Tissue-restoring biodegradable composite structure having uniform porosity

Abstract

The present invention provides a tissue-restoring biodegradable composition comprising microspheres that contain poly-L-lactic acid (PLLA) and hydroxyapatite (HAp). In addition, the present invention provides a method for preparing a tissue-restoring biodegradable composition, comprising the steps of: preparing microspheres by mixing poly-L-lactic acid and hydroxyapatite; and adding hyaluronic acid to the microspheres, wherein a collagen tissue regeneration effect is maintained from the initial stage to the final stage after a filler injection.

Description

Biodegradable composite structure for tissue restoration having uniform porosity

[0001] The present invention relates to a biodegradable composition for tissue restoration comprising microspheres composed of poly-L-lactic acid (PLLA) and hydroxyapatite (HAp). More specifically, the microspheres are a biodegradable composite structure for tissue restoration having uniform porosity, which can maintain the collagen tissue regeneration effect from the early stages of filler injection to the later stages.

[0002]

[0003] Fillers are used in cosmetic and reconstructive medicine to fill wrinkles, increase volume, and improve the appearance of the skin. Existing filler technologies include natural low- and high-molecular-weight fillers, calcium-based fillers, and synthetic polymer-based fillers. While natural low- and high-molecular-weight fillers utilize naturally derived ingredients, they have issues such as causing allergic reactions or low durability due to rapid biodegradation. Calcium-based fillers last longer but often cause stiffness at the injection site, while synthetic polymer-based fillers pose risks of inflammatory reactions and side effects, and are difficult to see effects in the early stages. To address these problems, the inventors have made diligent efforts to develop a new type of filler that is sustainable, effective from the initial stages to the mid-to-long term, minimizes pain, and reduces inflammatory responses.

[0004] The effect of the filler has been maximized by using a combination of various biocompatible materials, and this composite filler provides immediate effects after initial injection and can maintain stable effects over the long term.

[0005] The composite filler of the present invention is expected to significantly improve patient satisfaction in the fields of cosmetic and reconstructive medicine and set a new standard in the filler market.

[0006]

[0007] One objective of the present invention is to provide a biodegradable composition for tissue restoration comprising microspheres containing poly-L-lactic acid (PLLA) and hydroxyapatite (HAp).

[0008] Another objective of the present invention is to provide a method for preparing a biodegradable composition for tissue restoration, comprising the steps of: preparing microspheres by mixing poly-L-lactic acid and hydroxyapatite; and adding hyaluronic acid to the microspheres.

[0009]

[0010] Various embodiments described herein are described with reference to the drawings. In the following description, for a complete understanding of the invention, various specific details, such as specific forms, compositions, and processes, are described. However, specific embodiments may be practiced with or without one or more of these specific details, or in combination with other known methods and forms. In other examples, known processes and manufacturing techniques are not described as specific details so as not to unnecessarily obscure the invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that the particular features, forms, compositions, or characteristics described in association with the embodiment are included in one or more embodiments of the invention. Accordingly, the context of “one embodiment” or “an embodiment” expressed at various places throughout this specification does not necessarily represent the same embodiment of the invention. Additionally, particular features, forms, compositions, or characteristics may be combined in any suitable way in one or more embodiments. Unless otherwise specifically defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.

[0011]

[0012] In this invention, the term "filler" refers to a substance injected into the body primarily to restore or improve the volume of skin or tissues. Fillers are used for cosmetic purposes to smooth wrinkles or increase facial volume, and are generally composed of substances such as hyaluronic acid (HA), collagen, and polylactic acid (PLLA). Fillers are injected via syringe and provide an immediate volume-increasing effect at the injection site. The duration and characteristics of the effect vary depending on the type of filler used, and important features include high biocompatibility and the ability to safely decompose or be absorbed within the body.

[0013] In the present invention, the term “tissue restoration” refers to a process of inducing the morphological and functional recovery of biological tissues (e.g., skin, soft tissue, muscle, connective tissue, etc.) that have been damaged or degenerated due to trauma, aging, surgery, or disease. More specifically, it may include, but is not limited to, a process of restoring the tissue to a form similar to its original structure by promoting the generation of new cells and matrix at the damaged site; a process of restoring the elasticity, thickness, and strength of the tissue through cell proliferation, collagen synthesis, and ECM (extracellular matrix) rearrangement at the damaged site; and a process of improving functional deficits by supplementing or replacing the damaged site with biocompatible polymers, cells, or bioactive substances.

[0014] In this invention, the term “Poly-L-lactic acid (PLLA)” refers to a type of Polylactic acid (PLA) that is a biodegradable polymer produced by the polymerization of L-lactic acid. Poly-L-lactic acid is a polymer derived from plants that possesses high biocompatibility and biodegradability. Poly-L-lactic acid induces collagen synthesis and fibroblast proliferation through inflammatory responses while minimizing the risk of immune reactions. Furthermore, it is gradually decomposed within the body, converted into lactic acid, and naturally excreted.

[0015] In this invention, the term hydroxyapatite (HAp) refers to a naturally occurring mineral having the chemical formula Ca5(PO4)3(OH). It consists of two crystal unit cells and is found in teeth and bones within the body. Hydroxyapatite exhibits excellent biocompatibility, high bone similarity, and high osteoconductivity and biocompatibility; consequently, it is commonly used as a coating agent to promote bone regeneration, such as in fillers to replace severed bones or in artificial implants. Furthermore, hydroxyapatite is used in dermal fillers, primarily mixed with polymers such as poly-L-lactic acid, and also serves as a base material for drug delivery systems that stably bind bioactive molecules and allow for their slow release. In particular, the porous structure of hydroxyapatite encapsulates drugs, providing a long-term release effect.

[0016] In this invention, the term porosity refers to the property of a material having many small, regular or irregular holes within it. Porous materials are lightweight due to their numerous pores and possess a large surface area; they play an important role in catalysts, adsorbents, and drug delivery systems because they allow substances to be absorbed or pass through these pores.

[0017] In the present invention, the term "microsphere" refers to a spherical fine particle with a diameter ranging from 1 μm to 1000 μm. It can be produced from various materials such as polymers, ceramics, glass, and metals using natural or synthetic raw materials. Microspheres may be solid or hollow, or may have a porous structure with pores. In the case of a porous structure with pores, this is advantageous for the loading and sustained release of drugs or bioactive molecules, and a sustained therapeutic effect can be expected.

[0018] In this invention, the term hyaluronic acid (HA) refers to a polyanionic mucopolysaccharide that was first isolated from the vitreous humor of the eye by Meyer and Palmer in 1934 and is a bio-derived polymer widely present in nature. Hyaluronic acid is distributed in various molecular weights (1 to 10 million Daltons) in almost all tissues of animals, including skin, muscle, skeleton, blood, lymph, placenta, eye, cartilage, and synovial fluid, with the highest concentration found in skin tissue. As a polysaccharide, it connects collagen, elastin, and fibrous tissues within tissues such as cartilage and skin in mammals, including humans. Hyaluronic acid has been developed and used as a medical component for tissue repair (for the replacement and reconstruction of human tissue) both domestically and internationally, and is also widely used in the fields of cosmetic dermatology and plastic surgery.

[0019] In the present invention, the term "polynucleotide" refers to a biopolymer composed of 13 or more nucleotide units. It constitutes genetic material such as DNA or RNA and performs various physiological roles, including the storage of genetic information, regulation of biological functions, and tissue regeneration. Furthermore, polynucleotides have the function of promoting cell growth and tissue regeneration, so they are effectively used for wound healing and skin regeneration, and are used in filler products for skin regeneration and wrinkle improvement.

[0020] In the present invention, the term "bioactive molecule" refers to a molecule that induces specific physiological reactions within the body or interacts with biological systems to regulate the function of cells or tissues. These molecules are involved in various life activities of the human body and may include drugs, proteins, peptides, polynucleotides, etc. Bioactive molecules may influence biological functions such as cell growth, tissue regeneration, regulation of immune responses, and reduction of inflammation. Although not limited thereto, the bioactive molecules may include polynucleotides, EVs, growth factors, PDRN, etc.

[0021] In the present invention, the term extracellular vesicles (EVs) refers to small membrane-bound particles secreted by cells that play an important role in intercellular information transfer and the regulation of the extracellular environment. They contain various biomolecules such as proteins, lipids, RNA, and DNA. In particular, since extracellular vesicles may contain molecules generated or altered under specific disease conditions, they can be used as biomarkers in cancer diagnosis or research on degenerative diseases, and are also being studied as drug delivery systems due to their excellent biocompatibility. Although not limited thereto, the extracellular vesicles include exosomes, microvesicles, and apoptotic vesicles.

[0022] In the present invention, the term exosomes refers to small vesicles with a size of approximately 30 to 150 nm that originate from multivesicles and are produced inside a cell and then secreted outside the cell. They are responsible for intercellular signaling and regulating the cellular environment and are studied as targets for treatment and diagnosis.

[0023] In the present invention, the term microvesicles refers to vesicles that originate directly from the cell membrane and are released outside the cell, with a size of approximately 100 to 1000 nm. They are involved in interactions with the extracellular environment, intercellular material transport, and signal transduction.

[0024] In the present invention, the term apototic bodies refers to large vesicles formed by decomposition during the process of apoptosis with a size of about 500 to 2000 nm, which serve to remove cell debris after apoptosis.

[0025] In the present invention, the term growth factor (GF) refers to a naturally occurring substance capable of stimulating cell proliferation, wound healing, and cell differentiation. Growth factors regulate cell behavior by binding to receptors on the cell surface and activating cell signaling pathways.

[0026] The above growth factors include, but are not limited to, epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), and bone morphogenetic protein (BMP).

[0027] In this invention, the term Epidermal Growth Factor (EGF) refers to a growth factor that promotes the regeneration of skin and epithelial tissues and is primarily involved in wound healing and skin regeneration.

[0028] In the present invention, the term PDRN (Polydeoxyribonucleotide) refers to a DNA complex based on salmon germ cells and organs. It is a tissue regeneration material similar to human DNA and exists in cells to physiologically stimulate regeneration and metabolic activity. PDRN is classified as a polynucleotide and is a low-molecular-weight substance composed of nucleotide sequences that constitute DNA. PDRN possesses characteristics such as promoting growth factor production, promoting angiogenesis, inducing fibrous differentiation, reducing inflammation, and inducing the production and secretion of growth factors to achieve tissue regeneration, skin regeneration, and whitening effects, and is utilized in various medical and cosmetic fields.

[0029] In this invention, the term "biodegradability" refers to the ability to naturally decompose in the natural environment or within the body through microorganisms, enzymes, chemical reactions, etc. Biodegradable materials break down into small molecules over time and are ultimately converted into water, carbon dioxide, inorganic substances, or biometabolites. This process reduces the impact on the environment, and when used within the body, it can naturally decompose and disappear without the need for additional removal surgery.

[0030] The above biodegradable materials include, but are not limited to, poly-L-lactic acid, PLGA (Poly(lactic-co-glycolic acid)), hydrogel, and cellulose.

[0031] In this invention, the terms tissue restoration or tissue repair refer to the process in which damaged tissue is regenerated or repaired to restore its original function and structure. This is primarily achieved through physiological processes such as cell growth, differentiation, and collagen production, and involves the operation of biological mechanisms that facilitate the recovery of wounds or damaged tissues.

[0032] In this invention, the term "adjuvant" refers to a substance added to enhance or complement the function of the main component. These adjuvants improve the physical, chemical, and physiological properties of the material, thereby helping to effectively achieve objectives such as tissue restoration, drug delivery, and skin regeneration.

[0033] The above auxiliary agents include, but are not limited to, trehalose, sodium carboxymethyl cellulose (CMC), polyvinyl alcohol (PVA), polyethylene glycol (PEG), etc.

[0034] In the present invention, the term trehalose refers to a disaccharide that stabilizes the structure of cells or proteins during the freeze-drying process and enhances the stability of bioactive molecules due to its excellent ability to retain moisture. Additionally, it reduces cell damage during skin regeneration or wound healing processes and promotes tissue restoration and healing by retaining moisture.

[0035] In this invention, the term Sodium Carboxymethyl Cellulose (CMC) is highly soluble in water, increases viscosity, and regulates the slow release of drugs or bioactive molecules in vivo. This enhances injectability, provides sustained effects during tissue restoration and drug delivery, and plays a role in maintaining long-term effects by controlling sustained release in fillers, wound healing agents, and regenerative medicine.

[0036] In this invention, the term polyvinyl alcohol (PVA) refers to a polymeric material that dissolves in water and aids in particle formation during microsphere manufacturing, such as through solvent evaporation, while increasing physical stability. It acts as an emulsifier and stabilizer, stabilizing the structure of microspheres or porous fillers to ensure that drugs or bioactive molecules are properly loaded and decomposed stably within the body.

[0037] In this invention, the term polyethylene glycol (PEG) refers to a material with excellent biocompatibility that allows for the slow release of a drug in a drug delivery system. Additionally, it possesses properties that reduce immune responses and enable safe use within the body. Consequently, it enhances the sustained release of drugs in regenerative medicine, tissue engineering, and drug delivery systems, thereby enabling long-term effects in vivo.

[0038] In this invention, the term dopamine is primarily known as a neurotransmitter, but it also plays an important role in the field of biomaterials as a linker with surface binding and adhesive properties. Dopamine contains a catechol structure, possessing excellent adhesive strength capable of binding to various types of surfaces, and spontaneously oxidizes in weakly acidic or basic environments to form polydopamine. Polydopamine can stably bind to the surfaces of various materials, thereby enhancing biocompatibility and biological function. Since dopamine acts as a linker that forms a strong coating layer on the surface of various biomaterials and binds bioactive substances thereto, it can be widely applied to biomaterials and drug delivery systems.

[0039] In this invention, the term L-lysine refers to one of the essential amino acids, an important nutrient that must be consumed through food or supplements because the human body cannot synthesize it on its own. L-lysine performs various physiological roles, such as protein synthesis, growth promotion, and immune function enhancement, and is also used as a chemical linker. L-lysine can be used as a chemical linker because it possesses two functional groups: an amino group and a carboxyl group. It can form peptide or ester bonds with various molecules, making it advantageous for binding with macromolecular substances or bioactive molecules; it also has excellent biocompatibility and the characteristic of promoting collagen production.

[0040] In the present invention, the term collagen refers to one of the major proteins that constitute the body's connective tissues and plays an important structural role in various tissues such as skin, bone, tendons, cartilage, and muscle. Collagen is produced by fibroblasts within the body and forms a triple helix structure with amino acids such as proline, glycine, and hydroxyproline as its main components. Furthermore, collagen plays a particularly important role in wound healing and tissue restoration processes and is widely utilized in various fields such as cosmetics and regenerative medicine.

[0041] In the present invention, the term "anionic polymer" refers to a polymer that carries a negative charge when dissolved in water. Such polymers generally contain functional groups capable of carrying a negative charge, such as carboxyl groups (-COOH) or sulfonyl groups (-SO3H), which ionize in water to form anions. Anionic polymers are primarily used in various fields such as biomaterials, pharmaceuticals, and environmental remediation, and exhibit specific functionality through interaction with water.

[0042] The above anionic polymers may include, but are not limited to, hyaluronic acid (HA), alginate, carboxymethylcellulose (CMC), polyacrylic acid, and pectin.

[0043] In the present invention, the term alginate refers to a polysaccharide extracted from seaweed, mainly composed of galacturonic acid and mannuronic acid. It is an anionic substance when dissolved in water and is utilized in food, pharmaceuticals, and biomaterials due to its gel-forming properties.

[0044] In the present invention, the term carboxymethyl cellulose (CMC) refers to an anionic polymer produced by substituting the hydroxyl groups of cellulose with carboxymethyl groups. It is mainly used as a thickening agent or stabilizer in food, cosmetics, pharmaceuticals, etc., and is highly soluble in water.

[0045] In the present invention, the term polyacrylic acid refers to a polymer made by the polymerization of acrylic acid, and when dissolved in water, the carboxyl groups ionize and carry a negative charge. It is mainly used in hygroscopic materials, dental adhesives, cosmetics, etc.

[0046] In the present invention, the term pectin refers to a polysaccharide found mainly in fruits, which has galacturonic acid as its main component and possesses gel-forming properties. It is primarily used as a gelling agent and thickener in food, and is also utilized as a health functional food.

[0047] In the present invention, the term gelatin refers to a protein produced by heat-treating collagen obtained from animal skin, bone, cartilage, etc. It is a colorless or light yellowish-brown transparent solid substance that does not dissolve in cold water but dissolves well in warm water, allowing for the creation of various viscosities or shapes.

[0048] In this invention, the term dichloromethane (DCM) refers to a colorless, volatile liquid with the chemical formula CH2Cl2, which is one of the chlorinated methane derivatives. It is primarily used as a solvent and has a characteristic sweet odor at room temperature. Although it is almost insoluble in water, it is highly soluble in organic solvents and mixes well with materials such as plastics and rubber.

[0049]

[0050] In one embodiment of the invention, the first aspect provides a biodegradable composition for tissue restoration comprising microspheres containing poly-L-lactic acid (PLLA) and hydroxyapatite (HAp).

[0051] In the first embodiment above, the second embodiment provides a biodegradable composition for tissue restoration in which a polynucleotide (PN) is bonded to the surface of the hydroxyapatite, and in either the first or second embodiment, the third embodiment provides a biodegradable composition for tissue restoration in which the weight ratio (HAp:PN) of the hydroxyapatite to the polynucleotide is 4:1 to 6:1.

[0052] In the present invention, the weight ratio (HAp:PN) of the hydroxyapatite and polynucleotide may be 4.1:1 to 5.9:1, specifically 4.2:1 to 5.8:1, 4.3:1 to 5.7:1, 4.4:1 to 5.6:1, 4.5:1 to 5.5:1, 4.6:1 to 5.4:1, 4.7:1 to 5.3:1, 4.8:1 to 5.2:1, 4.9:1 to 5.1:1, more specifically 4.95:1 to 5.05:1, and most specifically 5:1.

[0053] The weight ratio of hydroxyapatite to polynucleotide (HAp:PN) of 5:1 mentioned above corresponds to a critical point where loading efficiency reaches saturation while minimizing the use of HAp. In this respect, the 5:1 ratio is not merely a random choice, but has significance as a technical limit where loading performance is maximized.

[0054] In any one of the first to third embodiments above, the fourth embodiment provides a biodegradable composition for tissue restoration that further comprises a bioactive molecule.

[0055] In any one of the first to fourth embodiments above, the fifth embodiment provides a biodegradable composition for tissue restoration in which the bioactive molecule is one or more selected from the group consisting of EVs, polynucleotides, growth factors, and PDRN.

[0056] In any one of the first to fifth embodiments above, the sixth embodiment provides a biodegradable composition for tissue restoration, wherein the composition is intended for injection into the skin.

[0057] In any one of the first to sixth embodiments above, the seventh embodiment provides a filler comprising the biodegradable composition for tissue restoration.

[0058] In any one of the first to seventh embodiments above, the eighth embodiment provides a biomaterial for cosmetics, diet health foods, biomedical materials, medical materials, or nanocomposite materials comprising the tissue restoration biodegradable composition.

[0059] In one embodiment of the present invention, the ninth aspect provides a method for preparing a biodegradable composition for tissue restoration, comprising the steps of: preparing microspheres by mixing poly-L-lactic acid and hydroxyapatite; and adding hyaluronic acid to the microspheres.

[0060] In the ninth embodiment above, the tenth embodiment provides a method for preparing a biodegradable composition for tissue restoration, further comprising the step of adding a bioactive molecule to the microspheres.

[0061] In any one of the ninth and tenth embodiments above, the eleventh embodiment provides a method for preparing a biodegradable composition for tissue restoration, wherein the bioactive molecule is one or more of EVs, polynucleotides, growth factors, and PDRN.

[0062] In any one of the ninth to eleventh embodiments above, the twelfth embodiment further comprises the step of adding an auxiliary agent to the microspheres, thereby providing a method for preparing a biodegradable composition for tissue restoration.

[0063] In any one of the 9th to 12th embodiments above, the 13th embodiment provides a method for preparing a biodegradable composition for tissue restoration, wherein the auxiliary agent is one or more of trehalose, CNC, PVA, and PEG.

[0064] In one embodiment of the present invention, the 14th aspect provides a pharmaceutical composition for tissue restoration comprising hyaluronic acid, poly-L-lactic acid, and hydroxyapatite as active ingredients, and in the 14th aspect, the 15th aspect provides a pharmaceutical composition for tissue restoration comprising one or more of EVs, polynucleotides, growth factors, PDRN, trehalose, CNC, PVA, and PEG.

[0065] In one embodiment of the present invention, the 16th aspect provides a microsphere for tissue restoration produced by the manufacturing method of any one of the 9th to 13th aspects.

[0066]

[0067] The present invention can reduce pain during filler injection by providing a tissue restoration composition containing microspheres having uniform porosity.

[0068] The microspheres of the present invention serve as delivery systems for active ingredients by loading bioactive substances into pores of a specific size.

[0069] The present invention provides a bio-restorative composition containing microspheres, thereby enabling the release of hyaluronic acid and bioactive substances from the microspheres during the initial stage of filler injection to produce a collagen tissue regeneration effect, the degradation of poly-L-lactic acid and bioactive substances during the middle stage of filler injection to maintain the collagen tissue regeneration effect, and the degradation of hydroxyapatite during the later stage of filler injection to maintain the collagen tissue regeneration effect.

[0070] Furthermore, the effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

[0071]

[0072] Figure 1 is a scanning electron microscope (SEM) image of the HAp / PLLA microsphere (HAp / PLLA MS) of the present invention.

[0073] Figure 2 shows the results of infrared spectral (FT-IR) analysis of the HAp-PN complex.

[0074] Figure 3 is a scanning electron microscope (SEM) image of a PN / HAp / PLLA microsphere (PN / HAp / PLLA MS).

[0075] Figure 4 shows the PN emission curve of a PN / HAp / PLLA microsphere (PN / HAp / PLLA MS).

[0076] Figure 5 is a graph showing the results of the cell proliferation evaluation of PN / HAp / PLLA microspheres (PN / HAp / PLLA MS).

[0077]

[0078] The present invention will be described in more detail below through examples. These examples are intended solely to explain the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the invention.

[0079]

[0080] Preparation Example 1. Preparation of HAp / PLLA Microspheres and SEM Imaging

[0081] To obtain PLLA microparticles with uniform porosity, poly-L-lactic acid (PLLA) with a molecular weight of 50,000 g / mol, hydroxyapatite (Hap) with a size of 50 to 100 nm, an aqueous solution of 0.5 wt% polyvinyl alcohol (PVA), an aqueous solution of 10 wt% ammonium bicarbonate (AB), and dichloromethane (DCM) were prepared.

[0082] Poly-L-lactic acid was dissolved in a mixed solvent at 10 wt%, and then hydroxyapatite was added to the poly-L-lactic acid solution in ratios of 10:1, 4:1, and 2:1, respectively. Afterward, a 10 wt% aqueous ammonium bicarbonate solution and a 0.5 wt% aqueous polyvinyl alcohol solution were added and mixed homogeneously. Subsequently, the mixed solution was stirred for about a day to evaporate the organic solvent, and then the ammonium bicarbonate was removed by washing with sterile distilled water at 40°C to fabricate pores.

[0083]

[0084] Preparation Example 2. Preparation of PN-linked HAp

[0085] In order to confirm the suitable binding conditions of HAp and PN in the HAp / PLLA composite prepared in Preparation Example 1 above by dissolving poly-L-lactic acid in a mixed solvent at 10 wt% and then adding hydroxyapatite to the solution at a weight ratio of 2:1, the binding yield was determined by varying the ratio of HAp to PN. A 1% (w / v) PN solution was prepared using 0.1 M MES buffer, and HAp was also appropriately dispersed by adding 20, 60, and 100 mg, respectively, to 18 mL of 0.1 M MES buffer. After adding 2 mL of the PN solution to the HAp dispersion, the reaction was carried out at 4°C for 1 day. At this time, the weight ratios of HAp to PN were 1:1, 3:1, and 5:1, respectively. After the reaction was finished, the mixture was centrifuged at 3500 rpm for 15 minutes at 4°C, and the HAp / PLLA that settled at the bottom was lightly washed with sterile water, centrifuged to obtain the product, and then freeze-dried.

[0086]

[0087] Preparation Example 3. Preparation of PN / HAp / PLLA MS

[0088] To bind PN to the HAp(50wt)% / PLLA MS structure, which is a PLLA microparticle with the highest HAp content, 100 mg was dispersed in 18 mL of 0.1 M MES buffer. Subsequently, 2 mL of a 1% (w / v) PN solution was prepared using 0.1 M MES buffer based on a HAp:PN ratio of 5:1, and the mixture was reacted at 4°C for about one day. After the reaction was complete, the mixture was centrifuged (3500 rpm, 15 min, 4°C), and only the pellets settled at the bottom were collected. After being lightly washed with sterile water and centrifuged, the pellets were finally freeze-dried to obtain the PN / HAp / PLLA MS.

[0089]

[0090] Example 1. Analysis of PN Loading Amount and Loading Efficiency of HAp-PN Complex

[0091] To quantify the amount of PN bound to HAp, the supernatant used during the preparation of HAp-PN was obtained, and the amount of PN remaining in the supernatant was quantified by measuring the absorbance at 260 nm using NanoDrop. Through this, the amount of PN remaining after the reaction could be indirectly quantified, which is shown in Table 1 below.

[0092]

[0093] Sample (HAp:PN Ratio) Loading Amount (μg / mL) Loading Efficiency (%) 1:150.4±26.4 6:5.03±2.6 5:1474.2±13.5 8:47.42±1.3 6:1914.7±0.1 8:91.50±0.0 2:1908.4±54.2 9:0.84±5.4 3:10:1926.1±58.8 2:92.61±5.88

[0094] It was confirmed that the most effective result was achieved when the HAp:PN ratio was 5:1, with a loading amount of 914.7 μg / mL and a loading efficiency of 91.50%. This represents an increase of approximately 17.1 times in loading amount and 18.19 times in loading efficiency compared to the HAp:PN ratio of 1:1, and an increase of approximately 1.93 times in loading amount and loading efficiency compared to the HAp:PN ratio of 3:1. Additionally, the 7:1 and 10:1 ratios showed results similar to 5:1 (within ±5%), but were inefficient due to higher HAp usage. Therefore, it was confirmed that the 5:1 ratio represents the saturation point for loading performance, serving as the optimal condition for achieving maximum loading effect with minimal HAp. In other words, the 5:1 ratio holds technical significance as it corresponds to the threshold point where loading efficiency rapidly improves before reaching saturation.

[0095]

[0096] Example 2. Elemental analysis (FT-IR) for confirmation of PN / HAp bonding

[0097] The inventors performed infrared spectroscopic analysis (FT-IR spectrometer, vertex 70) to evaluate whether PN was properly bound in the HAp-PN prepared in Preparation Example 2. As a result of the analysis, the expression of characteristic peaks of HAp and PN was confirmed. Specifically, the characteristic phosphate group (PO4) of HAp 3- ) Absorption peak at 1040–1090 cm -1 and 562-602 cm -1 It was confirmed in the region. In addition, 1230–1250 cm⁻¹ was identified as the intrinsic absorption peak of PN. -1 and 1085-1060 cm -1 Phosphate-related peaks in the region, and 1700–1600 cm⁻¹ -1 Peaks corresponding to C=O and C=N bonds were observed (see Fig. 2).

[0098]

[0099] Example 3. Analysis of PN Loading Amount and Loading Efficiency of HAp-PN Complex

[0100] To quantify the amount of PN bound to HAp when the HAp:PN ratio is 5:1, the supernatant obtained during the HAp-PN preparation process was recovered. Subsequently, the amount of PN remaining in the supernatant was quantified by measuring the absorbance at 260 nm using NanoDrop. Through this, the amount of PN remaining after the reaction could be indirectly calculated, and the results are presented in Table 2.

[0101] Sample Loading Amount (μg / mL) Loading Efficiency (%) PN / HAp / PLLA MS8 10.6 ± 14.56 81.06 ± 1.46

[0102]

[0103] Example 4. PN / HAp / PLLA MS Drug Release Behavior Test

[0104] The polynucleotide drug release behavior from the microspheres prepared in Preparation Example 3 above was confirmed. Specifically, microspheres were placed in 1 ml of PBS buffer (pH 7.4) and stirred at 100 rpm at 37 ℃. The drug release amount was determined over time (1 hour, 3 hours, 6 hours, 10 hours, 1 day, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, and 28 days), and the buffer was replaced with a fresh one after measurement. The released drug was analyzed by measuring the absorbance at 260 nm (see Fig. 4).

[0105]

[0106] Example 5. Cell proliferation test to verify PN / HAp / PLLA MS performance

[0107] To confirm the skin regeneration and functionality of the microspheres prepared in Preparation Example 3 above, the degree of cell proliferation was tested. 1×10 5 Canine adult stem cells were placed in PLLA MS, HAp / PLLA MS, and PN / HAp / PLLA MS and cultured for 1, 3, and 7 days. After 1, 3, and 7 days of culture, the cells were washed with phosphate buffer. After washing, CCK-8 proliferation kit reagent (Tetrazolium salt reagent, Cell Counting kit-8, Dojindo, USA) was added and cultured for 1 hour. Then, the culture medium was transferred to a 96-well plate and the absorbance was measured at 450 nm (Fig. 5).

[0108]

[0109] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A biodegradable composition for tissue restoration comprising microspheres containing poly-L-lactic acid (PLLA) and hydroxyapatite (HAp).

2. In Paragraph 1, A biodegradable composition for tissue restoration in which a polynucleotide (PN) is bonded to the surface of the above-mentioned hydroxyapatite.

3. In Paragraph 2, A biodegradable composition for tissue restoration, wherein the weight ratio (HAp:PN) of the hydroxyapatite and polynucleotide is 4:1 to 6:

1.

4. In Paragraph 1 or 2, A biodegradable composition for tissue restoration that further comprises bioactive molecules.

5. In Paragraph 4, A biodegradable composition for tissue restoration, wherein the above-mentioned bioactive molecule is one or more selected from the group consisting of EVs, polynucleotides, growth factors, and PDRN.

6. In Paragraph 1, The above composition is a biodegradable composition for tissue restoration intended for injection into the skin.

7. A filler comprising a biodegradable composition for tissue restoration according to any one of claims 1 to 6.

8. A biomaterial for cosmetics, diet health foods, biomedical materials, medical materials, or nanocomposite materials comprising a biodegradable composition for tissue restoration according to any one of claims 1 to 6.

9. A step of preparing microspheres by mixing poly-L-lactic acid and hydroxyapatite; and A method for preparing a biodegradable composition for tissue restoration, comprising the step of adding hyaluronic acid to the microspheres.

10. In Paragraph 9, A method for preparing a biodegradable composition for tissue restoration, further comprising the step of adding a bioactive molecule to the microspheres.

11. In Paragraph 10, A method for preparing a biodegradable composition for tissue restoration, wherein the above-mentioned bioactive molecule is one or more of EVs, polynucleotides, growth factors, and PDRN.

12. In Paragraph 9, A method for preparing a biodegradable composition for tissue restoration, further comprising the step of adding an auxiliary agent to the microspheres.

13. In Paragraph 12, A method for preparing a biodegradable composition for tissue restoration, wherein the above-mentioned adjuvant is one or more of trehalose, CNC, PVA, and PEG.

14. Microspheres for tissue restoration manufactured by the manufacturing method of any one of claims 9 to 13.

15. A pharmaceutical composition for tissue restoration comprising polynucleotide (PN), poly-L-lactic acid, and hydroxyapatite as active ingredients.

16. In Paragraph 15, The above pharmaceutical composition is a pharmaceutical composition for tissue restoration that further comprises one or more of EVs, polynucleotides, growth factors, PDRN, trehalose, CNC, PVA, and PEG.

17. In Paragraph 15, A pharmaceutical composition for tissue restoration, wherein the weight ratio (HAp:PN) of the hydroxyapatite and polynucleotide is 4:1 to 6:1.

Images