltage of 14 kV, a collection distance of 10 cm, and a feed rate of 1.0 mL / h.

[0075] In this invention, the drying is preferably vacuum drying, and the vacuum drying temperature is preferably room temperature. In this invention, the vacuum drying is preferably used to remove residual organic solvents.

[0076] After obtaining the nanofiber membrane, the present invention first immerses the nanofiber membrane in a mixed solution of polylysine and bone morphogenetic peptide-1 to obtain a first composite nanofiber membrane.

[0077] In this invention, the mass concentration of polylysine in the mixed solution of polylysine and bone-forming peptide-1 is preferably 0.8-1.5%, more preferably 1%. In this invention, the concentration of bone-forming peptide-1 in the mixed solution of polylysine and bone-forming peptide-1 is preferably 80-120 μg / mL, more preferably 100 μg / mL.

[0078] In this invention, the temperature of the first soaking is preferably 22-27°C, more preferably 25°C; the time is preferably 6-10 hours, more preferably 8 hours.

[0079] In this invention, after the first soaking, it is preferable to further wash the nanofiber membrane after the first soaking three times with deionized water.

[0080] After obtaining the first composite nanofiber membrane, the present invention further immerses the first composite nanofiber membrane in a mixed solution of gelatin and bone morphogenetic peptide-1 to obtain the second composite nanofiber membrane.

[0081] In this invention, the mass concentration of gelatin in the mixed solution of gelatin and bone morphogenetic peptide-1 is preferably 0.8-1.5%, more preferably 1%; the concentration of bone morphogenetic peptide-1 in the mixed solution of gelatin and bone morphogenetic peptide-1 is preferably 80-120 μg / mL, more preferably 100 μg / mL.

[0082] In this invention, the temperature of the second soaking is preferably 22-27°C, more preferably 25°C; the time is preferably 10-20 min, more preferably 15 min.

[0083] In this invention, after the second soaking, it is preferable to further wash the nanofiber membrane after the second soaking three times with deionized water.

[0084] After obtaining the second composite nanofiber membrane, the present invention further immerses the second composite nanofiber membrane in a mixed solution of polylysine and bone morphogenetic peptide-1 to obtain the third composite nanofiber membrane.

[0085] In this invention, the temperature of the third soaking is preferably 22-27°C, more preferably 25°C; the time is preferably 10-20 min, more preferably 15 min.

[0086] In this invention, after the third soaking, it is preferable to further wash the nanofiber membrane after the third soaking three times with deionized water.

[0087] The second and third soaking processes were repeated 15 times in sequence to obtain a nanofiber membrane loaded with the first drug-loaded layer.

[0088] After obtaining the nanofiber membrane loaded with the first drug-loaded layer, the present invention further immerses the nanofiber membrane loaded with the first drug-loaded layer in a mixed solution of polylysine and aspirin to obtain a fourth composite nanofiber membrane.

[0089] In this invention, the mass concentration of polylysine in the mixed solution of polylysine and aspirin is preferably 0.8-1.5%, more preferably 1%. In this invention, the concentration of aspirin in the mixed solution of polylysine and aspirin is preferably 80-120 μg / mL, more preferably 100 μg / mL.

[0090] In this invention, the temperature of the fourth soaking is preferably 22-27°C, more preferably 25°C; the time is preferably 6-10 hours, more preferably 8 hours.

[0091] In this invention, after the fourth soaking, it is preferable to further wash the nanofiber membrane after the fourth soaking three times with deionized water.

[0092] After obtaining the fourth composite nanofiber membrane, the present invention further immerses the fourth composite nanofiber membrane in a mixed solution of gelatin and aspirin to obtain the fifth composite nanofiber membrane.

[0093] In this invention, the mass concentration of gelatin in the mixed solution of gelatin and aspirin is preferably 0.8-1.5%, more preferably 1%. In this invention, the concentration of aspirin in the mixed solution of gelatin and aspirin is preferably 80-120 μg / mL, more preferably 100 μg / mL.

[0094] In this invention, the temperature of the fifth soaking is preferably 22-27°C, more preferably 25°C; the time is preferably 10-20 min, more preferably 15 min.

[0095] In this invention, after the fifth soaking, it is preferable to further wash the nanofiber membrane after the fifth soaking three times with deionized water.

[0096] After obtaining the fifth composite nanofiber membrane, the present invention further soaks the fifth composite nanofiber membrane in a mixed solution of poly-lysine and aspirin to obtain the sixth composite nanofiber membrane.

[0097] In this invention, the temperature of the sixth soaking is preferably 22-27°C, more preferably 25°C; the time is preferably 10-20 min, more preferably 15 min.

[0098] In this invention, after the sixth soaking, it is preferable to further wash the nanofiber membrane after the sixth soaking three times with deionized water.

[0099] The fifth and sixth immersions were repeated five times in sequence to obtain a nanofiber membrane loaded with the second drug-loaded layer.

[0100] After obtaining the nanofiber membrane loaded with the second drug-loaded layer, the present invention mixes the nanofiber membrane loaded with the second drug-loaded layer with a genipin solution and performs a crosslinking reaction to obtain the ASP / BFP-1 sustained-release nanofiber membrane.

[0101] In this invention, the mass concentration of the genipin solution is preferably 0.8-1.2%, more preferably 1.0%.

[0102] In this invention, the temperature of the crosslinking reaction is preferably 20-30°C, more preferably 25°C, and the time is preferably 6-10 hours, more preferably 8 hours.

[0103] The present invention also provides the application of the above-described ASP / BFP-1 sustained-release nanofiber membrane or the ASP / BFP-1 sustained-release nanofiber membrane prepared by the above-described preparation method in the preparation of materials for treating bone defects and repairing them.

[0104] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0105] Example 1

[0106] Preparation of biomimetic nano-hydroxyapatite:

[0107] 3.7068g of CaCl2 and 7.1628g of Na2HPO4·12H2O were weighed and dissolved in deionized water to obtain CaCl2 solution and Na2HPO4 solution, respectively.

[0108] 0.8 g of dopamine hydrochloride was weighed and added to a CaCl2 solution, resulting in a final dopamine concentration of 2 mg / mL. Na2HPO4 was then slowly added dropwise to the dopamine-dissolved CaCl2 solution. During the addition of Na2HPO4, the pH of the reaction solution was maintained at 8.5 using Tris buffer, and the mixture was continuously stirred at 60 °C for 12 h. Using dopamine as a template, apatite growth occurred through the chelation of calcium ions by dopamine hydrochloride.

[0109] After the reaction was complete, the mixed solution was aged at room temperature for 24 hours and then centrifuged. The precipitate obtained by centrifugation was washed alternately with deionized water and anhydrous ethanol until the supernatant became clear. Then, the precipitate was placed in a 60℃ oven and dried for 48 hours to obtain biomimetic nano-hydroxyapatite (tHA).

[0110] Polycaprolactone (PVA) with a weight-average molecular weight of 80,000 g / mol was dissolved in hexafluoro-2-propanol to obtain a PVA solution with a mass concentration of 10%. Then, total hydroxylamine (tHA) was suspended in the PVA solution by ultrasonication and vigorous stirring, resulting in a tHA mass concentration of 10% in the electrospinning solution. The electrospinning solution was then subjected to electrospinning with the following parameters: applied voltage of 14 kV, collection distance of 10 cm, and feed rate of 1.0 ml / h. The resulting electrospun fiber web was vacuum dried at room temperature to remove residual organic solvents, yielding a nanofiber membrane.

[0111] Polylysine was added to a bone morphogenetic peptide-1 solution, the beaker was covered with plastic wrap, and stirred overnight (8 h) to obtain a mixed solution of polylysine and bone morphogenetic peptide-1. The mass fraction of PLL in the mixed solution was 1%, and the concentration of bone morphogenetic peptide-1 was 100 μg / mL.

[0112] The nanofiber membrane was immersed in a mixed solution of polylysine and bone morphogenetic peptide-1 for 8 hours and then washed three times with deionized water to obtain the first composite nanofiber membrane.

[0113] The first composite nanofiber membrane was immersed in a mixed solution of gelatin and bone morphogenetic peptide-1 for 15 min, and then washed three times with deionized water to obtain the second composite nanofiber membrane. The mass concentration of gelatin in the mixed solution of gelatin and bone morphogenetic peptide-1 was 1%, and the concentration of bone morphogenetic peptide-1 was 100 μg / mL.

[0114] The second composite nanofiber membrane was immersed in a mixed solution of polylysine and bone morphogenetic peptide-1 for 15 minutes, and then washed three times with deionized water to obtain the third composite nanofiber membrane.

[0115] The second and third soaking processes were repeated 15 times in sequence to obtain a nanofiber membrane loaded with the first drug-loaded layer.

[0116] The nanofiber membrane loaded with the first drug-loaded layer was immersed in a mixed solution of polylysine and aspirin for 8 hours and then washed three times with deionized water to obtain the fourth composite nanofiber membrane. The mass fraction of PLL in the mixed solution was 1% and the concentration of aspirin was 100 μg / mL.

[0117] The fourth composite nanofiber membrane was immersed in a mixed solution of gelatin and aspirin for 15 min, and then washed three times with deionized water to obtain the fifth composite nanofiber membrane. The mass concentration of gelatin in the mixed solution of gelatin and aspirin was 1%, and the concentration of aspirin was 100 μg / mL.

[0118] The second composite nanofiber membrane was immersed in a mixed solution of poly-L-lysine and aspirin for 15 minutes, and then washed three times with deionized water to obtain the sixth composite nanofiber membrane.

[0119] The fifth and sixth immersions were repeated five times in sequence to obtain a nanofiber membrane loaded with the second drug-loaded layer.

[0120] Comparative Example 1

[0121] Preparation of biomimetic nano-hydroxyapatite:

[0122] 3.7068g of CaCl2 and 7.1628g of Na2HPO4·12H2O were weighed and dissolved in deionized water to obtain CaCl2 solution and Na2HPO4 solution, respectively.

[0123] 0.8 g of dopamine hydrochloride was weighed and added to a CaCl2 solution, resulting in a final dopamine concentration of 2 mg / mL. Na2HPO4 was then slowly added dropwise to the dopamine-dissolved CaCl2 solution. During the addition of Na2HPO4, the pH of the reaction solution was maintained at 8.5 using Tris buffer, and the mixture was continuously stirred at 60 °C for 12 h. Using dopamine as a template, apatite growth occurred through the chelation of calcium ions by dopamine hydrochloride.

[0124] After the reaction was complete, the mixed solution was aged at room temperature for 24 hours and then centrifuged. The precipitate obtained by centrifugation was washed alternately with deionized water and anhydrous ethanol until the supernatant became clear. Then, the precipitate was placed in a 60℃ oven and dried for 48 hours to obtain biomimetic nano-hydroxyapatite (tHA).

[0125] Polycaprolactone (PVA) with a weight-average molecular weight of 80,000 g / mol was dissolved in hexafluoro-2-propanol to obtain a PVA solution with a mass concentration of 10%. Then, total hydroxylamine (tHA) was suspended in the PVA solution by ultrasonication and vigorous stirring, resulting in a tHA mass concentration of 10% in the electrospinning solution. The electrospinning solution was then subjected to electrospinning with the following parameters: applied voltage of 14 kV, collection distance of 10 cm, and feed rate of 1.0 ml / h. The resulting electrospun fiber web was vacuum dried at room temperature to remove residual organic solvents, yielding a nanofiber membrane.

[0126] Polylysine was added to a bone morphogenetic peptide-1 solution, the beaker was covered with plastic wrap, and stirred overnight (8 h) to obtain a mixed solution of polylysine and bone morphogenetic peptide-1. The mass fraction of PLL in the mixed solution was 1%, and the concentration of bone morphogenetic peptide-1 was 100 μg / mL.

[0127] The nanofiber membrane was immersed in a mixed solution of polylysine and bone morphogenetic peptide-1 for 8 hours and then washed three times with deionized water to obtain the first composite nanofiber membrane.

[0128] The first composite nanofiber membrane was immersed in a mixed solution of gelatin and bone morphogenetic peptide-1 for 15 min, and then washed three times with deionized water to obtain the second composite nanofiber membrane. The mass concentration of gelatin in the mixed solution of gelatin and bone morphogenetic peptide-1 was 1%, and the concentration of bone morphogenetic peptide-1 was 100 μg / mL.

[0129] The second composite nanofiber membrane was immersed in a mixed solution of polylysine and bone morphogenetic peptide-1 for 15 minutes, and then washed three times with deionized water to obtain the third composite nanofiber membrane.

[0130] The second and third soaking processes were repeated 15 times in sequence to obtain a nanofiber membrane loaded with the first drug-loaded layer.

[0131] Comparative Example 2

[0132] Preparation of biomimetic nano-hydroxyapatite:

[0133] Weigh out 3.7068g of CaCl2 and 7.1628g of Na2HPO4·12H2O respectively and dissolve them in deionized water to obtain CaCl2 solution and Na2HPO4 solution.

[0134] 0.8 g of dopamine hydrochloride was weighed and added to a CaCl2 solution, resulting in a final dopamine concentration of 2 mg / mL. Na2HPO4 was then slowly added dropwise to the dopamine-dissolved CaCl2 solution. During the addition of Na2HPO4, the pH of the reaction solution was maintained at 8.5 using Tris buffer, and the mixture was continuously stirred at 60 °C for 12 h. Using dopamine as a template, apatite growth occurred through the chelation of calcium ions by dopamine hydrochloride.

[0135] After the reaction was complete, the mixed solution was aged at room temperature for 24 hours and then centrifuged. The precipitate obtained by centrifugation was washed alternately with deionized water and anhydrous ethanol until the supernatant became clear. Then, the precipitate was placed in a 60℃ oven and dried for 48 hours to obtain biomimetic nano-hydroxyapatite (tHA).

[0136] Polycaprolactone (PVA) with a weight-average molecular weight of 80,000 g / mol was dissolved in hexafluoro-2-propanol to obtain a PVA solution with a mass concentration of 10%. Then, total hydroxylamine (tHA) was suspended in the PVA solution by ultrasonication and vigorous stirring, resulting in a tHA mass concentration of 10% in the electrospinning solution. The electrospinning solution was then subjected to electrospinning with the following parameters: applied voltage of 14 kV, collection distance of 10 cm, and feed rate of 1.0 ml / h. The resulting electrospun fiber web was vacuum dried at room temperature to remove residual organic solvents, yielding a nanofiber membrane.

[0137] Comparative Example 3

[0138] The only difference from Example 1 is that bone morphogenetic peptide-1 and aspirin are not added.

[0139] Comparative Example 4

[0140] The only difference from Example 1 is that aspirin is not added.

[0141] Test Example 1

[0142] Bone marrow mesenchymal stem cells (BMSCs) were seeded onto the nanofiber membranes prepared in Examples 1 and 1-2, and cultured for 6 hours. The culture medium was then discarded, and the cells were washed three times with PBS. Cells were fixed with 2.5% glutaraldehyde solution and placed in a 4°C freezer. After dehydration with a gradient of ethanol solutions, the samples were critically dried, sputter-coated with gold, and observed and imaged using a scanning electron microscope. (See attached image). Figure 1 ,from Figure 1 It can be seen that the cells can extend pseudopodia more fully on the surface of the ASP / BFP-1 sustained-release nanofiber membrane prepared in Example 1, indicating that it has better adhesion and bonding.

[0143] Test Example 2

[0144] BMSCs were seeded onto the nanofiber membranes prepared in Examples 1 and 1-2 and cultured for 3 h and 6 h, respectively. The culture medium was discarded, and the cells were washed three times with PBS. Cells were fixed with 4% paraformaldehyde for 10 minutes, the liquid was discarded, and the cells were washed with PBS. Triton X-100 was added for 5 min, followed by PBS washing. Phalloidin labeled with fluorescein isothiocyanate was added and incubated at room temperature in the dark for 30 minutes for staining. The liquid was discarded, and the cells were washed three times with PBS. The cells were mounted with a fluorescent mounting medium containing DAPI, observed, and photographed. Results are shown in [Figure number missing]. Figure 2 , Figure 2 In the figures, ① represents the test results of the nanofiber membrane prepared in Comparative Example 2, ② represents the test results of the nanofiber membrane prepared in Comparative Example 1, and ③ represents the test results of the nanofiber membrane prepared in Example 1; from Figure 2 It can be seen that the cytoskeleton on the surface of the nanofiber membrane prepared in Example 1 is more extended.

[0145] Test Example 3

[0146] Cell proliferation was assessed using a cell counting kit (CCK-8). Cells were seeded onto different nanofiber membrane surfaces and cultured for 1, 4, and 7 days. The culture medium was replaced with CCK-8 working solution, and the cells were cultured at 37°C for 2 hours. 100 μl of supernatant from each well was transferred to a new 96-well plate, and absorbance was measured at 450 nm using a microplate reader. The results are shown below. Figure 3 , Figure 3 In the figures, ① represents the test results of the nanofiber membrane prepared in Comparative Example 2, ② represents the test results of the nanofiber membrane prepared in Comparative Example 1, and ③ represents the test results of the nanofiber membrane prepared in Example 1. Figure 3 It can be seen that as the culture time is extended, the proliferation of cells on the surface of the nanofiber membrane prepared in Example 1 is significantly better than that of the simple nanofiber membrane, showing a promoting effect on cell growth. Cells achieve better adhesion and growth on the surface of the nanofiber membrane prepared in Example 1, and the nanofiber membrane prepared in Example 1 has good biocompatibility.

[0147] Test Example 4

[0148] After general anesthetizing SD rats with isoflurane, the rat scalp was prepared and disinfected with povidone-iodine. A linear incision was made along the midline of the skull, and the skin and periosteum were separated using a periosteal elevator to expose the bone surface. A circular bone defect was created using a 5mm diameter trephine at 800 rpm. After removing the free bone tissue in the center, the nanofiber membranes prepared in Example 1 and Comparative Examples 1-4 were implanted into the defective areas, with a control group (without implantation) to completely fill the defect area. The periosteum and skin were sutured in layers with interrupted sutures. Six weeks after implantation, samples were collected by cutting the rat cranial parietal bone with tissue scissors and a blade, fixing it in 4% paraformaldehyde for 48 hours, and then using it for subsequent imaging examinations.

[0149] Fixed rat skull samples were analyzed using microCT. Scans were performed at 70 kV and 112 μA with a resolution of 1024 and a thickness of 0.048 mm. The bone definition range was 315-543 Hounsfield units. After 3D reconstruction, a circular bone defect with a diameter of 5 mm was defined as the region of interest. Bone volume fraction (BV / TV) and bone mineral density (BMD) were calculated using an image analysis system as quantitative analysis indicators. The 3D reconstructed image of the skull defect is shown below. Figure 4 Semi-quantitative analysis charts of bone volume fraction (BV / TV) and bone mineral density (BMD) are shown below. Figure 5 .from Figures 4-5 It can be seen that the nanofiber membrane prepared in Example 1 has a larger area for new bone formation, completely covering the defect area, with the highest bone volume fraction and normal bone density. This proves that the nanofiber membrane prepared in Example 1 has superior bone regeneration promotion performance.

[0150] Test Example 5

[0151] The fixed rat skull samples were dehydrated and cleared, then embedded in paraffin to form a paraffin block. The samples were cut into sections with a thickness of about 6 μm using a paraffin microtome. After spreading the sections in a 38°C water bath, the sections were removed using a glass slide and placed in a preheated 58°C slide oven for baking. After 2 hours, the following staining was performed (the sections need to be heated in a 60°C oven for 2 hours before staining).

[0152] ①H&E dyeing

[0153] Paraffin sections were sequentially immersed in xylene solutions I, II, and III for 15 minutes to ensure complete dewaxing; then placed in a gradient of alcohols (anhydrous ethanol I and II for 5 minutes each, 95% ethanol for 5 minutes each, 80% ethanol for 5 minutes each, and 70% ethanol for 5 minutes each) and tap water for 5 minutes to hydrate; the dewaxed sections were then immersed in hematoxylin staining solution for 2 minutes; after rinsing with running water, they were differentiated in 1% hydrochloric acid alcohol for a few seconds, then rinsed with tap water for 15 minutes to regain blue color; the sections were then placed in eosin staining solution for 2.5 minutes, and rinsed with tap water for 2 minutes; then dehydrated and cleared (immersed in 95% ethanol I and II for 2 seconds each, anhydrous ethanol I and II for 2 seconds each, and xylene I and II for 1 minute each); finally, they were mounted with neutral resin and observed under a microscope.

[0154] ②Masson staining

[0155] Sections were routinely dewaxed to water, stained with prepared Weigert iron hematoxylin for 10 min; then differentiated with acidic ethanol differentiation solution for 10 seconds, rinsed with running water for 10 min; blued with Masson's blue solution for 2 min, rinsed with deionized water for 1 min; stained with Ponceau Flavour Stain for 5 min (in the above operation, a weak acid working solution was prepared according to the ratio of distilled water: weak acid solution = 2:1), then washed with weak acid working solution for 1 min, washed with distilled water for 1 min, washed with phosphomolybdic acid solution for 1 min; washed with the prepared weak acid working solution for 1 min; directly stained with Fast Green staining solution for 1 min; washed with the prepared weak acid working solution for 1 min; placed in 95% ethanol and anhydrous ethanol I and II for rapid dehydration for 10 seconds; soaked in xylene I and II for 1 minute each; mounted with neutral resin, and observed under a microscope.

[0156] ③Sirius Red staining

[0157] Sections were routinely dewaxed to water and stained with Sirius red staining solution; rinsed slightly with running water to remove staining solution from the surface of the sections; cell nuclei were stained with Mayer hematoxylin staining solution; rinsed with running water for 10 min; routinely dehydrated and cleared, mounted with neutral resin, and observed under a polarized light microscope.

[0158] Figure 6 To test the microscopic observations of different staining methods in Example 5, from... Figure 6 It can be seen that the nanofiber membrane prepared in Example 1 better promotes the formation and maturation of bone collagen, the regenerated bone tissue has a higher degree of maturity, and the bone regeneration performance is more excellent.

[0159] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An ASP / BFP-1 sustained-release nanofiber membrane, characterized in that, It includes a nanofiber membrane, a first drug-loaded layer loaded on the surface and pores of the nanofibers, and a second drug-loaded layer loaded on the first drug-loaded layer; The preparation method of the ASP / BFP-1 sustained-release nanofiber membrane includes the following steps: A solution of biomimetic nano-hydroxyapatite and polycaprolactone was mixed to obtain an electrospinning solution. The electrospinning solution was sequentially electrospinned and dried to obtain a nanofiber membrane. The nanofiber membrane was first immersed in a mixed solution of poly-L-lysine and bone morphogenetic peptide-1 to obtain the first composite nanofiber membrane. The first composite nanofiber membrane was immersed in a mixed solution of gelatin and bone morphogenetic peptide-1 to obtain the second composite nanofiber membrane. The second composite nanofiber membrane was immersed in a mixed solution of poly-L-lysine and bone morphogenetic peptide-1 to obtain the third composite nanofiber membrane. The second and third immersions were repeated 15 times each to obtain a nanofiber membrane loaded with the first drug-loaded layer. The nanofiber membrane loaded with the first drug-carrying layer was immersed in a mixed solution of poly-L-lysine and aspirin to obtain a fourth composite nanofiber membrane. The fourth composite nanofiber membrane was immersed in a mixed solution of gelatin and aspirin to obtain the fifth composite nanofiber membrane. The fifth composite nanofiber membrane was immersed in a mixed solution of poly-L-lysine and aspirin to obtain the sixth composite nanofiber membrane. The fifth and sixth immersions were repeated 5 times each to obtain a nanofiber membrane loaded with the second drug-loaded layer. The nanofiber membrane loaded with the second drug-carrying layer was mixed with genipin solution and subjected to a cross-linking reaction to obtain the ASP / BFP-1 sustained-release nanofiber membrane.

2. The method for preparing the ASP / BFP-1 sustained-release nanofiber membrane according to claim 1, characterized in that, Includes the following steps: A solution of biomimetic nano-hydroxyapatite and polycaprolactone was mixed to obtain an electrospinning solution. The electrospinning solution was sequentially electrospinned and dried to obtain a nanofiber membrane. The nanofiber membrane was first immersed in a mixed solution of poly-L-lysine and bone morphogenetic peptide-1 to obtain the first composite nanofiber membrane. The first composite nanofiber membrane was immersed in a mixed solution of gelatin and bone morphogenetic peptide-1 to obtain the second composite nanofiber membrane. The second composite nanofiber membrane was immersed in a mixed solution of poly-L-lysine and bone morphogenetic peptide-1 to obtain the third composite nanofiber membrane. The second and third immersions were repeated 15 times each to obtain a nanofiber membrane loaded with the first drug-loaded layer. The nanofiber membrane loaded with the first drug-carrying layer was immersed in a mixed solution of poly-L-lysine and aspirin to obtain a fourth composite nanofiber membrane. The fourth composite nanofiber membrane was immersed in a mixed solution of gelatin and aspirin to obtain the fifth composite nanofiber membrane. The fifth composite nanofiber membrane was immersed in a mixed solution of poly-L-lysine and aspirin to obtain the sixth composite nanofiber membrane. The fifth and sixth immersions were repeated 5 times each to obtain a nanofiber membrane loaded with the second drug-loaded layer. The nanofiber membrane loaded with the second drug-carrying layer was mixed with genipin solution and subjected to a cross-linking reaction to obtain the ASP / BFP-1 sustained-release nanofiber membrane.

3. The preparation method according to claim 2, characterized in that, The solvent in the polycaprolactone solution is hexafluoro-2-propanol; the mass concentration of polycaprolactone in the polycaprolactone solution is 8-15%; and the mass concentration of biomimetic nano-hydroxyapatite in the electrospinning solution is 8-12%.

4. The preparation method according to claim 2, characterized in that, The mass concentration of polylysine in the mixed solution of polylysine and bone morphogenetic peptide-1 is 0.8-1.5%; the concentration of bone morphogenetic peptide-1 in the mixed solution of polylysine and bone morphogenetic peptide-1 is 80-120 μg / mL.

5. The preparation method according to claim 2, characterized in that, The mass concentration of gelatin in the mixed solution of gelatin and bone morphogenetic peptide-1 is 0.8-1.5%; the concentration of bone morphogenetic peptide-1 in the mixed solution of gelatin and bone morphogenetic peptide-1 is 80-120 μg / mL.

6. The preparation method according to claim 2, characterized in that, The concentration of aspirin in the mixed solution of polylysine and aspirin is 80-120 μg / mL; the concentration of aspirin in the mixed solution of gelatin and aspirin is 80-120 μg / mL.

7. The preparation method according to claim 2, characterized in that, The cross-linking reaction is carried out at a temperature of 20-30°C for 6-10 hours.

8. The application of the ASP / BFP-1 sustained-release nanofiber membrane according to claim 1 or the ASP / BFP-1 sustained-release nanofiber membrane prepared by the preparation method according to any one of claims 2 to 7 in the preparation of materials for treating bone defects.

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