KR102385695B1 - Novel hyaluronic acid -amino hydrogel complex composition with semi-IPN structure for soft tissue repair and augmentation and an use thereof - Google Patents

Abstract

The present invention relates to a biopolymer which forms a weak interpenetration network (semi-IPN) structure by a secondary annealing effect of complex amino acids in which a specific component content is changed in a high-density hyaluronic acid structure, and a method for preparing the same.

Description

Novel hyaluronic acid-amino hydrogel complex composition with semi-IPN structure for soft tissue repair and augmentation and an use thereof }

The present specification relates to a novel hyaluron-complex amino hydrogel composition for soft tissue repair and enlargement forming a semi-IPN structure, a method for preparing the same, and a use thereof.

Skin aging is a gradual and irreversible phenomenon. Skin aging occurs over time and is influenced by factors such as alcohol intake, tobacco use, and lifestyle such as exposure to UV rays. Aging of the facial skin is mainly caused by three main causes: atrophy, loss of elasticity, and swelling of the skin. First, atrophy is caused by a rapid decrease in skin tissue thickness. Second, the loss of elasticity is caused by sagging around the eyes and cheeks due to the decrease in the subcutaneous tissue. Third, the expansion of the skin is mainly caused by tissue expansion due to overweight. Due to this phenomenon, it acts as a major cause of skin aging. Cross-linked hyaluron is commonly used for more efficient repair of soft tissues that have been lost due to the above reasons.

Although hyaluron has excellent biocompatibility, it has a problem of having a short decomposition period due to low physical properties and expression of hyaluronidase (HYAL) in the body. General methods for preparing commercially available hyaluron are well known. Various methods of binding hyaluron and crosslinking of hyaluron are also known to reduce the water solubility and diffusivity of hyaluron and increase the viscosity of hyaluron. U.S. Pat. Nos. No. 5,356,883 is incorporated herein by reference. In addition, many forms of hyaluron are used, for example, as an adjuvant for preventing adhesions in surgery, an adjuvant for joints, an adjuvant for ophthalmic procedures, and a support for tissue engineering. U.S. Pat. Nos. 6,013,679 is incorporated herein by reference.

However, there are several defects when such a hyaluron product is applied to a soft tissue area. In particular, a compromise between in vivo properties and surgical utility is required. For example, hyaluron that has been sufficiently chemically modified or crosslinked to have desirable in vivo mechanical and biological stability properties may be very viscous and difficult or impossible to inject through a microneedle. Conversely, injectable hyaluron may have low in vivo biostability and mechanical properties.

For example, in general, extensive literature contains 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether ( BDDE) is crosslinked by adding 100 to 300ul. If the crosslinking time is reduced when the crosslinking agent is added, it is in a sol state. Conversely, as the crosslinking time is increased, a hard gel state is formed. In the sol state, a dialysis membrane is used to remove the crosslinking agent, but the crosslinking agent is not removed in the desired direction from the crosslinked hyaluronic acid network. Therefore, in order to solve this problem, the residual crosslinking agent is removed by increasing the dialysis time. However, this method has a problem of reducing the viscoelasticity by diluting the cross-linked hyaluron. In addition, when removing the crosslinking agent in the gel state, it is removed by washing with a large amount of PBS and sterile water. However, in this state, the crosslinking agent on the surface of the gel can be removed, but the crosslinking agent inside the gel cannot be removed.

To overcome this problem, a special crosslinking technology of hyaluron is required. The reason is that when a high content of the crosslinking agent is injected, the physical, chemical and biological properties of the hyaluron gel are modified, and when injected into the body, it attacks immune cells, etc., and there is a high probability of causing side effects such as inflammation and granuloma formation. Therefore, there is a need for a process technology for hybrid hyaluronic acid products that can minimize the content of the crosslinking agent.

That is, in order to solve the above-mentioned cross-linked hyaluronic acid problem, the intermediate morphology of a monophasic in a sol state and a biphasic in a gel crush state And hybrid-type hyaluronic acid products with characteristics are required. Currently commercialized hyaluron-based representative Dermal Fillers products are Galderma’s Restylane with NASHA and OBT technology and Allergan’s Juvederm with VYCROSS technology. It occupies about 70% of the market. The advantage of these hyaluron filler-based dermal fillers is that they are manufactured using their own process technology to realize high density of the final product by injecting a relatively small amount of crosslinking agent compared to other products. is different from the product of

However, although hyaluron is excellent for stable volume formation, questions are raised about the efficient repair of lost soft tissue.

In order to solve this problem, research on blending or chemically combining hyaluron with more effective substances and components for tissue reparing is being actively conducted, but commercialization is insufficient.

U.S. Patent No. 5,356,883 U.S. Patent No. 6,013,679 International Patent Publication No. WO 2008-008857 US Patent No. 8,071,722 US Patent No. 7,674,882 U.S. Patent No. 8,178,656

Bioconjugate Chemistry, 2010, 21, 240-247: Joem Y., et al., Effect of cross-linking reagents for hyaluronic acid hydrogel dermal fillers on tissue augmentation and regeneration. Carbohydrate Polymers, 2007, 70,251-257: Jeon, O., et al., Mechani cal properties and degradation behaviors of hyaluronic acid hydrogels cross-linked at various cross-linking densities. J. Am. Chem. Soc., 1955, 77 (14), 3908-3913: Schroeder W., et al., The amino acid composition of Bombyx mori silk fibroin and of Tussah silk fibroin. Biomacromolecules, 2010, 11 (9), 2230-2237: Serban, M., et. Al. Modular elastic patches: mechani cal and biological effects. Journal of Materials Chemistry, 2009, 19,6443-6450: Murphy A., et al., Biomedical applications of chemically modified silk fibroin. Biomacromolecules, 2004, 5, 751-757: Sohn, S., et al., Phase behavior and hydration of silk fibroin. Marsh, R. E., R. B. Corey, and L. Pauling (1995) An investigation of the stucture of silk fibroin. Biochim. Biophys. Acta. 16: 1-34 Lee, K-G., Y.-W. Lee, J.-H. Yeo, J. Nam, H.-Y. Kweon, and Y.-H. Park (1999) Structural characteristics of silk fibroin gel on the preparation conditions

Accordingly, the present inventors conducted research to solve the above problems, and as a result, a high-density cross-linked hyaluron using VLT (Vector Linker Technology) and a silk composition with a specified component content were blended without mutual chemical cross-linking after blending with a weak interpenetrating network ( It relates to a composite composition that compensates for the shortcomings of hyaluron by forming a semi-IPN) polymer, its process, and its use.

More specifically, the hydrogel of the present invention relates to a hyaluron-silk composite composition useful for more efficiently improving tissue graft viability and soft tissue enlargement.

The object of the present invention is not limited to the object mentioned above. The object of the present invention will become clearer from the following description, and will be realized by means and combinations thereof described in the claims.

In order to achieve the above object, the present invention provides means for solving the following problems.

One aspect of the present invention provides a method for preparing a hyaluronic acid-based hydrogel composition, the method comprising: a) preparing a low molecular weight hyaluronic acid composition having an average molecular weight of 10 kDa to 1500 kDa; b) preparing a high molecular weight hyaluronic acid composition having an average molecular weight of 1600 kDa to 4500 kDa; c) preparing a fibroin composition in which the contents of glycine, serine and alanine are changed; d) chemically primary crosslinking the low molecular weight hyaluronic acid composition of a) and the high molecular weight hyaluronic acid composition of b); And e) blending the random copolymer generated in step d) with the fibroin composition of step c) to form a semi-IPN structure; it provides a method for producing a hyaluron-based hydrogel composition, comprising a.

In one aspect of the present invention, the average molecular weight of the hyaluronic acid in step a) is 30 kDa to 1,000 kDa, to provide a method for producing a hyaluronic-based hydrogel composition.

In one aspect of the present invention, for the low molecular weight hyaluronic acid in step a), the crosslinking agent is added in an amount of 15ul to 35ul per gram of hyaluronic acid input, pH 12 to pH 14, temperature conditions of 278K to 298K, 4 hours to It provides a method for preparing a hyaluron-based hydrogel composition, which is prepared by reacting for a time of 10 hours.

In one embodiment, the crosslinking agent is 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether, BDDE), provides a method for preparing a hydrogel composition.

In one aspect of the present invention, the average molecular weight of the hyaluronic acid in step b) is 2,000 kDa to 4,000 kDa, it provides a method for preparing a hyaluronic-based hydrogel composition.

In one aspect of the present invention, for the high molecular weight hyaluronic acid in step b), the crosslinking agent is added in an amount of 15ul to 75ul per gram of hyaluronic acid input, pH 12 to pH 14, temperature conditions of 278K to 298K, 24 hours to It provides a method for preparing a hyaluron-based hydrogel composition, which is prepared by reacting for 50 hours.

In one embodiment, the crosslinking agent is 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether, BDDE) provides a method for preparing a hyaluron-based hydrogel composition.

In one aspect of the present invention, the fibroin composition of step c) has 10 to 14 mol% of serine, 42 to 47 mol% of glycine, and 27 to 32 mol% of alanine. Serine is reduced to 40% or less with respect to fibroin. And, glycine is increased by 5% or more, and alanine is increased by 5% or more.

In one embodiment, the fibroin composition of step c) has an average molecular weight of 100 kDa or less, the content of glycine is 40 to 50% by weight based on the total weight of the fibroin composition, and the glycine is glycine (β) and glycine ( The weight ratio of α) is in the range of 0.1: 99.9 to 10: 90, the content of alanine is 25 to 35% by weight based on the total weight of the fibroin composition, and the weight ratio of alanine (β) to alanine (α) is 0.1: It ranges from 99.9 to 10: 90, and the mol ratio of the heavy chain and the light chain in the fibroin is 0.1: 99.9 to 10: 90 It provides a method for preparing a hyaluron-based hydrogel composition.

In one aspect of the present invention, the amount of the fibroin composition is added in a weight ratio of 0.1: 9.9 to 3: 7 compared to hyaluron, to provide a method for preparing a hyaluron-based hydrogel composition.

In one aspect of the present invention, in step d), the pH 12 to 14, the reaction temperature of 278K to 298K, the reaction time of 24 hours to 50 hours, the content of hyaluronic acid in the solid content of the total composition is 10 to 20% by weight , provides a method for preparing a hyaluron-based hydrogel composition.

In one aspect of the present invention, in step e), a semi-IPN structure is formed by giving a physical annealing effect under a pressure condition of 0.1 atm to 2 atm, a temperature condition of 370 K to 400 K, and a reaction condition of 10 minutes to 100 minutes. It provides a method for producing a hyaluron-based hydrogel composition, characterized in that.

Another aspect of the present invention provides a hydrogel composition prepared according to any one of the aspects of the present invention.

In another aspect of the present invention, the composition is used as a soft tissue filler, provides a hydrogel composition.

In another aspect of the present invention, the composition is used as a cartilage filler, provides a hydrogel composition.

In another aspect of the present invention, the composition provides a hydrogel composition further comprising lidocaine or a derivative thereof.

The hydrogel composition prepared according to one aspect of the present invention forms a weak interpenetration (semi-IPN) network structure by the secondary annealing effect (Annealing Effect) of complex amino acids in which specific component content is changed in the high-density hyaluron structure.

The hydrogel composition prepared according to one aspect of the present invention significantly reduces the risk of side effects during application and the quality of the product due to the injection of a small amount of crosslinking agent.

The hydrogel composition prepared according to one aspect of the present invention provides a hybrid type hyaluron-silk peptide composition that improves the disadvantages of these products in the field of hyaluronic acid, which is a dermal filler used for soft tissue repair.

The hydrogel composition prepared according to one aspect of the present invention can promote the survival or growth of cells and other components of soft tissues during injection or support an optimal environment.

The hydrogel composition prepared according to one aspect of the present invention results in reducing the porosity of the gel structure by controlling the ratio between the crosslinking rate and the hydrolysis rate while increasing the physical properties compared to the existing product.

The gel structure of the hydrogel composition prepared according to one aspect of the present invention has properties that can improve the ability to induce enzyme resistance and cellular responses in the body.

The hydrogel composition prepared according to one aspect of the present invention is effective not only for stable volume formation, but also for efficient repair of lost soft tissue (soft tissue).

The effects of the present invention are not limited to the above-mentioned effects. It should be understood that the effects of the present invention include all effects that can be inferred from the following description.

1A is SEM data after primary refining of Bombryx mori. Figure 1b is the data measured by FT-IR ATR after dissolving in LiBr through the primary refining process. Figure 1c is the data measured by FT-IR ATR after dissolving CaCl ₂ -EtOH-H2O through the primary refining process.
2a is FT-IR ATR data measured after blending hyaluron-fibroin. Figure 2b is FT-IR ATR data through the annealing process of the blended hyaluron-fibroin composite hydrogel.
3 is SEM data measured after imparting the effect of blending and annealing cross-linked hyaluronic acid and silk fibroin. Figure 3a is SEM data measured by blending two materials prepared by the conventional hyaluronic acid crosslinking method and the conventional fibroin extraction method. Figure 3b is SEM data measured by blending the conventional hyaluronic acid and fibroin used in the present invention. 3c is SEM data measured by blending high-density hyaluronic acid and two materials prepared by the conventional fibroin extraction method. Figure 3d is SEM data measured by blending high-density hyaluronic acid and fibroin used in the present invention.
Figure 4 is a morphologically (morphologically) after giving the effect of blending and annealing with the high-density cross-linked hyaluronic acid, the ratio percent of the fibroin used in the present invention was measured through SEM. it is data Figure 4a is SEM data of the hydrogel containing 0.1 wt% fibroin. Figure 4b is SEM data of the hydrogel containing 10 wt% fibroin. Figure 4c is the SEM data of the hydrogel containing 20 wt% fibroin. Figure 4d is SEM data of the hydrogel containing 30 wt% fibroin. Figure 4e is SEM data of the hydrogel containing 100 wt% fibroin, that is, the hydrogel does not contain hyaluronic acid.
5 is data obtained by measuring the morphology before and after the annealing effect during the process of the present invention. 5A is an SEM measurement data of a crush state before the annealing effect is applied. FIG. 5B is an SEM data measured by enlarging FIG. 5A. Figure 5c is the SEM measurement data of the hybrid gel (hybrid gel) state after the application of the annealing effect. FIG. 5d is an SEM data measured by enlarging FIG. 5c.
6 shows the preparation of a hybrid gel of the present invention using each hyaluronic acid having a different molecular weight, freeze-drying, precipitation in 1X PBS (pH 7.2 to 7.6), and then in an incubator at 37° C. for 5 days. Weighed by filtering and drying. I has an average molecular weight (Mw) of 0.6 X 10 ⁶ Da, II has an average molecular weight of 1.6 (Mw) X 10 ⁶ Da, and III has an average molecular weight of 2.6 (Mw) X 10 ⁶ Da.
7 is XRD data measured by lyophilization after preparing the hybrid gel of the present invention. Figure 7a is data for confirming the internal crystallinity of fibroin using a general extraction method. 7b is data for confirming the crystallinity inside the hybrid gel measured by giving an annealing effect after blending the fibroin and high-density hyaluron gel using a general extraction method. 7c is data for confirming the crystallinity inside the hybrid gel after imparting an annealing effect during the process using the fibroin disclosed herein.
8 is a differential scanning calorimetry data measured after preparing the hybrid gel of the present invention. Figure 8a is the thermal analysis data of the hybrid gel before the annealing effect. Figure 8b is the thermal analysis data of the hybrid gel after the annealing effect.
9 is an in vitro measurement of the cell viability of the hybrid gel prepared in the present invention. (a : 1 day, b : 3 days, c : 7 days)
10 is a data showing the hydrogel degradation behavior by the enzyme of Test Example 9.
11 is the actual data of the hybrid gel prepared in the present invention, and is the actual data for visually confirming the adhesive force and viscoelasticity of the hybrid gel.
12 is a morphology data measured when manufacturing the high-density hyaluron gel of the present invention, outside the temperature and molecular weight ranges among the specific factors presented herein. 12a is a temperature of 278K or less when manufacturing a high-density hyaluron gel, and in more detail, SEM morphology data measured after preparation at 277K. Figure 12b is a high-density hyaluron gel produced at a temperature of 289K or higher, and in more detail, SEM morphology data measured after production at 290K. FIG. 12c shows a first molecular weight of 2 000 kDa to 4 000 kDa when preparing a high-density hyaluron gel, more specifically, an average molecular weight of 2 500 kDa, a second molecular weight of 30 kDa to 1,000 kDa, and more specifically, an average molecular weight of 450 kDa. These are SEM morphology data measured after manufacturing with a furnace. 12d shows a first molecular weight of 30 kDa to 1 000 kDa, more specifically, an average molecular weight of 450 kDa, a second molecular weight of 2 000 kDa to 4 000 kDa, and more specifically, an average molecular weight of 2 when the high-density hyaluron gel is prepared. These are SEM morphology data measured after manufacturing at or below each range of 500 kDa.
13 is a data obtained by measuring the molecular weight distribution of stabilized fibroin used herein. Figure 13a is data obtained by measuring the molecular weight distribution of stabilized fibroin used herein. Figure 13b is data obtained by measuring the stabilized fibroin used in the present application as a chromatogram in a pH 2.5 aqueous solution. 13c is data obtained by measuring the stabilized fibroin used in the present application as a chromatogram in a pH 3.0 aqueous solution. 13d is data obtained by measuring the stabilized fibroin used in the present application as a chromatogram in a pH 3.5 aqueous solution.
14 is data confirming the pH stability of fibroin used herein through Raman analysis.

Unless otherwise specified, all numbers, values, and/or expressions expressing ingredients, reaction conditions, and amounts of ingredients used herein refer to a variety of measures that may occur in obtaining such values, among others, in which such numbers are inherently different. Since they are approximations reflecting uncertainty, it should be understood as being modified by the term “about” in all cases. Also, where the disclosure discloses numerical ranges, such ranges are continuous and inclusive of all values from the minimum to the maximum inclusive of the range, unless otherwise indicated. Furthermore, when such ranges refer to integers, all integers inclusive from the minimum to the maximum inclusive are included, unless otherwise indicated.

In this specification, when a range is described for a variable, the variable will be understood to include all values within the stated range including the stated endpoints of the range. For example, a range of “5 to 10” includes the values of 5, 6, 7, 8, 9, and 10, as well as any subranges such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc. It will be understood to include any value between integers that are appropriate for the scope of the recited range, such as 5.5, 6.5, 7.5, 5.5 to 8.5 and 6.5 to 9, and the like. Also for example, ranges from "10% to 30%" include values of 10%, 11%, 12%, 13%, etc. and all integers up to and including 30%, as well as 10% to 15%, 12% to It will be understood to include any subranges such as 18%, 20% to 30%, etc., as well as any value between reasonable integers within the scope of the recited ranges, such as 10.5%, 15.5%, 25.5%, and the like.

Hereinafter, the present invention will be described in detail.

The present invention provides a formula with improved stability of a high-density hyaluron gel-based composite composition. One aspect of the present invention relates to a cross-linked biopolymer and an overall manufacturing method thereof. One aspect of the present invention relates to a biopolymer forming a weak interpenetration (semi-IPN) network structure by the secondary annealing effect of complex amino acids in which a specific component content is changed in a high-density hyaluron structure, and a manufacturing process thereof will be. One aspect of the present invention relates to the use of cross-linked biopolymers as dermal fillers.

Hereinafter, the relevant knowledge to help understand the technology of the present invention will be described.

raw material

Hyaluronic acid (hyaluron or hyaluronate) is a naturally occurring glycosaminoglycan and is used as a component of a dermal filler for wrinkle reduction and tissue volume. Hyaluron is an anionic nonsulfated glycosaminoglycan widely distributed throughout connective tissue, epithelial tissue, and nervous tissue. The polymer, hyaluron, has a molecular weight of several million daltons. The human body generally has about 15 grams of hyaluron in the body, and about 30% of it is broken down within a few hours or days by the body's enzymes and free radicals and replaced with newly synthesized hyaluron in the body. is made up of

Silk is a natural protein made from high-strength fibroin fibers and has mechanical properties comparable to or better than many synthetic high-performance fibers. Silk is also stable at physiological temperatures over a wide pH range and is insoluble in most aqueous and organic solvents. The degradation products of silk (eg peptides, amino acids) are biocompatible. And since ancient times, it has been widely used as an early suture. Silk is of non-mam-malian origin and has the advantage of much less immune rejection when applied in vivo than other similar natural biomaterials (eg, bovine or porcine collagen). Silk has a fibrous structure morphologically. In addition, the inside is formed of fibroin, and sericin has a structure surrounding the fibroin. Fibroin itself does not have an immune rejection reaction, but sericin causes an inflammatory reaction when it comes into contact with blood. Silk refers to a fibrous product in which secretions from specific organs of silkworms or spiders are solidified. There are many variations of natural silk. Fibroin is produced and secreted by the silk glands of the silkworm. When fibroin leaves the sweat glands, the adhesive component sericin is coated to make up the silk. And fibroin produced in spider is an immunogenic protein and is produced as a single filament without sericin. Currently, many techniques related to the extraction of relatively high molecular weight fibroin are known. However, due to the chemical shift of fibroin, the range of use is extremely limited. The cause is structurally alpha-helix when fibroin is extracted for the first time, but structural transition to insoluble beta-sheet occurs due to the surrounding environment (time, temperature, humidity). This reason is caused by the interaction of 18 amino acids and light chains and heavy chains present in fibroin. Therefore, in order to attenuate this chemical shift, fibroin with improved stability by changing the content of amino acids present in fibroin should be used in the present invention.

technical part

In general, the reference point applied to crosslink hyaluron is the rate of modification (MoD). However, there is a clear limitation in commercializing the theoretically optimal crosslinking rate based on the modification rate (MoD) used to crosslink hyaluron. The strain rate is expressed by Equation 1 below.

[Formula 1]

Modification (MoD)% = (number of moles of bound crosslinking agent/number of moles of repeating hyaluronic acid) x 100

That is, it may contain the risk of side effects when applied and the quality of the product due to excessive injection of the crosslinking agent. Therefore, it is necessary to implement a high-density hyaluronic acid gel with a minimum amount of cross-linking agent input based on the theoretical kinetics. To this end, in the present invention, the theoretical background of the present invention was implemented based on the chemical reaction rate model of Arrhenius Law. However, the content of the present invention is not entirely dependent on the chemical reaction rate equation of Arrhenius Law, but is applied to the process based on the theory.

The chemical reaction rate equation of Arrhenius Law applied to the present invention is shown in Equation 2 below.

[Equation 2]

In K = -(Ea/R)(1/T) + In A

K : rate constant

T : absolute temperature

A : Arrhenius constant (pre exponential factor)

Ea: activation energy

R : gas constant

Based on Equation 2, when the decomposition reaction and the crosslinking reaction occur simultaneously during crosslinking of hyaluron, the decomposition reaction rate that affects the reaction rate is K1, and the crosslinking reaction rate is K2.

Therefore, assuming that the decomposition reaction and the crosslinking reaction are a first-order reaction rate function model, assuming that the function Y1 by decomposition, the function Y2 by the crosslinking, and the function Y1Y2 occurring at the same time, the functions of the decomposition reaction and the crosslinking reaction are respectively: Same as Equations 3 and 4

[Equation 3]

Y ₁ = A exp (-K ₁ T)

[Equation 4]

Y ₂ = -B exp (-K ₂ T) + C

Assuming that Equation 3 and Equation 4 occur simultaneously, Equation 5 appears.

[Equation 5]

Y = -B exp (-(K ₁ + K ₂ )T) + C exp (-K ₁ T)

Y : Y ₁ Y ₂

In Equations 3 and 4, B and C are constants. That is, assuming that the initial modulus of elasticity of the hydrogel is G ₀ and the modulus of elasticity at the end of the reaction is G, G/G ₀ due to the decomposition reaction immediately before the initial reaction is 1, So A becomes 1. In addition, G/G ₀ by the cross-linking reaction immediately before the initial cross-linking reaction is 1. Therefore, the sum of B and C appears to be 1. And, the constant C must have a constant value regardless of the temperature, the maximum G/G ₀ reached when only the cross-linking reaction occurs without decomposition. That is, the constant B must also have a constant value regardless of the temperature.

Therefore, the temperature range applied among the methods to be implemented in the present invention is a theoretical basis that can be set based on Arrhenius's first-order reaction kinetics model.

Accordingly, G/G ₀ to be implemented in the present invention is at least 1, and the present invention can be implemented within the range of a maximum value of 80 or less.

However, the present invention does not rely entirely on Arrhenius's first-order reaction kinetics model. However, the disadvantage of hydrogel production based on the above theory is a big problem in terms of production efficiency.

Therefore, the method to be implemented in the present invention is an addition reaction of two or more hyaluronic acids having different molecular weights within the above range of (elastic modulus at the end of the reaction)/(initial modulus) of the above model. By applying the present invention can be implemented.

manufacturing process part

In the description of VLT terms used in this specification, a processing method of a CPU having an instruction for processing a plurality of data is referred to as a vector process or an array processor. VLT (Vector Linker Technology) is a process method in which the bonding of two or more substances to one final substance is not chemically interconnected, but affects the properties of the final substance.

In other words, based on the chemical reaction rate model of Arrhenius Law mentioned in the technical section, hyaluronic acids having different molecular weights or different reaction rates are blended in a random method instead of a block method and then crosslinked. . In addition, fibroin, which is not affected by the crosslinking agent, is added at the same time to achieve primary chemical crosslinking. After that, an annealing effect is given to form a secondary physical network structure using the stabilized properties of fibroin, and the structural morphology of the final product is high-density hyaluronic acid with weak interpenetration ( It is a technical principle to create a hydrogel with semi-IPN) linkages.

Accordingly, the content of the present invention is summarized as follows.

The present invention relates to a hybrid-type hyaluron-silk peptide composition that improves the disadvantages of these products in the field of hyaluronic acid, a dermal filler used for soft tissue repair, and a manufacturing process thereof.

The present invention relates to a composite hydrogel useful for soft tissue repair and a composition thereof, and these hydrogel compositions can promote the survival or growth of cells and other components of soft tissue during injection or support an optimal environment.

In general, according to literature, crosslinking of hyaluron is affected by crosslinking rate and decomposition rate and temperature. In the theoretical aspect presented in the present invention, the decomposition rate of hyaluron is more sensitive to temperature than the cross-linking rate. However, when the above theory is applied, theoretically, the crosslinking density increases but the reaction rate decreases, so it can be implemented experimentally, but from an industrial point of view, there is a problem in that production efficiency is decreased. Therefore, in order to improve the mechanical properties of hyaluron, which is the basis of the present invention, two or more reactions are sequentially carried out in a random format to prepare a hyaluron hydrogel having a high-density network structure.

This reduces the input content of butanediol diglycidyl ether (hereinafter referred to as BDDE) compared to the existing hyaluron dermal filler, thereby increasing the physical properties, which are the results characterized in different directions during crosslinking of the hyaluron hydrogel, while simultaneously increasing the crosslinking speed and In theory, the ratio between the hydrolysis rates can be controlled to achieve the result of reducing the porosity of the gel structure.

Therefore, such a gel structure has properties that can improve the ability to induce enzyme resistance and cellular responses in the body.

The complex amino acid presented in the present invention uses regenerated fibroin.

In general, according to some embodiments, the primary amino acid sequence of regenerated fibroin is Serine (Serine or Ser, HO ₂ CCH(NH ₂ )CH ₂ OH), Glycine or Gly, HO ₂ CCH ₂ NH ₂ ), Alanine (Alanine or Ala, HO ₂ CCH(NH ₂ )CH ₃ ) In the combined general content, the composition ratio is 10.61 (mol%), 43.42 (mol%), 30.17 (mol%).

However, in the above content, structurally, it is easily changed to β-sheet. This reason is caused by the interaction of 18 amino acids and light chains and heavy chains present in fibroin.

Therefore, the complex amino acids presented in the present invention were applied to the present invention by changing the content of the primary amino acid sequence in fibroin.

For example, in the combined content of serine, glycine and alanine, the composition ratio was reduced by 40% or less for serine, increased by 5% or more for glycine, and increased by 5% or more for alanine The present invention was achieved using regenerated fibroin.

In addition, the present invention was achieved using an average molecular weight of 100 kDa or less by reducing or removing the bond between the light chain and the heavy chain of the fibroin.

Therefore, the present invention blends the high-density cross-linked hyaluron with fibroin with a specific component changed, and gives a primary chemical cross-linking and secondary annealing effect, thereby providing a weak interpenetration (semi-IPN) structure on the hydrogel. It was invented so that it could be used more extensively by giving characteristics of an intermediate form between monophasic and biphasic, which are existing dermal fillers.

Hereinafter, various aspects of the present invention will be described.

One aspect of the present invention provides a method for preparing a hyaluronic acid-based hydrogel composition, the method comprising: a) preparing a low molecular weight hyaluronic acid composition having an average molecular weight of 10 kDa to 1500 kDa; b) preparing a high molecular weight hyaluronic acid composition having an average molecular weight of 1600 kDa to 4500 kDa; c) preparing a fibroin composition in which the contents of glycine, serine and alanine are changed; d) chemically first crosslinking the low molecular weight hyaluronic acid composition of a) and the high molecular weight hyaluronic acid composition of b); And e) blending the random copolymer generated in step d) with the fibroin composition of step c) to form a semi-IPN structure; it provides a method for producing a hyaluron-based hydrogel composition comprising a.

In one embodiment, the input timing of the fibroin composition in step c) is mixed by adding low molecular weight and high molecular weight hyaluron at an early stage during the primary crosslinking process. By reducing the content of serine containing a number of hydroxy functional groups in fibroin, the possible addition reaction by BDDE with the hydroxy group of hyaluronic acid was suppressed. After uniformly mixing low molecular weight and high molecular weight hyaluronic acid, fibroin is added to the crosslinking process by BDDE and blended. After that, cross-linking of only fibroin, excluding the cross-linked hyaluron network, is made by heat and pressure to form an interpenetrating polymer network structure.

In one aspect of the present invention, the average molecular weight of the hyaluronic acid in step a) is 30 kDa to 1,000 kDa, to provide a method for producing a hyaluronic-based hydrogel composition.

In one aspect of the present invention, for the low molecular weight hyaluronic acid in step a), the crosslinking agent is added in an amount of 15ul to 35ul per gram of hyaluronic acid input, pH 12 to pH 14, temperature conditions of 278K to 298K, 4 hours to It provides a method for preparing a hyaluron-based hydrogel composition, which is prepared by reacting for a time of 10 hours.

In one embodiment, the crosslinking agent is 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether, BDDE), provides a method for preparing a hydrogel composition.

A low molecular weight partially crosslinked hyaluron is prepared through step a).

In one aspect of the present invention, the average molecular weight of the hyaluronic acid in step b) is 2,000 kDa to 4,000 kDa, it provides a method for preparing a hyaluronic-based hydrogel composition.

In one aspect of the present invention, for the high molecular weight hyaluronic acid in step b), the crosslinking agent is added in an amount of 15ul to 75ul per gram of hyaluronic acid input, pH 12 to pH 14, temperature conditions of 278K to 298K, 24 hours to It provides a method for preparing a hyaluron-based hydrogel composition, which is prepared by reacting for 50 hours.

In one embodiment, the crosslinking agent is 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether, BDDE) provides a method for preparing a hyaluron-based hydrogel composition.

Prepare high molecular weight partially cross-linked hyaluron through step b).

In one aspect of the present invention, the fibroin composition of step c) has 10 to 14 mol% of serine, 42 to 47 mol% of glycine, and 27 to 32 mol% of alanine. Serine is reduced to 40% or less with respect to fibroin. And, glycine is increased by 5% or more, and alanine is increased by 5% or more.

In one aspect of the present invention, the fibroin composition of step c) is a fibroin composition containing 3 to 10 mol% of serine, 45 to 50 mol% of glycine, and 30 to 35 mol% of alanine, a hyaluron-based hydrogel A method of making the composition is provided.

In one embodiment, the fibroin composition of step c) has an average molecular weight of 100 kDa or less, the content of glycine is 40 to 50% by weight based on the total weight of the fibroin composition, and the glycine is glycine (β) and glycine ( The weight ratio of α) is in the range of 0.1: 99.9 to 10: 90, the content of alanine is 25 to 35% by weight based on the total weight of the fibroin composition, and the weight ratio of alanine (β) to alanine (α) is 0.1: It ranges from 99.9 to 10: 90, and the mol ratio of the heavy chain and the light chain in the fibroin is 0.1: 99.9 to 10: 90 It provides a method for preparing a hyaluron-based hydrogel composition.

In one aspect of the present invention, the low molecular weight hyaluronic acid composition of step b) may be included in an amount of 300 to 600 parts by weight based on 100 parts by weight of the low molecular weight hyaluronic acid composition of step a). Preferably, the low molecular weight hyaluronic acid composition of step b) may be included in an amount of 400 to 500 parts by weight based on 100 parts by weight of the low molecular weight hyaluronic acid composition of step a).

In one aspect of the present invention, the amount of the fibroin composition is added in a weight ratio of 0.1: 9.9 to 3: 7 compared to hyaluron, to provide a method for preparing a hyaluron-based hydrogel composition.

In one aspect of the present invention, the fibroin composition of step c) is 200 parts by weight to 9900 parts by weight based on 100 parts by weight of the total hyaluronic acid of the low molecular weight hyaluronic acid of step a) and the high molecular weight hyaluronic acid of step b) may be included as a part.

In one aspect of the present invention, in step d), the pH 12 to 14, the reaction temperature of 278K to 298K, the reaction time of 24 hours to 50 hours, the content of hyaluronic acid in the solid content of the total composition is 10 to 20% by weight , provides a method for preparing a hyaluron-based hydrogel composition.

In one aspect of the present invention, the crosslinking agent in step d) may be added in an amount of 20 to 70 ul with respect to 1 g of the hydrogel composition.

Through step d), a relatively low molecular weight partially crosslinked hyaluron formed in step a) and a relatively high molecular weight partially crosslinked hyaluron formed in step b) are formed into a random copolymer (co-polymer). The formed random copolymer (co-polymer), fibroin whose optional amino acid content of step c) is adjusted, is blended by adding a minimum of 0.1 weight percent to a maximum of 30 weight percent compared to hyaluron.

In one aspect of the present invention, in step e), a semi-IPN structure is formed by giving a physical annealing effect under a pressure condition of 0.1 atm to 2 atm, a temperature condition of 370 K to 400 K, and a reaction condition of 10 minutes to 100 minutes. It provides a method for producing a hyaluron-based hydrogel composition, characterized in that.

Another aspect of the present invention provides a hydrogel composition prepared according to any one of the aspects of the present invention.

In another aspect of the present invention, the composition is used as a soft tissue filler, provides a hydrogel composition.

In another aspect of the present invention, the composition is used as a cartilage filler, provides a hydrogel composition.

In another aspect of the present invention, the composition provides a hydrogel composition further comprising lidocaine or a derivative thereof.

Hereinafter, specific manufacturing examples of the present invention will be described.

The hybrid gel preparation disclosed herein can be processed by the following process example.

The crosslinking of the hyaluron disclosed herein is made through two or more steps.

In the present specification, the following process processes may correspond to each experimental example.

In one embodiment, the input amount of hyaluron in the first step of preparing the low molecular weight hyaluronic acid composition, the hyaluron concentration in a strong alkaline aqueous solution having a pH of 12 to 14 may have 5 to 20% by weight. In some embodiments, 5% by weight based on the weight percentage of the total hyaluron may be added. In some embodiments, 10% by weight based on the weight percentage of the total hyaluron may be added. In some embodiments, 15% by weight based on the weight percentage of the total hyaluron may be added. In some embodiments, 20 wt% may be added based on the weight percentage of the total hyaluron.

The average molecular weight of the hyaluron in the first step has an average molecular weight of 30 kDa to 1,000 kDa.

After the hyaluron input in the first step, the crosslinking temperature is maintained at 278K to 298K.

After the hyaluron input in the first step, the crosslinking time is maintained for 2 to 6 hours.

After the hyaluron input in the first step, the amount of the crosslinking agent is used in the range of 15ul to 35ul per gram of hyaluron input.

In one embodiment, the amount of hyaluron added in the second step of preparing the high molecular weight hyaluronic acid composition is 75 to 95 wt% based on the total weight of the low molecular weight and high molecular weight hyaluronic acid in the strong alkaline aqueous solution having a pH of 12 to 14. can have In some embodiments, 95% by weight based on the weight of the total hyaluronic acid may be added. In some embodiments, 90% by weight based on the weight of the total hyaluronic acid may be added. In some embodiments, 85% by weight based on the weight of the total hyaluronic acid may be added. In some embodiments, 80% by weight based on the weight of the total hyaluronic acid may be added.

The average molecular weight of the hyaluron in the second step has an average molecular weight of 2 000 kDa to 4 000 kDa.

After hyaluron input in the second step, the crosslinking temperature is maintained at 278K to 298K.

After the hyaluron input in the second step, the crosslinking time is maintained for 24 to 50 hours.

After the hyaluron input in the second step, the amount of the crosslinking agent is used in the range of 15ul to 75ul per gram of the second hyaluron input.

The morphology of the hyaluron gel prepared within the process range of the above process example shows the structure as shown in FIG. 3D.

That is, morphologically, the pore size is kept constant and the network shape of a uniform thickness is maintained at the same time, so that the viscoelasticity of the hydrogel is relatively increased.

On the other hand, when the process range of the above process example is higher or lower than the process range, a morphology as shown in FIG. 12 is formed.

That is, the above FIG. 12a is SEM morphology data measured after production at a temperature of 278K (Calvin temperature) or less, more specifically, at a temperature of 277K when the high-density hyaluron gel is manufactured. Although the density is high, the gap between the pores is It may have a random shape and affect the viscoelasticity of the final hybrid gel.

12b is SEM morphology data measured after production at a temperature of 289K or higher (Calvin temperature), more specifically, at a temperature of 290K during the production of high-density hyaluron gel, the density is relatively low compared to FIG. It can be seen that the size is relatively enlarged. Therefore, the viscoelasticity of the final hybrid gel will decrease.

12c is an SEM morphology measured after preparation of a high-density hyaluron gel, wherein the first molecular weight is 2 000 kDa to 4 000 kDa, more specifically 2500 kDa, and the second molecular weight is 30 kDa to 1 000 kDa, more specifically 450 kDa. (morphology) data. The total amount of hyaluron ?t was 15 wt% and 85 wt% of First and Second, and reacted for 40 hours at pH 13 and 285K conditions without fibroin. It is a structure in which two levels of hyaluron are intertwined, and the density is high, but the elasticity can be reduced.

FIG. 12d is SEM morphology data measured after the first molecular weight is 30 kDa to 1,000 kDa, more specifically 450 kDa, and the second molecular weight is 500 kDa when the high-density hyaluron gel is prepared. The total amount of hyaluron ?t was 15 wt% and 85 wt% of First and Second, and reacted for 40 hours at pH 13 and 285K conditions without fibroin. The same phenomenon as in FIG. 12C is seen. Finally, it shows a form close to biphasic rather than a hybrid gel with cloudiness, and elasticity may also decrease.

The third step disclosed in the present specification is a step of adding fibroin in which the contents of glycine, serine and alanine are changed.

The raw materials of fibroin used herein are shown in Table 1 below.

A B C D Asp 0.2 1.7 1.6 1.5 Thr 0.9 0.8 0.9 0.9 Ser 6.5 5.8 7.2 6.5 Glu 1.2 1.3 1.2 1.2 Gly 44.4 45.0 44.4 42.4 Ala 35.4 33.3 33.2 32.1 Gys 0.4 0.5 0.4 0.4 Val 2.6 2.6 2.5 2.4 Met 0.2 0.2 0.1 0.1 Lle 0.7 0.7 0.6 0.7 Leu 0.4 0.5 0.4 0.4 Tyr 5.1 5.0 5.0 5.1 Phe 0.8 0.9 0.8 0.7 Lys 0.5 0.6 0.6 0.5 His 0.3 0.3 0.3 0.2 Arg 0.5 0.5 0.5 0.5 Pro 0.1 0.2 0.1 0.1

All 4 breeds listed in Table 1 are Gajam dogs. In addition, Table 1 shows the amino acid content of each type of silk silkworm. In Table 1, A is Baekok jam, B is Hansang ll, C is Won jam 125, and D is won 126. In the present invention, Baekok jam of A was used among the provisional dogs.

In some embodiments, Bombyx mori, a raw material for fibroin used herein, is not limited to Bombyx mori silkworm and Tussah, Giant silkworm, but as shown in Table 1, fibroin using fibroin prefer to extract In other words, in the case of a dog, it is preferable to maintain a constant ratio of 42 to 47 mol% of glycine, 27 to 32 mol% of alanine, and 10 to 14 mol% of serine, but is not limited thereto.

The structure of FIG. 15 is data for easily explaining the shape of BM (bombyx mori silkworm), which is a yarn.

The SEM data of the fibroin yarn used in the present invention is shown in Figure 1a.

The composition of fibroin used in the present invention is as follows.

In some embodiments, the fibroin used is a double fibroin composed of a heavy chain, a light chain and a p25 protein, which consists of 11 domains in which glycine, alanine and serine form a repeating crystalline region, a midsection and a C- It consists of an amorphous region at the terminal N-terminus. (Fig. 16)

More specifically, the mol ratio of heavy chain and light chain is, for example, 0.1 : 99.9, at least 0.2 : 99.8, at least 0.3 : 99.7, at least 0.4 : 99.6, at least 0.5 : 99.5, at least 0.6 : 99.4, at least 0.7 : 99.3 , at least 0.8:99.2, at least 0.9:99.1, at least 1.0:99.0, at least 1.1:98.9, at least 1.2:98.8, at least 1.3:98.7, at least 1.4:98.6, at least 1.5:98.5, at least 1.6:98.4, at least 1.7:98.3 , at least 1.8:98.2, at least 1.9:98.1, at least 2.0:98.0, at least 2.1:97.9, at least 2.2:97.8, at least 2.3:97.7, at least 2.4:97.6, at least 2.5:97.5, at least 2.6:97.4, at least 2.7:97.3 , at least 2.8:97.2, at least 2.9:97.1, at least 3.0:97.0, at least 3.1:96.9, at least 3.2:96.8, at least 3.3:96.7, at least 3.4:96.6, at least 3.5:96.5, at least 3.6:96.4, at least 3.7:96.3 , at least 3.8:96.2, at least 3.9:96.1, at least 4.0:96.0, at least 4.1:95.9, at least 4.2:95.8, at least 4.3:95.7, at least 4.4:95.6, at least 4.5:95.5, at least 4.6:95.4, at least 4.7:95.3 , at least 4.8:95.2, at least 4.9:95.1, at least 5.0:95.0, at least, at least 5.1:94.9, at least 5.2:94.8, at least 5.3:94.7, at least 5.4:94.6, at least 5.5:94.5, at least 5.6:94.4, at least 5.7 : 94.3, at least 5.8 : 94.2, at least 5.9 : 94.1, at least 6.0:94.0, at least 6.1:93.9, at least 6.2:93.8, at least 6.3:93.7, at least 6.4:93.6, at least 6.5:93.5, at least 6.6:93.4, at least 6.7:93.3, at least 6.8:93.2, at least 6.9:93.1, at least 7.0:93.0, at least 7.1:92.9, at least 7.2:92.8, at least 7.3:92.7, at least 7.4:92.6, at least 7.5:92.5, at least 7.6:92.4, at least 7.7:92.3, at least 7.8:92.2, at least 7.9:92.1, at least 8.0:92.0, at least 8.1:91.9, at least 8.2:91.8, at least 8.3:91.7, at least 8.4:91.6, at least 8.5:91.5, at least 8.6:91.4, at least 8.7:91.3, at least 8.8:91.2, at least 8.9:91.1, at least 9.0:91.0, at least 9.1:90.9, at least 9.2:90.8, at least 9.3:90.7, at least 9.4:90.6, at least 9.5:90.5, at least 9.6:90.4, at least 9.7:90.3, at least 9.8:90.2, at least 9.9:90.1, at least 10.0 : 90.0.

In some embodiments, when Gly is 42 to 47% by weight relative to the total fibroin composition, the weight ratio of at least Gly(β) to Gly(α) is 0.1 : 99.9, at least 0.2 : 99.8, at least 0.3 : 99.7, at least 0.4 : 99.6, at least 0.5:99.5, at least 0.6:99.4, at least 0.7:99.3, at least 0.8:99.2, at least 0.9:99.1, at least 1.0:99.0, at least 1.1:98.9, at least 1.2:98.8, at least 1.3:98.7, at least 1.4: 98.6, at least 1.5:98.5, at least 1.6:98.4, at least 1.7:98.3, at least 1.8:98.2, at least 1.9:98.1, at least 2.0:98.0, at least 2.1:97.9, at least 2.2:97.8, at least 2.3:97.7, at least 2.4: 97.6, at least 2.5:97.5, at least 2.6:97.4, at least 2.7:97.3, at least 2.8:97.2, at least 2.9:97.1, at least 3.0:97.0, at least 3.1:96.9, at least 3.2:96.8, at least 3.3:96.7, at least 3.4: 96.6, at least 3.5:96.5, at least 3.6:96.4, at least 3.7:96.3, at least 3.8:96.2, at least 3.9:96.1, at least 4.0:96.0, at least 4.1:95.9, at least 4.2:95.8, at least 4.3:95.7, at least 4.4: 95.6, at least 4.5:95.5, at least 4.6:95.4, at least 4.7:95.3, at least 4.8:95.2, at least 4.9:95.1, at least 5.0:95.0, at least, at least 5.1:94.9, at least 5.2:94.8, at least 5.3:94.7, at least 5.4:94.6, at least 5.5:94.5, at least 5.6:94.4, at least 5.7:94.3, at least 5.8:9 4.2, at least 5.9:94.1, at least 6.0:94.0, at least 6.1:93.9, at least 6.2:93.8, at least 6.3:93.7, at least 6.4:93.6, at least 6.5:93.5, at least 6.6:93.4, at least 6.7:93.3, at least 6.8: 93.2, at least 6.9:93.1, at least 7.0:93.0, at least 7.1:92.9, at least 7.2:92.8, at least 7.3:92.7, at least 7.4:92.6, at least 7.5:92.5, at least 7.6:92.4, at least 7.7:92.3, at least 7.8: 92.2, at least 7.9:92.1, at least 8.0:92.0, at least 8.1:91.9, at least 8.2:91.8, at least 8.3:91.7, at least 8.4:91.6, at least 8.5:91.5, at least 8.6:91.4, at least 8.7:91.3, at least 8.8: 91.2, at least 8.9:91.1, at least 9.0:91.0, at least 9.1:90.9, at least 9.2:90.8, at least 9.3:90.7, at least 9.4:90.6, at least 9.5:90.5, at least 9.6:90.4, at least 9.7:90.3, at least 9.8: 90.2, at least 9.9:90.1, at least 10.0:90.0.

In some embodiments, when the molar ratio of Ala is 27 to 32%, the weight ratio of at least Ala(β) to Ala(α) is 0.1 : 99.9, at least 0.2 : 99.8, at least 0.3 : 99.7, at least 0.4 : 99.6, at least 0.5:99.5, at least 0.6:99.4, at least 0.7:99.3, at least 0.8:99.2, at least 0.9:99.1, at least 1.0:99.0, at least 1.1:98.9, at least 1.2:98.8, at least 1.3:98.7, at least 1.4:98.6, at least 1.5:98.5, at least 1.6:98.4, at least 1.7:98.3, at least 1.8:98.2, at least 1.9:98.1, at least 2.0:98.0, at least 2.1:97.9, at least 2.2:97.8, at least 2.3:97.7, at least 2.4:97.6, at least 2.5:97.5, at least 2.6:97.4, at least 2.7:97.3, at least 2.8:97.2, at least 2.9:97.1, at least 3.0:97.0, at least 3.1:96.9, at least 3.2:96.8, at least 3.3:96.7, at least 3.4:96.6, at least 3.5:96.5, at least 3.6:96.4, at least 3.7:96.3, at least 3.8:96.2, at least 3.9:96.1, at least 4.0:96.0, at least 4.1:95.9, at least 4.2:95.8, at least 4.3:95.7, at least 4.4:95.6, at least 4.5:95.5, at least 4.6:95.4, at least 4.7:95.3, at least 4.8:95.2, at least 4.9:95.1, at least 5.0:95.0, at least, at least 5.1:94.9, at least 5.2:94.8, at least 5.3:94.7, at least 5.4:94.6 , at least 5.5:94.5, at least 5.6:94.4, at least 5.7:94.3, at least 5.8:94.2, at least 5.9 : 94.1, at least 6.0:94.0, at least 6.1:93.9, at least 6.2:93.8, at least 6.3:93.7, at least 6.4:93.6, at least 6.5:93.5, at least 6.6:93.4, at least 6.7:93.3, at least 6.8:93.2, at least 6.9 : 93.1, at least 7.0:93.0, at least 7.1:92.9, at least 7.2:92.8, at least 7.3:92.7, at least 7.4:92.6, at least 7.5:92.5, at least 7.6:92.4, at least 7.7:92.3, at least 7.8:92.2, at least 7.9 : 92.1, at least 8.0:92.0, at least 8.1:91.9, at least 8.2:91.8, at least 8.3:91.7, at least 8.4:91.6, at least 8.5:91.5, at least 8.6:91.4, at least 8.7:91.3, at least 8.8:91.2, at least 8.9 : 91.1, at least 9.0:91.0, at least 9.1:90.9, at least 9.2:90.8, at least 9.3:90.7, at least 9.4:90.6, at least 9.5:90.5, at least 9.6:90.4, at least 9.7:90.3, at least 9.8:90.2, at least 9.9 : 90.1, at least 10.0 : 90.0.

For example, in the case of fibroin used in one aspect of the present invention, a combination of serine or Ser, HO2CCH(NH2)CH2OH), glycine (Glycine or Gly, HO2CCH2NH2), alanine (Alanine or Ala, HO2CCH(NH2)CH3) In the content, the composition ratio was reduced by 40% or less in the case of serine, increased by more than 5% in the case of glycine, and increased by more than 5% in the case of alanine, the present invention was achieved using regenerated fibroin.

In addition, the present invention was achieved using an average molecular weight of 100 kDa or less by reducing or removing the bond between the light chain and the heavy chain of the fibroin.

In one embodiment, the change in the content of fibroin can be implemented through a method of extracting through a protein purification process and chromatography process after first reducing the hydroxyl groups present in fibroin at a high pressure of 3 atm or less in a neutral salt state of pH 6 to pH 8 there is.

The content of fibroin used in the present invention is 0.1 to 30% by weight based on the total weight of the hybrid gel composition. If it is less than 0.1 wt%, the sem-IPN structure of the hybrid gel, which is the final material, is not formed, and the gel state is partially confirmed inside the hydrogel. And, at 30% by weight or more, there is a problem in that the entire hydrogel rapidly gels in a short time due to the mutual repulsion action of the anionic hyaluron. (Form a morphology similar to Fig. 4e)

Therefore, in the present invention, when fibroin is 0.1 wt% based on the total weight of the hybrid gel composition, the morphology as shown in Figure 4a is formed. When it is 10% by weight, a morphology as shown in FIG. 4B is formed. When it is 20% by weight, a morphology as shown in FIG. 4c is formed. When it is 30% by weight relative to the hyaluron content, a morphology as shown in FIG. 4D is formed.

In the fourth step disclosed in the present invention, a random copolymer is formed of the relatively low molecular weight partially cross-linked hyaluron formed in the first step and the relatively high molecular weight partially cross-linked hyaluron formed in the second step. make it

The formed random copolymer (co-polymer), the fibroin whose optional amino acid content of the third step is adjusted is blended by adding at least 0.1 wt% to up to 30 wt% based on the total weight of the hybrid gel composition.

The optimum environmental conditions in the above blending process include a process of reacting at least 24 to 50 hours at a strong alkaline condition of pH 12 to pH 14, and a temperature of at least 278 K to a maximum of 298 K.

In one embodiment, depending on the pH change of the aqueous solution of the present composition, the mutual binding time between molecular chains may vary. That is, although not limited to theory, in theory, proteins including fibroin form zwitter ions with the same positive and negative charges of heavy chain, light chain, and random coil molecular chains near the isoelectronic point, thereby minimizing the ionic repulsion between each other. The conditions for interaction between macromolecular chains are reduced and increased, and accordingly, a beta-sheet structure is formed by hydrophobic defects, hydrogen defects, ionic bonds, etc., which develops into a network structure, which affects the formation of gels.

In the composition produced in the present invention, when the pH is at least 12 to 14, the mutual repulsive force between molecular chains is reduced, thereby reducing the gel formation time. That is, as the pH increases, the mutual repulsive force between molecular chains increases and the gel formation time increases. At least at a pH of 12 or higher, aggregation does not occur between molecular chains due to strong ionic repulsion between molecules, which is caused by the dominant negative or positive charges between molecular chains.

The fifth step disclosed in the present invention is a step in which fibroin is mutually penetrated into the hyaluron randomized network structure formed primarily in the fourth step to form a fibroin network.

The optimum condition for this step is to maintain a pressure of 0.1 atm to 2 atm during the process of forming the semi-IPN structure, and at the same time maintain a temperature of at least 370K to 400K. In addition, the time is applied for a minimum of 10 minutes to a maximum of 100 minutes to achieve the secondary crosslinking step mentioned in the present invention.

Hereinafter, the preferred manufacturing method and scope presented above are described and described in detail in the present invention, but those skilled in the art can make various modifications, additions, and substitutions without departing from the scope of the present invention, which are defined in the claims. It is clear that they are contemplated within the scope of the present invention. It will be readily understood by those skilled in the art that the various features described and described in the present invention can be further modified by introducing the features shown in any one of the conditions set forth in the present invention.

Hereinafter, the present invention will be described in more detail through specific experimental examples. The following experimental examples are only examples to help the understanding of the present invention, and the scope of the present invention is not limited thereto.

Test Example 1. Semi-IPN structure analysis data of hybrid gel (FTIR-ATR)

This test is data confirming the chemical shift of fibroin before and after annealing of the hybrid gel.

As shown in amide I, amide II and amide III, amide I oscillations indicate CO stretching, whereas amide II oscillations indicate bending of NH bonds. CN stretching (Franks, 1993) is associated with amide III oscillations. FTIR-ATR spectra before (FIG. 2a) and after (FIG. 2b) chemical shift were respectively shown for the combination of NH strain and CN stretching vibration (Magnani et al., 1991). Without annealing, amide Ⅱ appeared at 1527 cm ^-1 and amide Ⅰ appeared at 1622 cm ^-1 . After annealing, the absorption bands were displayed at 1626 cm ^-1 , 1700 cm ^-1 for amide I and 1515 cm ^-1 for amide II.

These results showed that the beta-sheet form in the form of random coils and alpha-helix after annealing treatment in the secondary structure of silk fibroin (data from literature analysis: Franks, 1993; Freddi et al., 1999; Um et al., 2001; Chen et al. ., 2001a; Cardenas et al., 2002; Li et al., 2002). In addition, the band at 1700 cm ^-1 can be predicted to show the fibroin arrangement in the antiparallel beta-sheet region.

That is, in conclusion, chemical shift does not occur in the fibroin used herein in the hybrid gel before annealing, but chemical shift occurs after the annealing process, so that high-density hyaluronic acid and fibroin form a semi-IPN structure through spectroscopic analysis. Confirmed.

Test Example 2. Structural analysis data (13C-NMR) of the silk peptide before and after annealing of the hybrid gel.

In this test, as data analyzing the stability of the silk peptide used herein, the structural similarity to the silk fibroin presented herein was confirmed through 13C NMR analysis.

In the sample before annealing of the silk protein composition used in the present invention, as a result of spectroscopic analysis, Gly(Cα) 44.6ppm, Gly(C=O) 169.5ppm, Ala(Cα)49.8ppm, Ala(Cβ) 21.4ppm, Ala(C= O) A peak was observed at 173.0 ppm, and in the composition after annealing, Gly (Cα) 45.6 ppm, Gly (C=O) 170.4 ppm, Ala (Cα) 50.7 ppm, Ala (Cβ) 22.1 ppm, Ala (C =O) A peak was observed at 173.9 ppm.

In conclusion, it is found that the peak caused by the Ala Beta carbon, which is characteristic of the structure of silk Ⅱ, appears around 20 ppm, so that chemical shift occurs before and after annealing in the present invention, which is found to indicate structurally semi-IPN.

That is, it was confirmed through 13C-NMR that chemical shift occurred by annealing without chemical bonding with high-density hyaluronic acid.

Test Example 3. Semi-IPN structure analysis data of hybrid gel (1H-NMR)

In this test, it was confirmed through nuclear magnetic resonance analysis (1H-NMR) that cross-linking of hyaluron inside the hybrid gel proceeds without fibroin interference.

In general, when hyaluron is cross-linked, specific peaks are detected in A and B in FIG. 17 .

In 1H-NMR analysis of the annealed hybrid gel prepared herein, the N-acetyl group (A) of hyaluronic acid was detected at 1.66 ppm, and the alkyl group of BDDE was detected at 2.05 ppm.

Therefore, through Test Example 1, Test Example 2, and Test Example 3, it was verified that the hybrid gel prepared in the present application had a semi-IPN structure.

Test Example 4. Measurement of molecular weight distribution of fibroin

To measure the molecular weight distribution, a 4M silk aqueous solution was prepared by adding a lyophilized specimen to urea, and then measured by Fast Protein Liquid Chromatography (FPLC) (AKTA Purifier, Amersham Biosciences). The column superdex 200 10/300 was used, and the flow rate was 0.5 ml/min.

As shown in FIG. 13 , the fibroin sample in an aqueous solution state showed a sharp peak at 10 min, which corresponds to the upper fractionation limit of molecular weight, and a broad peak in a wide range from 15 min to 30 min in the chromatogram. That is, it was confirmed that fibroin in aqueous solution has a wide range of molecular weight distribution from a heavy chain peptide of about 350 kDa to 30 kDa or less, the fractionation limit of the column.

Fibroin in an aqueous solution forms an emulsion and forms a micelle structure with a diameter of 100 to 200 nm to form a molecule of a macroprotein [Marsh, R. E., R. B. Corey, and L. Pauling (1995) An investigation of the stucture of silk fibroin. Biochim. Biophys. Acta. 16: 1-34] [Lee, K-G., Y.-W. Lee, J.-H. Yeo, J. Nam, H.-Y. Kweon, and Y.-H. Park (1999) Structural characteristics of silk fibroin gel on the preparation conditions.].

The molecular weight of the heavy chain calculated from the gene sequence was 340 kDa. Therefore, based on the above analysis data, it was confirmed that fibroin was more stabilized by inhibiting the formation of a semi-solid gel in a dispersed system that forms a three-dimensional network that is connected to each other while suppressing the formation of protein colloids by aggregating existing molecular chains. .

Test Example 5. pH stability test during the process

This test is a test on the stability of fibroin by pH during the process.

The hybrid gel used in this test was prepared and used within the range presented herein.

0.43 g of hyaluronic acid having a molecular weight of 100 kDa, 2 g of hyaluronic acid having a molecular weight of 3000 kD, and 50ul of BDDE are mixed. After that, 30 weight percent of the fibroin used herein is added relative to the total weight of hyaluron to confirm the cohesiveness of fibroin at each pH (pH 2.5, 3.0, 3.5, 4.0).

As shown in FIG. 14 , as the pH is lowered to 4 or less, aggregation may occur and partial gelation may occur in the process.

Therefore, it was confirmed by FTIR that it is possible to proceed with the primary cross-linking reaction by maintaining the pH of 12 to 14 in the primary process.

Test Example 6. Decomposition experiment of hybrid gel cross-linked with hyaluron of different molecular weight

This test measured the reduction rate of the hybrid gel according to the molecular weight of hyaluron.

The purpose of this experiment is to cross-link hyaluron of different molecular weights and decompose them in PBS at 37°C.

In this test, a hybrid gel prepared within the scope of the present application was prepared as follows.

Preparation I in Fig. 6

0.43 g of hyaluronic acid having a molecular weight of 100 kDa, 2 g of hyaluronic acid having a molecular weight of 0.6 X 10 ⁶ Da, and 50ul of BDDE are mixed and cross-linked. Thereafter, 0.1 wt% of fibroin used based on the total weight of the hybrid gel composition is added to perform secondary crosslinking. Conditions for primary crosslinking are maintained at 288K, pH 13.5, and crosslinking time of 30 hours. The secondary annealing process was maintained at 1.5 atm, 380 K, and 30 minutes to prepare a final hybrid gel.

Preparation II of Fig. 6

0.43 g of hyaluronic acid having a molecular weight of 100 kDa, 2 g of hyaluronic acid having a molecular weight of 1.6 (M _w ) X 10 ⁶ Da, and 50ul of BDDE are mixed and cross-linked. Thereafter, 0.1 wt% of fibroin used based on the total weight of the hybrid gel composition is added to perform secondary crosslinking. Conditions for primary crosslinking are maintained at 288K, pH 13.5, and crosslinking time of 30 hours. The secondary annealing process was maintained at 1.5 atm, 380 K, and 30 minutes to prepare a final hybrid gel.

Ⅲ Preparation of Figure 6

0.43 g of hyaluronic acid having a molecular weight of 100 kDa, 2 g of hyaluronic acid having a molecular weight of 2.6 (M _w ) X 10 ⁶ Da, and 50ul of BDDE are mixed and cross-linked. Thereafter, 0.1 wt% of fibroin used based on the total weight of the hybrid gel composition is added to perform secondary crosslinking. Conditions for primary crosslinking are maintained at 288K, pH 13.5, and crosslinking time of 30 hours. The secondary annealing process was maintained at 1.5 atm, 380 K, and 30 minutes to prepare a final hybrid gel.

As shown in FIG. 6 , the weight reduction rate of the three types of hybrid gels by 30 days was about 20%, and showed the same decomposition behavior as in Test Example 9.

Test Example 7. Morphology analysis according to the fibroin content

This test is the morphology data of the hybrid gel according to the content of fibroin within the range presented herein.

0.43 g of hyaluronic acid having a molecular weight of 100 kDa, 2 g of hyaluronic acid having a molecular weight of 3000 kD, and 50ul of BDDE are mixed and cross-linked. Thereafter, 0.1% by weight, 10% by weight, 20% by weight, 30% by weight, and 100% by weight of the fibroin used in the present application based on the total weight of the hybrid gel composition is added to perform secondary crosslinking. Conditions for primary crosslinking are maintained at 288K, pH 13.5, and crosslinking time of 30 hours. The secondary annealing process was maintained at 1.5 atm, 380 K, for 30 minutes to prepare a final hybrid gel, and then the morphology was measured in the aqueous phase through SEM.

The test results were as shown in FIG. 4 . Figure 4a is SEM data of the hydrogel containing 0.1 wt% fibroin. Figure 4b is SEM data of the hydrogel containing 10 wt% fibroin. Figure 4c is the SEM data of the hydrogel containing 20% by weight fibroin. Figure 4d is the SEM data of the hydrogel containing 30% by weight fibroin. Figure 4e is the SEM data of the hydrogel containing 100% by weight fibroin.

As shown in FIG. 4, as the fibroin content increases, the gel density and the network structure of the hydrogel are very closely connected, but when it contains 30% by weight or more, the morphological characteristics of fibroin are exhibited.

Test Example 8. Evaluation of swelling degree

Two different types of hydrogels within the range presented herein were freeze-dried and swollen at 37°C for 24 hours. (%) and sate content (%) are measured data.

[Equation 6]

Swelling ratio (%) = (Ws - Wd)/Wd X 100

[Equation 7]

Sater content(%) = (Ws - Wd)/Ws X 100

Ws: Specimen weight after swelling, Wd: Specimen weight before swelling

However, the products of other companies used for comparison in this test are not absolute evaluation data, but were used as data for self-comparison experiments.

Sample 1: Galderma's Restylane

Sample 2: Allergan's Juvederm

Sample 3: First cross-linking by mixing 0.43 g of hyaluronic acid with a molecular weight of 100 kDa, 2 g of hyaluronic acid with a molecular weight of 3 000 kD, and 50ul of BDDE. Thereafter, 0.1% by weight of the fibroin used herein is added to the hybrid gel composition at an amount of 2% by weight based on the total weight of the hybrid gel composition. Conditions for primary crosslinking are maintained at 288K, pH 13.5, and crosslinking time of 30 hours. The secondary annealing process was maintained at 1.5 atm, 380 K, and 30 minutes to prepare a final hybrid gel.

Sample 4: First cross-linking by mixing 0.43 g of hyaluronic acid with a molecular weight of 100 kDa, 2 g of hyaluronic acid with a molecular weight of 3 000 kD, and 50ul of BDDE. Thereafter, 30% by weight of the fibroin used herein is added to the hybrid gel composition to perform secondary crosslinking. Conditions for primary crosslinking are maintained at 288K, pH 13.5, and crosslinking time of 30 hours. The secondary annealing process was maintained at 1.5 atm, 380 K, and 30 minutes to prepare a final hybrid gel.

The swelling degree evaluation results are shown in Table 2 below.

Sample 1 Sample 2 Sample 3 Sample 4 Swelling ratio (%) 49.51 47.31 44.84 46.48 Sater content (%) 97.12 96.51 97.81 97.85

Test Example 9. Enzymatic degradation behavior evaluation test of hybrid gel

To analyze the degradation behavior by the enzyme, a hyaluronidase solution at a concentration of 10 u/ml was prepared in PBS, and the mass change of the hydrogel was measured in 10 ml of hyaluronidase solution per 1 g of the hybrid gel, and the result is shown in FIG. it was

10A shows a low molecular weight (600 kDa) to a high molecular weight (2 000 kDa) mixed weight ratio of 1 to 4.65, and the BDDE used for the low molecular weight was 40ul compared to 1g of low molecular weight hyaluronic acid, and high molecular weight hyaluronic acid BDDE used for ronic acid was used in 100ul of high molecular weight hyaluronic acid compared to 1g. And fibroin was used in 30% by weight of the hydrogel solids (hyaluronic acid, fibroin). The reaction conditions were pH 13, 300K, cross-linking and neutralization for 40 hours, and then a hybrid gel prepared by annealing at 1.5atm, 380K, and 10 minutes.

B of FIG. 10 shows that the mixed weight ratio of low molecular weight (600 kDa) to high molecular weight (2 000 kDa) is 1: 4.65, and the BDDE used for low molecular weight is 19ul compared to 1g of low molecular weight hyaluronic acid, and high molecular weight hyaluronic acid BDDE used for ronic acid was used in 25ul of high molecular weight hyaluronic acid compared to 1g. And fibroin was used in 10% by weight of the hydrogel solids (hyaluronic acid, fibroin). The reaction conditions were a hybrid gel prepared by annealing at 1.5atm, 380K, and 10 minutes after crosslinking and neutralization at pH 13, 280K, 40 hours.

In FIG. 10C, a mixed weight ratio of low molecular weight (600 kDa) to high molecular weight (2 000 kDa) was used in a ratio of 1: 4.65, and the BDDE used for low molecular weight was 19ul compared to 1g of low molecular weight hyaluronic acid, and high molecular weight hyaluronic acid BDDE used for ronic acid was used in 25ul of high molecular weight hyaluronic acid compared to 1g. And fibroin was used 0.1% by weight of the hydrogel solids (hyaluronic acid, fibroin). The reaction conditions were a hybrid gel prepared by annealing at 1.5atm, 380K, and 10 minutes after crosslinking and neutralization at pH 13, 280K, 40 hours.

As a result of the experiment, almost all of C was decomposed within 40 hours, whereas A and B lasted 2 to 5 times longer. As a result, A and B, which have high crosslinking efficiency and high elastic modulus, have high resistance to decomposition because they contain a lot of crosslinked hyaluronic acid, and A and B with low swelling degree have a small amount of permeation of body fluids, so the penetration of enzymes is also less. It can be seen that the decomposition rate is slow. However, in the case of A, the crosslinking agent content is 140ul per gram of hyaluronic acid, the reaction temperature is above the above range (300K), and the fibroin content is 30% by weight. The hybrid (intermediate form between mono and biphasic) implemented in this application could not be implemented. In addition, structurally, a semi-IPN network was formed, indicating relatively resistance to degrading enzymes.

Test Example 10. Cell viability test in vitro

In this test, chondrocytes were evaluated on days 1, 3, and 7 using the L/D (live/dead) analysis technique in which chondrocytes were stained with calcein-AM (Ca-AM) and dead cells.

The sample was prepared by freeze-drying a hybrid gel to prepare a gel disk (height 2mm, diameter 4mm), washed and swollen with PBS, and then incubated in an incubator at 17° C. and 5% CO2 conditions in a darkened state. And it was measured through the LSM510 laser scanning microscope.

The hybrid gel used in this test had a low molecular weight (600 kDa) to high molecular weight (2000 kDa) mixed weight ratio of 1 to 4.65. 280K, conditions for 5 hours, BDDE used for high molecular weight hyaluronic acid was used in 25ul compared to 1g of high molecular weight hyaluronic acid. In addition, as for the added fibroin, 5 mol% of serine, 48 mol% of glycine, and 32 mol% of alanine were added to 12% by weight of the total fibroin hydrogel solids (hyaluronic acid + fibroin). And after crosslinking and neutralization at pH 13, 280K, 40 hours, 1.5atm, 380K, 10 minutes annealing process, freeze-dried at -350K or less, -320K was prepared by freeze-drying for 48 hours.

The experimental results were as shown in FIG. 9 . It is the result of measuring the cell viability of the hybrid gel prepared in the present invention in vitro. (a : 1 day, b : 3 days, c : 7 days)

The results showed that the chondrocyte viability of the chondrogenic medium in the cell-containing hydrogel decreased slightly from 93% at day 1 to 86% at day 7. This phenomenon is expected to be the effect of TGF-β1 on chondrocyte viability in the hydrogel, and it can be seen that the proportion of viable cells is not affected by the cell culture medium composition and culture period. In addition, it was confirmed through Fig. 9 that chondrocytes in the hybrid gel looked elongated, resembling a fibroblast-like morphology, which could indicate the onset of chondrocyte dedifferentiation.

Test Example 11. Stability test of hybrid gel (XRD)

After preparing a hybrid gel, it was lyophilized and XRD was measured. In Figure 7, Figure 7a is data for confirming the internal crystallinity of fibroin using a general extraction method. 7b is data for confirming the crystallinity inside the hybrid gel measured by giving an annealing effect after blending the fibroin and high-density hyaluron gel using a general extraction method. Figure 7c is data for confirming the crystallinity inside the hybrid gel after imparting an annealing effect during the process using the fibroin disclosed herein. As the fibroin, fibroin containing 5 mol% of serine, 48 mol% of glycine, and 32 mol% of alanine among fibroin molar (mol) concentrations was used.

As shown in FIGS. 2A and 7A , before the hybrid gel was given an annealing effect, it is structurally close to amorphous, and a wide amorphous halo centered at about 22° is characteristic. And the weak diffraction peak at 28.3° indicates the structure of silk I induced by hyaluron.

After the annealing effect was applied, a structural transition to a weakly insoluble crystalline structure appeared as shown in FIGS. 2b, 7b, and 7c, and a specific silk II peak at 20.2° and a silk I peak at 24.5° were characteristically.

As shown in FIGS. 1A and 1B , absorption peaks according to the amorphous structure of pure silk before annealing are shown.

And, as shown in Figs. 2a and 2b, after imparting the annealing effect of the hybrid gel, a structural absorption peak suggesting that it is typical silk II was shown.

Therefore, based on the FT-IR ATR and XRD results, it was confirmed that the hyaluronic acid-silk complex mainly had a water-stable crystal structure after the annealing effect was imparted to the hybrid gel of the present invention.

Above, the test examples of the present invention have been described, but those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. . Therefore, it should be understood that the test examples described above are illustrative in all respects and not restrictive.

Claims (16)

In the method for preparing a hyaluronic acid-based hydrogel composition, the method comprises:
a) preparing a low molecular weight hyaluronic acid composition having an average molecular weight of 10 kDa to 1500 kDa;
b) preparing a high molecular weight hyaluronic acid composition having an average molecular weight of 1600 kDa to 4500 kDa;
c) preparing a fibroin composition in which the contents of glycine, serine and alanine are changed;
d) chemically primary crosslinking the low molecular weight hyaluronic acid composition of a) and the high molecular weight hyaluronic acid composition of b); and
e) forming a semi-IPN structure by blending the random copolymer generated in step d) with the fibroin composition of step c);
includes,
The fibroin composition of step c) includes serine, glycine and alanine, and serine is 40 mol% with respect to fibroin containing 10 to 14 mol% of serine, 42 to 47 mol% of glycine, and 27 to 32 mol% of alanine is decreased to less than or equal to, glycine is increased by 5 mol% or more, and alanine is increased by 5 mol% or more, and the weight ratio of glycine (β) to glycine (α) is 0.1: 99.9 to 10: 90, and the alanine The weight ratio of silver alanine (β) and alanine (α) ranges from 0.1: 99.9 to 10: 90, and the mol ratio of heavy chain and light chain in fibroin is 0.1: 99.9 to 10: 90,
In step d), the pH 12 to 14, the reaction temperature of 278K to 298K, the reaction time of 24 hours to 50 hours, the content of hyaluronic acid in the solid content of the total composition is 10 to 20% by weight,
In step e), a semi-IPN structure is formed by giving a physical annealing effect under a pressure condition of 0.1atm to 2atm, a temperature condition of 370K to 400K, and a reaction condition of 10 minutes to 100 minutes,
A method for preparing a hyaluron-based hydrogel composition.
The method of claim 1,
The average molecular weight of the hyaluronic acid in step a) is 30 kDa to 1,000 kDa, the method for producing a hyaluronic-based hydrogel composition.
3. The method of claim 2,
The low molecular weight hyaluronic acid in step a) is prepared by adding a crosslinking agent in an amount of 15 μl to 35 μl per gram of hyaluronic acid input, and reacting for a time of 4 hours to 10 hours at a temperature of pH 12 to pH 14, 278K to 298K, and 4 hours to 10 hours. The method for producing a hyaluron-based hydrogel composition to be.
4. The method of claim 3,
The crosslinking agent is 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether, BDDE), a method of producing a hydrogel composition.
The method of claim 1,
The average molecular weight of the hyaluronic acid in step b) is 2,000 kDa to 4,000 kDa, a method for producing a hyaluronic-based hydrogel composition.
6. The method of claim 5,
The high molecular weight hyaluronic acid in step b) is prepared by adding a crosslinking agent in an amount of 15 ul to 75 ul per gram of hyaluronic acid input, and reacting for 24 hours to 50 hours under a temperature condition of pH 12 to pH 14, 278K to 298K, and 24 hours to 50 hours A method for preparing a phosphorus, hyaluron-based hydrogel composition.
7. The method of claim 6,
The crosslinking agent is 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether, BDDE), a method of producing a hyaluron-based hydrogel composition.
delete The method of claim 1,
The fibroin composition of step c) is,
An average molecular weight of 100 kDa or less,
The content of glycine is 40 to 50% by weight based on the total weight of the fibroin composition,
The content of alanine is 25 to 35% by weight based on the total weight of the fibroin composition,
A method for preparing a hyaluron-based hydrogel composition.
The method of claim 1,
The input amount of the fibroin composition is 0.1: 9.9 to 3: 7 compared to hyaluron, the method for producing a hyaluron-based hydrogel composition is added in a weight ratio of 7.
delete delete A hydrogel composition prepared according to any one of claims 1 to 7 and 9 to 10.
14. The method of claim 13,
The composition is used as a soft tissue filler, a hydrogel composition.
14. The method of claim 13,
The composition is used as a cartilage filler, a hydrogel composition.
14. The method of claim 13,
The composition further comprises lidocaine or a derivative thereof, a hydrogel composition.

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