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 in particular to the preparation of an exosome and electrospinning composite material for promoting bone regeneration and its application in bone tissue regeneration. Background Art

[0002] In recent years, the research on bone regeneration by tissue engineering technology has made significant progress. First, from the perspective of biomaterials: the commonly used biomaterials for 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 needs by adjusting the chemical structure 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. Secondly, stem cells play an important role in bone regeneration. Mesenchymal stem cells (MSCs) are one of the most studied types of stem cells, which 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 into the bone defect site, the regeneration of bone tissue can be promoted. 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 using genetic engineering techniques to make cells express growth factors can enhance the effect of bone regeneration.

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

[0004] Bone regeneration is a complex process that requires multidisciplinary collaboration, including biology, medicine, materials science, engineering, etc. Experts from different fields work together to research and develop new technologies and methods, which will help promote the development of the field of bone regeneration. The current status of bone regeneration in tissue engineering technology is encouraging, with research continuing to deepen and new technologies and methods emerging. However, to achieve widespread clinical application, many problems need to be solved, and further research and innovation are needed.

[0005] The use of stem cells has certain ethical issues and the problem 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 bionic materials provides a new idea for tissue engineering bone regeneration. Summary of the invention

[0006] Exosomes have shown great potential in the field of tissue regeneration due to their many advantages such as low immunogenicity, easy acquisition and storage, and high safety.

[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 membrane materials to prepare a kind of exosomes that can stably release, give full play to the good stability and low immunogenicity of exosomes, and achieve the effect of accurately promoting local bone regeneration, so as to achieve the repair and functional reconstruction of defects.

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

[0009] A preparation method for an exosome-electrospinning composite material for promoting bone regeneration comprises 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 multidirectional 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: different proportions of PLLA and gelatin are dissolved in a co-solvent trifluoroethanol, composite fibers are prepared by electrospinning, and spinning is performed using a receiver to obtain a non-woven and parallel-arranged fiber membrane, wherein the receiver is one of a metal flat plate and a roller;

[0012] S3. Load the exosomes onto 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, fat, 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 extraction and identification of human periodontal ligament stem cells PDLSC and extraction of exosomes from PDLSC.

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

[0016] The periodontal ligament of orthodontic reduction teeth was obtained, the tissue was cut into pieces under sterile conditions, digested with 0.2% type I collagenase at 37°C for 45 minutes, the digested cells were suspended in α-MEM 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. Osteogenic, adipogenic, and chondrogenic induction culture media were used to induce differentiation of stem cells, and Alizarin red, Oil Red O, Alcian blue and other staining were performed after 2-3 weeks to detect whether the stem cells have differentiation potential, and the differentiation potential includes osteogenic, adipogenic and chondrogenic.

[0017] As a further technical solution of the present invention, the exosome extraction of PDLSC comprises the following steps:

[0018] Two days before extracting exosomes, the induction medium was replaced with exosome-free serum medium. Exosomes were extracted using 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 cell 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 an appropriate amount of PBS to obtain an exosome suspension for further use; the total exosome protein was extracted and quantitatively identified; an appropriate amount of the exosome suspension was taken for transmission electron microscopy observation.

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

[0020] Poly-L-lactic acid (molecular weight of 100,000) 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 (freezing strength value of 250 gBloom) 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;

[0021] The polymer solution C is loaded into a sterile syringe, and the syringe is fixed on a constant flow 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 with randomly arranged fibers. The composite fiber membrane is placed in a vacuum drying oven, kept at a constant temperature of 37°C, and dried at 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, whose morphology SEM image is shown as follows Figure 2 shown.

[0022] As a further technical solution of the present invention, 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.

[0023] As a further technical solution of the present invention, the step of loading exosomes on the surface of the PLLA / gelatin composite fiber membrane includes: the PLLA / gelatin composite fiber membrane prepared in step S2 is placed in the exosome suspension overnight, and the loading concentration is 45ug 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 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 (PLLA / Gel-P), and then the same concentration of exosomes was loaded.

[0026] Another object of the present invention is to provide an application of a PLLA / gelatin composite fiber membrane prepared by providing exosomes promoting bone regeneration and an electrospinning composite material 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 and combines them with membrane materials to prepare a kind of exosomes that can stably release, giving full play to the good stability and low immunogenicity of exosomes, and achieving the effect of accurately promoting local bone regeneration, thereby achieving defect repair and functional reconstruction. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1This is a transmission electron microscopy of human PDLSC exosomes extracted in Example 1.

[0030] Figure 2 This is a scanning electron microscope image of the electrospinning in Example 1 before and after loading exosomes.

[0031] Figure 3 The material in Example 3 was placed in the maxillary bone defect of the rat.

[0032] Figure 4 This is the Micro-ct comparison result of the animal experiment in Example 3. DETAILED DESCRIPTION

[0033] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] The present invention provides a method for preparing an exosome-electrospinning composite material for promoting bone regeneration, comprising the following steps:

[0035] S1. Extraction of exosomes from human mesenchymal stem cells: The source tissues of human mesenchymal stem cells include bone marrow, fat, umbilical cord, periodontal ligament and dental pulp. Obtain the source tissues 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 from human mesenchymal stem cells;

[0036] S2. Preparation of PLLA / gelatin composite fiber membrane: different proportions of PLLA and gelatin are dissolved in a co-solvent trifluoroethanol, composite fibers are prepared by electrospinning, and spinning is performed using a receiver to obtain a non-woven and parallel-arranged fiber membrane, wherein the receiver is one of a metal flat 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 an application of a PLLA / gelatin composite fiber membrane prepared by providing exosomes promoting bone regeneration and an electrospinning composite material in promoting bone tissue regeneration.

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

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

[0041] Furthermore, after the fiber is prepared, in order to facilitate the attachment of exosomes, the membrane can be modified with polydopamine, and the adsorption loading and controlled release of exosomes can be achieved by using natural bioadhesion molecules, while increasing the roughness of the material, further promoting the adhesion and value-added of cells and promoting the local osteogenesis effect.

[0042] After the electrospun fiber scaffold provided by the present invention is stably combined with exosomes, the regeneration process can be further accurately regulated by regulating the source of exosomes or performing functional processing on exosomes. The preparation method is simple and effective, the process is stable, the repeatability is strong, and it is conducive to large-scale production.

[0043] Example 1.

[0044] The present invention provides a method for preparing an exosome-electrospinning composite material for promoting bone regeneration, comprising the following steps:

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

[0046] The periodontal ligament of orthodontic reduction teeth was obtained, the tissue was cut into pieces under sterile conditions, digested with 0.2% type I collagenase at 37°C for 45 minutes, the digested cells were suspended in α-MEM 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. Osteogenic, adipogenic, and chondrogenic induction culture media were used to induce differentiation of stem cells, and Alizarin red, Oil Red O, Alcian blue and other staining were performed after 2-3 weeks to detect whether the stem cells have differentiation potential, and the differentiation potential includes osteogenic, adipogenic and chondrogenic.

[0047] Exosome extraction from PDLSCs:

[0048] Two days before extracting exosomes, the induction medium was replaced with exosome-free serum medium. Exosomes were extracted by 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 cell 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 an appropriate amount of PBS to obtain an exosome suspension for further use; the total exosome protein was extracted and quantitatively identified; an appropriate amount of the exosome suspension was taken for transmission electron microscopy observation, such as Figure 1 shown.

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

[0050] Poly-L-lactic acid (molecular weight of 100,000) 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 (freezing strength value of 250 gBloom) 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;

[0051] The polymer solution C is loaded into a sterile syringe, and the syringe is fixed on a constant flow 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 with randomly arranged fibers. The composite fiber membrane is placed in a vacuum drying oven, kept at a constant temperature of 37°C, and dried at 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, whose morphology SEM image is shown as follows Figure 2 The PLLA / gelatin composite fiber membrane was placed in the exosome suspension overnight, with a loading concentration of 45ug exosomes per mg of material.

[0052] In this embodiment, 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.

[0053] Example 2.

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

[0055] The thickness of the PLLA / gelatin composite fiber membrane was prepared by referring to the steps in Example 1. 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 fiber membrane (PLLA / Gel-P), and then the same concentration of exosomes was loaded.

[0056] Example 3.

[0057] PLLA / gelatin fiber membrane loaded with human PDLSC exosomes was used to promote maxillary bone regeneration in rats.

[0058] Ordinary PLLA / gelatin fiber membrane (PLLA / gel), the exosome-functionalized PLLA / gelatin composite fiber membrane (PLLA / gel+exo) in Example 1, and the polarized exosome-functionalized PLLA / gelatin composite fiber membrane (PLLA / gel-P+exo) in Example 2 were implanted into a rat maxillary defect model. The bone defect is shown in FIG. Figure 3 The bone repair effect was observed after 4 and 8 weeks, respectively. The Micro-CT reconstruction results are shown in Figure 4 As shown in the figure, more new bone was formed in the defect areas of both groups after the addition of exosomes, indicating that the prepared exosome-functionalized scaffold has a good effect in guiding bone tissue regeneration and has broad application prospects in the field of bone tissue engineering.

[0059] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0060] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes 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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