Preparation of exosome and electrostatic spinning composite material for promoting bone regeneration and application of exosome and electrostatic spinning composite material in bone tissue regeneration

Abstract

The invention relates to the technical field of tissue engineering, and particularly discloses preparation of an exosome and electrostatic spinning composite material for promoting bone regeneration and application of the exosome and electrostatic spinning composite material in bone tissue regeneration. Identifying the multiplication capacity, the surface marker and the multidirectional differentiation capacity of the cells after the cells are separated and cultured, and finally extracting and identifying the exosome; pLLA and gelatin are dissolved in a cosolvent trifluoroethanol, composite fibers are prepared through electrostatic spinning, and a composite fiber membrane is obtained through spinning by adopting a receiver; the exosome is loaded on the surface of the PLLA / gelatin composite fiber membrane. According to the invention, the exosome derived from human mesenchymal stem cells is combined with the membrane material to prepare the exosome capable of stably releasing, the good stability and low immunogenicity of the exosome are exerted, and the effect of accurately promoting local bone regeneration is realized. And defect repair and functional reconstruction are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue engineering, and particularly relates to the preparation of an exosome and electrospun composite material for promoting bone regeneration and its application in bone tissue regeneration. Background Art

[0002] In recent years, significant progress has been made in the research of tissue engineering technology in bone regeneration. First, from the perspective of biomaterials: Commonly used biomaterials in bone tissue engineering include natural materials and synthetic materials. Natural materials such as collagen and chitosan have good biocompatibility and biodegradability; synthetic materials such as polylactic acid and polyglycolic acid can meet different requirements by adjusting their chemical structures and physical properties. In addition, the research on composite materials has also attracted much attention, combining the advantages of different materials to further improve the effect of bone regeneration. Second, stem cells play an important role in bone regeneration. Mesenchymal stem cells (MSCs) are one of the most studied types of stem cells at present and can differentiate into osteoblasts, chondrocytes, etc. By extracting the patient's own MSCs, amplifying them in vitro and combining them with biomaterials, and then transplanting them to the bone defect site, it can promote the regeneration of bone tissue. Many growth factors have also been proven to promote bone regeneration: Growth factors such as bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) can regulate cell proliferation, differentiation and migration, and play a key role in bone regeneration. Combining growth factors with biomaterials or enabling cells to express growth factors through genetic engineering technology can enhance the effect of bone regeneration.

[0003] The clinical application of tissue engineering technology in the field of bone regeneration is increasing continuously. Some studies have achieved encouraging results, such as using tissue-engineered bone to repair defects in the skull, jawbone and other parts. However, the current clinical applications still face some challenges, such as problems of cell source, safety and effectiveness of biomaterials, etc., and further research and verification are needed.

[0004] Bone regeneration is a complex process that requires multidisciplinary cooperation, including biology, medicine, materials science, engineering, etc. Experts in different fields jointly researching and developing new technologies and methods will help promote the development of the field of bone regeneration. The current situation of bone regeneration in tissue engineering technology is encouraging, with continuous in-depth research and the emergence of new technologies and new methods. However, to achieve wide clinical applications, many problems still need to be solved and further research and innovation are required.

[0005] The use of stem cells has certain ethical issues and problems of insufficient cell sources. In recent years, scientific research has found that exosomes extracted from functional stem cells can play a similar role in promoting tissue regeneration. Therefore, combining exosomes with biomimetic materials provides a new idea for tissue-engineered bone regeneration. Summary of the Invention

[0006] Due to its many advantages such as low immunogenicity, easy access and storage, and high safety, exosomes show great potential in the field of tissue regeneration.

[0007] Based on the above background and technical advantages, the purpose of the present invention is to use exosomes derived from human mesenchymal stem cells and combine them with a membranous material to prepare a material that can stably release exosomes, exert the good stability and low immunogenicity of exosomes, and achieve the effect of precisely promoting local bone regeneration, realizing the repair of defects and functional reconstruction.

[0008] The present invention adopts the following technical solutions:

[0009] Preparation of an exosome and electrospun composite material for promoting bone regeneration, comprising the following steps:

[0010] S1. Extraction of exosomes from human mesenchymal stem cells: Obtain the source tissue of human mesenchymal stem cells, identify their proliferation ability, surface markers, and multi-directional differentiation ability after cell isolation and culture, and finally extract and identify exosomes derived from human mesenchymal stem cells;

[0011] S2. Preparation of PLLA / gelatin composite fiber membrane: Dissolve different ratios of PLLA and gelatin in the co-solvent trifluoroethanol, prepare composite fibers by electrospinning, and use a receiver to perform electrospinning to obtain non-woven and parallel-arranged fiber membranes, and the receiver is one of a metal plate and a roller;

[0012] S3. Load the exosomes on the surface of the PLLA / gelatin composite fiber membrane.

[0013] As a further technical solution of the present invention, the source tissues of human mesenchymal stem cells include bone marrow, adipose tissue, umbilical cord, periodontal ligament, and dental pulp.

[0014] As a further technical solution of the present invention, the extraction of exosomes from human mesenchymal stem cells includes the extraction and identification of human periodontal ligament stem cells (PDLSCs) and the extraction of exosomes from PDLSCs.

[0015] As a further technical solution of the present invention, the extraction and identification of human periodontal ligament stem cells (PDLSCs) include the following steps:

[0016] Obtain the periodontal ligament of orthodontic extraction teeth. Under sterile conditions, cut the tissue into pieces, digest with 0.2% type I collagenase at 37°C for 45 minutes. The digested cells are suspended in α-MEM medium containing 20% serum, inoculated in a T25 culture flask, and cultured in an incubator at 37°C and 5% CO2. After cell confluence, digest with 0.1% trypsin / 0.1% EDTA solution for subculture. The cells cultured for 3 - 5 passages are cryopreserved in liquid nitrogen for later use. Identify the stem cells by flow cytometry analysis, analyze the cell surface markers CD105, CD90, CD146, CD34 and cell purity. Detect the proliferation ability of stem cells by CCK-8 and colony formation efficiency experiments. Induce and differentiate the stem cells with osteogenic, adipogenic, chondrogenic and other induction media respectively. After 2 - 3 weeks, perform alizarin red, oil red O, alcian blue and other stainings respectively to detect whether the stem cells have differentiation potential, and the differentiation potential includes osteogenesis, adipogenesis and chondrogenesis.

[0017] As a further technical solution of the present invention, the extraction of exosomes from PDLSC includes the following steps:

[0018] Two days before exosome extraction, replace the induction medium with exosome-free serum medium. Extract exosomes by kit method and gradient centrifugation method respectively. Collect the cell culture supernatant, centrifuge at 4°C and 300×g for 10 minutes to remove cell impurities, centrifuge at 4°C and 1000×g for 15 minutes and at 10,000×g for 30 minutes, and centrifuge at 4°C and 100,000×g for 2 hours with an ultracentrifuge. Resuspend the centrifuged exosomes in an appropriate amount of PBS to obtain an exosome suspension for further use; extract the total protein of exosomes and perform quantitative identification; take an appropriate amount of exosome suspension for transmission electron microscopy observation.

[0019] As a further technical solution of the present invention, the preparation of the PLLA / gelatin composite fiber membrane includes the following steps:

[0020] Dissolve poly-L-lactic acid (molecular weight 100,000) in trifluoroethanol and stir magnetically at room temperature for 24 hours to obtain polymer solution A with a concentration of 0.1 g / mL; dissolve gelatin (gel strength value 250 g Bloom) in trifluoroethanol and stir magnetically at room temperature for 24 hours to obtain polymer solution B with a concentration of 0.1 g / mL; mix polymer solution A and polymer solution B in a volume ratio of 1:1, and stir magnetically at room temperature for 4 hours to mix evenly to obtain polymer solution C;

[0021] Load the polymer solution C into a sterile syringe, fix the syringe on a constant flow pump, and perform electrospinning with a stainless-steel roller as the receiver. The roller speed is 400 rpm, the voltage is 15 kV, the receiving distance is 15 cm, the extrusion speed is 0.8 mL / h, and continuous spinning is carried out for 10 hours to obtain a composite fiber membrane with randomly arranged fibers. Place the composite fiber membrane in a vacuum drying oven, keep the temperature at 37 °C, and dry it under a vacuum of 30 Pa for 24 hours. Then place the dried composite fiber membrane in a 90% ethanol solution of 1,3-dimethylaminopropyl-3-ethylcarbodiimide / N-hydroxysuccinimide and perform cross-linking treatment at 4 °C for 24 hours, followed by freeze-drying for 24 hours to obtain a cross-linked composite fiber membrane, namely the PLLA / gelatin composite fiber membrane. Its SEM morphology diagram is as shown in Figure 2 shown.

[0022] As a further technical solution of the present invention, the total concentration of 1,3-dimethylaminopropyl-3-ethylcarbodiimide and N-hydroxysuccinimide is 0.02 g / mL, and the mass ratio of 1,3-dimethylaminopropyl-3-ethylcarbodiimide to N-hydroxysuccinimide is 2.5:1.

[0023] As a further technical solution of the present invention, the step of loading exosomes onto the surface of the PLLA / gelatin composite fiber membrane includes: placing the PLLA / gelatin composite fiber membrane prepared in step S2 in the exosome suspension overnight, and the loading concentration is 45 μg of exosomes per mg of material.

[0024] As a further technical solution of the present invention, the following steps are also included:

[0025] Referring to the thickness of the PLLA / gelatin composite fiber membrane prepared in step S2, the dried PLLA / gelatin composite fiber membrane is subjected to corona polarization treatment at a polarization voltage of 12 kV at room temperature for 30 minutes to obtain an electrode-polarized PLLA / gelatin composite fiber membrane (PLLA / Gel-P), and then exosome loading of the same concentration is carried out.

[0026] Another object of the present invention is to provide an application of the PLLA / gelatin composite fiber membrane prepared by the preparation of an exosome and electrospinning composite material for promoting bone regeneration in promoting bone tissue regeneration.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention uses exosomes derived from human mesenchymal stem cells, combines with a membranous material to prepare a composite material that can stably release exosomes, exerts the good stability and low immunogenicity of exosomes, and achieves the effect of precisely promoting local bone regeneration. Realize the repair of defects and functional reconstruction. Brief Description of the Drawings

[0029] Figure 1Transmission electron microscopy of human PDLSC exosomes extracted in Example 1.

[0030] Figure 2 Scanning electron microscopy images of electrospinning before and after loading exosomes in Example 1.

[0031] Figure 3 Placing the material at the rat maxillary bone defect site in Example 3.

[0032] Figure 4 Results of Micro-ct contrast images in the animal experiment of Example 3. Detailed implementation manners

[0033] The following combines specific embodiments to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0034] The present invention provides a preparation of an exosome and electrospinning composite material for promoting bone regeneration, including the following steps:

[0035] S1. Extraction of exosomes from human mesenchymal stem cells: The source tissues of human mesenchymal stem cells include bone marrow, adipose tissue, umbilical cord, periodontal ligament, and dental pulp. Obtain the source tissues of human mesenchymal stem cells, identify their proliferation ability, surface markers, and multi-directional differentiation ability after cell isolation and culture, and finally extract and identify exosomes derived from human mesenchymal stem cells;

[0036] S2. Preparation of PLLA / gelatin composite fiber membranes: Dissolve different ratios of PLLA and gelatin in the co-solvent trifluoroethanol, prepare composite fibers by electrospinning, and use a receiver to obtain non-woven and parallel arranged fiber membranes. The receiver is one of a metal plate and a roller;

[0037] S3. Load the exosomes onto the surface of the PLLA / gelatin composite fiber membrane.

[0038] Another object of the present invention is to provide the application of a PLLA / gelatin composite fiber membrane prepared by a method for preparing an exosome and electrospinning composite material for promoting bone regeneration in promoting bone tissue regeneration.

[0039] Based on the above technical solutions, the present invention can also be improved as follows

[0040] Further, when preparing the composite fiber membrane, the fiber packing density can be adjusted to characterize the effects of fiber composition / arrangement / membrane thickness, etc. on the micro-morphology, mechanical properties, and degradation properties.

[0041] Furthermore, after the fiber preparation, to facilitate the attachment of exosomes, the membrane can be modified with polydopamine, using natural bioadhesive molecules to achieve the adsorption, loading, and controlled release of exosomes. At the same time, the roughness of the material is increased, further promoting cell adhesion and proliferation, and promoting the local osteogenic effect.

[0042] The present invention provides that after the electrospun fiber scaffold is stably combined with exosomes, by regulating the source of exosomes or functionalizing exosomes, the regeneration process can be further precisely regulated. The preparation method is simple and effective, the process is stable, and the repeatability is strong, which is conducive to large-scale production.

[0043] Example 1.

[0044] The present invention provides a preparation of an exosome and electrospun composite material for promoting bone regeneration, comprising the following steps:

[0045] Extraction and identification of human periodontal ligament stem cells PDLSC:

[0046] Obtain the periodontal ligament of orthodontic extraction teeth. Under sterile conditions, cut the tissue into pieces, digest with 0.2% type I collagenase at 37°C for 45 min. The digested cells are suspended in α-MEM medium containing 20% serum, inoculated in a T25 culture flask, and cultured in a 37°C, 5% CO2 incubator. After cell confluence, digest with 0.1% trypsin / 0.1% EDTA solution for subculture. The cells cultured for 3 - 5 passages are cryopreserved in liquid nitrogen for later use. Flow cytometry analysis is used to identify the stem cells, and the cell surface markers CD105, CD90, CD146, CD34 and cell purity are analyzed. CCK-8 and colony formation efficiency experiments are used to detect the proliferation ability of the stem cells. The stem cells are induced to differentiate and cultured with osteogenic, adipogenic, chondrogenic and other induction media respectively. After 2 - 3 weeks, alizarin red, oil red O, alcian blue and other stains are performed respectively to detect whether the stem cells have differentiation potential, and the differentiation potential includes osteogenesis, adipogenesis and chondrogenesis.

[0047] Extraction of exosomes from PDLSC:

[0048] Two days before exosome extraction, replace the induction medium with exosome-free serum medium. Extract exosomes by the kit method and gradient centrifugation method respectively. Collect the cell culture supernatant, centrifuge at 4°C, 300×g for 10 min to remove cell impurities, centrifuge at 4°C, 1000×g for 15 min and 10,000×g for 30 min, and centrifuge at 4°C, 100,000×g for 2 h with an ultracentrifuge. Resuspend the centrifuged exosomes in an appropriate amount of PBS to obtain an exosome suspension for further use; extract the total protein of the exosomes and perform quantitative identification; take an appropriate amount of the exosome suspension for transmission electron microscopy observation, as Figure 1 shown.

[0049] Preparation and exosome loading of PLLA / gelatin composite fiber membranes:

[0050] Dissolve poly-L-lactic acid (molecular weight 100,000) in trifluoroethanol and stir magnetically at room temperature for 24 hours to obtain polymer solution A with a concentration of 0.1 g / mL; dissolve gelatin (gel strength 250 g Bloom) in trifluoroethanol and stir magnetically at room temperature for 24 hours to obtain polymer solution B with a concentration of 0.1 g / mL; mix polymer solution A and polymer solution B in a volume ratio of 1:1 and stir magnetically at room temperature for 4 hours to mix evenly to obtain polymer solution C;

[0051] Load polymer solution C into a sterile syringe, fix the syringe on a peristaltic pump, and perform electrospinning using a stainless-steel roller as the receiver. The roller speed is 400 rpm, the voltage is 15 kV, the receiving distance is 15 cm, and the extrusion speed is 0.8 mL / h. Continuously electrospin for 10 hours to obtain a composite fiber membrane with randomly arranged fibers; place the composite fiber membrane in a vacuum drying oven and dry it at a constant temperature of 37°C and a vacuum of 30 Pa for 24 hours. Place the dried composite fiber membrane in a 90% ethanol solution of 1,3-dimethylaminopropyl-3-ethylcarbodiimide / N-hydroxysuccinimide and crosslink it at 4°C for 24 hours, and then freeze-dry it for 24 hours to obtain a crosslinked composite fiber membrane, that is, the PLLA / gelatin composite fiber membrane. Its SEM image of the morphology is as Figure 2 shown. Place the PLLA / gelatin composite fiber membrane in the exosome suspension overnight, and the loading concentration is 45 μg exosomes per mg of the material.

[0052] In this example, the total concentration of 1,3-dimethylaminopropyl-3-ethylcarbodiimide and N-hydroxysuccinimide is 0.02 g / mL, and the mass ratio of 1,3-dimethylaminopropyl-3-ethylcarbodiimide to N-hydroxysuccinimide is 2.5:1.

[0053] Example 2.

[0054] Preparation of polarized PLLA / gelatin fiber membranes loaded with human periodontal ligament stem cell exosomes:

[0055] Refer to the steps in Example 1 to prepare the PLLA / gelatin composite fiber membrane. After drying, corona polarize the PLLA / gelatin composite fiber membrane at a polarization voltage of 12 kV at room temperature for 30 minutes to obtain a polarized PLLA / gelatin fiber membrane (PLLA / Gel-P), and then perform exosome loading with the same concentration.

[0056] Example 3.

[0057] Effect of PLLA / gelatin fiber membranes loaded with human PDLSC exosomes on promoting maxillary bone regeneration in rats.

[0058] The ordinary PLLA / gelatin fiber membrane (PLLA / gel), the exosome-functionalized PLLA / gelatin composite fiber membrane in Example 1 (PLLA / gel+exo), and the polarized exosome-functionalized PLLA / gelatin composite fiber membrane in Example 2 (PLLA / gel-P+exo) were implanted into the maxillary bone defect model of rats. The bone defect images are as shown in Figure 3 shown. The bone repair effects were observed at 4 weeks and 8 weeks respectively. The Micro-CT reconstruction results are as shown in Figure 4 shown. There was more new bone formation in the defect areas of the two groups after adding exosomes, indicating that the prepared exosome-functionalized fiber scaffolds have a good effect on guiding bone tissue regeneration and have broad application prospects in the field of bone tissue engineering.

[0059] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention.

[0060] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A preparation of an exosome-electrospinning composite material for promoting bone regeneration, characterized in that: The following steps are involved: S1. Extraction of exosomes from human mesenchymal stem cells: Obtain the source tissue of human mesenchymal stem cells, identify their proliferation ability, surface markers and multidirectional differentiation ability after cell isolation and culture, and finally extract and identify exosomes derived from human mesenchymal stem cells; S2. Preparation of PLLA / gelatin composite fiber membrane: different proportions of PLLA and gelatin were dissolved in a co-solvent trifluoroethanol, composite fibers were prepared by electrospinning, and a receiver was used for spinning to obtain non-woven and parallel-arranged fiber membranes; S3. Load the exosomes onto the surface of the PLLA / gelatin composite fiber membrane.

2. The preparation of an exosome-electrospinning composite material for promoting bone regeneration according to claim 1, characterized in that: The source tissues of human mesenchymal stem cells include bone marrow, fat, umbilical cord, periodontal ligament and dental pulp.

3. The preparation of a composite material of exosomes and electrospinning for promoting bone regeneration according to claim 1, characterized in that: The exosome extraction of human mesenchymal stem cells includes extraction and identification of human periodontal ligament stem cells PDLSC and exosome extraction of PDLSC.

4. The preparation of a composite material of exosomes and electrospinning for promoting bone regeneration according to claim 3, characterized in that: The extraction and identification of human periodontal ligament stem cells PDLSC comprises the following steps: The periodontal ligament of orthodontic reduction teeth was obtained, the tissue was minced under sterile conditions, digested with 0.2% type I collagenase at 37°C for 45 minutes, the digested cells were suspended in α-MEM culture medium containing 20% ​​serum, inoculated in a T25 culture flask, and cultured in a 37°C, 5% CO2 incubator. After the cells were confluent, they were digested and subcultured with 0.1% trypsin / 0.1% EDTA solution, and the cells of culture 3-5 generations were frozen in liquid nitrogen for later use. Flow cytometry was used to identify stem cells, and cell surface markers CD105, CD90, CD146, CD34 and cell purity were analyzed. CCK-8 and clone formation efficiency experiments were used to detect the proliferation ability of stem cells. Induction culture medium was used to induce differentiation of stem cells, and staining was performed after 2-3 weeks to detect whether the stem cells had differentiation potential, which included osteoblastogenesis, adipogenesis and chondrogenesis.

5. The preparation of a composite material of exosomes and electrospinning for promoting bone regeneration according to claim 4, characterized in that: The exosome extraction of PDLSC comprises the following steps: Two days before the extraction of exosomes, the induction medium was replaced with exosome-free serum medium, and exosomes were extracted by the kit method and gradient centrifugation method, respectively. The cell culture supernatant was collected and centrifuged at 4°C, 300 × g for 10 min to remove cellular impurities, 4°C, 1000 × g for 15 min and 10,000 × g for 30 min, and ultracentrifuged at 4°C, 100,000 × g for 2 h. The exosomes obtained by centrifugation were resuspended in PBS to obtain an exosome suspension.

6. The preparation of a composite material of exosomes and electrospinning for promoting bone regeneration according to claim 1, characterized in that: The preparation of the PLLA / gelatin composite fiber membrane comprises the following steps: Poly-L-lactic acid was dissolved in trifluoroethanol, and magnetically stirred at room temperature for 24 hours to obtain a polymer solution A with a concentration of 0.1 g / mL; gelatin was dissolved in trifluoroethanol, and magnetically stirred at room temperature for 24 hours to obtain a polymer solution B with a concentration of 0.1 g / mL; polymer solution A and polymer solution B were mixed at a volume ratio of 1:1, and magnetically stirred at room temperature for 4 hours to obtain a polymer solution C; The polymer solution C is loaded into a sterile syringe, and the syringe is fixed on a constant current pump. Electrospinning is performed using a stainless steel drum as a receiver. The drum speed is 400 rpm, the voltage is 15 kV, the receiving distance is 15 cm, and the extrusion speed is 0.8 mL / h. The spinning is continued for 10 hours to obtain a composite fiber membrane. The composite fiber membrane is placed in a vacuum drying oven, kept at a constant temperature of 37°C and a vacuum degree of 30 Pa for 24 hours. The dried composite fiber membrane is placed in a 90% ethanol solution of 1,3-dimethylaminopropyl-3-ethyldiimide / N-hydroxysuccinimide, cross-linked at 4°C for 24 hours, and then freeze-dried for 24 hours to obtain a cross-linked composite fiber membrane, namely, a PLLA / gelatin composite fiber membrane.

7. The preparation of a composite material of exosomes and electrospinning for promoting bone regeneration according to claim 6, characterized in that: The total concentration of the 1,3-dimethylaminopropyl-3-ethyldiimide and N-hydroxysuccinimide is 0.02 g / mL, and the mass ratio of 1,3-dimethylaminopropyl-3-ethyldiimide to N-hydroxysuccinimide is 2.5:

1.

8. The preparation of a composite material of exosomes and electrospinning for promoting bone regeneration according to claim 6, characterized in that: The step of loading exosomes on the surface of the PLLA / gelatin composite fiber membrane includes: placing the PLLA / gelatin composite fiber membrane prepared in step S2 in an exosome suspension overnight, and the loading concentration is 45ug exosomes per mg material.

9. The preparation of a composite material of exosomes and electrospinning for promoting bone regeneration according to claim 1, characterized in that: The following steps are also included: Referring to the thickness of the PLLA / gelatin composite fiber membrane prepared in step S2, the dried PLLA / gelatin composite fiber membrane was corona polarized at room temperature with a polarization voltage of 12 kV for 30 minutes to obtain an electrically polarized PLLA / gelatin composite fiber membrane, and then exosomes were loaded.

10. Use of a PLLA / gelatin composite fiber membrane prepared by combining the exosomes for promoting bone regeneration and an electrospinning composite material as claimed in any one of claims 1 to 9 in promoting bone tissue regeneration.

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