Multilayer electrospun micro / nano fiber membranes based on marine sulfated polysaccharides for periodontal tissue / bone regeneration

Abstract

The present invention relates to a two- or three-layer film composition for periodontal use, comprising a hydrophobic polymer outer layer and one or two inner layers in the form of electrospun micro / nanofibers based on marine sulfated polysaccharides and calcium.

Description

Technical Field

[0001] The present invention relates to a composition in the form of a two - layer or three - layer film for use in the periodontium, comprising an outer layer of a hydrophobic polymer and one or two inner layers in the form of electrospun micro / nanofibers based on marine sulfated polysaccharides and calcium.

Background Art

[0002] Periodontitis is an inflammation of the periodontal tissues induced by bacteria [D. Fraser, et al. Periodontal Wound Healing and Regeneration: Insights for Engineering New Therapeutic Approaches. Front. Dent. Med. 2022, 3, 815810], characterized by the destruction of the periodontal ligament (PDL) and alveolar bone [V. Uskokovic et al. Polymeric nanotechnologies for the treatment of periodontitis: A chronological review. Int. J. Pharm. 2022, 625, 122065], and is a major cause of tooth loss in adults.

[0003] The optimal goal of restoring tooth function is achieved through periodontal regeneration procedures aimed at restoring the periodontal apparatus to its initial state [Uskokovic V. et al. Polymeric nanotechnologies for the treatment of periodontitis: A chronological review. Int. J. Pharm. 2022, 625, 122065; S. Mirzaeei, et al. An overview of guided tissue regeneration (GTR) systems designed and developed as drug carriers for management of periodontitis. J. Drug Deliv. Sci. Technol. 2022, 71, 103341]. Guided tissue / bone regeneration (GTR / GBR) membranes act as a physical barrier that isolates rapidly growing epithelium, providing sufficient time and space for PDL cells and osteoblasts to proliferate and counteract attachment loss [D. Fraser, et al. Periodontal Wound Healing and Regeneration: Insights for Engineering New Therapeutic Approaches. Front. Dent. Med. 2022, 3, 815810; S. Mirzaeei, et al. An overview of guided tissue regeneration (GTR) systems designed and developed as drug carriers for management of periodontitis. J. Drug Deliv. Sci. Technol. 2022, 71, 103341].

[0004] However, the main drawbacks of currently available GTR / GBR membranes are their low adhesion rates and the unsuccessful regeneration of osteocytes.

[0005] Nanofiber scaffolds / membranes designed to mimic the natural structure of the extracellular matrix (ECM) have been shown to enhance cell proliferation and differentiation, and are now being developed as carriers, growth factors, antimicrobial agents, and other bioactive substances for cells [S. Zupancic, et al. Sustained release of antimicrobials from double-layer nanofiber mats for local treatment of periodontal disease, evaluated using a new micro flow through apparatus. J. Control. Release 2019, 316, 223-235]. Therefore, nanotechnology can contribute to periodontal tissue engineering [Uskokovic V. et al. Polymeric nanotechnologies for the treatment of periodontitis: A chronological review. Int. J. Pharm. 2022, 625, 122065; S. Mirzaeei, et al. An overview of guided tissue regeneration (GTR) systems designed and developed as drug carriers for management of periodontitis. J. Drug Deliv. Sci. Technol. 2022, 71, 103341].Electrospinning, using a charged polymer solution jet, is the most successful method for preparing fiber scaffolds with fiber diameters ranging from microscale to nanoscale (on the order of μm to nm), and shows great potential in the development of membranes for periodontal regeneration [SL. Bee, et al. Asymmetric resorbable-based dental barrier membrane for periodontal guided tissue regeneration and guided bone regeneration: A review. J. Biomed. Mater. Res. 2022, 110, 2157-2182]. This is an efficient, versatile, easy, cost-effective, and scalable method for producing polymer fibers, and by varying a wide range of parameters (e.g., applied voltage, polymer flow rate, distance from tip to collector), it is possible to produce fibers with desired properties and diverse morphologies.

[0006] The main advantages of micro / nanofiber membranes are their high porosity, increased surface area, adjustable size, fiber orientation and diameter, and adjustable micromechanical properties [M. Zafar, et al. Potential of Electrospun Nanofibers for Biomedical and Dental Applications. Materials 2016, 9, 1-21]. Furthermore, their inherent interconnectivity and topography can facilitate angiogenesis and the transport of nutrients and bioactive agents [J. Park, et al. Nanosize and vitality: TiO2 nanotube diameter directs cell fate. Nano Lett. 2007, 7, 1686-1691]. In particular, nanotopography of these fibrous structures increases the total surface area, surface-to-volume ratio, and roughness, thereby enhancing adhesion between the bone-cementum interface and the underlying scaffold surface. Meanwhile, microtopography facilitates cell penetration, angiogenesis, and nutrient diffusion, resulting in better spatial organization for cell growth [X. Xu, et al. Biodegradable engineered fiber scaffolds fabricated by electrospinning for periodontal tissue regeneration. J. Biomater. Appl. 2021, 36, 55-75].

[0007] However, the main drawback of currently available micro / nanofiber membranes is that they do not promote the regeneration of osteocytes and periodontal ligament cells.

[0008] Document US2018 / 0078346A1 refers to a composition of a three-layer dental membrane from electrospun nanofibers of biodegradable polymers having pores of different diameters. However, the formulation in document US2018 / 0078346A1 is based on synthetic polymers and hydrophilic agents that do not have the ability to promote the regeneration of osteocytes and periodontal ligament cells. Furthermore, the formulation in document US2018 / 0078346A1 does not contain marine sulfated polysaccharides or calcium.

[0009] Marine sulfated polysaccharides are highly biocompatible and biodegradable materials characterized by diverse structures and functions [MJ Cardoso, et al. Marine origin polysaccharides in drug delivery systems. Mar. Drugs 2016, 14, 34]. Due to the wide range of bioactivities exhibited by marine sulfated polysaccharides, these materials are ideal biomaterials for the development of novel systems for applications in tissue engineering, wound healing, and drug delivery [M. Claveri, et al. Marine-derived polymeric materials and biomimetics: An overview. Polymers 2020, 12, 1002]. Carrageenan, urban, and fucan possess anticoagulant, antioxidant, antitumor, antihyperlipidemia, and immunomodulatory activities, while also promoting osteoblast adhesion and proliferation, thus exhibiting bone formation effects [K. Iliou, et al, Marine Biopolymers as Bioactive Functional Ingredients of Electrospun Nanofibrous Scaffolds for Biomedical Applications. Mar. Drugs 2022, 20, 314]. Electrospun nanofiber scaffolds based on marine sulfated polysaccharides possess multifunctional and tunable mechanical properties, attracting increasing interest in the development of advanced systems in the biomedical field [K. Iliou, et al, Marine Biopolymers as Bioactive Functional Ingredients of Electrospun Nanofibrous Scaffolds for Biomedical Applications. Mar. Drugs 2022, 20, 314].

[0010] Conventional technologies do not mention dental micro / nanofiber membrane compositions that simultaneously contain marine sulfated polysaccharides and calcium, have the ability to promote bone and periodontal ligament regeneration, and may contain antibacterial agents. [Overview of the project] [Problems that the invention aims to solve]

[0011] The present invention relates to a two- or three-layer film composition for periodontal use, comprising a hydrophobic polymer outer layer and one or two inner layers in the form of electrospun micro / nanofibers based on marine sulfated polysaccharides and calcium. [Means for solving the problem]

[0012] The problems mentioned above are solved by the present invention as defined below:

[0013] Definition 1. A composition in the form of a two- or three-layer film for periodontal use, wherein the composition comprises A and B. A is a hydrophobic polymer, B is selected from the following four options b1 and / or b2 and / or b3 and / or b4: b1 is a calcium salt of marine sulfated polysaccharide in the form of electrospun micro / nanofibers. b2 is a mixture of a calcium salt of a marine sulfated polysaccharide and a calcium salt of a hydrophilic polymer in the form of electrospun micro / nano fibers. b3 is a mixture of marine sulfated polysaccharides and calcium salts of marine sulfated polysaccharides in the form of electrospun micro / nano fibers. b4 is a mixture of marine sulfated polysaccharides and calcium salts of hydrophilic polymers in the form of electrospun micro / nano fibers. A composition characterized by the following features.

[0014] Definition 2. The composition according to Definition 1, characterized in that the marine sulfated polysaccharides and / or calcium salts thereof contained in b1, b2, b3, or b4 for the production of electrospun micro / nanofibers are selected from carrageenan, urban, and fucan, and / or calcium salts thereof, preferably from carrageenan and / or calcium salts of carrageenan, and more preferably from calcium salts of carrageenan.

[0015] The composition according to Definition 1 or 2, characterized in that the calcium salt of the hydrophilic polymer contained in Definition 3.b2 or b4 is selected from the calcium salt of poly(L-glutamic acid) and the calcium salt of poly(L-aspartic acid), and preferably the calcium salt of poly(L-glutamic acid).

[0016] A composition according to any one of Definitions 1 to 3, characterized in that Definition 4.A is selected from polycaprolactone, poly(L-lactide), and poly(DL-lactide), preferably polycaprolactone or poly(DL-lactide), and more preferably polycaprolactone.

[0017] Definition 5. A composition according to any one of Definitions 1 to 4, characterized in that it is formulated in two layers, i) the outer layer comprises A, and ii) the inner layer comprises b1 and / or b2 and / or b3 and / or b4.

[0018] Definition 6. The composition according to Definition 5, characterized in that the average diameter of the electrospun micro / nano fibers in the inner layer is in the range of 100 nm to 1100 nm, preferably 300 nm to 900 nm, more preferably 500 nm to 700 nm, for example, 632 nm.

[0019] Definition 7. A composition according to Definition 5 or 6, characterized in that it is compounded into two layers, i) the outer layer comprises polycaprolactone, for example in the form of a cast film, and ii) the inner layer comprises a calcium salt of poly(L-glutamic acid) and a calcium salt of carrageenan in the form of electrospun micro / nano fibers.

[0020] Definition 8. A composition according to any one of Definitions 1 to 4, characterized in that it is formulated in three layers, i) the outer layer comprises A, ii) the intermediate layer comprises b1 and / or b2 and / or b3 and / or b4, or a calcium salt of a hydrophilic polymer, and iii) the inner layer comprises b1 and / or b2 and / or b3 and / or b4.

[0021] Definition 9. The composition according to Definition 8, characterized in that the average diameter of the electrospun micro / nanofibers in the middle layer and / or the inner layer is in the range of 40 nm to 600 nm, preferably 100 nm to 500 nm, more preferably 200 nm to 400 nm, for example, 292 nm.

[0022] Definition 10. The composition according to Definition 8 or 9, characterized in that it is formulated into three layers, wherein i) the outer layer contains polycaprolactone, for example, in the form of a cast film, ii) the middle layer contains a calcium salt of poly(L-glutamic acid) in the form of electrospun micro / nanofibers, and iii) the inner layer contains a calcium salt of carrageenan in the form of electrospun micro / nanofibers.

[0023] Definition 11. The composition according to any one of Definitions 1 to 10, further comprising one or more pharmaceutically active substances, preferably antibacterial agents, more preferably metronidazole or ciprofloxacin, most preferably metronidazole and / or other non-pharmaceutically active substances and / or other excipients.

[0024] Definition 12. The composition according to any one of Definitions 1 to 11, for use in the treatment of periodontitis.

[0025] Prior to the present invention, there is no mention in the prior art of the use of a unique combination comprising an outer layer of a hydrophobic polymer and one or two inner layers in the form of electrospun micro / nanofibers based on marine sulfated polysaccharides and calcium in a specific form of the previous definitions.

[0026] Surprisingly, it has been found that the compositions of the present invention as defined by Definitions 1 to 12, comprising an outer layer of a hydrophobic polymer and one or two inner layers in the form of electrospun micro / nanofibers based on marine sulfated polysaccharides and calcium, exhibit osteogenic activity by the use of marine sulfated polysaccharides and accelerate bone formation by calcium.

[0027] Furthermore, surprisingly, it has been found that compositions of the present invention, as defined by definitions 1-12, comprising a hydrophobic polymer outer layer and one or two inner layers in the form of electrospun micro / nanofibers based on marine sulfated polysaccharides and calcium, can promote cell adhesion of periodontal ligament cells, osteogenic differentiation of periodontal ligament cells, and proliferation of osteoblasts, thereby enhancing bone and periodontal ligament regeneration.

[0028] Surprisingly, it has been found that compositions of the present invention, as defined by definitions 1 to 12, comprising a hydrophobic polymer outer layer and one or two inner layers in the form of electrospun micro / nanofibers based on marine sulfated polysaccharides and calcium, exhibit enhanced mechanical strength and act as a barrier that prevents the proliferation of epithelial cells, for example, in the form of a nonporous polymer cast film, when present in the hydrophobic polymer outer layer.

[0029] Finally, and surprisingly, it was found that the more specific the claims, the stronger the effects and advantages of the present invention.

[0030] According to the present invention, a composition in the form of a two- or three-layer film for periodontal use comprises an outer layer of a hydrophobic polymer and one or two inner layers in the form of electrospun micro / nano fibers based on marine sulfated polysaccharides and calcium.

[0031] According to the present invention, in a composition in the form of a two- or three-layer film for periodontal use, the calcium salt of the hydrophilic polymer for the production of electrospun micro / nanofibers is selected from the calcium salt of poly(L-glutamic acid) and the calcium salt of poly(L-aspartic acid).

[0032] In a preferred embodiment, in a composition of the present invention in the form of a two- or three-layer film for periodontal use, the calcium salt of the hydrophilic polymer for the production of electrospun micro / nanofibers is a calcium salt of poly(L-glutamic acid).

[0033] In a preferred embodiment, in a composition of the present invention in the form of a two- or three-layer film for periodontal use, the calcium salt of the hydrophilic polymer for the production of electrospun micro / nanofibers is the calcium salt of poly(L-aspartic acid).

[0034] According to the present invention, in a composition in the form of a two- or three-layer film for periodontal use, the marine sulfated polysaccharides or their calcium salts for the production of electrospun micro / nanofibers are selected from carrageenan, urban, fucan, and / or salts thereof.

[0035] In a preferred embodiment, in a composition of the present invention in the form of a two- or three-layer film for periodontal use, the marine sulfated polysaccharide for the production of electrospun micro / nano fibers is carrageenan.

[0036] In a preferred embodiment, in a composition of the present invention in the form of a two- or three-layer film for periodontal use, the salt of marine sulfated polysaccharide for the production of electrospun micro / nanofibers is a calcium salt of carrageenan.

[0037] In a preferred embodiment, in a composition of the present invention in the form of a two- or three-layer film for periodontal use, the marine sulfated polysaccharide for the production of electrospun micro / nanofibers is Urban.

[0038] In a preferred embodiment, in a composition of the present invention in the form of a two- or three-layer film for periodontal use, the salt of marine sulfated polysaccharide for the production of electrospun micro / nanofibers is the calcium salt of Urban.

[0039] In a preferred embodiment, in a composition of the present invention in the form of a two- or three-layer film for periodontal use, the marine sulfated polysaccharide for the production of electrospun micro / nano fibers is fukan.

[0040] In a preferred embodiment, in a composition of the present invention in the form of a two- or three-layer film for periodontal use, the salt of marine sulfated polysaccharide for the production of electrospun micro / nanofibers is a calcium salt of Fucan.

[0041] According to the present invention, in a composition in the form of a two- or three-layer film for periodontal use, the hydrophobic polymer forms the outer layer of the composition, for example, in the form of a cast film, and is selected from polycaprolactone, poly(L-lactide), and poly(DL-lactide).

[0042] In a preferred embodiment, the hydrophobic polymer in the two- or three-layer film composition for periodontal use of the present invention is polycaprolactone.

[0043] In a preferred embodiment, the hydrophobic polymer in the two- or three-layer film composition for periodontal use of the present invention is poly(L-lactide).

[0044] In preferred embodiments, the hydrophobic polymer in the two- or three-layer film composition for periodontal use of the present invention is poly(DL-lactide).

[0045] According to the present invention, the composition in the form of a film for periodontal use is formulated in two layers, the outer layer comprising a hydrophobic polymer and the inner layer comprising marine sulfated polysaccharides and calcium in the form of electrospun micro / nano fibers.

[0046] In a preferred embodiment, the composition in the form of a film for periodontal use of the present invention is formulated in two layers, the outer layer comprising a hydrophobic polymer and the inner layer comprising a calcium salt of marine sulfated polysaccharide in the form of electrospun micro / nanofibers.

[0047] In a preferred embodiment, the composition in the form of a film for periodontal use of the present invention is formulated in two layers, the outer layer comprising a hydrophobic polymer and the inner layer comprising a calcium salt of marine sulfated polysaccharide and a calcium salt of hydrophilic polymer in the form of electrospun micro / nano fibers.

[0048] In a preferred embodiment, the composition in the form of a film for periodontal use of the present invention is formulated in two layers, the outer layer comprising a hydrophobic polymer and the inner layer comprising marine sulfated polysaccharides and their calcium salts in the form of electrospun micro / nano fibers.

[0049] In a preferred embodiment, the composition in the form of a film for periodontal use of the present invention is formulated in two layers, the outer layer comprising a hydrophobic polymer and the inner layer comprising marine sulfated polysaccharides and calcium salts of hydrophilic polymers in the form of electrospun micro / nanofibers.

[0050] In a particularly preferred embodiment, the composition in the form of a film for periodontal use of the present invention is formulated in two layers, the outer layer comprising polycaprolactone and the inner layer comprising a calcium salt of carrageenan in the form of electrospun micro / nanofibers.

[0051] In a particularly preferred embodiment, the composition in the form of a film for periodontal use of the present invention is formulated in two layers, the outer layer comprising polycaprolactone and the inner layer comprising calcium salts of carrageenan and poly(L-glutamic acid) in the form of electrospun micro / nanofibers.

[0052] In a particularly preferred embodiment, the composition in the form of a film for periodontal use of the present invention is formulated in two layers, the outer layer comprising polycaprolactone and the inner layer comprising carrageenan and its calcium salts in the form of electrospun micro / nanofibers.

[0053] In a particularly preferred embodiment, the composition in the form of a film for periodontal use of the present invention is formulated in two layers, the outer layer comprising polycaprolactone and the inner layer comprising carrageenan and a calcium salt of poly(L-glutamic acid) in the form of electrospun micro / nanofibers.

[0054] According to the present invention, in a two-layer composition of the present invention for use as a film for periodontal use, the average diameter of the electrospun micro / nano fibers in the inner layer is in the range of 100 nm to 1100 nm.

[0055] In a preferred embodiment, the composition of the present invention for periodontal use is in the form of a two-layer film, wherein the average diameter of the electrospun micro / nano fibers in the inner layer is in the range of 300 nm to 900 nm.

[0056] In a particularly preferred embodiment, in a composition of the present invention in the form of a film for periodontal use, which is composed of two layers, the average diameter of the electrospun micro / nano fibers of the inner layer is in the range of 500 nm to 700 nm, for example, 632 nm.

[0057] According to the present invention, the composition in the form of a film for periodontal use is formulated into three layers, the outer layer comprising a hydrophobic polymer, and the middle and inner layers comprising marine sulfated polysaccharides and calcium in the form of electrospun micro / nano fibers.

[0058] In a preferred embodiment, the composition in the form of a film for periodontal use of the present invention is formulated in three layers, the outer layer comprising a hydrophobic polymer, the middle layer comprising a calcium salt of marine sulfated polysaccharide in the form of electrospun micro / nano fibers, and the inner layer comprising marine sulfated polysaccharide in the form of electrospun micro / nano fibers.

[0059] In a preferred embodiment, the composition in the form of a film for periodontal use of the present invention is formulated in three layers, the outer layer comprising a hydrophobic polymer, the middle layer comprising a calcium salt of a hydrophilic polymer in the form of electrospun micro / nanofibers, and the inner layer comprising marine sulfated polysaccharides in the form of electrospun micro / nanofibers.

[0060] In a preferred embodiment, the composition in the form of a film for periodontal use of the present invention is formulated in three layers, the outer layer comprising a hydrophobic polymer, the middle layer comprising calcium salts of marine sulfated polysaccharides and calcium salts of hydrophilic polymers in the form of electrospun micro / nanofibers, and the inner layer comprising marine sulfated polysaccharides in the form of electrospun micro / nanofibers.

[0061] In a preferred embodiment, the composition in the form of a film for periodontal use of the present invention is formulated in three layers, the outer layer comprising a hydrophobic polymer, the middle layer comprising calcium salts of marine sulfated polysaccharides in the form of electrospun micro / nanofibers, and the inner layer comprising calcium salts of marine sulfated polysaccharides in the form of electrospun micro / nanofibers.

[0062] In a preferred embodiment, the composition in the form of a film for periodontal use of the present invention is formulated in three layers, the outer layer comprising a hydrophobic polymer, the middle layer comprising a calcium salt of a hydrophilic polymer in the form of electrospun micro / nanofibers, and the inner layer comprising a calcium salt of marine sulfated polysaccharide in the form of electrospun micro / nanofibers.

[0063] In a preferred embodiment, the composition in the form of a film for periodontal use of the present invention is formulated in three layers, the outer layer comprising a hydrophobic polymer, the middle layer comprising calcium salts of marine sulfated polysaccharides and calcium salts of hydrophilic polymers in the form of electrospun micro / nanofibers, and the inner layer comprising calcium salts of marine sulfated polysaccharides in the form of electrospun micro / nanofibers.

[0064] In a particularly preferred embodiment, the composition in the form of a film for periodontal use of the present invention is formulated in three layers, the outer layer comprising polycaprolactone, the middle layer comprising calcium salt of carrageenan in the form of electrospun micro / nanofibers, and the inner layer comprising calcium salt of carrageenan in the form of electrospun micro / nanofibers.

[0065] In a particularly preferred embodiment, the composition in the form of a film for periodontal use of the present invention is formulated in three layers, the outer layer comprising polycaprolactone, the middle layer comprising a calcium salt of poly(L-glutamic acid) in the form of electrospun micro / nanofibers, and the inner layer comprising a calcium salt of carrageenan in the form of electrospun micro / nanofibers.

[0066] In a particularly preferred embodiment, the composition in the form of a film for periodontal use of the present invention is formulated in three layers, the outer layer comprising polycaprolactone, the middle layer comprising calcium salt of poly(L-glutamic acid) and calcium salt of carrageenan in the form of electrospun micro / nanofibers, and the inner layer comprising calcium salt of carrageenan in the form of electrospun micro / nanofibers.

[0067] In a particularly preferred embodiment, the composition in the form of a film for periodontal use of the present invention is formulated into three layers, the outer layer comprising polycaprolactone, the middle layer comprising a calcium salt of carrageenan in the form of electrospun micro / nanofibers, and the inner layer comprising carrageenan in the form of electrospun micro / nanofibers.

[0068] In a particularly preferred embodiment, the composition in the form of a film for periodontal use of the present invention is formulated in three layers, the outer layer comprising polycaprolactone, the middle layer comprising a calcium salt of poly(L-glutamic acid) in the form of electrospun micro / nanofibers, and the inner layer comprising carrageenan in the form of electrospun micro / nanofibers.

[0069] In a particularly preferred embodiment, the composition in the form of a film for periodontal use of the present invention is formulated in three layers, the outer layer comprising polycaprolactone, the middle layer comprising calcium salt of poly(L-glutamic acid) and calcium salt of carrageenan in the form of electrospun micro / nanofibers, and the inner layer comprising carrageenan in the form of electrospun micro / nanofibers.

[0070] According to the present invention, in a composition in the form of a film for periodontal use, which is compounded into three layers, the average diameter of the electrospun micro / nano fibers in the intermediate layer and / or inner layer is in the range of 40 nm to 600 nm.

[0071] In a preferred embodiment, in a composition of the present invention in the form of a film for periodontal use, which is compounded into three layers, the average diameter of the electrospun micro / nano fibers of the intermediate and / or inner layers is in the range of 100 nm to 500 nm.

[0072] In a particularly preferred embodiment, in a composition of the present invention in the form of a film for periodontal use, which is compounded into three layers, the average diameter of the electrospun micro / nano fibers of the intermediate and / or inner layers is in the range of 200 nm to 400 nm, for example, 292 nm.

[0073] According to the present invention, the composition in the form of a two- or three-layer film for periodontal use comprises one or more pharmaceutically active substances and / or other non-pharmaceutically active substances and / or other excipients.

[0074] In a preferred embodiment, the composition of the present invention for periodontal use in the form of a two- or three-layer film contains an antibacterial agent.

[0075] In a particularly preferred embodiment, the composition of the present invention for periodontal use, in the form of a two- or three-layer film, comprises metronidazole.

[0076] In a particularly preferred embodiment, the composition of the present invention for periodontal use, in the form of a two- or three-layer film, comprises ciprofloxacin.

[0077] According to the present invention, the two- or three-layer composition for periodontal use is suitable for use in the treatment of periodontitis or other oral diseases. [Modes for carrying out the invention]

[0078] The present invention will be further illustrated by the following exemplary, non-limiting embodiments.

[0079] [Example 1] Preparation of a three-layer micro / nano fiber membrane for periodontal use (GTR1) The outer layer contains a cast polycaprolactone film, the intermediate layer contains poly(L-glutamic acid) calcium salt in the form of electrospun micro / nanofibers, while the inner layer contains carrageenan calcium salt in the form of electrospun micro / nanofibers. In the case of electrospinning of poly(L-glutamic acid) calcium salt (PG-Ca) and carrageenan calcium salt (CG-Ca), polyethylene oxide (PEO) was used as a hydrophilic polymer carrier to enhance their electrospinnability in the nonwoven film.

[0080] 1. Preparation of spinning solution All spinning solutions were prepared with stirring for 24 hours to ensure their homogeneity. Polycaprolactone (PCL) spinning solutions were prepared at room temperature, while all aqueous spinning solutions were prepared at 60°C. To prepare the PCL spinning solution, PCL (molecular weight 80,000) was dissolved in DCM / DMF8:2 at a concentration of 10% w / v (e.g., 0.27 g of PCL in 2.7 mL of solvent). CG-Ca / PEO spinning solutions were prepared by dissolving CG-Ca in distilled H2O at a concentration of 1% w / v, followed by the addition of PEO (molecular weight 900,000) at a concentration of 4% w / v (e.g., 0.05 g of CG-Ca and 0.2 g of PEO in 5 mL of solvent). A PG-Ca / PEO spinning solution was prepared by dissolving PG-Ca in distilled H2O at a concentration of 4% w / v, followed by the addition of PEO (molecular weight 7,000,000) at a concentration of 4% w / v (for example, 0.2 g of PG-Ca and 0.2 g of PEO in 5 mL of solvent).

[0081] Electrospinning was performed using a γ-High Voltage Research DC power generator with a maximum voltage of 50kV. The spinning solution was filled into 10mL disposable syringes equipped with a 23-gauge (23G) stainless steel blunt needle. The syringes were mounted on a horizontally positioned, programmable syringe pump (Harvard PHD 2000, Harvard Apparatus), and the produced micro / nano fibers were deposited onto an RC-6000 (NaBond Technologies) rotating drum collector at a rotational speed of 400 rpm. The temperature and relative humidity were 21±2℃ and 60±5%, respectively.

[0082] 2. Preparation of the outer layer To prepare the outer layer, a cast film was generated on the surface of a rotating drum according to the solvent casting process. To prepare the outer layer (cast film), PCL (molecular weight 80,000) was dissolved in benzene at a concentration of 8% w / v while stirring at room temperature for 48 hours (e.g., 24 g of PCL in 300 mL of solvent) to ensure the homogeneity of the solution. An appropriate volume of the PCL solution was transferred to a container. The drum was then immersed in the polymer solution and rotated until the entire surface of the cylinder was covered with the polymer solution. The container containing the PCL solution was then removed, and the drum was rotated in air until the solvent had completely evaporated and a thin film had formed on its surface. The outer layer served as a control sample (CTRL) in various analyses of the fabricated multilayer films.

[0083] 3. Construction of the central layer To fabricate the central layer of the three-layer GTR1 film, the central layer was electrospun onto the surface of the outer layer (PCL cast film). To obtain the central layer, PCL and PG-Ca / PEO spinning solutions were co-electrospun using an antiparallel setup with syringes mounted on two horizontally opposed programmable syringe pumps to ensure homogeneity of the blended polymer fibers (a small volume of the PCL electrospun solution was first electrospun separately, and then the electrospun solution was co-electrospun to ensure cohesion between the deposited fibers and the cast film). The supply rate and tip-to-collector distance of the PCL spinning solution were fixed at 2.5 mL / h and 16 cm, respectively, while the supply rate and tip-to-collector distance of the PG-Ca / PEO spinning solution were adjusted to 5 mL / h and 27 cm, respectively. Electrospinning was performed with a fixed applied voltage of 27 kV.

[0084] 4. Fabrication of the inner layer To fabricate the inner layer of the three-layer GTR1 membrane, the inner layer was electrospun onto the surface of the central layer. To obtain the inner layer, the CG-Ca / PEO spinning solution was electrospun with the applied voltage, supply rate, and distance from the tip to the collector fixed at 27kV, 3mL / h, and 30cm, respectively.

[0085] [Example 2] Preparation of a two-layer micro / nano fiber membrane for periodontal use (GTR2) The outer layer contains a cast film of polycaprolactone, while the inner layer contains calcium salts of poly(L-glutamic acid) and carrageenan in the form of electrospun micro / nanofibers. In the case of electrospinning of calcium salts of poly(L-glutamic acid) (PG-Ca) and carrageenan (CG-Ca), polyethylene oxide (PEO) was used as a hydrophilic polymer carrier to enhance their electrospinnability in the nonwoven film.

[0086] 1. Preparation of spinning solution All spinning solutions were prepared with stirring for 24 hours to ensure their homogeneity. The PCL spinning solution was prepared at room temperature, while all aqueous spinning solutions CG-Ca / PG-Ca / PEO were prepared at 60°C. To prepare the PCL spinning solution, PCL (molecular weight 80,000) was dissolved in DCM / DMF8:2 at a concentration of 10% w / v (e.g., 0.27 g of PCL in 2.7 mL of solvent). The CG-Ca / PG-Ca / PEO spinning solution was prepared by dissolving CG-Ca in distilled H2O at a concentration of 1% w / v, followed by the addition of PG-Ca at a concentration of 4% w / v and PEO (molecular weight 7,000,000) at a concentration of 4% w / v (e.g., 0.05 g of CG-Ca, 0.2 g of PG-Ca, and 0.2 g of PEO in 5 mL of solvent).

[0087] Electrospinning was performed using a γ-High Voltage Research DC power generator with a maximum voltage of 50kV. The spinning solution was filled into 10mL disposable syringes equipped with a 23-gauge (23G) stainless steel blunt needle. The syringes were mounted on a horizontally positioned, programmable syringe pump (Harvard PHD 2000, Harvard Apparatus), and the produced micro / nano fibers were deposited onto an RC-6000 (NaBond Technologies) rotating drum collector at a rotational speed of 400 rpm. The temperature and relative humidity were 21±2℃ and 60±5%, respectively.

[0088] 2. Preparation of the outer layer The outer layer of the two-layered film is prepared in the same manner as described above for the three-layered film of Example 1.

[0089] 3. Fabrication of the inner layer To fabricate the inner layer of the bilayer GTR2 film, the inner layer was electrospun onto the surface of the outer layer (PCL cast film). To obtain the inner layer, PCL and CG-Ca / PG-Ca / PEO spinning solutions were co-electrospun using an antiparallel setup with syringes mounted on two horizontally opposed programmable syringe pumps to ensure homogeneity of the blended polymer fibers (small volumes of the PCL electrospun solution were first electrospun separately to ensure cohesion between the deposited fibers and the cast film, and then the electrospun solutions were co-electrospun). Electrospinning was performed with a fixed applied voltage of 27kV. The supply rate of the PCL spinning solution and the distance from the tip to the collector were fixed at 2.5mL / h and 16cm, respectively, while the supply rate of the CG-Ca / PG-Ca / PEO spinning solution and the distance from the tip to the collector were adjusted to 5mL / h and 27cm, respectively.

[0090] The micro / nanofiber membranes GTR1 and GTR2 of Examples 1 and 2 were characterized for their morphology, diameter range, and average diameter of the micro / nanofibers. Morphological characterization of the micro / nanofiber membranes GTR1 and GTR2 of Examples 1 and 2 (Figure 1) was performed using a PhenomWorld (Thermo Fischer Scientific) desktop scanning electron microscope (SEM) equipped with a tungsten filament (10kV) and a charge reduction sample holder. It was found that a non-porous surface was obtained in the outer layer, while a uniform micro / nanofiber network, with cylindrical morphological fibers, was successfully obtained in the electrospun layer inside both fabricated membranes. The diameter range and average diameter of the fabricated micro / nanofibers are shown in Table 1.

[0091] Table 1. Diameter range and average diameter of manufactured micro / nano fibers [Table 1]

[0092] The micro / nano fiber membranes GTR1 and GTR2 of Examples 1 and 2 were characterized for their thermal stability. The thermal stability of the micro / nano fiber membranes GTR1 and GTR2 of Examples 1 and 2 was investigated by thermogravimetric analysis (TGA) using a TA thermogravimetric analyzer (TGA55, TA Instruments). As shown in the TGA thermogram (Figure 2), the CTRL, GTR1, and GTR2 membranes showed similar thermal profiles characterized by the main alteration stages in their thermogravimetric curves. Specifically, the alteration of CTRL began at 335°C and was completed at 401°C. The decomposition of GTR1 began at 319°C and was completed at 403°C, while GTR2 began to decompose at 309°C, and its alteration was completed at approximately 391°C.

[0093] The key mechanical properties of the micro / nano fiber membranes GTR1 and GTR2 of Examples 1 and 2, as well as the membrane-referenced CTRL, were tested using specially prepared specimens. The determined mechanical properties included the modulus of elasticity (E), ultimate tensile strength (UTS), plastic deformation, and percentile reduction of initial force (RAS). For each membrane, seven dumbbell-shaped specimens of specific dimensions were cut and placed in a tensile strength meter (Tensometer Aegis 10, Monsanto, UK) (Figure 3). The crosshead speed was set to 25 mm / min. The results were recorded, stress-strain diagrams were created, and from these, the membrane resistance to elastic deformation, as well as tensile strength and plastic deformation, were calculated. For determining the reduction of force under constant strain (relaxation), for each membrane, seven orthogonal parallelogram-shaped specimens were prepared and then placed in a low-force load cell machine connected to a PC that measured force every 30 seconds (Figure 4). The initial applied force was set to 3N, and its decrease over time was recorded for 48 hours, as earlier studies have shown that the plateau is reached much earlier. Data collected from both trials were statistically evaluated using one-way ANOVA and Tukey's test.

[0094] Table 2 presents the results for the mechanical properties of the micro / nano fiber films GTR1 and GTR2 of Examples 1 and 2, and the corresponding figures are shown in Figures 5 and 6. The two-layer film GTR2 is characterized by the highest elastic modulus value (128.3 MPa), followed by the CTRL film which serves as a control, and finally the three-layer film GTR1. The CTRL film shows the lowest average ultimate tensile strength, but this is not significantly different from the other two. From the stress-strain curves, it is clear that placing an additional layer on the CTRL film results in a dramatic increase in plastic deformation in the range of 1.6 to 3.5 times. Regarding relaxation tests, no statistically significant differences were recorded between the films. The percentile decrease of the initial force under constant strain at 48 hours reached 35%, of which 80% occurred in the first few hours.

[0095] In conclusion, membrane GTR2 exhibited the highest average elastic modulus. The differences in average elastic modulus and ultimate tensile strength between the different membranes were not critically large. The control membrane CTRL is the main factor influencing the fundamental mechanical properties of E and UTS. Adding further layers on top of the CTRL membrane increased plastic deformation. The membrane percentile decrease of initial force under constant strain was concentrated in the first few hours, and after 48 hours, no statistically significant difference was observed between the averages of the different membranes.

[0096] Table 2. Mechanical properties of the micro / nano fiber films GTR1 and GTR2, and the film CTRL, of Examples 1 and 2, expressed as mean values ​​(standard deviation). [Table 2]

[0097] E (elastic modulus), UTS (ultimate tensile strength), and RAS (percentile reduction of initial force under constant strain). The presence of different abbreviations indicates statistically significant differences between the means of different films (p<0.05).

[0098] The micro / nanofiber membranes GTR1 and GTR2 of Examples 1 and 2 were evaluated for their cell adhesion and the morphology of PDL cells seeded on the fabricated membranes. The micro / nanofiber membranes GTR1 and GTR2 of Examples 1 and 2 were evaluated in vitro using human PDL cells cultured in Dulbecco's Modified Eagle Medium (DMEM) isolated from the periodontal ligament of healthy teeth. In passage 4, PDL cells were seeded on membrane GTR1, GTR2, and CTRL, or on a standard culture plate (control), and cultured in the same medium.

[0099] Cell adhesion on the membranes was examined by scanning electron microscopy. The biocompatibility of these membranes with respect to PDL cells was investigated by MTT assay from day 1 to day 7. The bone induction properties of the membranes were studied by alizarin red staining.

[0100] All the membranes examined were biocompatible with PDL cells and non-cytotoxic (Figure 7). The membranes were found to promote the adhesion of seeded cells (Figures 8 and 9).

[0101] Cells seeded on a membrane exhibit extension of lamellar and filopodia, which indicates good cell adhesion and migration.

[0102] Alizarin red staining revealed that membrane GTR1 and GTR2 in Examples 1 and 2 (the former being more pronounced than the latter) could promote the osteogenic differentiation of PDL cells more rapidly (within one week in bone induction medium) compared to membrane CTRL and the control plate (Figure 10).

[0103] From the previous examples, it can be seen that the compositions exhibit increased mechanical strength, particularly a dramatic increase in plastic deformation, compared to the reference film, and that they have advantages such as being biocompatible with periodontal ligament cells, being non-cytotoxic, and having the ability to promote the adhesion and migration of periodontal ligament cells, as well as the ability to promote the osteogenic differentiation of these cells. The calcium supply from the compositions of Examples 1 and 2 contributes considerably favorably to the regeneration and osteogenic differentiation of periodontal ligament cells.

[0104] The results mentioned above are explained in more detail and better understood by referring to Figures 1-10 attached below. [Brief explanation of the drawing]

[0105] [Figure 1] (a) SEM image and diameter distribution histogram of film GTR1 with an average diameter of 292 nm, (b) SEM image and diameter distribution histogram of film GTR2 with an average diameter of 632 nm, and (c) SEM image of film CTRL. [Figure 2] These are TGA thermograms of CTRL, GTR1, and GTR2 membranes. [Figure 3] (a, b) Diagrams illustrating a dumbbell-shaped membrane test specimen prepared using an acrylic mold of specific dimensions for tensile testing, and (c) a membrane placed on tensometer grabbers that move at a speed of 25 mm / min. [Figure 4] (a) A relaxation membrane test specimen (3 × 50 mm) in the shape of an orthogonal parallelogram, and (b) a low-force load cell that applies strain to the membrane until an initial force of 3 N is reached, and then records the decrease in strain over time. [Figure 5] These are exemplary stress-strain curves for the micro / nano fiber films GTR1 and GTR2 of Examples 1 and 2, as well as the reference film CTRL. [Figure 6] (a) Curves of the stress relaxation test of the micro / nano fiber membrane GTR1 of Example 1, (b) micro / nano fiber membrane GTR2 of Example 2, and (c) reference membrane CTRL. [Figure 7] (a) proliferation of PDL cells cultured for 1 day and (b) for 7 days on a standard culture plate (control), as well as seeded on CTRL, GTR1, and GTR2 membranes. [Figure 8] These are SEM images of (a) GTR1, (b) GTR2, and (c) PDL cells seeded on a CTRL membrane after 4 days of culture in DMEM. [Figure 9]These are SEM images of (a) GTR1, (b) GTR2, and (c) PDL cells seeded on a CTRL membrane after 4 days of culture in osteogenic medium. [Figure 10] These are images of PDL cells stained with alizarin red (dark gray in the image) after 7 days of bone formation induction, showing (a) control, (b) CTRL membrane, (c) GTR1 membrane, and (d) GTR2 membrane.

Claims

1. A composition in the form of a two- or three-layer film for periodontal use, wherein the composition comprises A and B. A is a hydrophobic polymer, B has the following four options b １ and / or b ２ and / or b ３ and / or b ４ Selected from, b １ However, in the form of electrospun micro / nano fibers, they are calcium salts of marine sulfated polysaccharides, b ２ However, in the form of electrospun micro / nanofibers, it is a mixture of calcium salts of marine sulfated polysaccharides and calcium salts of hydrophilic polymers. b ３ However, in the form of electrospun micro / nano fibers, it is a mixture of marine sulfated polysaccharides and calcium salts of marine sulfated polysaccharides. b ４ However, in the form of electrospun micro / nanofibers, it is a mixture of marine sulfated polysaccharides and calcium salts of hydrophilic polymers. A composition characterized by the following features.

2. b for the production of the electrospun micro / nanofibers １ or b ２ or b ３ or b ４ The composition according to claim 1, characterized in that the marine sulfated polysaccharides and / or their calcium salts contained in b are selected from carrageenan, ulvan and fucoidan, and / or their calcium salts, preferably from carrageenan and / or the calcium salt of carrageenan, more preferably the calcium salt of carrageenan.

3. b ２ or b ４ The composition according to claim 1 or 2, characterized in that the calcium salt of the hydrophilic polymer contained therein is selected from the calcium salt of poly(L-glutamic acid) and the calcium salt of poly(L-aspartic acid), and preferably the calcium salt of poly(L-glutamic acid).

4. The composition according to any one of claims 1 to 3, characterized in that A is selected from polycaprolactone, poly(L-lactide), and poly(DL-lactide), preferably polycaprolactone or poly(DL-lactide), and more preferably polycaprolactone.

5. It is formulated in two layers: i) the outer layer contains A, and ii) the inner layer contains b １ and / or b ２ and / or b ３ and / or b ４ A composition according to any one of claims 1 to 4, characterized by containing the following:

6. The composition according to claim 5, characterized in that the average diameter of the electrospun micro / nano fibers of the inner layer is in the range of 100 nm to 1100 nm, preferably 300 nm to 900 nm, more preferably 500 nm to 700 nm, for example, 632 nm.

7. The composition according to claim 5 or 6, characterized in that it is formulated in two layers, i) the outer layer comprises polycaprolactone, for example in the form of a cast film, and ii) the inner layer comprises a calcium salt of poly(L-glutamic acid) and a calcium salt of carrageenan in the form of electrospun micro / nanofibers.

8. It is formulated in three layers: i) the outer layer contains A, and ii) the middle layer contains b １ and / or b ２ and / or b ３ and / or b ４ , or containing a calcium salt of a hydrophilic polymer, iii) inner layer is b １ and / or b ２ and / or b ３ and / or b ４ A composition according to any one of claims 1 to 4, characterized by containing the following:

9. The composition according to claim 8, characterized in that the average diameter of the electrospun micro / nano fibers in the intermediate layer and / or the inner layer is in the range of 40 nm to 600 nm, preferably 100 nm to 500 nm, more preferably 200 nm to 400 nm, for example, 292 nm.

10. The composition according to claim 8 or 9, characterized in that it is formulated in three layers, i) the outer layer comprises polycaprolactone, for example in the form of a cast film; ii) the intermediate layer comprises a calcium salt of poly(L-glutamic acid) in the form of electrospun micro / nano fibers; and iii) the inner layer comprises a calcium salt of carrageenan in the form of electrospun micro / nano fibers.

11. The composition according to any one of claims 1 to 10, further comprising one or more pharmaceutically active substances, preferably antibacterial agents, more preferably metronidazole or ciprofloxacin, most preferably metronidazole and / or other non-pharmaceutically active substances and / or other excipients.

12. A composition according to any one of claims 1 to 11 for use in the treatment of periodontitis.

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