A bone biomembrane and a preparation method and application thereof

Abstract

The present application relates to the technical field of medical materials, and particularly relates to a bone formation promoting biomembrane, a preparation method and application thereof. The present application uses methacrylic anhydride (MA) and gelatin (Gelatin) to prepare methacrylated gelatin (GelMA), and grafts phosphate ions on the GelMA to obtain phosphorylated GelMA (denoted as P-GelMA); then, polycaprolactone (PCL) and P-GelMA are obtained into nanofiber membranes through electrospinning; finally, a cerium source solution is sprayed on the surface of the membrane, and a biomembrane is obtained after photocrosslinking. The biomembrane has good biocompatibility and osteogenic activity, and the ability to promote bone regeneration and bone healing in vivo, which provides a new choice for the clinical treatment of bone defects and expands the application prospect of bone biomembrane.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, and in particular to a bone-promoting biomembrane, its preparation method, and its application. Background Technology

[0002] Cerium is a chemical element whose biological functions are not fully understood. Studies have shown that cerium can play a protective role within cells through its antioxidant properties. Cerium can also participate in the regulation of some biological processes, such as cell proliferation and differentiation, and plays an important role in the catalytic activity of some enzymes, such as peroxidase and lipid peroxidase. In recent years, research has shown that cerium not only plays an important role in cell protection and regulation, but also promotes bone repair, primarily by increasing the proliferation and differentiation of osteocytes, thereby promoting bone tissue regeneration. In addition, cerium can inhibit inflammatory responses, reducing the inflammatory response caused by bone injury, thus accelerating the bone repair process. Studies have also found that cerium can promote calcium deposition in osteocytes, enhancing bone strength and stability. Therefore, cerium, as a novel bone repair agent, has broad application prospects and can be used in the treatment of bone diseases such as fractures and osteoporosis.

[0003] Membrane induction is a commonly used method to promote bone repair by using biomembranes to help bone tissue regrowth and repair. The core of this technique is to cover the bone defect area with a biomimetic periosteum, utilizing the bioactivity of the biomembrane to promote the proliferation and differentiation of osteocytes, thereby accelerating bone tissue growth and repair. Simultaneously, it protects the defect area from external interference, contributing to the formation of intact bone tissue. The advantages of membrane induction include its simplicity, minimal patient trauma, and relatively short recovery time. Furthermore, biomembranes can be customized to meet different clinical needs, such as material selection, thickness, and pore size, to satisfy diverse patient requirements. The most common method for preparing biomembranes is electrospinning, where a solution of polymers or biomaterials is sprayed from a needle tip and formed into nanofibers under the influence of an electric field, ultimately creating a fibrous film or nanofiber structure. Biomembranes prepared using this technique have high structure and surface area, providing a larger bioactive surface area and better cell adhesion properties.

[0004] As clinical needs become increasingly precise and efficient, simple biomembranes are no longer sufficient to provide effective repair. A growing body of research suggests that while optimizing biomembrane performance, endowing them with good biological activity is becoming increasingly important. However, current technologies often introduce a large amount of organic materials to improve mechanical strength and drug release. Excessive introduction of functional groups not only increases the complexity of biomembrane preparation but also introduces potential risks of in vivo degradation. Therefore, there is an urgent need to develop more effective and demand-specific biomembrane preparation strategies that enhance biological function while ensuring safety, thereby increasing the prospects for clinical application. Summary of the Invention

[0005] In view of this, the present invention provides a bone-promoting biomembrane, its preparation method, and its application. This bone-promoting biomembrane possesses good biocompatibility and osteogenic activity, as well as the ability to promote bone regeneration and healing in vivo, providing a new option for the clinical treatment of bone defects and expanding the application prospects of bone biomembranes.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] The present invention provides a bone-promoting biomembrane, which is a hydrogel fiber membrane with cerium ions loaded on its surface; the hydrogel fiber membrane is made by electrospinning phosphorylated methacrylic acid modified gelatin and biodegradable materials.

[0008] Preferably, the mass ratio of the phosphorylated methacrylic acid modified gelatin to the biodegradable material is (1-3):(1-3), specifically 1:1, 1:2, 1:3, 2:1, 2:3, 3:1 or 3:2; the biodegradable material includes one or more of polycaprolactone, polylactic acid, and polyurethane.

[0009] The present invention also provides a method for preparing the aforementioned osteogenic biofilm, comprising:

[0010] a) Phosphorylated methacrylic acid modified gelatin and biodegradable materials are mixed and dissolved in hexafluoroisopropanol to obtain a spinning solution, and the spinning solution is spun to obtain a hydrogel fiber membrane.

[0011] b) The cerium source solution is sprayed onto the surface of the hydrogel fiber membrane and photocrosslinked to obtain a bone-promoting biomembrane.

[0012] Preferably, the phosphorylated methacrylic acid modified gelatin described in step a) is prepared by the following method:

[0013] 1) Sodium hexametaphosphate was reacted in an EDC·HCl solution containing imidazole. The reaction product was purified and lyophilized to obtain imidazole-modified sodium polyphosphate.

[0014] 2) The imidazole-modified sodium polyphosphate is mixed with an aqueous solution of methacrylamide-modified gelatin and reacted at 48-52°C for 4-5 hours. After dialysis, filtration and freeze-drying, phosphorylated methacrylic acid-modified gelatin is obtained.

[0015] Preferably, in step 1):

[0016] The solution containing imidazole and EDC·HCl has an imidazole concentration of 44.5-45.5 mM and an EDC·HCl concentration of 15.5-16.5 mM.

[0017] The final concentration of sodium hexametaphosphate in the reaction system is 3.2-3.4 mM;

[0018] The reaction process is maintained at pH 5.8-6.2.

[0019] Preferably, in step 2):

[0020] The concentration of the methacrylic acid modified gelatin aqueous solution is 3.4-3.6 mg / mL;

[0021] In the reaction system, the concentration of imidazole-modified sodium polyphosphate is 10-40 mg / mL;

[0022] The reaction process is maintained at pH 8.2-8.6.

[0023] The present invention does not impose any special restrictions on the source of the methacrylic acid modified gelatin, which can be commercially available or prepared according to conventional methods in the art. In specific embodiments of the present invention, the methacrylic acid modified gelatin is prepared by the following method:

[0024] Methacrylic anhydride was slowly added dropwise to a gelatin aqueous solution and the mixture was shaken in a constant temperature shaker at 48-52℃ for 3-4 hours. During the reaction, the pH of the solution was kept weakly alkaline (7.5-8.5). After the reaction was completed, deionized water was added to dilute the solution and the pH was adjusted to neutral. After dialysis, filtration and freeze-drying, methacrylamide gelatin was obtained.

[0025] Preferably, in step a), the mass-to-volume ratio of the phosphorylated methacrylic acid modified gelatin, the biodegradable material, and hexafluoroisopropanol, in mg:mg:ml, is (250-750):(250-750):(5-20), more preferably 250:750:10 or 500:500:10; the biodegradable material includes one or more of polycaprolactone, polylactic acid, and polyurethane, and in a specific embodiment of the present invention, the biodegradable material is polycaprolactone.

[0026] Preferably, the concentration of the cerium source solution in step b) is 495-505 μM, and the cerium source solution is a cerium chloride solution or a cerium nitrate solution.

[0027] Preferably, the photocrosslinking in step b) specifically involves irradiation with a 10mW / cm² UV lamp for 10 minutes.

[0028] The present invention also provides the application of the osteogenic biomembrane described in the present invention or the osteogenic biomembrane prepared by the preparation method described in the present invention in the preparation of bone repair materials.

[0029] The present invention also provides a bone repair material, characterized in that it includes the bone-promoting biomembrane described in the present invention or the bone-promoting biomembrane prepared by the preparation method described in the present invention.

[0030] The beneficial effects of this invention are as follows:

[0031] (1) The present invention can prepare an electrospun hydrogel biomembrane with positive feedback to amplify the osteogenic effect. The biomembrane has good biocompatibility and can also release cerium ions and phosphate ions in response to alkaline phosphatase, thereby further enhancing the activity of alkaline phosphatase, thus forming a virtuous cycle and amplifying the osteogenic effect in situ in the osteogenic region, which has application value in repairing bone defects.

[0032] (2) Experimental results show that when the biomembrane prepared in this invention is implanted into the skull defect site of rats, and samples are taken for observation after a period of time, the repair effect of the biomembrane group is much better than that of the control group. It can be seen that the biomembrane can be used as a highly efficient ossification system, providing a new reference for the clinical treatment of bone defects. Attached Figure Description

[0033] Figure 1 The macroscopic morphology of the spun hydrogel biofilm is shown;

[0034] Figure 2 The microstructure of the spun hydrogel biofilm is shown.

[0035] Figure 3 Elemental analysis of the spun hydrogel biofilm;

[0036] Figure 4 Results showing the biocompatibility of the spun hydrogel biofilm;

[0037] Figure 5 Demonstrates the in vitro osteogenic effect of spun hydrogel biomembranes;

[0038] Figure 6 This study demonstrates the repair of skull defects using spun hydrogel biomembranes. Detailed Implementation

[0039] This invention provides a method for promoting bone biofilm formation, its preparation, and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0040] Unless otherwise specified, the test materials used in this invention are all commercially available products that can be purchased on the market.

[0041] This invention provides a bone-promoting biomembrane that enhances osteogenic effects, its preparation method, and its application. The bone-promoting biomembrane (specifically referred to as a spun hydrogel biomembrane) is a hydrogel fiber membrane with cerium ions loaded on its surface; the hydrogel fiber membrane is prepared by electrospinning phosphorylated methacrylic acid-modified gelatin and biodegradable materials.

[0042] Polycaprolactone (PCL) is a biodegradable material with a high concentration of hydroxyl and ester bonds in its molecular structure, exhibiting excellent biocompatibility and mechanical properties. It has been widely used in the preparation of sutures, repair materials, and implants, and can naturally degrade within the human body. Gelatin is a natural protein that can be extracted from animal skin, bones, fish scales, and plants. It also possesses good biocompatibility and biodegradability and is easy to process and prepare. A biomimetic periosteum prepared from PCL and gelatin possesses a hierarchical porous structure, similar to the structure and function of natural bone tissue.

[0043] This invention grafts phosphate ions onto methacrylamide gelatin to obtain phosphorylated GelMA. Then, the phosphorylated GelMA is mixed with polycaprolactone and dissolved in hexafluoroisopropanol. The resulting hydrogel fiber membrane is obtained by electrospinning. Finally, a cerium source solution is sprayed onto the surface of the nanofiber membrane and photocrosslinked to obtain a cerium-loaded osteogenic biomembrane.

[0044] In some embodiments, the mass ratio of phosphorylated methacrylic acid modified gelatin to biodegradable material is (1-3):(1-3), specifically 1:1, 1:2, 1:3, 2:1, 2:3, 3:1, or 3:2. The biodegradable material includes one or more of polycaprolactone, polylactic acid, and polyurethane; in a specific embodiment of the present invention, the biodegradable material is polycaprolactone.

[0045] In some implementation schemes, the preparation process of phosphorylated methacrylic acid modified gelatin (hereinafter referred to as phosphorylated GelMA) includes:

[0046] 1) Prepare a solution containing imidazole and EDC·HCl in advance, and adjust the pH to 5.8-6.2. Then add sodium hexametaphosphate. During the reaction, pay attention to maintaining the pH at 5.8-6.2. After purification and lyophilization, the obtained product is imidazole-modified polyphosphate (hereinafter referred to as PolyP-Im); wherein, EDC·HCl is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0047] 2) Mix PolyP-Im with an aqueous solution of methacrylamide gelatin and react at 48-52℃ for 4-5 hours, maintaining the pH at 8.2-8.6 during the reaction. Then, after dialysis, filtration, and freeze-drying, phosphorylated methacrylic acid modified gelatin (referred to as phosphorylated GelMA) is obtained.

[0048] In some implementations, in step 1), the concentration of imidazole in the solution containing imidazole and EDC·HCl is 44.5-45.5 mM, and the concentration of EDC·HCl is 15.5-16.5 mM; the concentration of sodium hexametaphosphate in the solution obtained after adding sodium hexametaphosphate is 3.2-3.4 mM, where the concentration unit mM represents mmol / L.

[0049] In some implementations, the concentration of the aqueous solution of methacrylamide gelatin is 3.4-3.6 mg / mL, and the concentration of imidazole-modified sodium polyphosphate in the resulting solution after mixing with the aqueous solution of methacrylamide gelatin is 10-40 mg / mL.

[0050] In some implementation schemes, the preparation process of the methacrylated gelatin used to prepare the aqueous solution of methacrylated gelatin is as follows: first, gelatin is dissolved in water to obtain a gelatin solution, then methacrylic anhydride is slowly added dropwise to the gelatin solution, and the reaction is carried out in a constant temperature shaker at 48-52℃ for 3-4 hours. During the reaction, the pH value of the solution is kept weakly alkaline (7.5-8.5). After the reaction is completed, deionized water is added to dilute and the pH value is adjusted to neutral. Then, after dialysis, filtration, and freeze-drying, methacrylated gelatin is obtained.

[0051] In some implementations, in step a), the mass-volume ratio of the phosphorylated methacrylic acid modified gelatin, biodegradable material, and hexafluoroisopropanol, in mg:mg:ml, is (250-750):(250-750):(5-20), specifically 500:500:10, 250:750:10, 750:250:10, 250:750:5, or 250:750:20.

[0052] In some implementations, the cerium source solution is a cerium chloride solution or a cerium nitrate solution, and the concentration of cerium ions is 495-505 μM, specifically 495 μM, 500 μM, or 505 μM; where the concentration unit μM represents μmol / L.

[0053] In some implementations, the conditions for photocrosslinking are: the UV lamp irradiation time is 10-15 minutes, specifically 10, 11, 12, 13, 14, or 15 minutes.

[0054] The present invention also provides a spun hydrogel biomembrane that amplifies osteogenic effects, which is prepared by the above-described preparation method.

[0055] Experiments show that the spun hydrogel biomembrane obtained by this invention has stable biocompatibility and the ability to promote osteogenic differentiation and accelerate calcium deposition in vitro, and has good therapeutic effect in the repair of bone defects in vivo.

[0056] This invention also provides the application of osteogenic biomembranes with enhanced osteogenic effects in the preparation of bone repair materials.

[0057] The present invention also provides a bone repair material, including the bone-promoting biomembrane or the bone-promoting biomembrane prepared by the preparation method.

[0058] The present invention will be further illustrated below with reference to the embodiments:

[0059] Example 1: Preparation of spun hydrogel biomembrane

[0060] (1) Weigh 10g of gelatin and add it to 100mL of deionized water. Stir in a 60℃ constant temperature water bath until completely dissolved to obtain a gelatin solution. Add 10mL of methacrylic anhydride dropwise to the above gelatin solution at a rate of 1mL / min. Shake in a 50℃ constant temperature shaker for 3 hours, ensuring the pH of the solution is 8.0 during the reaction. After the reaction is complete, add 400mL of 50℃ deionized water to dilute the reaction solution and adjust the pH to neutral. After dialysis, filtration, and lyophilization, solid GelMA can be obtained for later use.

[0061] (2) Prepare a solution containing 45 mM imidazole and 16 mM EDC·HCl in advance, and adjust the pH to 6.0. Then add sodium hexametaphosphate to make the final concentration of sodium hexametaphosphate 3.3 mM, and keep the pH at 6.0 during the reaction. The product is purified by ethanol precipitation and lyophilized to obtain a white powder PolyP-Im.

[0062] (3) 143 mL of GelMA aqueous solution (3.5 mg / mL) was mixed with PolyP-Im to obtain a solution with a PolyP-Im concentration of 40 mg / mL. The mixture was then reacted at 50 °C for 5 hours, with the pH maintained at 8.5 during the reaction. After dialysis, filtration, and lyophilization, a white powder P-GelMA was obtained.

[0063] (4) Dissolve 250 mg P-GelMA and 750 mg PCL together in 10 mL of hexafluoroisopropanol. After complete dissolution, inject the solution into the nozzle, adjust the voltage of the high-voltage power supply and the distance from the nozzle to 10 cm, and the voltage to 20 kV. Under the action of the electric field, the polymer solution forms extremely fine fibers and falls into the collector.

[0064] (5) Spray a 500 μM cerium chloride solution evenly onto the spun membrane and irradiate it with a 10 mW / cm2 UV lamp for 10 minutes. After photocrosslinking is completed, rinse repeatedly with deionized water to remove uncrosslinked cerium chloride and other impurities to obtain a spun hydrogel biomembrane loaded with cerium phosphate (denoted as PCL-GCP).

[0065] Example 2: Preparation of spun hydrogel biomembrane

[0066] (1) Weigh 10g of gelatin and add it to 100mL of deionized water. Stir in a 60℃ constant temperature water bath until completely dissolved to obtain a gelatin solution. Add 10mL of methacrylic anhydride dropwise to the above gelatin solution at a rate of 1mL / min. Shake in a 50℃ constant temperature shaker for 3 hours, ensuring the pH of the solution is 8 during the reaction. After the reaction is complete, add 400mL of 50℃ deionized water to dilute the reaction solution and adjust the pH to neutral. After dialysis, filtration, and lyophilization, solid GelMA can be obtained for later use.

[0067] (2) Prepare a solution containing 45 mM imidazole and 16 mM EDC·HCl in advance, and adjust the pH to 6.0. Then add sodium hexametaphosphate to make the final concentration of sodium hexametaphosphate 3.3 mM, and keep the pH at 6.0 during the reaction. The product is purified by ethanol precipitation and lyophilized to obtain a white powder PolyP-Im.

[0068] (3) 143 mL of GelMA aqueous solution (3.5 mg / mL) was mixed with PolyP-Im, and the concentration of PolyP-Im in the resulting solution was 20 mg / mL. The mixture was then reacted at 50 °C for 5 hours, with the pH maintained at 8.5 during the reaction. After dialysis, filtration, and lyophilization, a white powder, P-GelMA, was obtained.

[0069] (4) Dissolve 500 mg of P-GelMA and 500 mg of PCL (polycaprolactone) together in 10 mL of hexafluoroisopropanol. After complete dissolution, inject the solution into the nozzle, adjust the voltage of the high-voltage power supply and the distance from the nozzle to 10 cm, and set the voltage to 20 kV. Under the action of the electric field, the polymer solution forms extremely fine fibers and falls into the collector.

[0070] (5) Spray a 500 μM cerium chloride solution evenly onto the spun membrane and irradiate it with a 10 mW / cm2 UV lamp for 10 minutes. After photocrosslinking is completed, rinse repeatedly with deionized water to remove uncrosslinked cerium chloride and other impurities to obtain a spun hydrogel biomembrane loaded with cerium phosphate.

[0071] Comparative Example 1

[0072] 1000 mg of PCL was dissolved in 10 mL of hexafluoroisopropanol. After complete dissolution, the solution was injected into the nozzle, and the voltage of the high-voltage power supply and the distance from the nozzle were adjusted to 10 cm, with a voltage of 20 kV. Under the action of the electric field, the polymer solution formed extremely fine fibers and fell into the collector, yielding a PCL spun membrane (denoted as PCL).

[0073] Comparative Example 2

[0074] Take 250 mg of GelMA obtained in step (1) of Example 1 and 750 mg of PCL, and dissolve them together in 10 mL of hexafluoroisopropanol. After complete dissolution, inject the solution into the nozzle, adjust the voltage of the high-voltage power supply and the distance from the nozzle to 10 cm, and the voltage to 20 kV. Under the action of the electric field, the polymer solution forms extremely fine fibers and falls into the collector, obtaining a PCL and GelMA mixed spun membrane (denoted as PCL-GelMA).

[0075] Comparative Example 3

[0076] Take 250 mg of P-GelMA obtained in step (3) of Example 1 and 750 mg of PCL, and dissolve them together in 10 mL of hexafluoroisopropanol. After complete dissolution, inject the solution into the nozzle, adjust the voltage of the high-voltage power supply and the distance from the nozzle to 10 cm, and the voltage to 20 kV. Under the action of the electric field, the polymer solution forms extremely fine fibers and falls into the collector, resulting in a PCL and phosphorylated GelMA mixed spun membrane (denoted as PCL-GP).

[0077] Comparative Example 4

[0078] 125 mg of P-GelMA obtained in step (3) of Example 1, 125 mg of GelMA obtained in step (1) of Example 1, and 750 mg of PCL were dissolved together in 10 mL of hexafluoroisopropanol. After complete dissolution, the solution was injected into the nozzle, and the voltage of the high-voltage power supply and the distance from the nozzle were adjusted to 10 cm, with a voltage of 20 kV. Under the action of the electric field, the polymer solution formed extremely fine fibers and fell into the collector, resulting in a mixed spun film of PCL and half-phosphorylated GelMA. At this time, the amount of cerium ions that the half-phosphorylated GelMA could bind was also half that in Example 1.

[0079] In in vitro experiments, its ability to promote calcium nodule deposition was significantly weaker than that in Example 1.

[0080] Test case

[0081] The macroscopic morphology of the spun hydrogel biomembrane prepared in Example 1 is as follows: Figure 1As shown, taking the spun hydrogel biomembrane prepared in Example 1 as an example (denoted as PCL-GCP), its material characterization, in vitro biocompatibility determination, in vitro osteogenic capacity determination, and repair of skull defects by the biomembrane are as follows.

[0082] 1. Materials Characterization

[0083] 1.1. Microscopic morphology of biofilm was detected using SEM. The specific method was to dehydrate the prepared biofilm, perform critical point drying, place it on a stage, sputter gold for 45 seconds using a plasma sputtering instrument, and then detect it under SEM.

[0084] Specific results are as follows Figure 2 As shown, from Figure 2 As can be seen, the disordered fibers form a porous structure suitable for cell adhesion and growth, and cerium phosphate can be observed attached in the pores.

[0085] 1.2. Elemental composition of biofilms was determined using an XPS analyzer. The sample was placed on the XPS analyzer's sample stage, and the vacuum pump was activated to create a vacuum. Maintaining a vacuum state between the sample and the instrument's interior was crucial to avoid X-ray scattering and interference from impurity molecules. The sample was irradiated with an X-ray source, ionizing the atoms and generating photoelectrons. The energy of these photoelectrons was used to determine the electronic structure of the atoms, thus obtaining information such as the elemental composition and chemical bond states of the sample surface.

[0086] Specific results are as follows Figure 3 As shown, the elemental composition of the spinning membrane includes cerium ions, proving that cerium ions were successfully modified onto the surface of the spinning membrane.

[0087] 2. In vitro biocompatibility assay

[0088] 2.1. Using rat bone marrow mesenchymal stem cells, the effect of composite scaffolds on cell proliferation at different time points was detected using CCK-8 cell proliferation technology. Specifically, cells were seeded in culture flasks and cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody. When the cells reached passage P1, they were digested with 0.25% trypsin, centrifuged at 1,500 rpm for 5 min, and the cell count was determined. Cells were then divided into groups of 1000 / cm³. 2The biofilm was seeded in 96-well plates. The groups were: (1) PCL spun membrane group of Comparative Example 1, labeled PCL; (2) PCL and GelMA mixed spun membrane group of Comparative Example 2, labeled PCL-GelMA; (3) PCL and phosphorylated GelMA mixed spun membrane group of Comparative Example 3, labeled PCL-GP; (4) Cerium ion modified PCL and phosphorylated GelMA mixed spun membrane group of Example 1, labeled PCL-GCP. Groups (1)-(3) served as control groups, and group (4) was the experimental group. In the experiment, the biofilm was fixed to the bottom of the plate, with 6 wells per group. Complete culture was used, and the biofilm was cultured at 37°C in 5% CO2 for 1 week. The culture medium was changed every 3 days. On days 1, 3, 5, and 7, serum-free DMEM / F12 solution containing 10% CCK-8 reagent was added, and the plates were incubated in a 37°C, 5% CO2 incubator for 1 hour in the dark. After incubation, the absorbance (OD) of each group was measured at 450 nm using a spectrophotometer.

[0089] Depend on Figure 4 The results showed that there was no significant difference in cell proliferation rate between the control group (comparative examples 1-4) and the experimental group (Example 1), indicating that the biomembrane does not change the cell proliferation capacity, and the spun hydrogel biomembrane obtained by this invention has stable biocompatibility.

[0090] 3. In vitro osteogenic capacity assay

[0091] 3.1. Alkaline phosphatase staining: The biomembrane was fixed to the bottom of the culture plate, and bone marrow mesenchymal stem cells were seeded on the membrane and cultured. After 7 days of osteogenic induction intervention, the cells were fixed with 4% paraformaldehyde for 30 minutes. The fixed cells were then immersed in alkaline phosphatase staining solution and incubated at room temperature for 30 minutes. Finally, the stained cells were washed to remove excess staining agent and other impurities, and the stained cells were observed under a microscope to observe their ALP staining status, assess their ALP activity and differentiation potential.

[0092] 3.2. Alizarin Red Staining: The biomembrane was fixed to the bottom of the culture plate, and bone marrow mesenchymal stem cells were seeded onto the membrane and cultured. After 14 days of osteogenic induction intervention, the cells were fixed with 4% paraformaldehyde for 30 minutes. The fixed cells were then immersed in alizarin red staining solution and incubated at room temperature for 15 minutes. Finally, the stained cells were washed to remove excess staining agent and other impurities, and the alizarin red staining was observed under a microscope.

[0093] The results are as follows Figure 5As shown, the cerium-loaded biomembrane of the present invention can accelerate ALP secretion and promote calcium nodule formation in vitro, with significantly better effects than the fibrous membranes of comparative examples 1-4. It can be seen that the spun hydrogel biomembrane obtained by the present invention has a significant ability to promote osteogenic differentiation and accelerate calcium deposition in vitro.

[0094] 4. Repair of skull defects by biomembranes

[0095] 4.1. Anesthetize the rats and perform procedures such as disinfection and shaving. Prepare surgical instruments and a biomembrane. After the anesthesia is stable, make an incision in the center of the rat's skull to expose the skull. Drill holes using a 5mm diameter electric drill, taking care to avoid touching the dura mater and brain tissue. After creating the defect, cover the skull defect with the prepared biomembrane, fix the biomembrane, and suture it. Harvest tissue 4 weeks after surgery.

[0096] 4.2. After decalcification, dehydration, paraffin infiltration, and embedding, the obtained bone tissue was cut into 7μm thick tissue sections using a microtome, and the degree of bone tissue repair was assessed using H&E staining and Masson staining.

[0097] The results are as follows Figure 6 As shown, CTRL represents the blank group, where no biomembrane is used. (From...) Figure 6 It is evident that the cerium-loaded biomembrane can enhance osteoconductivity and promote new bone formation in vivo, with significant effects. Compared with the biomembranes of Comparative Examples 1 to 3, it is clear that the spun hydrogel biomembrane obtained in this invention has good therapeutic effects in the repair of bone defects in vivo.

[0098] In summary, this invention innovatively proposes a method for preparing an electrospun hydrogel biomembrane with positive feedback amplifying osteogenic effect and applies it to the field of bone repair. Preliminary results demonstrate that the biomembrane has good biocompatibility and osteogenic activity, as well as the ability to promote bone regeneration and bone healing in vivo, showing promising clinical translation prospects and providing a new option for the clinical treatment of bone defects.

[0099] It should be understood that the specific embodiments described above are merely illustrative or explanatory of the principles of the invention and do not constitute a limitation thereof. Therefore, any modifications, equivalent substitutions, improvements, etc., made without departing from the spirit and scope of the invention should be included within the protection scope of the invention. Furthermore, the appended claims are intended to cover all variations and modifications falling within the scope and boundaries of the appended claims, or equivalent forms of such scope and boundaries.

Claims

1. A bone-biofilm-promoting composition, characterized by, It is a hydrogel fiber membrane with cerium ions loaded on its surface; the hydrogel fiber membrane is made by electrospinning phosphorylated methacrylic acid modified gelatin and biodegradable materials; The cerium ions react with the phosphate groups on the hydrogel fiber membrane to form cerium phosphate.

2. The osteoconductive biomembrane according to claim 1, wherein, The mass ratio of the phosphorylated methacrylic acid modified gelatin to the biodegradable material is (1~3):(1~3); the biodegradable material includes one or more of polycaprolactone, polylactic acid, and polyurethane.

3. The method for preparing an osteoconductive biomembrane according to claim 1 or 2, wherein include: a) Phosphorylated methacrylic acid modified gelatin and biodegradable materials are mixed and dissolved in hexafluoroisopropanol to obtain a spinning solution, and the spinning solution is spun to obtain a hydrogel fiber membrane. b) The cerium source solution is sprayed onto the surface of the hydrogel fiber membrane and photocrosslinked to obtain a bone-promoting biomembrane.

4. The preparation method according to claim 3, characterized in that, The phosphorylated methacrylic acid modified gelatin described in step a) is prepared by the following method: 1) Sodium hexametaphosphate was reacted with an imidazole-containing EDC·HCl solution. The reaction product was purified and lyophilized to obtain imidazole-modified sodium polyphosphate. 2) The imidazole-modified sodium polyphosphate is mixed with an aqueous solution of methacrylamide-modified gelatin and reacted at 48-52°C for 4-5 hours. After dialysis, filtration and freeze-drying, phosphorylated methacrylic acid-modified gelatin is obtained.

5. The production method according to claim 4, characterized by, In step 1): The solution containing imidazole and EDC·HCl has an imidazole concentration of 44.5-45.5 mM and an EDC·HCl concentration of 15.5-16.5 mM. The final concentration of sodium hexametaphosphate in the reaction system is 3.2-3.4 mM; The reaction process is maintained at pH 5.8-6.

2.

6. The preparation method according to claim 4, characterized in that, In step 2): The concentration of the methacrylic acid modified gelatin aqueous solution is 3.4-3.6 mg / mL; In the reaction system, the concentration of imidazole-modified sodium polyphosphate is 10-40 mg / mL; The reaction process is maintained at pH 8.2-8.

6.

7. The preparation method according to claim 3, characterized in that, In step a), the mass-to-volume ratio of the phosphorylated methacrylic acid modified gelatin, the biodegradable material, and hexafluoroisopropanol is (250~750) mg : (250~750) mg : (5~20) mL.

8. The preparation method according to claim 7, characterized in that, The mass-to-volume ratio of the phosphorylated methacrylic acid modified gelatin, the biodegradable material, and hexafluoroisopropanol is 500 mg: 500 mg: 10 mL.

9. The preparation method according to claim 3, characterized in that, The concentration of the cerium source solution in step b) is 495~505µM, and the cerium source solution is a cerium chloride solution or a cerium nitrate solution.

10. The preparation method according to claim 3, characterized in that, The photocrosslinking described in step b) specifically involves irradiating with a 10mW / cm² UV lamp for 10 minutes.

11. The use of the osteogenic biomembrane according to claim 1 or 2 or the osteogenic biomembrane prepared by the preparation method according to any one of claims 3 to 10 in the preparation of bone repair materials.

12. A bone repair material, characterized in that, Includes the osteogenic biofilm as described in claim 1 or 2, or the osteogenic biofilm prepared by the preparation method described in any one of claims 3 to 10.

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