A preparation method of composite nanofibers promoting neuralization and mediating phase separation function and a composite nanofiber and application thereof

Abstract

This invention discloses a method for preparing composite nanofibers that promote neurotransmission and mediate phase separation, as well as a composite nanofiber and its application. Using coaxial electrospinning technology, coaxial nanofibers (MBG-NH2 / K63@SF / NGF) were prepared, consisting of an amino-modified mesoporous bioactive glass (MBG-NH2) with adsorbed K63 polyubiquitin chains as the "core" and silk fibroin (SF) with adsorbed nerve growth factor (NGF) as the "shell." The NGF in these nanofibers provides an effective supply for neurotransmission during bone repair, while the liposome-encapsulated K63 polyubiquitin chains promote autophagy induced by intracellular phase separation, thereby promoting osteogenic processes and ultimately achieving bone defect repair. In other words, this material can mediate phase separation and autophagy, and induce neurogenesis to promote bone defect repair. This also indicates that the bone repair scaffold material of the composite nanofibers prepared in this invention, which promotes neurotransmission and mediates phase separation-autophagy, has high application prospects in the field of bone tissue engineering.

Description

Technical Field

[0001] This invention relates generally to the field of biomedical engineering, and specifically to a method for preparing composite nanofibers that promote neurotransmission and mediate phase separation, and a composite nanofiber and its application. Background Technology

[0002] Large bone defects have a severe negative impact on skeletal physiology and can lead to damage to the neural network. Therefore, the generation of new nerves during osteogenic processes is indispensable, and a healthy neural network is equally important. Neuronalization of bone defect sites can regulate bone formation and osteoinduction, thereby better promoting bone defect repair. Therefore, in addition to excellent osteoinduction properties, promoting the reconstruction and functional recovery of the neural network at bone defect sites is also a function that an ideal biological scaffold should possess.

[0003] Mature bone is richly innervated by a nervous system; therefore, a lack of neural networks during bone growth can affect the quality of subsequent new bone formation. Peribular nerves participate in bone development and repair through the secretion of neurotransmitters, neuropeptides, axonal guidance factors, and neurotrophic factors. Correspondingly, bone provides mechanical support and protection to its internal nerves, achieving a fusion of the skeletal and neural microenvironments. This signaling and material transport between bone and periosseous nerves can be used to design bone tissue engineering scaffolds for repairing bone defects or treating diseases related to bone and peripheral nerves. Studies have shown that neurotrophic factors promote osteogenic and neurogenesis of bone mesenchymal stem cells (BMSCs) in vitro and in vivo, and can indirectly promote osteogenic formation by increasing neurogenesis, thereby improving bone formation in tissue engineering.

[0004] Phase separation, through the formation of relatively independent spatial domains, selectively enriches molecules and forms unique structures, playing a crucial role in various physiological and signal transduction processes in plant and animal cells. Autophagy is a highly regulated degradation mechanism in eukaryotic cells. Autophagy activity levels are related to osteoblast transformation and the occurrence and function of osteoclasts. Mesenchymal-derived osteoblasts are responsible for the synthesis, secretion, and mineralization of the bone matrix, and further differentiate into osteocytes upon embedding in the mineralized matrix. Osteocytes embed themselves in the bone matrix, forming a cellular network that regulates skeletal remodeling. Multinucleated osteoclasts derived from hematopoietic stem cells undergo bone resorption during remodeling. Osteoclasts secrete degradative enzymes onto the bone surface to dissolve minerals and digest the bone matrix, and recycle the degraded contents through endocytosis. These three cell types are closely interconnected, coordinating the homeostasis of the skeletal system. In recent years, researchers have discovered a close link between autophagy and osteogenic differentiation. Furthermore, autophagy can promote the degradation of liquid condensates, and the pre-autophagosome structure also undergoes phase separation to regulate autophagosome formation. Therefore, constructing bone tissue engineering scaffolds that can promote phase separation and acting on bone defects to promote autophagy through phase separation, thereby promoting the proliferation, differentiation and mineralization of osteoblasts and ultimately promoting the bone repair process, is an urgent problem that needs to be solved in current bone tissue engineering.

[0005] Studies have shown that the p62 protein recognizes K63 polyubiquitinated chains, forming droplets, i.e., p62 bodies, through a phase separation mechanism, and mediating the formation of autophagosomes. Phase separation-mediated assembly of p62-polyubiquitinated proteins is crucial for misfolding and the autophagic degradation of unwanted proteins. Research indicates that a large number of undegraded autophagic vacuoles or autophagosomes aggregate within the cell bodies of undifferentiated or early osteogenic bone MSCs, suggesting that autophagosomes likely play a promoting role in the differentiation of BMSCs during early osteogenic processes.

[0006] In recent years, bone tissue engineering technology has been gradually developing and improving. We have discovered the importance and necessity of simultaneous bone and nerve repair, that is, nerve repair should be carried out at the same time as bone repair to restore sensation and improve the quality of life of patients after bone defect recovery. At the same time, nerve repair can also promote bone defect repair. However, research in this field and the issue of improving the quality of life of patients after bone defect recovery have only recently been given attention by researchers. Moreover, the important life process of phase separation-autophagy has not yet been incorporated into the research of bone tissue engineering. We hope to develop a bone replacement scaffold with good biocompatibility and good osteogenic effect based on the phase separation-autophagy life process and the nerve repair process. The scaffold can be implanted into the bone defect site to promote bone tissue repair and the restoration of normal physiological function, providing a better method for bone repair. Summary of the Invention

[0007] This invention provides a method for preparing composite nanofibers that promote neurotransmission and mediate phase separation, as well as a composite nanofiber and its application. The composite nanofiber is used to construct a composite nanofiber scaffold that can induce cells to enter the phase separation-autophagy pathway to promote bone repair and nerve repair, ultimately promoting bone repair by promoting the phase separation-autophagy life process and the nerve repair process.

[0008] To achieve the above objectives, this invention proposes a method for preparing composite nanofibers that promote neurotransmission and mediate phase separation, comprising the following steps:

[0009] S1. Preparation of amino-modified mesoporous bioactive glass, silk fibroin solution, liposomes and electrospinning substrate solution;

[0010] S2. K63 polyubiquitin chain, liposomes, amino-modified mesoporous bioactive glass and electrospinning substrate solution are mixed to obtain MBG-NH2 / K63 electrospinning solution. Nerve growth factor and silk fibroin solution are mixed and adsorbed to obtain SF / NGF electrospinning solution.

[0011] S3. Using MBG-NH2 / K63 electrospinning solution as the core and SF / NGF electrospinning solution as the shell, coaxial electrospinning was performed to obtain MBG-NH2 / K63@SF / NGF nanofiber scaffolds.

[0012] To achieve the above objectives, the present invention also proposes a composite nanofiber that promotes neurotransmission and mediates phase separation, which is prepared by the above-described preparation method.

[0013] To achieve the above objectives, the present invention also proposes an application of composite nanofibers that promote neurotransmission and mediate phase separation, using the composite nanofibers prepared by the above method as a bone repair scaffold.

[0014] This invention utilizes coaxial electrospinning technology to prepare coaxial nanofibers (MBG-NH2 / K63@SF / NGF) with an amino-modified mesoporous bioactive glass (MBG-NH2) adsorbed with K63 polyubiquitin chains as the "core" and silk fibroin (SF) adsorbed with nerve growth factor (NGF) as the "shell." The NGF in these nanofibers effectively supplies neurotransmission during bone repair, while the liposome-encapsulated K63 polyubiquitin chains promote autophagy induced by intracellular phase separation, thereby promoting osteogenic processes and ultimately achieving bone defect repair. In other words, this material can mediate phase separation and autophagy, as well as induce neurogenesis to promote bone defect repair. This demonstrates that the composite nanofiber bone repair scaffold material prepared in this invention, which promotes neurotransmission and mediates phase separation-autophagy, has high application potential in the field of bone tissue engineering. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0016] Figure 1 A comparison of the effects of different concentrations of K63 polyubiquitin chains on autophagy mediated by BMSCs.

[0017] Figure 2 These are scanning electron microscope images of three groups of bioactive scaffold materials—MBG-NH2, MBG-NH2 / K63, and bone repair material—prepared in Example 1 of this invention; wherein, Figure 2 (a) is a scanning electron microscope image of MBG-NH2 at the 1 μm scale. Figure 2 (d) is a scanning electron microscope image of MBG-NH2 at a scale of 500 nm; Figure 2 (b) is a scanning electron microscope image of MBG-NH2 / K63 at the 1 μm scale. Figure 2 (e) is a scanning electron microscope image of MBG-NH2 / K63 at a scale of 500 nm; Figure 2 (c) is a scanning electron microscope image of the bone repair material at the 1 μm scale. Figure 2 (f) is a scanning electron microscope image of the bone repair material at the 500nm scale;

[0018] Figure 3 The following are the FTIR spectra of three groups of bioactive scaffold materials, namely MBG-NH2, MBG-NH2 / K63 and bone repair material, prepared in Example 1 of this invention.

[0019] Figure 4 These are scanning electron microscope images of three groups of bioactive scaffold materials—MBG-NH2, MBG-NH2 / K63, and bone repair material—obtained in simulated body fluid for 14 days after immersion in simulated body fluid, as shown in Example 1 of this invention. Figure 4 (a) is a scanning electron microscope image at a scale of 500 nm after MBG-NH2 immersion for 14 days. Figure 4 (d) is a scanning electron microscope image at the 1 μm scale after MBG-NH2 soaking for 14 days; Figure 4 (b) is a scanning electron microscope image at a scale of 500 nm after MBG-NH2 / K63 immersion for 14 days. Figure 4 (e) is a scanning electron microscope image at the 1 μm scale after MBG-NH2 / K63 soaking for 14 days; Figure 4(c) A scanning electron microscope image of the bone repair material at a scale of 500 nm after 14 days of immersion. Figure 4 (f) is a scanning electron microscope image of the bone repair material at the 1 μm scale after 14 days of immersion;

[0020] Figure 5 The mechanical property test results of the support material in Embodiment 1 of the present invention;

[0021] Figure 6 This is an image of alizarin red staining of mineralized nodules after 14 days of mineralization induction culture of osteoblasts on the scaffold material in Example 1 of the present invention.

[0022] Figure 7 This is a diagram showing the autophagy results of BMSCs cultured on the scaffold material in Example 1 of the present invention;

[0023] Figure 8 This is a diagram showing the phase separation results of BMSCs cultured on the scaffold material in Example 1 of the present invention;

[0024] Figure 9 This is a diagram showing the neuralization results of BMSCs cultured on the scaffold material in Example 1 of the present invention.

[0025] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0028] This invention proposes a method for preparing composite nanofibers that promote neurotransmission and mediate phase separation, characterized by comprising the following steps:

[0029] S1. Preparation of amino-modified mesoporous bioactive glass, silk fibroin solution, liposomes and electrospinning substrate solution;

[0030] S2. K63 polyubiquitin chain, liposomes, amino-modified mesoporous bioactive glass and electrospinning substrate solution are mixed to obtain MBG-NH2 / K63 electrospinning solution. Nerve growth factor and silk fibroin solution are mixed and adsorbed to obtain SF / NGF electrospinning solution.

[0031] S3. Using MBG-NH2 / K63 electrospinning solution as the core and SF / NGF electrospinning solution as the shell, coaxial electrospinning was performed to obtain MBG-NH2 / K63@SF / NGF nanofiber scaffolds.

[0032] It is worth noting that published studies only indicate that K63 polyubiquitin chains can mediate autophagy, thereby promoting osteoblast differentiation. However, no research has shown that it can promote bone repair in bone defects, and these studies have only been conducted at the cellular level. This invention hypothesizes that by inducing cells to enter the phase separation-autophagy pathway, it can promote osteogenic differentiation and bone repair. Therefore, further research was conducted to discover its role in promoting bone repair in bone defects, and to prepare it into a scaffold material suitable for bone growth for application in bone repair. This research focuses on its function at the bone tissue level. Furthermore, in this invention, K63 polyubiquitin chains are directly delivered into cells by encapsulating them in liposomes.

[0033] Amino-modified mesoporous bioactive glass is a good material for inducing osteoblast attachment and extension, but how to make it play a better role is a hot topic for scientists. This invention utilizes its adsorption and slow release properties to adsorb and release K63 polyubiquitin chains that can promote autophagy, so that K63 polyubiquitin chains and mesoporous bioactive glass synergistically promote the bone repair process.

[0034] Preferably, in step S1, the step of preparing amino-modified mesoporous bioactive glass is as follows:

[0035] Mesoporous bioactive glass powder was synthesized using the sol-gel method and template self-assembly method.

[0036] The synthesized mesoporous bioactive glass powder was activated by calcination at 120-160℃ for 4-8 h, then mixed with 0.05-0.15 M APTES and reacted for 12-20 h. The resulting mixture was then washed and dried to obtain amino-modified mesoporous bioactive glass powder.

[0037] APTES refers to 3-aminopropyltriethoxysilane.

[0038] Preferably, in step S1, the step of preparing the silk fibroin solution is as follows:

[0039] Silkworm cocoons are degummed, dried, dissolved, dialyzed, and concentrated to obtain a silk fibroin solution of 80-120 mg / mL.

[0040] Preferably, in step S1, the liposome preparation step is as follows:

[0041] Prepare a solution by mixing DOTAP, cholesterol, and chloroform solvent at a ratio of [1:(0.5-1.5)]mol:1mL, and then evaporate the resulting solution in a vacuum rotary evaporator for 15-20 minutes. DOTAP refers to (2,3-dioleoxypropyl)trimethylammonium chloride.

[0042] After rotary evaporation, PBS buffer was added to the solution and the mixture was placed in an ice bath in an ultrasonic cleaner for 1-2 hours. The mixture was then vortexed and ultrasonically broken up for 15 minutes to obtain liposomes with a concentration of 5-8 mg / mL.

[0043] The ratio of DOTAP, cholesterol, and PBS buffer is [1:(0.5-1.5)]mol:(2-5)mL; wherein, the ultrasonic disruptor is set to operate at 10-20% power in a mode of working for 3-5 seconds and stopping for 7-12 seconds.

[0044] Preferably, in step S1, the step of preparing the electrospinning substrate solution is as follows:

[0045] An electrospinning substrate solution was prepared by mixing poly(ε-caprolactone) and hexafluoroisopropanol at a ratio of 1 g:(8-12) mL.

[0046] Preferably, in step S2, the concentration of K63 polyubiquitin chains in the MBG-NH2 / K63 electrospinning solution is 0.3-0.8 μmol / L. If the concentration exceeds 0.8 μmol / L, the cell phase separation-autophagy level will decrease, while if the concentration is below 0.3 μmol / L, the cell phase separation-autophagy level is very low and insufficient to induce subsequent cellular processes, such as... Figure 1The image shows a comparison of the effects of different concentrations of K63 polyubiquitin chains on autophagy mediated by BMSCs. The liposome concentration was 1.5-2.0 mg / mL. If the concentration was below 1.5 mg / mL, it was insufficient to encapsulate the K63 polyubiquitin chain, failing to efficiently activate phase separation-autophagy and subsequent life processes. If the concentration exceeded 2.0 mg / mL, it would cause cell death, as liposomes themselves are harmful to cells. Therefore, K63 polyubiquitin chains and liposomes need to be at appropriate concentrations. The encapsulation of bovine serum albumin by liposomes was measured using classical methods. The encapsulation efficiency of (BSA) was 66.04% (see Table 1 below). Based on the comparison of the size, spatial shape, and hydrophilic and hydrophobic groups of BSA and K63 polyubiquitin chains, the dosage range of liposomes can be obtained. The concentration of amino-modified mesoporous bioactive glass is 8-12 mg / mL. If the concentration is lower than 8 mg / mL, the mechanical properties of the final nanofiber scaffold are very weak and insufficient to play a load-bearing role in bone repair. If the concentration is higher than 12 mg / mL, MBG is not easy to pass through the coaxial spinneret. Coaxial electrospinning is not easy due to too much insoluble material.

[0047] Table 1 Encapsulation efficiency of BSA protein by liposomes

[0048]

[0049] Preferably, in the SF / NGF electrospinning solution in step S2, the final concentration of nerve growth factor is 30-60 μg / mL, and the concentration of silk fibroin solution is 20-40 mg / mL.

[0050] The time for mixed adsorption is 10-18 hours.

[0051] Preferably, step S3 specifically includes:

[0052] The MBG-NH2 / K63 electrospinning solution and the SF / NGF electrospinning solution were mixed uniformly by high-speed magnetic stirring for 0.5-1.5 hours; where high speed means a rotation speed of not less than 2000 rpm.

[0053] Using a homogeneously mixed MBG-NH2 / K63 electrospinning solution as the core and an SF / NGF electrospinning solution as the shell, the solutions were transferred into two syringes. The two syringes were connected to the inner and outer conduits of a coaxial spinning head, which was connected to the positive electrode. The injection pump rate was set to 0.5-1.0 mL / h, and coaxial electrospinning was performed to obtain MBG-NH2 / K63@SF / NGF nanofiber scaffolds.

[0054] This invention also proposes a composite nanofiber that promotes neurotransmission and mediates phase separation, which is prepared using the above-described method.

[0055] This invention also proposes the application of composite nanofibers that promote neurotransmission and mediate phase separation, using the composite nanofibers prepared by the above method as a bone repair scaffold.

[0056] Example 1

[0057] Mesoporous bioactive glass MBG, synthesized by self-assembly of sol-gel and template, was activated by calcination at 150°C for 8 h, then mixed with 0.1 M APTES and reacted for 20 h to obtain MBG-NH2.

[0058] Silkworm cocoons were degummed, dried, dissolved, dialyzed, and concentrated to obtain a 100 mg / mL silk fibroin solution;

[0059] DOTAP: cholesterol: chloroform solvent was dissolved in a ratio of [1:1] mol: 1 mL and then vacuum evaporated for 20 min. 3 mL of PBS buffer was added and placed in an ultrasonic cleaner in an ice bath for 2 h. The mixture was then vortexed and ultrasonically broken up for 15 min. The ultrasonic cleaner was set to 10% power and operated in a 5 s working / 10 s stopping mode.

[0060] Prepare a PCL electrospinning substrate solution by mixing the components in a 1:12 ratio;

[0061] K63 polyubiquitin chain (concentration 0.3 μmol / L), liposomes (concentration 1.8 mg / mL) and MBG-NH2 (concentration 10 mg / mL) were mixed (MBG-NH2 / K63) as the core, and NGF (concentration 60 μg / mL) and SF (concentration 30 mg / mL) were mixed and adsorbed for 18 h (SF / NGF) as the shell. The injection pump rate was set to 0.5 mL / h, and coaxial electrospinning was performed to obtain MBG-NH2 / K63@SF / NGF nanofiber scaffold, i.e. bone repair material.

[0062] Example 2

[0063] Mesoporous bioactive glass MBG, synthesized by self-assembly of sol-gel and template, was activated by calcination at 120°C for 8 h, then mixed with 0.15 M APTES and reacted for 20 h to obtain MBG-NH2.

[0064] Silkworm cocoons were degummed, dried, dissolved, dialyzed, and concentrated to obtain a 120 mg / mL silk fibroin solution;

[0065] DOTAP: cholesterol: chloroform was dissolved in a solvent of [1:1.5] mol: 1 mL. The mixture was then evaporated in a vacuum rotary evaporator for 15 min. 2 mL of PBS buffer was added to an ultrasonic cleaner and placed in an ice bath for 1 h. The mixture was then vortexed and ultrasonically broken up for 15 min. The ultrasonic cleaner was set to 10% power and operated in a 5 s working / 12 s stopping mode.

[0066] Prepare the PCL electrospinning substrate solution by mixing them in a 1:8 ratio;

[0067] K63 polyubiquitin chain (concentration of 0.8 μmol / L), liposomes (concentration of 2.0 mg / mL), and MBG-NH2 (concentration of 12 mg / mL) were mixed (MBG-NH2 / K63) as the core, and NGF (final concentration of 30 μg / mL) and SF (concentration of 20 mg / mL) were mixed and adsorbed for 18 h (SF / NGF) as the shell. The injection pump rate was set to 0.75 mL / h, and coaxial electrospinning was performed to obtain MBG-NH2 / K63@SF / NGF nanofiber scaffold, i.e. bone repair material.

[0068] Example 3

[0069] Mesoporous bioactive glass MBG, synthesized by self-assembly of sol-gel and template, was activated by calcination at 160°C for 4 h, then mixed with 0.05 M APTES and reacted for 12 h to obtain MBG-NH2.

[0070] Silkworm cocoons were degummed, dried, dissolved, dialyzed, and concentrated to obtain a silk fibroin solution of 80 mg / mL.

[0071] DOTAP: cholesterol: chloroform was dissolved in a solvent of [1:0.5] mol: 1 mL. The mixture was then evaporated in a vacuum rotary evaporator for 20 min. 5 mL of PBS buffer was added to an ultrasonic cleaner and placed in an ice bath for 1 h. The mixture was then vortexed and ultrasonically broken up for 15 min. The ultrasonic cleaner was set to 10% power and operated in a 3 s working / 7 s stopping mode.

[0072] Prepare a PCL electrospinning substrate solution by mixing the components in a 1:12 ratio;

[0073] K63 polyubiquitin chain (concentration of 0.3 μmol / L), liposomes (concentration of 1.5 mg / mL), and MBG-NH2 (concentration of 8 mg / mL) were mixed (MBG-NH2 / K63) as the core, and NGF (final concentration of 60 μg / mL) and SF (concentration of 40 mg / mL) were mixed and adsorbed for 10 h (SF / NGF) as the shell. The injection pump rate was set to 1.0 mL / h, and coaxial electrospinning was performed to obtain MBG-NH2 / K63@SF / NGF nanofiber scaffold, i.e. bone repair material.

[0074] Figure 2 SEM images show the surface morphology of three nanofiber scaffolds: MBG-NH2, MBG-NH2 / K63, and a bone repair material. The images reveal that all three nanofiber scaffolds form a continuous, uniformly sized, stacked three-dimensional network structure, exhibiting extracellular matrix-like properties that facilitate cell adhesion, proliferation, and differentiation.

[0075] Figure 3 The FTIR spectra of three nanofiber scaffolds—MBG-NH2, MBG-NH2 / K63, and bone repair material—are shown. The results indicate that PCL (poly-ε-caprolactone), MBG-NH2, MBG-NH2 / K63, and the bone repair material scaffold all exhibit similar characteristic peaks at 1500 cm⁻¹. -1 and 1600cm -1 Both peaks at 3290 cm⁻¹ differ from those of MBG-NH₂ and MBG-NH₂ / K₆₃. These two peaks correspond to amide bonds in the β-sheet structure of SF, specifically type II and type I, respectively. Furthermore, at 3290 cm⁻¹... -1 and 3400cm -1 The peaks at the specified locations indicate that the bone repair scaffold and the MBG-NH2 / K63 scaffold contain amide groups, pointing to SF and K63 (liposomes), respectively. This result suggests that the introduction of SF and K63 (liposome encapsulation) did not significantly alter the chemical structure of the scaffolds, but rather they were adsorbed into the scaffolds through physical forces.

[0076] Figure 4 The figures show the SBF mineralization results of three nanofiber scaffolds: MBG-NH2, MBG-NH2 / K63, and a bone repair material. Simulating biomineralization through immersion in simulated body fluids is a common method in bone tissue engineering for evaluating the osteoinductive and osteotransfer properties of scaffolds. The figures show that after 14 days of immersion in simulated body fluids, the MBG-NH2 scaffold showed almost no deposit nodules on the fibers; the MBG-NH2 / K63 scaffold formed rough, near-cubic deposit nodules at certain locations on the nanofibers; and larger deposit nodules were observed in the bone repair material scaffold compared to the MBG-NH2 / K63 scaffold. This demonstrates that the bone repair material scaffold possesses good in vitro mineralization capabilities and exhibits high bioactivity.

[0077] Figure 5The figures show the mechanical property test results of three nanofiber scaffolds: MBG-NH2, MBG-NH2 / K63, and a bone repair material. The results show that the elastic modulus of the MBG-NH2 / K63 scaffold and the bone repair material scaffold are significantly higher than that of the MBG-NH2 scaffold, and the elastic modulus of the bone repair material scaffold is significantly higher than that of the MBG-NH2 / K63 scaffold. This indicates that the introduction of liposome-encapsulated K63 polyubiquitin chains and SF can improve the scaffold's toughness and provide better support for bone defect repair. **P<0.01, ***P<0.001

[0078] Figure 6 This study presents the results of Alizarin Red staining of mineralized nodules in the extracellular matrix of BMSCs cultured on three bioactive scaffold materials: MBG-NH2, MBG-NH2 / K63, and bone repair material, after 14 days of mineralization induction culture. The results showed that, compared to the sporadic mineralized nodules formed by BMSCs cultured on the MBG-NH2 scaffold, the other two scaffolds produced larger and more numerous mineralized nodules. Among these, the bone repair material scaffold exhibited a higher number and density of mineralized nodules, indicating that the bone repair material scaffold has a better ability to promote BMSC mineralization.

[0079] Figure 7 This image illustrates the effects of three nanofiber scaffolds—MBG-NH2, MBG-NH2 / K63, and bone repair material—on autophagy mediated by BMSCs. The results show that BMSCs cultured on the MBG-NH2 / K63 and bone repair material scaffolds exhibited significant autophagy, with observed formation of autophagosomes within the cells. BMSCs cultured on the MBG-NH2 / K63 scaffold showed stronger LC3-CFP and LAMP1-mCherry fluorescence, while the LC3-CFP fluorescence intensity of BMSCs cultured on the bone repair material scaffold was similar to that on the MBG-NH2 / K63 scaffold, but the LAMP1-mCherry fluorescence intensity was slightly weaker. This is because the coaxial structure allows for a longer process of K63 release from the nanofiber "core" and its action on BMSCs compared to the MBG-NH2 / K63 scaffold, thus delaying the autophagy process in BMSCs. Therefore, it can be concluded that the MBG-NH2 / K63 and bone repair material scaffolds can mediate autophagy in BMSCs.

[0080] Figure 8The images show the effects of phase separation mediated by three nanofiber scaffolds: MBG-NH2, MBG-NH2 / K63, and bone repair material. The results show that BMSCs cultured on the MBG-NH2 / K63 and bone repair material scaffolds exhibited significant phase separation behavior, with numerous and large-area co-localization of p62 and LC3 proteins (circled in black) in spherical droplets. In contrast, BMSCs cultured on the MBG-NH2 scaffold showed no significant co-localization, and the fluorescence of both p62 and LC3 proteins was dimmer than in the latter two groups, indicating a lower degree of phase separation autophagy. Therefore, the MBG-NH2 / K63 and bone repair material scaffolds can mediate the phase separation of p62 and LC3B proteins in BMSCs, forming phase-separating bodies.

[0081] Figure 9 Immunofluorescence images of neural cells on three nanofiber scaffolds: MBG-NH2, MBG-NH2 / K63, and bone repair material. The results showed that the MBG-NH2 and MBG-NH2 / K63 scaffolds exhibited low levels of NSE and GFAP expression, indicating a lower degree of neural differentiation in BMSCs cultured on these scaffolds. In contrast, the bone repair material scaffold clearly showed neuron-like cells, with strong NSE fluorescence (circled in black), exhibiting axonal neuron morphology. Simultaneously, the weaker GFAP fluorescence demonstrated that the BMSCs cultured on this scaffold differentiated more significantly towards neurons, exhibiting more active neurofibrillary-like activity.

[0082] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing composite nanofibers that promote neurotransmission and mediate phase separation, characterized in that, Includes the following steps: S1. Preparation of amino-modified mesoporous bioactive glass, silk fibroin solution, liposomes and electrospinning substrate solution; S2. K63 polyubiquitin chain, liposomes, amino-modified mesoporous bioactive glass and electrospinning substrate solution are mixed to obtain MBG-NH2 / K63 electrospinning solution. Nerve growth factor and silk fibroin solution are mixed and adsorbed to obtain SF / NGF electrospinning solution. S3. Using MBG-NH2 / K63 electrospinning solution as the core and SF / NGF electrospinning solution as the shell, coaxial electrospinning was performed to obtain MBG-NH2 / K63@SF / NGF nanofiber scaffolds.

2. The preparation method according to claim 1, characterized in that, In step S1, the steps for preparing amino-modified mesoporous bioactive glass are as follows: Mesoporous bioactive glass powder was synthesized using the sol-gel method and template self-assembly method. The synthesized mesoporous bioactive glass powder was activated by calcination at 120-160℃ for 4-8 h, then mixed with 0.05-0.15 M APTES and reacted for 12-20 h. The resulting mixture was then washed and dried to obtain amino-modified mesoporous bioactive glass powder.

3. The preparation method according to claim 1, characterized in that, In step S1, the steps for preparing the silk fibroin solution are as follows: Silkworm cocoons are degummed, dried, dissolved, dialyzed, and concentrated to obtain a silk fibroin solution with a concentration of 80-120 mg / mL.

4. The preparation method according to claim 1, characterized in that, In step S1, the steps for preparing liposomes are as follows: Prepare a solution by mixing DOTAP, cholesterol, and chloroform solvent at a ratio of [1:(0.5-1.5)]mol:1mL, and then rotary evaporate the resulting solution in a vacuum rotary evaporator for 15-20 minutes. After rotary evaporation, PBS buffer was added to the solution and the mixture was placed in an ice bath in an ultrasonic cleaner for 1-2 hours. The mixture was then vortexed and ultrasonically broken up for 15 minutes to obtain liposomes with a concentration of 5-8 mg / mL. The ratio of DOTAP, cholesterol, and PBS buffer is [1:(0.5-1.5)]mol:(2-5)mL; The ultrasonic breaker is set to operate at 10-20% power in a mode that runs for 3-5 seconds and stops in 7-12 seconds.

5. The preparation method according to claim 1, characterized in that, In step S1, the steps for preparing the electrospinning substrate solution are as follows: An electrospinning substrate solution was prepared by mixing poly(ε-caprolactone) and hexafluoroisopropanol at a ratio of 1 g:(8-12) mL.

6. The preparation method according to claim 1, characterized in that, In the MBG-NH2 / K63 electrospinning solution in step S2, the concentration of K63 polyubiquitin chains is 0.3-0.8 μmol / L, the concentration of liposomes is 1.5-2.0 mg / mL, and the concentration of amino-modified mesoporous bioactive glass is 8-12 mg / mL.

7. The preparation method according to claim 1, characterized in that, In the SF / NGF electrospinning solution in step S2, the concentration of nerve growth factor is 30-60 μg / mL, and the concentration of silk fibroin solution is 20-40 mg / mL. The time for mixed adsorption is 10-18 hours.

8. The preparation method according to claim 1, characterized in that, Step S3 specifically includes: The MBG-NH2 / K63 electrospinning solution and the SF / NGF electrospinning solution were mixed uniformly by high-speed magnetic stirring for 0.5-1.5 hours; where high speed means a rotation speed of not less than 2000 rpm. Using a homogeneously mixed MBG-NH2 / K63 electrospinning solution as the core and an SF / NGF electrospinning solution as the shell, the solutions were transferred into two syringes. The two syringes were connected to the inner and outer conduits of a coaxial spinning head, which was connected to the positive electrode. The injection pump rate was set to 0.5-1.0 mL / h, and coaxial electrospinning was performed to obtain MBG-NH2 / K63@SF / NGF nanofiber scaffolds.

9. A composite nanofiber that promotes neurotransmission and mediates phase separation, characterized in that, It is prepared by the preparation method according to any one of claims 1-8.

10. An application of a composite nanofiber that promotes neurotransmission and mediates phase separation, characterized in that, The composite nanofibers prepared by any one of claims 1-8 or the composite nanofibers of claim 9 are used as bone repair scaffolds.

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