KR102521838B1 - Methods for preparing biomaterials for tissue repair having excellent elasticity and safety - Google Patents

Abstract

The present invention relates to a method for manufacturing a biomaterial for tissue repair in which the amount of crosslinking agent is significantly reduced by undergoing a crosslinking reaction through a differentiated mixing and transformation process compared to conventional crosslinking techniques. By lowering the content of the cross-linking agent, toxicity is reduced and elastic physical properties are improved, enabling large-capacity procedures for shaping not only the face but also the body.

Description

Methods for preparing biomaterials for tissue repair having excellent elasticity and safety}

The present invention relates to a method for manufacturing a biomaterial for tissue repair having excellent stability and physical properties such as durability in the body, a biomaterial for tissue repair prepared by the method, and various uses thereof.

As a person ages, the effects of prolonged facial muscle movements such as gravity, sun-exposure, laughing, frowning, chewing and squinting appear on the face. The underlying tissue that keeps the skin looking young begins to break down, often resulting in laugh lines, smile lines, crow's feet, and facial creases, commonly referred to as " called "the effects of aging". In addition, in addition to the facial area, body parts also cause a decrease in elasticity, wrinkles, and volume decrease due to the influence of aging.

In an effort to treat or correct these effects of aging, various types of fillers are available to help fill in wrinkles and depressions on the body, including the face, while at the same time restoring fat loss-related tissue volume loss. are being developed

The first dermal fillers approved by the US Food and Drug Administration (FDA) over 20 years ago were bovine collagen-based fillers, which are bovine-based and have the potential to cause allergic reactions in humans, with approximately 3 to 5% of people experiencing severe allergic reactions to bovine collagen. It has been reported to cause allergic reactions. Human-derived collagen filler compositions then received FDA approval in February 2003. Although the human-derived collagen has the advantage of significantly reducing the risk of allergic reaction, it still has problems such as the rapid decomposition of the injected filler component. Accordingly, hyaluronic acid (HA)-based products were developed through research on fillers that do not cause allergic reactions and maintain a smoother and more youthful appearance, and HA-based fillers were approved by the FDA in December 2003. was first approved by Since then, development of other HA-based fillers has been rapid.

Hyaluronic acid (HA), a type of GAG (glycosaminoglycan), is a natural, water-soluble polysaccharide, especially glucosaminoglycan, which is the main component of the extra-cellular matrix and is widely used in connective tissue and epithelial tissue of animals. distributed HA, a human-derived component, has excellent biocompatibility and shows low immune response, such as not causing allergic reactions. In addition, since HA has the ability to bind a large amount of moisture up to 1000 times its own volume, it is an excellent volumizer that can improve skin structure and maintain elasticity.

The ideal filler requires no harmful side effects, long-lasting, soft, smooth, and natural looking, while having degrading enzymes to correct problems when they occur. In addition, it is required that discomfort to the patient is minimized, a large-capacity procedure is possible, and the procedure result is excellent while the operation cost is low. However, dermal fillers containing collagen or hyaluronic acid as a main component are expensive, but the duration of the effect is too short, and side effects due to harmful ingredients have also been reported.

Therefore, it has been possible to perform large-capacity procedures at a lower cost and have further developed a technique capable of manufacturing a composition for filler with enhanced elasticity.

One object of the present invention is to provide a method for manufacturing a tissue repair type biomaterial having high elasticity properties.

Another object of the present invention is to provide a tissue repair type biomaterial prepared by the method of the present invention.

Another object of the present invention relates to various uses of the tissue repair type biomaterial provided in the present invention.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, numerous specific details are set forth, such as specific forms, compositions and processes, etc., in order to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well known processes and manufacturing techniques have not been described in specific detail in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, the appearances of "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, particular features, forms, compositions, or properties may be combined in one or more embodiments in any suitable way.

Unless there is a specific definition within the specification, all scientific and technical terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention belongs.

According to one embodiment of the present invention, preparing an aqueous solution containing hyaluronic acid, a salt thereof or a mixture thereof;

hydrating the hyaluronic acid aqueous solution; and

It relates to a method for producing a biomaterial for tissue repair, comprising the step of cross-linking hyaluronic acid by adding a cross-linking agent to an aqueous solution of hydrated hyaluronic acid.

In the present invention, the "biomaterial for tissue repair" refers to a living material used for replacement, restoration, or reconstruction of human tissues and organs, such as blood vessels, heart, diaphragm, fascia, and skin (dermal, cutaneous, etc.). The biomaterial is used in a broad sense that includes human-derived components that are secondary processed by mixing additives to improve functions.

In the present invention, the "hyaluronic acid" is a linear high-molecular polysaccharide in which N-acetyl-D-glucosamine and D-glucuronic acid are alternately bonded. It is known that hyaluronic acid is present in connective tissues such as subcutaneous tissue and cartilage tissue of mammals, as well as in the vitreous body of the eye, the umbilical cord, and the like, and also in the capsular membranes of streptococci and bacilli. Common methods for obtaining hyaluronic acid include a method of extracting from chicken combs, umbilical cords, etc., and a method of culturing streptococcus and bacillus followed by extraction and purification therefrom. In the present invention, the hyaluronic acid may be used as a meaning including all salts, derivatives, or aqueous solutions thereof as well as hyaluronic acid itself.

In the present invention, the hyaluronic acid salt includes both inorganic salts such as sodium hyaluronate, magnesium hyaluronate, zinc hyaluronate, and cobalt hyaluronate, and organic salts such as tetrabutylammonium hyaluronate. A combination of the two may be used, and is not limited thereto as long as it corresponds to the hyaluronic acid salt used in the art.

In the present invention, the hyaluronic acid aqueous solution may be obtained by adding hyaluronic acid, a salt thereof, or a mixture thereof to a buffer.

In the present invention, the buffer may be an alkaline aqueous solution, and the type is not particularly limited, but, for example, sodium hydroxide (NaOH) aqueous solution, potassium hydroxide (KOH) aqueous solution, ammonium hydroxide (NH ₄ OH) aqueous solution, lithium hydroxide ( LiOH) aqueous solution and calcium hydroxide (Ca(OH) ₂ ) may be at least one selected from the group consisting of an aqueous solution.

In the present invention, the buffer, preferably the aqueous alkali solution, may have a concentration of 0.01 to 3 N, preferably 0.1 to 0.5 N.

In the present invention, the hydration step may be performed by adding a solvent to the hyaluronic acid aqueous solution and performing at least two steps of stirring.

In the present invention, the solvent may be one or more solvents selected from the group consisting of phosphate buffered saline (PBS), distilled water, deionized water and ultrapure water, but is not limited thereto.

The stirring process in the present invention, (1) 500 to 1,000 rpm, preferably 600 to 800 rpm for 5 to 20 minutes, preferably 8 to 15 minutes of stirring for 1-1st stirring step; And a 1-1st cooling step of cooling while stirring at 300 to 600 rpm, preferably 400 to 550 rpm for 2 to 10 minutes, preferably 3 to 7 minutes; A first step comprising; and

(2) 2-1 stirring step of stirring at 500 to 1,000 rpm, preferably 600 to 800 rpm for 5 to 20 minutes, preferably 8 to 15 minutes; 2-1st cooling step of cooling while stirring at 300 to 600 rpm, preferably 500 to 550 rpm for 2 to 10 minutes, preferably 3 to 7 minutes; a 2-2 stirring step of stirring at 500 to 1,000 rpm, preferably 600 to 800 rpm for 5 to 20 minutes, preferably 8 to 15 minutes; A second step including a 2-2 cooling step of cooling while stirring at 1,500 to 2,000 rpm, preferably 1,600 to 1,900 rpm for 5 to 20 minutes, preferably 10 to 15 minutes; may include.

In the first step in the present invention, each of the 1-1 stirring step and the 1-1 cooling step is repeated at least once, preferably 1 to 5 times, more preferably 2 to 4 times. can Here, when the 1-1 stirring step and the 1-1 cooling step are repeatedly performed, after the 1-1 stirring step is repeatedly performed, the 1-1 cooling step is repeatedly performed. Alternatively, the 1-1st stirring step and the 1-1st cooling step may be alternately performed.

In the present invention, the first step may be repeated 1 to 5 times, preferably 2 to 4 times, and most preferably 3 times.

In the present invention, the second step may be repeated 1 to 5 times, preferably 1 to 3 times, and most preferably 2 times.

In the present invention, the hydration concentration may be 12 to 24% by weight, preferably 15 to 20% by weight, and most preferably 18% by weight.

In the present invention, the stirring process in the hydration step is 500 to 1,000 rpm, preferably 600 to 800 rpm for 5 to 20 minutes, preferably 8 to 15 minutes for stirring 1-1st stirring step; and 300 to 600 rpm, preferably 400 to 550 rpm for 2 to 10 minutes, preferably 3 to 7 minutes, after repeating the 1-1 cooling step of cooling while alternating three times, 500 to 1,000 2-1 stirring step of stirring at rpm, preferably 600 to 800 rpm for 5 to 20 minutes, preferably 8 to 15 minutes; 2-1st cooling step of cooling while stirring at 300 to 600 rpm, preferably 500 to 550 rpm for 2 to 10 minutes, preferably 3 to 7 minutes; a 2-2 stirring step of stirring at 500 to 1,000 rpm, preferably 600 to 800 rpm for 5 to 20 minutes, preferably 8 to 15 minutes; The 2-2 cooling step of cooling while stirring at 1,500 to 2,000 rpm, preferably 1,600 to 1,900 rpm for 10 to 15 minutes may be sequentially repeated twice.

In the present invention, each step of the stirring process may be performed at intervals of 20 to 40 minutes, preferably each step may be performed at intervals of 30 minutes.

In the present invention, the total stirring process in the hydration step may be performed for 2 to 3 hours, preferably for 150 minutes.

In the present invention, after the hydration step, the step of defoaming the hydrated hyaluronic acid aqueous solution may be further included.

In the present invention, the degassing step may be performed for 10 to 20 minutes, preferably 13 to 17 minutes, at a speed of 1,500 to 2,000 rpm, preferably 1,700 to 1,900 rpm of hydrated sodium hyaluronate.

In the present invention, after the defoaming step, a crosslinking reaction of hyaluronic acid may be performed by adding a crosslinking agent to the degassed aqueous solution of hyaluronic acid.

In the present invention, the crosslinking agent is polyethylene glycol diglycidyl ether (PEGDE), 1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl Ether (ethylene glycol diglycidyl ether: EGDGE), 1,6-hexanediol diglycidyl ether, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1- Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), polyethylene glycol (PEG), propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether ), polytetramethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglyc Diglycerol polyglycidyl ether, glycerol polyglycidyl ether, tri-methylpropane polyglycidyl ether, bisepoxypropoxyethylene (1,2-(bis(2 ,3-epoxypropoxy)ethylene), pentaerythritol polyglycidyl ether and sorbitol polyglycidyl ether, N-hydroxysuccinimide (NHS) and It may be at least one selected from the group consisting of DVS (Divinylsulfone), preferably polyethylene glycol diglycidyl ether (PEGDE), but is not limited thereto.

In the present invention, during the crosslinking reaction, the crosslinking agent has excellent crosslinking ability even when added in a small amount of 2.0 mL/L or less, preferably 0.5 to 1.5 mL/L.

In one example of the present invention, the crosslinking agent may be polyethylene glycol diglycidyl ether (PEGDE), and the crosslinking agent is 2.0 mL/L or less, preferably in an amount of 0.5 to 1.5 mL/L. may be added.

In the present invention, the crosslinking reaction may be performed such that the crosslinking degree of hyaluronic acid is in the range of 0.01 to 50%, preferably in the range of 0.05 to 30%.

In the present invention, the "degree of crosslinking" is defined as the % weight ratio of the crosslinking agent to the hyaluronic acid monomer units in the crosslinking portion of the hyaluronic acid-based composition. It is measured as the weight ratio of crosslinking agent to the weight ratio of hyaluronic acid monomers.

In the present invention, by performing the stirring process of the 1-1st cooling step, the 2-1st cooling step and the 2-2nd cooling step in the hydration step, the molecular weight reduction of hyaluronic acid is minimized and at the same time high concentration of hyaluronic acid can be hydrated, so the amount of crosslinking agent required for filler production can be reduced by 60 to 70% compared to before. Accordingly, when the biomaterial for tissue repair produced in the present invention is applied to a living body, it has low toxicity and excellent safety, so that it can be operated not only on the face but also on a wide range of bodies, and can be mass-produced at low cost.

According to another embodiment of the present invention, it relates to a biomaterial for tissue repair produced by the manufacturing method of the present invention.

The biomaterial for tissue repair of the present invention is to be administered to a subject, and the subject may mean all animals including humans. At this time, the animal is preferably not only humans but also mammals such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, and cats that require tissue repair similar thereto, but are not limited thereto.

When the biomaterial composition for tissue repair according to the present invention is injected into the skin, the injection force is at an extrusion rate of about 10 mm/min using a standard 1 mL syringe (eg, 1.0 mL BD syringe) ( For example, as measured through a MECESIN) 27G Υ 1/2" needle, it may generally range from 5 N to 40 N, but is not limited thereto.

The biomaterial composition for tissue repair of the present invention may be used for the purpose of a pharmaceutical composition or a cosmetic composition, but is not limited thereto.

According to another embodiment of the present invention, it relates to a pharmaceutical composition for tissue repair comprising the biomaterial for tissue repair of the present invention as an active ingredient.

In the present invention, the pharmaceutical composition includes lidocaine, ambucaine, amolanone, amylocaine, benoxinate, benzocaine, betoxycaine ( betoxycaine), biphenamine, bupivacaine, butacaine, butamben, butanilicaine, butethamine, butoxycaine, carti Carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine, dibucaine, dimethysoquin, dimethocaine ), diperodon, dycyclonine, ecgonidine, ecgonine, ethyl chloride, etidocaine, beta-eucaine eucaine), euprocin, phenalcomine, formocaine, hexylcaine, hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate, levoxadrol, mepivacaine, meprylcaine, metabutoxycaine, methyl chloride, myrtecaine ), naepaine, octacaine, orthocaine, oxethazaine, parethoxycaine, phenacaine, phenol, piperocaine (piperocaine), pyridocaine, polidocanol, pramoxine, prilocaine, procaine, propanocaine, proparacaine, Propipocaine, propoxycaine, pseudococaine, pyrrocaine, ropivacaine, salicyl alcohol, tetracaine, tolylcaine ( tolycaine), trimecaine (trimecaine), zolamine (zolamine) and may further include a local anesthetic selected from the group consisting of salts thereof, but is not limited thereto.

In the present invention, the pharmaceutical composition may further include polyols, buffers, antioxidants, bacteriostats, vitamins, amino acids, metals, antioxidants, diluents, dispersants, surfactants, binders, lubricants, or mineral salts, but is limited thereto it is not going to be

In the present invention, the pharmaceutical composition is a composition for filling or replacing biological tissue, filling wrinkles, remodeling of the face, or increasing lip volume. , compositions for use in the treatment of rehydration of the skin by mesotherapy, compositions for replacement or temporary replenishment of synovial fluid in arthritis, increasing the volume of a sphincter or urethra in urology or gynecology in ophthalmology, as an adjuvant in cataract surgery or as a composition for the treatment of glaucoma, in pharmaceuticals as a gel releasing an active substance, in surgery as a bone reconstruction, in increasing the volume of vocal cords, or as a surgical tissue ) may be a composition for the preparation of

The route of administration of the pharmaceutical composition according to the present invention is not limited to these, but is oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical , sublingual or rectal. Parenteral administration is preferred.

In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intracapsular, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

According to another embodiment of the present invention, it relates to a cosmetic composition for skin improvement comprising the biomaterial for tissue repair of the present invention as an active ingredient.

The biomaterial for tissue repair provided in the present invention not only has excellent skin improvement effects such as skin moisturizing, skin whitening, wrinkle improvement and anti-aging when absorbed into the skin, but also has no irritation or side effects on the skin and is safe.

In the cosmetic composition of the present invention, the active ingredient and the biomaterial for tissue repair are overlapped with those described in the manufacturing method, so the detailed description thereof will be omitted.

In the present invention, "improvement" refers to all improvement of skin wrinkles by irradiation of the composition of the present invention, more specifically, improvement of appearance or beneficial change such as maintenance of skin volume or restoration of skin volume through physical repair. Actions can be included without limitation.

In the present invention, the cosmetic composition is cosmetic water, nutrient lotion, nutrient essence, massage cream, beauty bath water additive, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, sunscreen Screen cream, suntan cream, skin lotion, skin cream, sunscreen cosmetics, cleansing milk, depilatory makeup, face and body lotion, face and body cream, skin whitening cream, hand lotion, hair lotion, cosmetic cream, jasmine oil , bath soap, water soap, beauty soap, shampoo, hand sanitizer (hand cleaner), medicated soap, medical soap, cream soap, facial wash, body cleanser, scalp cleanser, hair rinse, cosmetic soap, tooth whitening gel, toothpaste, etc. can be manufactured with To this end, the composition of the present invention may further include a solvent or an appropriate carrier, excipient or diluent commonly used in the preparation of cosmetic compositions.

In the present invention, the type of solvent that can be further added to the cosmetic composition is not particularly limited, but, for example, water, saline, DMSO, or a combination thereof may be used. In addition, carriers, excipients or diluents include purified water, oil, wax, fatty acids, fatty alcohols, fatty acid esters, surfactants, humectants, thickeners, antioxidants, viscosity stabilizers, chelating agents, buffers, lower alcohols, and the like. Includes, but is not limited to. In addition, whitening agents, moisturizing agents, vitamins, sunscreens, perfumes, dyes, antibiotics, antibacterial agents, and antifungal agents may be included as necessary.

In the present invention, as the oil, hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, coconut oil, jojoba oil, and avocado oil may be used, and as the wax, beeswax, spermaceti, carnauba, candelilla, montan, ceresin , liquid paraffin, lanolin may be used.

In the present invention, stearic acid, linoleic acid, linolenic acid, and oleic acid may be used as the fatty acid, and cetyl alcohol, octyl dodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol, hexa Decanol may be used, and isopropyl myristate, isopropyl palmitate, and butyl stearate may be used as the fatty acid ester. As the surfactant, cationic surfactants, anionic surfactants, and nonionic surfactants known in the art can be used, and surfactants derived from natural products are preferred as much as possible. In addition, it may include a moisture absorbent, thickener, antioxidant, etc. widely known in the field of cosmetics, and the type and amount thereof are known in the art.

In the case of using the manufacturing method of the present invention, it is possible to prepare a biomaterial for tissue repair having excellent physical properties even with a small amount of a crosslinking agent, and accordingly, when the biomaterial for tissue repair is injected into the human body, toxicity is remarkably low, not only for the face but also for the face. It has the advantage of enabling large-capacity procedures for body plastic surgery.

1 is a graph showing the amount of a tissue repair type biomaterial prepared according to an embodiment of the present invention and a crosslinking agent used in a comparative example.
Figure 2 is a graph showing the elasticity of the tissue repair type biomaterial manufactured according to an embodiment of the present invention and the physical properties of a comparative example.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent 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 present invention. .

Example 1: Preparation of cross-linked composite of the present invention (biomaterial for tissue repair)

1.1. Hyaluronic Acid Hydration Step and Crosslinking Step

In a process of mixing and stirring a predetermined amount of sodium hyaluronate in a sodium hydroxide solution, the sodium hydroxide solution is first introduced into a reaction tank, and the sodium hydroxide solution added to the reaction tank is added. It is carried out in the step of stirring so that sodium hyaluronate is completely dissolved. More specifically, after dissolving 40 g of sodium hyaluronate (20% (w / v)) in 160 mL of the sodium hydroxide solution prepared above, the centrifugal mixer is operated to homogeneously mix the sodium hyaluronate in the sodium hydroxide solution was allowed to dissolve completely.

In the step of hydration, the first stage of stirring is stirring at a speed of 700 rpm for 15 minutes, stirring at a speed of 500 rpm for 5 minutes while cooling, stirring at a speed of 700 rpm for 10 minutes, and finally stirring at a speed of 500 rpm. A cross-stirring process was performed by stirring and cooling for 5 minutes. Thereafter, the above process was repeated two more times and mixing and stirring was performed for a total of 105 minutes. Further, in a two-step stirring process, stirring was performed at a speed of 700 rpm for 10 minutes, stirring at a speed of 500 rpm for 5 minutes while cooling, stirring at a speed of 700 rpm for 10 minutes, and finally stirring at a speed of 1,800 rpm for 12 minutes. The alternating stirring process was performed by cooling with stirring for 10 minutes. Thereafter, the above process was additionally repeated once to further agitate for a total of 74 minutes, and a process of degassing the sodium hyaluronate hydrated by the above process was performed at 1800 rpm for 15 minutes.

1 mL/L of polyethylene glycol diglycidyl ether (PEGDE), a cross-linking agent, was added to the mixed solution prepared above, and the cross-linking agent (PEGDE) was added to the reactor and stirred to mix evenly to obtain sodium hyaluronate gel. raw materials were created. The method for preparing a crosslinked composite according to the present invention is characterized in that a cooling step is performed during the initial hydration reaction. The cooling step in this hydration step is an improvement of the existing method of manufacturing a cross-linking agent complex, and when hyaluronic acid is injected into the body, it plays a role in maintaining high elastic properties and also has an effect of reducing the amount of cross-linking agent used, so it is decomposed in vivo. It lowers the level of toxicity to the body when injecting the cross-linked complex, which is indispensable for the manufacture of tissue repair materials for inhibition, so that large-capacity procedures can be performed more safely.

1.2 Step of preparing the product after the crosslinking step

After performing the initial crosslinking reaction in Example 1.1, the sodium hyaluronate gel raw material was filled in a container and further crosslinked in an incubator at 35° C. for 24 hours. Thereafter, the sodium hyaluronate gel raw material was washed with sodium chloride solution and PBS, and each washing process was repeated three or more times. The washed sodium hyaluronate gel raw material was put into a preparation tank, PBS solution was added so that the sodium hyaluronate gel raw material had a concentration of 20 mg/mL, and then pulverization was performed using a grinder. The sodium hyaluronate gel tissue repair biomaterial filled in the filling tank was filled into a pre-filled syringe by 10.0 mL using a charger, and the filling of the sodium hyaluronate gel tissue repair biomaterial was completed to increase the stability of the product After sterilization was performed by loading the prefilled syringe into a high-pressure sterilizer, product manufacturing was completed through foreign matter inspection and packaging.

Comparative Example 1. Preparation of cross-linked composite (biomaterial for tissue repair) through conventional hydration process

After dissolving 20 g of sodium hyaluronate (10% (v/v)) in 180 mL of sodium hydroxide solution, put in a stirrer and stir at 500 rpm for 60 to 90 minutes. In the hydration method that is usually performed, the form of hydration with an impeller-type stirrer is such that polymer molecules such as hyaluronic acid are aligned in one direction by receiving force in the direction perpendicular to gravity in the solvent, and there is no cooling and degassing process for a long time. When hydrated, the viscosity is lowered by heat and oxidation. In contrast, in the present invention, during hydration, the molecules of the polymer are aligned in various directions (gravity, opposite direction) to increase twist between molecules. Increased intermolecular twist can increase the polymer's viscosity, resulting in highly elastic products. The addition of cooling and defoaming processes during hydration in order to prevent the decrease in viscosity due to heat and oxidation generated during polymer hydration is a distinctive feature from existing methods.

Experimental Example 1. Analysis of mechanical properties of the cross-linked composite of the present invention

The storage modulus of the crosslinked composites prepared in Example 1 and Comparative Example 1 was measured by a rheological method, and the results are shown in FIG. 1 . Here, Comparative Example 1 is indicated in red (existing process), and Example 1 is indicated in blue (development process).

As shown in FIG. 1, unlike the conventional crosslinking technology, in the case of the crosslinked composite of the present invention, which additionally undergoes a mixing process of 150 minutes and a deformation process of 15 minutes in the initial crosslinking step, the content of the PEGDE crosslinking agent is 2.5 From mL/L to 1 mL/L, it was confirmed that the amount of cross-linking agent was reduced by about 60% while maintaining the same physical properties from 550 Pa·s to 545 Pa·s with a viscoelastic coefficient. Specific figures are shown in Table 1 below.

item Comparative Example 1 Example 1 PEGDE (mL/L) 2.5 1.0 G'(Pa) 300 310 G" (Pa) 25.2 30.07 P/A (°) 5.6 5.64 C V (Pa s) 550 545

From this, it was found that in the case of the method of the present invention, even though the amount of crosslinking agent used was reduced by 60% or more compared to the previous method, it was possible to secure physical properties equivalent to or higher than those of the crosslinked composite prepared by the conventional method.

Experimental Example 2. Comparative measurement of physical properties of commercially available products and products of the present invention

The present inventors performed measurement of rheological properties in order to compare and measure the physical properties of commercially available filler products and the product of the present invention. Rheological properties are one of the important factors representing physical properties of cross-linked composites. The composition of the cross-linked composite prepared in Example 1 (development product) and four commercially available PEG fillers were compared and measured for physical properties using a rheometer. It was measured in the range of 0.01 to 100 Hz using 25 ℃, 4 mm Plate Geometry, and the complex viscosity value measured at 0.1 Hz was converted into a graph and shown in FIG. 2.

Referring to FIG. 2, it can be seen that the cross-linked composite (development product) of Example 1 prepared according to the present invention has very high elasticity compared to commercially available products.

Through the above results, the tissue repair type biomaterial, which is a cross-linked composite prepared by the method of the present invention, has excellent stability in the body by lowering the content of toxic cross-linking agents compared to conventional composites cross-linked in a conventional manner, and has excellent elastic properties. It has an improvement effect, so it is possible to perform large-capacity procedures for plastic surgery not only on the face but also on the body.

Furthermore, the present invention uses a standardized container to sequentially carry out a crosslinking reaction involving mixing and transformation and a washing process during the initial crosslinking reaction, so that only the number of containers to be used in the process needs to be increased when the manufacturing unit of the raw material is increased. It is easy to expand the unit and can provide the advantages of convenient operation.

Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and variations are possible without departing from the technical spirit of the present invention described in the claims. It will be obvious to those skilled in the art.

Claims (21)

Preparing an aqueous solution containing hyaluronic acid, a salt thereof or a mixture thereof;
hydrating the hyaluronic acid aqueous solution;
Defoaming the hydrated hyaluronic acid aqueous solution; and
A method for producing a biomaterial for tissue repair, comprising the step of cross-linking hyaluronic acid by adding a cross-linking agent to an aqueous solution of hydrated hyaluronic acid,
The hydration step is performed by adding a solvent to hyaluronic acid and performing the following two-step stirring process,
a) the first stage of the stirring process is a 1-1st stirring step of stirring for 5 to 20 minutes at 500 to 1,000 rpm; A 1-1st cooling step of cooling while stirring at 300 to 600 rpm for 2 to 10 minutes; and
b) the second stage of the stirring process is a 2-1st stirring step of stirring at 500 to 1,000 rpm for 5 to 20 minutes; A 2-1st cooling step of cooling while stirring at 300 to 600 rpm for 2 to 10 minutes; a 2-2 stirring step of stirring at 500 to 1,000 rpm for 5 to 20 minutes; 2-2 cooling step of cooling while stirring at 1,500 to 2,000 rpm for 5 to 20 minutes; consisting of, a manufacturing method. According to claim 1,
The hyaluronic acid aqueous solution is obtained by adding hyaluronic acid, a salt thereof, or a mixture thereof to a buffer, a manufacturing method. According to claim 2,
The buffer is at least one selected from the group consisting of sodium hydroxide (NaOH) aqueous solution, potassium hydroxide (KOH) aqueous solution, ammonium hydroxide (NH ₄ OH) aqueous solution, lithium hydroxide (LiOH) aqueous solution, and calcium hydroxide (Ca(OH) ₂ ) aqueous solution. , manufacturing method. According to claim 3,
The buffer is a concentration of 0.01 to 3 N, manufacturing method. delete According to claim 1,
Wherein the solvent is at least one selected from the group consisting of phosphate buffered saline (PBS), distilled water, deionized water and ultrapure water. delete According to claim 1,
Step 1 of the stirring process is repeated 1 to 5 times, the manufacturing method. delete According to claim 1,
Step 2 of the stirring process is repeated 1 to 5 times, the manufacturing method. According to claim 1,
Wherein the stirring process is carried out for 2 to 3 hours. delete According to claim 1,
In the degassing step, the manufacturing method is carried out by stirring the hydrated hyaluronic acid aqueous solution at 1,500 to 2,000 rpm for 10 to 20 minutes. According to claim 1,
The crosslinking agent is polyethylene glycol diglycidyl ether (PEGDE), 1,4-butandiol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether (ethylene glycol diglycidyl ether: EGDGE), 1,6-hexanediol diglycidyl ether, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1-Ethyl-3 -(3-dimethylaminopropyl)carbodiimide, EDC), polyethylene glycol (PEG), propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, poly Tetramethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether (diglycerol polyglycidyl ether), glycerol polyglycidyl ether, tri-methylpropane polyglycidyl ether, bisepoxypropoxyethylene (1,2-(bis(2,3- epoxypropoxy)ethylene), pentaerythritol polyglycidyl ether and sorbitol polyglycidyl ether, N-hydroxysuccinimide (NHS) and DVS (Divinylsulfone) ) At least one selected from the group consisting of, a manufacturing method. According to claim 1,
Wherein the crosslinking agent is added in an amount of 2.0 mL/L or less. Claims 1 to 4, 6, 8, 10, 13 to 15 of any one of the manufacturing method of a biomaterial for tissue repair produced by the manufacturing method. delete delete delete delete delete

Images