Nanofiber membrane for bone regeneration and manufacturing method therefor

Abstract

The present disclosure aims to provide a nanofiber membrane for bone regeneration manufactured by electrospinning a polymer solution containing a polyethylene oxide and bone morphogenetic protein. The nanofiber membrane, according to the present disclosure, may be applied to an area of a bone defect by means of electrospinning to promote bone regeneration.

Description

Nanofiber membrane for bone regeneration and method for manufacturing the same

[0001] The present invention relates to a nanofiber membrane for bone regeneration, a pharmaceutical composition for bone regeneration comprising the same, and a method for manufacturing a nanofiber membrane for bone regeneration.

[0002] Bone tissue is the only hard tissue in the body; when damaged by trauma, tumors, deformities, or physiological phenomena, it undergoes a process of forming new bone by filling the defect site to restore it. Currently, the most common methods for treating bone defects include autologous bone grafting, allogeneic bone grafting, and xenogeneic bone grafting. Autologous bone grafting, which involves harvesting and transplanting a portion of the patient's own bone, is the most effective method, but it has disadvantages such as requiring a secondary surgery and the difficulty of obtaining a sufficient amount of bone. Allogeneic bone grafting carries a risk of immune reactions and, although low, a risk of viral infections such as AIDS and hepatitis. Xenogeneic bone grafting may also face limitations in use due to immune reaction issues and the risk of mad cow disease. Accordingly, there is a demand for the development of biocompatible bone graft materials that can replace existing graft materials without the risk of disease transmission.

[0003] Biomaterials for bone tissue regeneration must possess good processability and moldability, remain securely in place over the wound, provide an environment for cell attachment, growth, and differentiation, and ensure that the byproducts generated upon degradation are also biocompatible. In particular, bone graft materials must possess appropriate compressive strength and yield value to facilitate positional fixation and shape retention during suturing or implantation following injection and filling. Conversely, if the material's adhesiveness is too high, it may easily adhere to instruments during the procedure, making it inconvenient to use. Therefore, there is a need to develop bone graft scaffolds that are biocompatible and maintain their shape for a certain period, suitable for grafting into bone defects.

[0004] Conventional bone defect treatments involved manually cutting a scaffold to fit the defect site and filling it. However, this method has limitations in precisely cutting the scaffold to the exact size required for bone damage and makes it difficult to maintain uniform osteoporosis. Accordingly, there is a need for the development of a nanofiber scaffold that possesses mechanical properties suitable for bone defects and can be manufactured easily.

[0005] The present disclosure aims to provide a nanofiber membrane for bone regeneration that can be applied to a bone defect site to promote bone regeneration.

[0006] The present disclosure aims to provide a method for manufacturing a nanofiber membrane for bone regeneration that can easily produce a support having mechanical properties suitable for bone defect sites.

[0007] The present disclosure provides a nanofiber membrane for bone regeneration prepared by electrospinning a polymer solution comprising polyethylene oxide and a bone-forming protein.

[0008] The above bone morphogenetic protein may include one or more selected from BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, and DBM (demineralized bone matrix).

[0009] The above-mentioned nanofiber membrane for bone regeneration may have an average diameter of 10 to 1000 nm.

[0010] The above-mentioned nanofiber membrane for bone regeneration may comprise polyethylene oxide and BMP-2.

[0011] The above polyethylene oxide may have a number average molecular weight of 10,000 to 2,000,000 g / mol.

[0012] The present disclosure provides a pharmaceutical composition for bone regeneration comprising a nanofiber membrane for bone regeneration according to one embodiment of the present disclosure.

[0013] The present disclosure provides a method for manufacturing a nanofiber membrane for bone regeneration, comprising the steps of: mixing polyethylene oxide; and one or more bone-forming proteins selected from BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, and DBM (demineralized bone matrix); to prepare a polymer solution; and electrospinning the polymer solution.

[0014] The above manufacturing method may further include the step of reacting a polymer solution at 40 to 80°C for 0.5 to 5 hours.

[0015] The above polymer solution may further include a water and ethanol mixed solvent.

[0016] The above mixed solvent may contain 1 to 10 parts by weight of ethanol per 1 part by weight of water.

[0017] The above polymer solution may contain 0.1 to 1 weight part of bone-forming protein per 1 weight part of polyethylene oxide.

[0018] The above polymer solution may include polyethylene oxide and BMP-2.

[0019] The above electrospinning may be performed under conditions of a voltage of 10 to 200 V and a radiation distance of 5 to 50 cm.

[0020] A nanofiber membrane for bone regeneration according to one embodiment of the present invention can be implanted into a bone defect site to promote bone regeneration.

[0021] In addition, the nanofiber membrane for bone regeneration according to one embodiment of the present invention has excellent formulation retention and physical properties, resulting in minimal damage due to bleeding when inserted into a bone defect site and excellent in vivo stability.

[0022] A method for manufacturing a nanofiber membrane for bone regeneration according to one embodiment of the present invention allows the nanofiber membrane to be implanted into a bone defect using a simple spraying method.

[0023] Specifically, the method for manufacturing a nanofiber membrane for bone regeneration according to one embodiment of the present invention is efficient because it allows for the manufacturing of the nanofiber membrane in a non-contact manner, thereby reducing the risk of infection and the time required for the procedure.

[0024] Figure 1 shows a spreadability evaluation in which a nanofiber membrane is immersed in water at 37°C and its shape is maintained over time.

[0025] Figure 2 shows the results of observing the defect site after one week by applying the nanofibers of a) Comparative Example 2, b) Comparative Example 1, c) Example 1, d) Example 2, and e) Comparative Example 3, respectively, to the damaged defect site after the progression of the skull defect.

[0026] The present invention will be described in detail below. Unless otherwise defined, terms used in this specification should be interpreted as generally understood by those skilled in the art. The drawings and embodiments of this specification are intended to enable those skilled in the art to easily understand and practice the present invention; therefore, details that may obscure the essence of the invention may be omitted from the drawings and embodiments, and the present invention is not limited to the drawings and embodiments.

[0027] The singular form used in this specification may be intended to include the plural form unless specifically indicated otherwise in the context.

[0028] Furthermore, the numerical range used in this invention includes lower and upper limits and all values ​​within the range, increments logically derived from the form and width of the defined range, all of the specified values, and all possible combinations of upper and lower limits of the numerical range defined in different forms. Unless otherwise specifically defined in the specification of this invention, values ​​outside the numerical range that may occur due to experimental error or rounding are also included in the defined numerical range.

[0029] In this specification, terms such as "include," "have," and "have" mean that the features or components described in the specification are present, and unless specifically limited, this does not preclude the possibility that one or more other features or components may be added.

[0030] As used herein, the terms “bone regeneration” or “bone formation” may refer to any phenomenon in which bone damage is treated, alleviated, or improved through processes such as the proliferation of bone cells or the differentiation of osteoblasts into bone cells in damaged bone tissue, but is not limited thereto; however, said bone regeneration may specifically be due to the promotion of bone differentiation.

[0031] The present disclosure provides a nanofiber membrane for bone regeneration that can be implanted into a bone defect site to promote bone regeneration and has excellent in vivo stability due to its excellent formulation retention.

[0032] Specifically, the bone regeneration nanofiber membrane of the present disclosure is prepared by electrospinning a polymer solution comprising polyethylene oxide (PEO); and bone morphogenetic proteins (BMPs).

[0033] Since the above-mentioned nanofiber membrane for bone regeneration is manufactured through electrospinning, it can be easily attached regardless of the shape of the bone defect site, and is advantageous for bone regeneration as it can efficiently deliver bone-forming proteins by targeting the bone defect site.

[0034] The above bone morphogenetic protein may comprise one or more selected from BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, and DBM (demineralized bone matrix), and specifically, may be selected from demineralized bone matrix, BMP-2, BMP-4, and BMP-7, etc.

[0035] According to one embodiment, the bone morphogenetic protein may be a demineralized bone matrix (DBM), and the demineralized bone matrix may include one or more selected from BMP-2, BMP-4, BMP-7, platelet-rich plasma (PRP), platelet-derived growth factor (PDGF), and TGF-beta growth factor.

[0036] According to one embodiment, the nanofiber membrane for bone regeneration may have an average diameter of 10 to 1000 nm, a lower limit of 50, 100, 150, 200, or 250 nm, and an upper limit of 900, 800, 700, 600, or 500 nm.

[0037] According to one embodiment, the nanofiber membrane for bone regeneration may comprise polyethylene oxide and BMP-2.

[0038] According to one embodiment, the polyethylene oxide may have a number average molecular weight of 10,000 to 2,000,000 g / mol, a lower limit of 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000 g / mol, and an upper limit of 2,000,000, 1,800,000, 1,600,000, 1,400,000, 1,200,000, or 1,000,000 g / mol.

[0039] The present disclosure provides a pharmaceutical composition for bone regeneration comprising a nanofiber membrane for bone regeneration according to one embodiment of the present disclosure. The pharmaceutical composition for bone regeneration of the present disclosure may be used for the treatment of bone-related diseases requiring bone differentiation and regeneration, exemplarily traumatic bone injury such as a fracture or bone defect occurring after surgery, but is not limited thereto.

[0040] The pharmaceutical composition for bone regeneration of the present disclosure may further comprise a pharmaceutically acceptable carrier, said pharmaceutically acceptable carrier being one commonly used in formulations and including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0041] The pharmaceutical composition for bone regeneration of the present disclosure may additionally include, in addition to the above components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc.

[0042] The pharmaceutical composition for bone regeneration disclosed herein may be administered orally or parenterally, specifically by parenteral administration. In addition, in the case of parenteral administration, it may be administered intravenously, subcutaneously, intramuscularly, intraperitoneally, endothelially, topically, intranasally, intrapulmonaryly, rectally, or intra-articularly.

[0043] Suitable dosages of the pharmaceutical composition for bone regeneration of the present disclosure vary by factors such as formulation method, mode of administration, age, body weight, gender, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity, and a physician of ordinary skill can easily determine and prescribe an effective pharmaceutical dosage effective for the desired treatment or prevention.

[0044] The pharmaceutical composition for bone regeneration of the present disclosure may be prepared in a unit volume form or contained in a multi-volume container by formulation using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. The formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0045] The pharmaceutical composition for bone regeneration of the present disclosure may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

[0046] The present disclosure provides a method for manufacturing a nanofiber membrane for bone regeneration, comprising the steps of: mixing polyethylene oxide; and one or more bone-forming proteins selected from BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, and DBM (demineralized bone matrix); to prepare a polymer solution; and electrospinning the polymer solution.

[0047] According to one embodiment, the bone morphogenetic protein may be any one selected from demineralized bone matrix, BMP-2, BMP-4, and BMP-7.

[0048] According to one embodiment, the bone morphogenetic protein may be a demineralized bone matrix (DBM), and the demineralized bone matrix may include one or more selected from BMP-2, BMP-4, BMP-7, platelet-rich plasma (PRP), platelet-derived growth factor (PDGF), and TGF-beta growth factor.

[0049] According to one embodiment, the polymer solution may be reacted at 40 to 80°C for 0.5 to 5 hours, the lower limit may be 45, 50, or 55°C, and the upper limit may be 75, 70, or 65°C.

[0050] According to one embodiment, the polymer solution may comprise a mixed solvent of water and ethanol.

[0051] According to one embodiment, the mixed solvent may contain 1 to 10 parts by weight of ethanol per 1 part by weight of water, and specifically, the lower limit of ethanol per 1 part by weight of water may be 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 10 parts by weight, and the upper limit may be 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 parts by weight.

[0052] According to one embodiment, the polymer solution may contain 0.1 to 1 weight part of bone-forming protein per 1 weight part of polyethylene oxide, and specifically, may contain 0.2 to 0.8 weight parts, specifically 0.3 to 0.7 weight parts, and more specifically 0.4 to 0.6 weight parts of the total bone-forming protein per 1 weight part of polyethylene oxide.

[0053] According to one embodiment, the polymer solution may include polyethylene oxide and BMP-2.

[0054] According to one embodiment, the electrospinning may be performed under conditions of a voltage of 10 to 200 V and a spinning distance of 5 to 50 cm, specifically under conditions of a voltage of 10 to 100 V. Additionally, the average voltage of the electrospinning may be 5 to 50 V, 10 to 40 V, or 15 to 30 V.

[0055] The nanofiber membrane for bone regeneration according to the present invention will be described in more detail below through specific examples. However, the following examples are merely references for the detailed explanation of the present invention and do not limit the invention to such examples, and the invention may be implemented in various forms. Furthermore, the terms used in the description of the present invention are intended only to effectively describe specific examples and are not intended to limit the invention.

[0056] [Examples 1 to 2 and Comparative Examples 1 to 3]

[0057] A polymer solution was prepared with the composition shown in Table 1 below, and for Comparative Example 1, Example 1, and Example 2, a nanofiber membrane was prepared by electrospinning the polymer solution onto a stainless steel panel at a voltage of 10 to 100 V (average 20 V) using a tribostatic electrostatic-based portable nanofiber manufacturing device disclosed in Korean Patent Publication KR 10-2021-0151311 A.

[0058] As shown in Table 1 below, it was confirmed that nanofibers were formed through electrospinning in Comparative Example 1, Example 1, and Example 2, while Comparative Example 2 and Comparative Example 3 were confirmed to be in the form of a viscous gel and liquid, respectively, as the polymer solution itself without electrospinning.

[0059] Comparative Example 1 Example 1 Example 2 Comparative Example 2 Comparative Example 3 Polyethylene Oxide (mg) 10 10 10 100 BMP-2 (mg) 0 0.5 1 11 Water (mg) 18.75 18.75 18.75 18.75 18.75 Alcohol (mg) 5 6.25 56.25 56.25 56.25 56.25 Frictional Electrostatic Nanofiber Formation OO OX (Gel) X (Liquid)

[0060] The results of measuring the diameter, density, and compressive strength of the nanofibers of the aforementioned Examples 1 and 2 and Comparative Example 1 are shown in Table 2 below.

[0061] Comparative Example 1 Example 1 Example 2 Nanofiber Diameter (nm) 150~350 nm 250~500 nm 250~500 nm Nanofiber Density (mg / cm³) 3 )1.36 1.41 1.38 Compressive strength (g / cm² 2 )304311309

[0062] [Evaluation Example 1] Spreadability Evaluation

[0063] When nanofibers are filled into a bone defect, the formulation may be released by surrounding blood flow. To investigate the spreading ability of the nanofiber membrane due to blood flow, Examples 1 and 2 and Comparative Examples 1 to 3 were immersed in water at 37°C, and the maintenance of the external shape over time was observed. At this time, as shown in Figure 1, the results of measuring the time required for each nanofiber membrane to spread in water are shown in Table 3 below.

[0064] Comparative Example 1 Example 1 Example 2 Comparative Example 2 Comparative Example 3 Spreading time 30 minutes 30 minutes 30 minutes 5 minutes Immediately

[0065] As shown in Table 3 above, it can be seen that Comparative Example 1, Example 1, and 2, which were prepared in the form of tribostatic nanofibers, maintain their appearance in water for a longer period compared to Comparative Example 2 and 3.

[0066] [Evaluation Example 2] Bone Regeneration Evaluation

[0067] Local anesthesia was administered to 2-week-old nude mice by intramuscularly injecting 2% lidocaine HCl, and a skull defect model was fabricated by perforating the rat skull by 1 cm. The polymer solutions of Examples 1 and 2 and Comparative Examples 1 and 3 were injected into the bone defect of the skull defect model using a needle and implanted. The results of CT imaging taken after 4 weeks are shown in Figure 2, and the results of calculating the bone regeneration volume after 4 weeks relative to the total volume of the initial bone defect before nanofiber implantation are shown in Table 4 below.

[0068] Comparative Example 1 Example 1 Example 2 Comparative Example 2 Comparative Example 3 Bone regeneration rate (%) 25±12.2% 75±8.3% 90±5.8% 45±9.5% 7.8±5.9%

[0069] As shown in Table 4 above, it can be confirmed that Examples 1 and 2 exhibit a bone regeneration rate more than twice that of the comparative example, and accordingly, it is believed that the bone regeneration nanofiber membrane of the present invention can achieve a significantly superior effect in promoting bone regeneration in bone defects.

[0070] As described above, the present invention has been explained by specific details, limited embodiments, and comparative examples; however, these are provided merely to aid in a more comprehensive understanding of the invention, and the invention is not limited to the above embodiments. Those skilled in the art can make various modifications and variations from this description.

[0071] Accordingly, the scope of the present invention is not limited to the described embodiments, and all things equivalent to or having equivalent variations to the claims set forth below, as well as the claims set forth below, shall be considered to fall within the scope of the concept of the present invention.

Claims

1. A nanofiber membrane for bone regeneration, prepared by electrospinning a polymer solution comprising polyethylene oxide; and bone-forming protein.

2. In Paragraph 1, A nanofiber membrane for bone regeneration, wherein the bone morphogenetic protein comprises one or more selected from BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, and DBM (demineralized bone matrix).

3. In Paragraph 1, The above-mentioned nanofiber membrane for bone regeneration is a nanofiber membrane for bone regeneration having an average diameter of 10 to 1000 nm.

4. In Paragraph 1, The above-mentioned nanofiber membrane for bone regeneration comprises polyethylene oxide and BMP-2.

5. In Paragraph 1, The above polyethylene oxide is a nanofiber membrane for bone regeneration having a number average molecular weight of 10,000 to 2,000,000 g / mol.

6. A pharmaceutical composition for bone regeneration comprising a nanofiber membrane for bone regeneration according to any one of claims 1 to 5.

7. Polyethylene oxide; and A step of preparing a polymeric solution by mixing one or more bone morphogenetic proteins selected from BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, and DBM (deminered bone matrix); and A method for manufacturing a nanofiber membrane for bone regeneration, comprising the step of electrospinning the above polymer solution.

8. In Paragraph 7, A method for manufacturing a nanofiber membrane for bone regeneration, further comprising the step of reacting the above polymer solution at 40 to 80°C for 0.5 to 5 hours.

9. In Paragraph 7, A method for manufacturing a nanofiber membrane for bone regeneration, wherein the above polymer solution further comprises a mixed solvent of water and ethanol.

10. In Paragraph 9, A method for manufacturing a nanofiber membrane for bone regeneration, wherein the above-mentioned mixed solvent contains 1 to 10 parts by weight of ethanol per 1 part by weight of water.

11. In Paragraph 7, A method for manufacturing a nanofiber membrane for bone regeneration, wherein the polymer solution comprises 0.1 to 1 weight part of bone-forming protein per 1 weight part of polyethylene oxide.

12. In Paragraph 7, A method for manufacturing a nanofiber membrane for bone regeneration, wherein the above polymer solution comprises polyethylene oxide and BMP-2.

13. In Paragraph 7, A method for manufacturing a nanofiber membrane for bone regeneration, wherein the above electrospinning is performed under conditions of a voltage of 10 to 200 V and a spinning distance of 5 to 50 cm.

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