Cartilage regeneration nanofibrous membrane and manufacturing method therefor

Abstract

An object of the present disclosure is to provide a cartilage regeneration nanofibrous membrane manufactured by electrospinning a polymer solution comprising: polyethylene oxide; at least one selected from hyaluronic acid, polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), alginic acid, carrageenan, chitin, chitosan, poloxamer, cellulose and carboxymethyl chitosan; and at least one selected from TGF-β, kartogenin, chondroitin sulfate, polydeoxyribonucleotide, polynucleotide and curcumin.

Description

Nanofiber membrane for cartilage regeneration and method for manufacturing the same

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

[0002] Cartilage is an important tissue that reduces friction between bones in the joints and protects the joint area. Due to the nature of cartilage tissue, which is difficult to heal naturally, appropriate treatment is essential when it is damaged; if left untreated, inflammation in the joint can become chronic and eventually progress to serious joint diseases. Therefore, although various treatment methods have been developed and are in use, methods to achieve the regeneration of the cartilage itself remain limited, and there is a need to develop a fundamental cure.

[0003] Chondrogenic cartilage disease is a representative chronic condition affecting over 65% of the global population, and it is one of the diseases that is difficult to treat completely once it develops. Cartilage lacks blood vessels, which limits the supply of nutrients and oxygen, resulting in very low regenerative capacity. Cartilage damage is primarily caused by factors such as strenuous physical activity, aging, and obesity, and can lead to chronic conditions like arthritis. Currently, general treatments for chondrogenic cartilage disease aim to alleviate pain and inflammation or prevent further damage, but they are unable to fully restore damaged cartilage.

[0004] To date, one of the representative methods for treating cartilage damage is microfracture surgery. Microfracture surgery is a technique that regenerates cartilage by creating tiny perforations in the damaged cartilage to stimulate the bone marrow, allowing autologous stem cells from the bone marrow to flow into the affected area. However, microfracture surgery is primarily applied to patients in the early stages, and while it is used to aid in cartilage regeneration, it has limitations in fundamentally regenerating the cartilage itself completely.

[0005] The present invention aims to provide a nanofiber membrane for cartilage regeneration that can remain in joint synovial fluid and protect the defective area.

[0006] The present invention aims to provide a nanofiber membrane for cartilage regeneration that promotes cartilage regeneration and exhibits high adhesion within the joint.

[0007] The present disclosure provides a nanofiber membrane for cartilage regeneration prepared by electrospinning a polymer solution comprising: polyethylene oxide; one or more selected from hyaluronic acid, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), alginic acid, carrageenan, chitin, chitosan, poloxamer, cellulose, and carboxymethyl chitosan; and one or more selected from TGF-β, cartogenin, chondroitin sulfate, polydeoxyribonucleotide, polynucleotide, and curcumin.

[0008] The above-mentioned nanofiber membrane for cartilage regeneration may have an average diameter of 10 to 1000 nm.

[0009] The above-mentioned nanofiber membrane for cartilage regeneration may comprise polyethylene oxide, hyaluronic acid, and TGF-β.

[0010] The above polyethylene oxide may have a number average molecular weight of 10,000 to 2,000,000 g / mol.

[0011] The present disclosure provides a pharmaceutical composition for cartilage regeneration comprising a nanofiber membrane for cartilage regeneration according to one embodiment of the present disclosure.

[0012] The present disclosure provides a method for manufacturing a nanofiber membrane for cartilage regeneration, comprising the steps of: mixing polyethylene oxide; one or more selected from hyaluronic acid, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), alginic acid, carrageenan, chitin, chitosan, poloxamer, cellulose, and carboxymethyl chitosan; and one or more selected from TGF-β, cartogenin, chondroitin sulfate, polydeoxyribonucleotide, polynucleotide, and curcumin to prepare a polymer solution; and electrospinning the polymer solution.

[0013] The above polymer solution may be reacted at 50 to 70°C for 0.5 to 5 hours.

[0014] The above polymer solution may include a mixed solvent of water and ethanol.

[0015] The above mixed solvent may contain 1 to 10 parts by weight of ethanol per 1 part by weight of water.

[0016] The above polymer solution may contain 0.1 to 1 weight part of one or more selected from hyaluronic acid, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), alginic acid, carrageenan, chitin, chitosan, poloxamer, cellulose, and carboxymethyl chitosan per 1 weight part of polyethylene oxide.

[0017] The above polymer solution may contain 0.01 to 0.5 parts by weight of TGF-β, cartogenin, chondroitin sulfate, polydeoxyribonucleotide, polynucleotide, and curcumin per 1 part by weight of polyethylene oxide.

[0018] The above polymer solution may include polyethylene oxide, hyaluronic acid, and TGF-β.

[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 cartilage regeneration according to one embodiment of the present invention can be implanted into a cartilage defect site to provide physical lubrication and cushioning, thereby alleviating joint pain and aiding cartilage regeneration.

[0021] A nanofiber membrane for cartilage regeneration according to one embodiment of the present invention can exhibit excellent biocompatibility, persistence in the body, and tissue adhesion.

[0022] The method for manufacturing a nanofiber membrane for cartilage regeneration according to one embodiment of the present invention is manufactured in a non-contact manner, which has a low risk of infection and is efficient as it can reduce the procedure time.

[0023] Figure 1 shows the results of observing the defect site after one week, after injecting polymer solutions of a) the control group, b) Comparative Example 2, c) Comparative Example 3, d) Example 1, and e) Example 2, respectively, into the damaged cartilage site following the progression of a defect in the knee cartilage.

[0024] 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.

[0025] The singular form used in this specification is intended to include the plural form unless specifically indicated in the context.

[0026] 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.

[0027] 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.

[0028] In the present invention, "cartilage" is a tissue that originates embryologically from the same mesoderm as bone tissue, forms an endoskeletal system together with bone, and is composed of chondrocytes surrounded by a large amount of extracellular matrix, and the chondrocytes can synthesize and secrete cartilage matrix within the cartilage.

[0029] In the present invention, "cartilage regeneration" refers to any state in which part or all of the tissue of a damaged, defective, or missing cartilage area is completely or partially restored or recovered to its original state, and in the present invention, regeneration may be used interchangeably with restoration, repair, or reconstruction.

[0030] The present disclosure provides a nanofiber membrane for cartilage regeneration that can be implanted into a cartilage defect site to increase the secretion of growth factors related to cartilage formation, matrix formation, and cartilage regeneration, thereby providing an environment suitable for cartilage regeneration.

[0031] Specifically, the nanofiber membrane for cartilage regeneration of the present disclosure is prepared by electrospinning a polymer solution comprising: polyethylene oxide (PEO); one or more selected from hyaluronic acid, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), alginic acid, carrageenan, chitin, chitosan, poloxamer, cellulose, and carboxymethyl chitosan; and one or more selected from TGF-β (Transforming Growth Factor-beta), kartogenin, chondroitin sulfate, polydeoxyribonucleotide, polynucleotide, and curcumin.

[0032] The nanofiber membrane for cartilage regeneration disclosed in the present disclosure can promote the differentiation of chondrocytes and cartilage synthesis. Furthermore, since the nanofiber membrane is manufactured by electrospinning a polymer solution of the aforementioned composition, it can be applied to a cartilage defect site with a large surface area and excellent adhesion, and can provide physical lubrication and cushioning to the damaged site.

[0033] According to one embodiment, the nanofiber membrane for cartilage 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. The diameter may represent the diameter of the nanofibers constituting the nanofiber membrane for cartilage regeneration.

[0034] According to one embodiment, the nanofiber membrane for cartilage regeneration may comprise polyethylene oxide, hyaluronic acid, and TGF-β, and specifically, TGF-β may be TGF-β3.

[0035] 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.

[0036] According to one embodiment, the extracellular matrix may be included in an amount of 1 to 20 weight% with respect to the entire polymer solution, the lower limit may be 0.1, 0.3, 0.5, 0.8, or 1 weight%, and the upper limit may be 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 weight%.

[0037] The present disclosure provides a pharmaceutical composition for cartilage regeneration comprising a nanofiber membrane for cartilage regeneration according to one embodiment of the present disclosure. The pharmaceutical composition for cartilage regeneration of the present disclosure may be used for the treatment of cartilage damage caused by trauma, degenerative arthritis, or rheumatoid arthritis, but is not limited thereto.

[0038] The pharmaceutical composition for cartilage 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.

[0039] The pharmaceutical composition for cartilage regeneration of the present disclosure may additionally include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc. in addition to the above components.

[0040] The pharmaceutical composition for cartilage regeneration of the present disclosure 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, specifically by intra-articular administration.

[0041] Suitable dosages of the pharmaceutical composition for cartilage 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.

[0042] The pharmaceutical composition for cartilage regeneration of the present disclosure may be prepared in a unit dose form or contained in a multi-dose 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.

[0043] The pharmaceutical composition for cartilage 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.

[0044] The present disclosure provides a method for manufacturing a nanofiber membrane for cartilage regeneration, comprising the steps of: mixing polyethylene oxide; one or more selected from hyaluronic acid, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), alginic acid, carrageenan, chitin, chitosan, poloxamer, cellulose, and carboxymethyl chitosan; and one or more selected from TGF-β, cartogenin, chondroitin sulfate, polydeoxyribonucleotide, polynucleotide, and curcumin to prepare a polymer solution; and electrospinning the polymer solution.

[0045] 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.

[0046] According to one embodiment, the polymer solution may comprise a mixed solvent of water and ethanol.

[0047] According to one embodiment, the mixed solvent may contain 1 to 10 parts by weight of ethanol per 1 part by weight of water. Specifically, the lower limit of ethanol per 1 part by weight of water may be 1, 1.5, 2, 2.5, or 2.8 parts by weight, and the upper limit may be 10, 9, 8, 7, 6, 5, 4, or 3.5 parts by weight, and the values ​​may be between these.

[0048] According to one embodiment, the polymer solution may comprise 0.1 to 1 weight part selected from hyaluronic acid, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), alginic acid, carrageenan, chitin, chitosan, poloxamer, cellulose, and carboxymethyl chitosan per 1 weight part of polyethylene oxide, and specifically, the total weight part of the aforementioned materials may comprise 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 per 1 weight part of polyethylene oxide.

[0049] According to one embodiment, the polymer solution may contain 0.01 to 0.5 parts by weight of TGF-β, cartogenin, chondroitin sulfate, polydeoxyribonucleotide, polynucleotide, and curcumin per 1 part by weight of polyethylene oxide, specifically, the total weight of the aforementioned materials per 1 part by weight of polyethylene oxide may be 0.01 to 0.4 parts by weight, 0.01 to 0.3 parts by weight, or 0.01 to 0.2 parts by weight, and more specifically, 0.03 to 0.15 parts by weight.

[0050] As the above polymer solution contains the aforementioned amount of cartilage regeneration-promoting material, it can achieve excellent physical properties and a superior cartilage regeneration effect during electrospinning.

[0051] According to one embodiment, the polymer solution may comprise polyethylene oxide, hyaluronic acid, and TGF-β.

[0052] 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.

[0053] The nanofiber membrane for cartilage regeneration according to the present invention will be described in more detail below through specific embodiments. However, the following embodiments are merely references for the detailed explanation of the present invention and the present invention is not limited thereto and may be implemented in various forms. Furthermore, the terms used in the description of the present invention are intended only to effectively describe specific embodiments and are not intended to limit the present invention.

[0054] [Examples 1 to 2 and Comparative Examples 1 to 3]

[0055] A polymer solution was prepared with the composition shown in Table 1 below, and 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-based portable nanofiber manufacturing device disclosed in Korean Patent Publication KR 10-2021-0151311 A.

[0056] Comparative Example 1 Comparative Example 2 Comparative Example 3 Example 1 Example 2 Polyethylene Oxide (mg) 10 10 10 10 10 Hyaluronic Acid (mg) 0 5 0 5 5 TGF-β (mg) 0 0 10.5 1 Water (mg) 18.7 5 18.7 5 18.7 5 18.7 5 18.7 5 Alcohol (mg) 5 6.2 5 5 6.2 5 6.2 5 6.2 5 6.2 5 6.2 5

[0057] The results of measuring the diameter and density of the nanofibers of the aforementioned Examples 1 to 2 and Comparative Examples 1 to 3 are shown in Table 2 below.

[0058] Comparative Example 1 Comparative Example 2 Comparative Example 3 Example 1 Example 2 Nanofiber Diameter (nm) 150~350 250~500 150~350 250~500 250~500 Nanofiber Density (mg / cm²) 3 )1.351.741.391.691.62

[0059] [Evaluation Example 1] Adhesion Strength Evaluation

[0060] A stainless steel plate of size 50 x 125 mm was prepared, and polymer solutions of Examples 1 and 2 and Comparative Examples 1 to 3 were electrospun onto the stainless steel plate in the same manner as in the examples and comparative examples to produce a nanofiber film with a thickness of 1 mm. Subsequently, the stainless steel plate with the nanofiber film was fixed at 180 degrees to a universal testing machine, and the load was measured by pulling the universal testing machine at a speed of 5 mm / s. The results are shown in Table 3 below.

[0061] A silicone pad with dimensions of 50 x 125 mm was prepared, and a nanofiber membrane was fabricated by electrospinning the polymer solutions of Examples 1 and 2 and Comparative Examples 1 to 3 onto the silicone pad, respectively, in the same manner as in the examples and comparative examples. The pad was then placed upright at 90 degrees inside a chamber at 37°C for 1 minute. The remaining percentage was determined by measuring the area of ​​the remaining amount relative to the total area, and the results are shown in Table 3.

[0062] Comparative Example 1 Comparative Example 2 Comparative Example 3 Example 1 Example 2 Load (N) 5.1±1.58 78.5±6.59 4.9±1.41 75.1±8.11 74.5±9.57 Residual Area (%) 82% 100% 80% 100% 100%

[0063] According to Table 3 above, it can be seen that excellent adhesive strength can be achieved in the case of Example 1 and Example 2.

[0064] [Evaluation Example 2] Cartilage Regeneration Evaluation

[0065] All New Zealand white rabbits were purchased from Damul Science (Daejeon, Korea), and a cartilage defect model was created in 15 rabbits (3–3.5 kg, 3 months old) to evaluate the regenerative effect of cartilage tissue (3 samples per group). After anesthesia, a cartilage defect with a diameter of 4 mm and a height of 1 mm was created on the femoral trochlear surface of the knee of each rabbit. For Examples 1 and 2 and Comparative Examples 2 and 3, the polymer solutions of Examples 1 and 2 and Comparative Examples 1 and 3 were injected into the damaged cartilage sites of the knee cartilage of 3 rabbits, respectively, using a syringe needle after the defect had progressed. The results of observing the defect sites one week later are shown in Figure 1, and the results of quantitatively measuring the cartilage regeneration rate one week after surgery compared to before surgery are shown in Table 4 below. The group that received no treatment after the knee cartilage defect progressed was designated as the control group.

[0066] Control Group Comparative Example 2 Comparative Example 3 Example 1 Example 2 Cartilage regeneration rate (%) -30±9.5% 80±4.5% 92±5.5% 100%

[0067] As shown in Table 4 above, it can be seen that in the case of Examples 1 and 2, a significantly superior cartilage regeneration effect can be achieved compared to the comparative example.

[0068] 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.

[0069] 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. Polyethylene oxide; One or more selected from hyaluronic acid, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), alginate, carrageenan, chitin, chitosan, poloxamer, cellulose, and carboxymethyl chitosan; and Nanofiber membrane for cartilage regeneration, prepared by electrospinning a polymer solution comprising one or more selected from TGF-β, cartogenin, chondroitin sulfate, polydeoxyribonucleotide, polynucleotide, and curcumin.

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

3. In Paragraph 1, The above-mentioned nanofiber membrane for cartilage regeneration comprises polyethylene oxide, hyaluronic acid, and TGF-β.

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

5. A pharmaceutical composition for cartilage regeneration comprising a nanofiber membrane for cartilage regeneration according to any one of claims 1 to 4.

6. Polyethylene oxide; One or more selected from hyaluronic acid, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), alginate, carrageenan, chitin, chitosan, poloxamer, cellulose, and carboxymethyl chitosan; and One or more selected from TGF-β, cartogenin, chondroitin sulfate, polydeoxyribonucleotide, polynucleotide, and curcumin; and a step of preparing a polymer solution by mixing A method for manufacturing a nanofiber membrane for cartilage regeneration, comprising the step of electrospinning the above polymer solution.

7. In Paragraph 6, A method for manufacturing a nanofiber membrane for cartilage regeneration, wherein the above polymer solution is reacted at 50 to 70 ℃ for 0.5 to 5 hours.

8. In Paragraph 6, A method for manufacturing a nanofiber membrane for cartilage regeneration, wherein the polymer solution comprises a mixed solvent of water and ethanol.

9. In Paragraph 8, A method for manufacturing a nanofiber membrane for cartilage regeneration, wherein the above-mentioned mixed solvent contains 1 to 10 parts by weight of ethanol per 1 part by weight of water.

10. In Paragraph 6, A method for manufacturing a nanofiber membrane for cartilage regeneration, wherein the polymer solution comprises 0.1 to 1 weight part selected from hyaluronic acid, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), alginic acid, carrageenan, chitin, chitosan, poloxamer, cellulose, and carboxymethyl chitosan per 1 weight part of polyethylene oxide.

11. In Paragraph 6, A method for preparing a nanofiber membrane for cartilage regeneration, wherein the polymer solution comprises 0.01 to 0.5 parts by weight of TGF-β, cartogenin, chondroitin sulfate, polydeoxyribonucleotide, polynucleotide, and curcumin per 1 part by weight of polyethylene oxide.

12. In Paragraph 6, A method for manufacturing a nanofiber membrane for cartilage regeneration, wherein the above polymer solution comprises polyethylene oxide, hyaluronic acid, and TGF-β.

13. In Paragraph 6, A method for manufacturing a medical nanofiber membrane, 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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