Method for producing self-crosslinked hyaluronic acid gel and product thereof

Abstract

To provide a method for manufacturing an auto-crosslinked hyaluronic acid gel.SOLUTION: The present disclosure provides a method comprising the steps for conducting auto-crosslinking reaction of a colloid containing hyaluronic acid continuously at low temperature in an acidic environment, and treating the reaction product with steam at high temperature to obtain the auto-crosslinked hyaluronic acid gel with high viscosity.SELECTED DRAWING: None

Description

[Technical field]

[0001] Detailed Description of the Invention The present disclosure relates to a method for producing hyaluronic acid, and in particular a self-crosslinked hyaluronic acid gel, and the product thereof. [Background technology]

[0002] Hyaluronic acid has a molecular weight of about 400 D and is a linear polysaccharide formed from disaccharide units composed of β-1,3-N-acetylglucosamine and β-1,4-glucuronic acid linked via alternating β-1-4 and β-1-3 glycosidic bonds. Commercial hyaluronic acid can be extracted from fermentation of bacteria, such as Streptococcus, or from animal tissues, such as cockscomb. Linear hyaluronic acid is rapidly degraded in vivo by certain enzymes (e.g., hyaluronidase) or free radical reactions, and therefore its half-life is shortened in vivo. Furthermore, the applications of linear hyaluronic acid are limited due to its lack of mechanical strength. Therefore, hyaluronic acid is often cross-linked for practical applications.

[0003] Self-crosslinked hyaluronic acid is an ester produced by esterification of intermolecular or intramolecular hydroxyl groups (-OH) and carboxyl groups (-COOH) of hyaluronic acid in the presence of an activator or catalyst. Since no crosslinking agent is used, no molecular fragments of crosslinking groups are formed on the resulting self-crosslinked hyaluronic acid, which therefore can maintain excellent biocompatibility and viscoelasticity, and is widely used in various medical applications. For example, self-crosslinked hyaluronic acid can be applied to treat arthropathy (e.g., as disclosed in US 6,251,876), to prevent postoperative adhesions (e.g., as disclosed in US 7,504,386), or as a subdermal filler (e.g., as disclosed in Andrea Alessandrini et al., Plastic and Reconstructive Surgery, Vol. 118, pp. 341-346 (2006)). Self-crosslinked hyaluronic acid is also widely used in tissue engineering applications. Self-crosslinking hyaluronic acid-based post-surgical adhesion prevention gels are commercially available as Hyalobarrier® and HYALOGLIDE®. Summary of the Invention [Problem to be solved by the invention]

[0004] To address one or more of the aforementioned problems, the present disclosure provides a low temperature self-crosslinking reaction process in the presence of acid to produce a high viscosity self-crosslinked hyaluronic acid gel that is steamed. [Means for solving the problem]

[0005] The present disclosure provides a method for producing an autocrosslinked hyaluronic acid gel, comprising: (a) preparing a colloid containing hyaluronic acid or a metal salt thereof; (b) carrying out the self-crosslinking reaction of the colloid containing hyaluronic acid or a metal salt thereof in an acidic environment at about -10°C to about -30°C for about 7 to about 42 days; and (c) treating the product of step (b) with steam to obtain a self-crosslinked hyaluronic acid gel.

[0006] Examples of salts of hyaluronic acid can be alkali metal salts, including but not limited to potassium, sodium, zinc or lithium salts of hyaluronic acid. For example, the metal salt of hyaluronic acid can be sodium hyaluronate.

[0007] In some embodiments of the present disclosure, the methods do not include the use of any cross-linking agents.

[0008] In some embodiments of the present disclosure, step (a) comprises providing a colloid containing hyaluronic acid or a metal salt thereof in the form of granules, stripes or strings.

[0009] In some embodiments of the present disclosure, in step (b), the acidic environment is provided with an acid at a concentration of about 0.5 N to about 1.5 N, such as about 0.51 N to about 1.4 N, about 0.52 N to about 1.3 N, about 0.53 N to about 1.2 N, about 0.54 N to about 1.1 N, about 0.55 N to about 1.0 N, about 0.56 N to about 0.9 N, about 0.57 N to about 0.8 N, about 0.58 N to about 0.7 N, or about 0.59 N to about 0.6 N. In some embodiments of the present disclosure, the acid is hydrochloric acid, nitric acid, and / or sulfuric acid.

[0010] In some embodiments of the present disclosure, step (b) comprises mixing hyaluronic acid or a colloid containing a metal salt thereof with an acid at room temperature to form a mixture, and allowing the mixture to stand at about -10°C to about -30°C for about 7 days to about 42 days. For example, the mixture is allowed to stand at about -12°C to about -28°C, about -14°C to about -26°C, about -16°C to about -24°C, about -18°C to about -22°C, about -10°C, about -20°C, or about -30°C; for about 9 to about 40 days, about 11 to about 40 days, about 13 to about 38 days, about 15 to about 36 days, about 17 to about 34 days, about 19 to about 32 days, about 21 to about 30 days, about 23 to about 28 days, about 25 to about 27 days, about 7 days, about 14 days, about 21 days, or about 28 days. In some embodiments of the present disclosure, the concentration of hyaluronic acid or a metal salt thereof in the mixture is from about 10 wt% to about 30 wt%, e.g., from about 11 wt% to about 28 wt%, from about 12 wt% to about 26 wt%, from about 13 wt% to about 24 wt%, from about 14 wt% to about 22 wt%, from about 15 wt% to about 20 wt%, from about 16 wt% to about 18 wt%, from about 10 wt% to about 20 wt%, or from about 10 wt% to about 14.5 wt%.

[0011] In some embodiments of the present disclosure, in step (b), The acidic environment is provided by an acid having a concentration of about 0.58N to about 1.0N, and the self-crosslinking reaction is carried out at about -10°C for about 7 to about 42 days. The acidic environment is provided by an acid having a concentration of about 0.58N to about 1.5N, and the self-crosslinking reaction is carried out at about -20°C for about 7 to about 35 days. The acidic environment is provided by an acid having a concentration of about 0.58N to about 1.0N, and the self-crosslinking reaction is carried out at about -20°C for about 7 to about 28 days. The acidic environment is provided by an acid having a concentration of about 1.0N to about 1.5N, and the self-crosslinking reaction is carried out at about -30°C for about 14 to about 35 days; or The acidic environment is provided with an acid at a concentration of about 0.58N to about 1.0N, and the self-crosslinking reaction is carried out at about -30°C for about 14 to about 35 days.

[0012] In some embodiments of the present disclosure, the method includes performing the self-crosslinking reaction only once, and the entire self-crosslinking reaction is performed continuously at about -10°C to about -30°C.

[0013] In some embodiments of the present disclosure, prior to step (c), the method further comprises homogenizing and / or washing the product of step (b).

[0014] In some embodiments of the present disclosure, the product of step (b) has an equilibrium swelling capacity of about 12 to about 30.

[0015] In some embodiments of the present disclosure, prior to step (c), the method further comprises adjusting the pH of the product of step (b) to about 6.0 to about 7.0. In some embodiments of the present disclosure, prior to step (c), the method further comprises adjusting the pH of the product of step (b) to about 6.3 to about 6.8. In some embodiments of the present disclosure, the step of adjusting the pH is after the homogenizing and / or washing steps.

[0016] The temperature and time of steam treatment are not limited in the present disclosure, provided that the self-crosslinked hyaluronic acid gel of the present disclosure can be correctly produced. In some embodiments of the present disclosure, step (c) comprises treating the product of step (b) with steam at about 110°C to about 130°C for about 10 minutes to about 60 minutes. For example, the temperature of the steam used in step (c) is about 112°C to about 128°C, about 114°C to about 126°C, about 116°C to about 124°C, about 118°C to about 122°C, or about 121°C.

[0017] In some embodiments of the present disclosure, the method is for producing an auto-crosslinked hyaluronic acid gel, wherein the auto-crosslinked hyaluronic acid gel exhibits at least one of the following: The tan δ of the self-crosslinked hyaluronic acid gel at a concentration of about 1.5 wt % to about 7.5 wt %, measured at 25° C. and at each and every frequency from 0.1 Hz to 10.0 Hz, is less than or equal to 1; The elastic modulus of the self-crosslinked hyaluronic acid gel at a concentration of about 1.5 wt % to about 7.5 wt %, measured at 25° C. and 1.0 Hz, is about 30 Pa to about 8000 Pa; The viscosity of the self-crosslinked hyaluronic acid gel at a concentration of about 1.5 wt % to about 7.5 wt %, measured at 25° C. and 1.0 Hz, is about 20 Pa to about 2000 Pa; or At 25℃, 1sec -1 The shear viscosity of the self-crosslinked hyaluronic acid gel at a concentration of about 1.5 wt% to about 7.5 wt% is about 20 Pa·s to about 900 Pa·s, measured at a shear rate of 1.5 wt%. In some embodiments of the present disclosure, the aforementioned concentration refers to the concentration of hyaluronic acid of a salt thereof in the self-crosslinked hyaluronic acid gel.

[0018] The present disclosure further provides an auto-crosslinked hyaluronic acid gel, wherein the tan δ of the auto-crosslinked hyaluronic acid gel at a concentration of about 1.5 wt% to about 7.5 wt% measured at 25°C and at each and every frequency from 0.1 Hz to 10.0 Hz is less than or equal to 1, and the auto-crosslinked hyaluronic acid gel is sterile.

[0019] In some embodiments of the present disclosure, the elastic modulus of the self-crosslinked hyaluronic acid gel at a concentration of about 1.5 wt % to about 7.5 wt % is about 30 Pa to about 8000 Pa measured at 25° C. and 1.0 Hz.

[0020] In some embodiments of the present disclosure, the viscosity of the self-crosslinked hyaluronic acid gel at a concentration of about 1.5 wt % to about 7.5 wt % is about 20 Pa to about 2000 Pa measured at 25° C. and 1.0 Hz.

[0021] In some embodiments of the present disclosure, the method is carried out at 25° C. and for 1 sec. -1 The shear viscosity of the self-crosslinked hyaluronic acid gel at a concentration of about 1.5 wt % to about 7.5 wt % measured at a shear rate of about 20 Pa·s to about 900 Pa·s, about 38.04 Pa·s to about 896.91 Pa·s, or about 55.69 Pa·s to about 896.91 Pa·s.

[0022] In some embodiments of the present disclosure, the method is carried out at 25° C. and for 50 sec. -1 The shear viscosity of the self-crosslinked hyaluronic acid gel at a concentration of about 1.5 wt % to about 7.5 wt % measured at a shear rate of about 0.31 Pa·s to about 35.66 Pa·s, about 1.97 Pa·s to about 35.66 Pa·s, or about 2.18 Pa·s to about 35.66 Pa·s.

[0023] The present disclosure further provides a method for promoting bone regeneration, treating bone defects, preventing post-surgical adhesions, subcutaneous filling, and / or treating arthropathy in a subject in need thereof, comprising administering to a subject in need thereof an autocrosslinking hyaluronic acid gel. [Brief description of the drawings]

[0024] [Figure 1] 1 is a diagram showing a comparison of the shear viscosity of self-crosslinked hyaluronic acid particles (before steam treatment) and self-crosslinked hyaluronic acid gel (after steam treatment). Self-crosslinked hyaluronic acid particles are prepared by the self-crosslinking reaction of hyaluronic acid or its salt-containing colloid (hyaluronic acid concentration 14.5 wt%) in the presence of 1.0N HCl at -10°C for 14 days. The pH of self-crosslinked hyaluronic acid particles (before steam treatment) is 6.5, and the self-crosslinked hyaluronic acid particles are treated with steam at 121°C for 15 minutes. [Diagram 2] Figure 1 shows the elastic modulus (G') and viscous modulus (G'') of self-crosslinked hyaluronic acid gel versus frequency of measurement. Self-crosslinked hyaluronic acid particles are prepared by the self-crosslinking reaction of hyaluronic acid or its salt-containing colloid (hyaluronic acid concentration 14.5 wt%) in the presence of 1.5N HCl at -30°C for 14 days. The pH of the self-crosslinked hyaluronic acid particles (before steam treatment) is 6.5, and the self-crosslinked hyaluronic acid particles are treated with steam at 121°C for 15 minutes. [Diagram 3]1 shows the shear viscosity vs. shear rate of the self-crosslinked hyaluronic acid gel. The self-crosslinked hyaluronic acid particles are prepared by the self-crosslinking reaction of hyaluronic acid or its salt-containing colloid (hyaluronic acid concentration 14.5 wt%) in the presence of 1.5N HCl at -30°C for 14 days. The pH of the self-crosslinked hyaluronic acid particles (before steam treatment) is 6.5, and the self-crosslinked hyaluronic acid particles are treated with steam at 121°C for 15 minutes. [Figure 4] 1 shows the effect of pH on the elastic modulus of self-crosslinked hyaluronic acid particles (before steam treatment) and self-crosslinked hyaluronic acid gel (after steam treatment). Self-crosslinked hyaluronic acid particles are prepared by the self-crosslinking reaction of hyaluronic acid or its salt-containing colloid (hyaluronic acid concentration 14.5 wt%) in the presence of 1.5N HCl at -20°C for 21 days. Self-crosslinked hyaluronic acid particles are treated with steam at 121°C for 20 minutes. [Diagram 5] Figure 1 shows the effect of pH on the viscosity of self-crosslinked hyaluronic acid particles (before steam treatment) and self-crosslinked hyaluronic acid gel (after steam treatment). Self-crosslinked hyaluronic acid particles are prepared by the self-crosslinking reaction of hyaluronic acid or its salt-containing colloid (hyaluronic acid concentration 14.5 wt%) in the presence of 1.5N HCl at -20°C for 21 days. Self-crosslinked hyaluronic acid particles are treated with steam at 121°C for 20 minutes. [Figure 6]FIG. 14 corresponds to Table 14 and shows the results of an experiment comparing the percentage of newly formed bone volume after cranial defect treatment with the disclosed self-crosslinking hyaluronic acid gel and a control group. No. 1: Blank group: After the skull defect surgery, the wound is directly covered with Bio-Gide membrane; No. 2: After the skull defect surgery, the gel of the control group is implanted in the wound and then covered with Bio-Gide membrane; No. 3: After the skull defect surgery, the gel of experimental group 1 is implanted in the wound and then covered with Bio-Gide membrane; No. 4: After the skull defect surgery, the gel of experimental group 2 is implanted in the wound and then covered with Bio-Gide membrane; No. 5: After the skull defect surgery, the gel of experimental group 3 is implanted in the wound and then covered with Bio-Gide membrane; No. 6: After the skull defect surgery, the gel of experimental group 4 is implanted in the wound and then covered with Bio-Gide membrane; No. 7: Positive control group: After the skull defect surgery, the wound is directly covered with CoreBone membrane. 1000 bone powder is implanted and then covered with Bio-Gide membrane. CoreBone 1000 used in the positive control group is a foreign commercial bone graft material in granular form, which is produced by the Israeli company CoreBone and certified by the European Union. CoreBone 1000 is a natural bone graft material and has the advantage of high biocompatibility. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] It is known that self-crosslinked hyaluronic acid can be obtained by a self-crosslinking reaction in the presence of an activator, 2-chloro-1-methylpyridinium iodide (CMPI) (disclosed in U.S. Pat. No. 5,676,964) or in the presence of an acid (e.g., nitric acid or hydrochloric acid) at low temperature (e.g., Japanese Patent No. 4460663), but both of these existing techniques have drawbacks.

[0026] The self-crosslinking transesterification of hyaluronic acid using CMPI as an activator is a complex reaction involving large amounts of organic solvents, ion exchange resins and toxic reagents. The reaction causes the conversion of sodium hyaluronate to tetramonium hyaluronate, which is soluble in polar organic solvents, using a column packed with the resin, where the resin must be treated with toxic TBAOH (tetrabutylammonium hydroxide). The resulting aqueous solution containing tetraammonium hyaluronate is freeze-dried and dissolved in an organic solvent, such as dimethyl sulfoxide (DMSO), and then CMPI is added to carry out the self-crosslinking reaction. During the reaction, triethylamine is added to neutralize the hydrochloric acid generated in the reaction, which reduces the pH of the solution. After the self-crosslinking reaction, an aqueous sodium chloride solution is added to the post-reaction mixture to remove the protecting groups, and then a solvent (e.g., ethanol) is used to precipitate the self-crosslinked hyaluronate, which is then dried to form the self-crosslinked hyaluronic acid. In the above process, when hyaluronic acid undergoes functional group substitution reaction in resin column, the concentration of hyaluronic acid during substitution is low (about 0.5wt% to 1.0wt% hyaluronic acid) due to the high viscosity of hyaluronic acid solution.Low concentration of hyaluronic acid produces a large volume of solution, which requires a large amount of solvent (e.g. ethanol) to precipitate.The tetraammonium salt of hyaluronate obtained by resin ion exchange has limited solubility in organic solvent, which also requires a large amount of organic solvent during esterification reaction, resulting in the problem of solvent recovery.

[0027] Another method for synthesizing self-crosslinked hyaluronic acid is carried out at low temperature and in the presence of acid without using organic solvents and toxic activators. The method is disclosed in Japanese Patent No. 4460663, and includes the steps of adjusting the pH of 1% hyaluronic acid solution to 2.0 or less with acidic solution (e.g., hydrochloric acid, nitric acid and sulfuric acid), placing the hyaluronic acid solution at -20°C for 120 hours, then washing the obtained product with a buffer solution of pH 7.0, and crushing the obtained product to obtain a hyaluronic acid gel. In the aforementioned washing step, soluble hyaluronic acid may be removed, and thus the viscosity of the obtained hyaluronic acid gel is relatively low, which is undesirable for practical medical use. Moreover, the gel obtained with such a low reaction concentration of hyaluronic acid cannot remain in a gel state after steam treatment or sterilization, making it difficult to maintain sterility for application.

[0028] In addition, when preparing a mixture containing high concentration of hyaluronic acid for reaction, if hyaluronic acid powder is directly mixed with a solution containing acid (e.g. sulfuric acid, hydrochloric acid, or nitric acid), the mixture tends to form aggregates containing hyaluronic acid powder inside. Such aggregates are difficult to disperse or dissolve by stirring, which prevents hyaluronic acid from being effectively mixed with acidic solution, or requires a long stirring time, which causes hydrolysis of hyaluronic acid. Therefore, the subsequent self-crosslinking reaction at low temperature cannot be carried out correctly, and a large amount of hyaluronic acid that does not self-crosslink or only self-crosslinks to a low degree may be lost in the subsequent washing step. Therefore, the self-crosslinking hyaluronic acid particles or gel with stable properties cannot be obtained through the above-mentioned method.

[0029] In order to improve the performance of the self-crosslinked hyaluronic acid gel as a biodegradable medical material, it is necessary to use hyaluronic acid with a high molecular weight and a high concentration (e.g., 10 wt% or more) in the reaction. However, the colloid of hyaluronic acid with a high molecular weight becomes very viscous as the concentration increases, so a long stirring time is required to properly mix the hyaluronic with the acid, which also results in hydrolysis of the hyaluronic acid. For example, Comparative Example 1 of Japanese Patent No. 4383035 states that 15 grams of nitric acid with a 1N concentration was placed in a mortar, and 6 grams of sodium hyaluronate powder with a molecular weight of 2 million Daltons was mixed with the nitric acid (the reaction concentration of sodium hyaluronate is 28.6 wt%), and the mixture was thoroughly kneaded until it was uniformly mixed. Since sodium hyaluronate immediately formed aggregates with nitric acid, it was difficult to mix the mixture uniformly even after about 30 minutes of continuous kneading. In the continuous kneading process, the molecular weight of hyaluronic acid was reduced by nitric acid, thus forming a liquid-like mixture. After a certain number of days of refrigeration and freezing at -20°C, when the frozen product was thawed in phosphate buffer, sodium hyaluronate was dissolved in phosphate buffer and could not even form hyaluronic acid gel. This patent proposes an improved method of mixing hyaluronic acid and acid solution by first freezing the acid solution into fine granular or pulverized form, and then mixing it with hyaluronic acid powder at a low temperature of 0°C to -20°C, so that the reaction concentration of hyaluronic acid is 10wt% or more. However, such a method requires the acid solution to be frozen into fine granular or pulverized form, and mixing to be carried out continuously at a low temperature. The method is complicated and unsuitable for industrial production.

[0030] U.S. Patent No. 9,216,193 discloses a self-crosslinked hyaluronic acid composition comprising self-crosslinked hyaluronic acid particles having an equilibrium swelling capacity of 3 to 10, a particle size of 10 μm to 100 μm, and a hyaluronic acid concentration of 3 wt% to 8 wt%. The self-crosslinked hyaluronic acid composition is -1400°C and 25±2°C, it has a shear viscosity of less than 300 mPa·s, which can improve the problem of excessive thrust in the production of high-concentration joint cavity injections. However, this patent follows the method used in Japanese Patent No. 4383035, in which the acidic solution is first frozen into a fine granular or pulverized form and then mixed with hyaluronic acid powder at -10°C. Therefore, this patent still has the disadvantage of mixing at low temperature, which is complicated and undesirable for industrial production. Meanwhile, the self-crosslinking hyaluronic acid composition obtained by this method is in a granular form with a viscosity of only 300 mPa·s or less, which is not suitable for medical products that require high viscosity, such as wound dressings or postoperative adhesion prevention products.

[0031] Other techniques for synthesizing self-crosslinked hyaluronic acid gels at low temperatures and in the presence of acid include US 6,387,413, US 6,790,461, US 6,635,267 and US 7,014,860, from which the gels obtained also show low viscosity after washing steps to remove the acid and neutralization steps. These patents also fail to demonstrate that the gels obtained can maintain the desired state and crosslinking properties required for medical applications after steam treatment or sterilization.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In case of conflict, the present document, including definitions, will control.

[0033] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise.

[0034] The term "or" as used in this specification and the appended claims means "and / or" unless the content clearly dictates otherwise. In the case of multiple dependent claims, the term "or" is intended to re-reference more than one of the preceding independent or dependent claims for replacement purposes only.

[0035] As used herein, the term "hyaluronic acid or metal salt colloid" refers to a colloid formed from a solid powder or solid wool-like material that swells by absorbing a liquid solvent, examples of which include, but are not limited to, distilled water, deionized water, or saline or buffer solutions.

[0036] The term "gel" as used herein refers to a material that has a storage or elastic modulus (G') equal to or greater than its loss or viscous modulus (G''). The storage or elastic modulus (G') and the loss or viscous modulus (G'') can be measured at a certain frequency by a rheometer (e.g., AR2000ex rheometer, TA instruments). For example, at 25°C and at each and every frequency from 0.1 to 10 Hz, the elastic modulus (G') of a "gel" can be equal to or greater than the viscous modulus (G'') and the loss tangent (tan δ, which is equal to viscous modulus G'' / elastic modulus G') is equal to or less than 1. In some embodiments of the present disclosure, the storage or elastic modulus (G') and the loss or viscous modulus (G'') can be equal to or greater than 1 when measured at 25°C and at each and every frequency from 0.1 to 10 Hz. -1 The shear viscosity of the “gel” measured at a shear rate of at least 20 Pa·s.

[0037] The present disclosure provides a method for synthesizing an autocrosslinked hyaluronic acid gel at low temperature and in the presence of an acid, which can be steamed at high temperature. The obtained autocrosslinked hyaluronic acid gel has high viscosity and is sterile, which is suitable for medical applications such as adhesion prevention, bone regeneration and repair, subcutaneous filler (or dermal filler), joint disease treatment, etc. Specifically, the present disclosure provides a method for producing an autocrosslinked hyaluronic acid gel, comprising: (a) preparing a colloid containing hyaluronic acid or a metal salt thereof; (b) carrying out the self-crosslinking reaction of the colloid containing hyaluronic acid or a metal salt thereof in an acidic environment at about -10°C to about -30°C for about 7 to about 42 days; and (c) treating the product of step (b) with steam to obtain a self-crosslinked hyaluronic acid gel.

[0038] In some embodiments of the present disclosure, the method for producing an autocrosslinked hyaluronic acid gel exhibits at least one of the following: The tan δ at 25°C and at a concentration of about 1.5 wt% to about 7.5 wt% measured at each and every frequency of 0.1 Hz to 10.0 Hz is 1 or less, the elastic modulus at 25°C and at a concentration of about 1.5 wt% to about 7.5 wt% measured at 1.0 Hz is about 30 Pa to about 8000 Pa, the viscosity at 25°C and at a concentration of about 1.5 wt% to about 7.5 wt% measured at 1.0 Hz is about 20 Pa to 2000 Pa, and the viscosity at 25°C and at a concentration of about 1.5 wt% to about 7.5 wt% measured at 1.0 Hz is about 1 sec -1 A shear viscosity of about 20 Pa·s to about 900 Pa·s at a concentration of about 1.5 wt % to about 7.5 wt % measured at a shear rate of about 100 Pa·s to about 900 Pa·s. In some embodiments of the present disclosure, the aforementioned concentrations refer to the concentration of hyaluronic acid of a salt thereof in the self-crosslinked hyaluronic acid gel.

[0039] In some embodiments of the present disclosure, prior to the self-crosslinking reaction, hyaluronic acid or its metal salt (which may be in powder or granular form) is immersed in or mixed with an aqueous solvent to absorb the aqueous solvent and thus form a hyaluronic acid or its metal salt-containing colloid with an approaching solidity. In some embodiments of the present disclosure, the aqueous solvent is distilled water or deionized water. Without intending to be bound by any theory, it is believed that the hyaluronic acid or its metal salt-containing powder absorbs the aqueous solvent and swells uniformly to form a colloid, so that the acid can be easily and thoroughly mixed with the hyaluronic acid or its metal salt-containing colloid by stirring. In some embodiments of the present disclosure, the hyaluronic acid or its metal salt-containing colloid can be mixed with the acid at room temperature without forming aggregates containing the hyaluronic acid powder, thereby overcoming the difficulties of aggregation and low-temperature mixing in the prior art. The mixture can be maintained at low temperature for a sufficient time to self-crosslink, and then can be optionally homogenized, washed, and neutralized. After removing aqueous solvent, self-crosslinked hyaluronic acid particles are obtained.Self-crosslinked hyaluronic acid particles can then be treated with high temperature steam to form sterile high viscosity self-crosslinked hyaluronic acid gel.The method disclosed herein improves the low viscosity of crosslinked hyaluronic acid produced by traditional low temperature synthesis, and can be used for medical applications such as postoperative adhesion prevention, bone regeneration and defect repair, dermal filler, and arthropathy treatment, etc.

[0040] In some embodiments of the present disclosure, the self-crosslinking of hyaluronic acid is an esterification reaction between the intermolecular or intramolecular hydroxyl group (-OH) and the carboxyl group (-COOH) of hyaluronic acid. The method of the present disclosure does not use a crosslinking agent and is achieved only through the self-crosslinking reaction mentioned.

[0041] The average molecular weight of hyaluronic acid or its metal salt according to the present disclosure is not particularly limited, provided that the hyaluronic acid or its metal salt can be used to form colloids and gels. In some embodiments of the present disclosure, the average molecular weight of hyaluronic acid or its metal salt is about 766,000 or more (intrinsic viscosity η=1.4 m 3 / kg; average molecular weight, Laurent's formula (molecular weight = {[η] × 10 5 / 3.6} 1 / 0.78 In some embodiments of the present disclosure, the average molecular weight of hyaluronic acid or a metal salt thereof is from about 983,000 to about 1,121,000 (about 1.7 m 3 / kg to about 2.0m 3 The intrinsic viscosity of hyaluronic acid or a metal salt thereof can be measured according to pharmacopoeias, for example those described in the European Pharmacopoeia 9.0.

[0042] In some embodiments of the present disclosure, step (a) includes providing hyaluronic acid or a colloid containing a metal salt thereof in the form of granules, stripes or strings. For example, the colloid containing hyaluronic acid or a colloid containing a metal salt thereof can be crushed into small pieces (e.g., granules, stripes or strings) before the self-crosslinking reaction in step (b). The method of crushing is not limited to the present disclosure. For example, the colloid containing hyaluronic acid or a colloid containing a metal salt thereof can be crushed, sieved or chopped through a syringe, a sieve or by a metal, e.g., a blade.

[0043] In some embodiments of the present disclosure, the reaction temperature in step (b) is from about -10°C to about -30°C, for example, from about -10°C to about -20°C.

[0044] In some embodiments of the present disclosure, the acidic environment can be provided with any type of acid at any concentration, provided that hyaluronic acid or its metal salt can sufficiently self-crosslink in the acidic environment to form a self-crosslinked hyaluronic acid gel. In some embodiments of the present disclosure, the acidic environment is provided with an acid at a concentration of about 0.5N to about 1.5N, the acid being selected from the group consisting of hydrochloric acid, nitric acid, and sulfuric acid.

[0045] In some embodiments of the present disclosure, step (b) includes mixing hyaluronic acid or a colloid containing a metal salt thereof with an acid at room temperature to form a mixture, and allowing the mixture to stand at about -10°C to about -30°C for about 7 to 42 days. The acid can be prepared as a solution (e.g., an aqueous solution) of any concentration before mixing, provided that the concentration of the acid in the mixture, for example, about 0.5N to 1.5N, can create a proper environment for the self-crosslinking reaction of hyaluronic acid. In some embodiments of the present disclosure, the mixture is prepared by mixing hyaluronic acid or a colloid containing a metal salt thereof with a sufficient amount of a 14N nitric acid solution, a 12N hydrochloric acid solution, or a 12N sulfuric acid solution. Alternatively, in some embodiments of the present disclosure, the mixture is prepared by mixing hyaluronic acid or a colloid containing a metal salt thereof with a sufficient amount of a hydrochloric acid solution, a nitric acid solution, or a sulfuric acid solution with a concentration of about 6N to about 12N.

[0046] In some embodiments of the present disclosure, hyaluronic acid or its metal salt reaches a predetermined concentration in the mixture. In some embodiments of the present disclosure, the concentration of hyaluronic acid or its metal salt in the mixture is about 10 wt% to about 30 wt%, for example about 10 wt% to about 20 wt%, or about 10 wt% to about 14.5 wt%.

[0047] The means of mixing the hyaluronic acid or its metal salt-containing colloid with the acid is not limited to the present disclosure, provided that a homogeneous mixture can be achieved. Mixing can be performed manually or mechanically, such as, but not limited to, a handheld mixing stick, or mechanically, such as, but not limited to, a kneader, stirrer, blender, or centrifugal mixer.

[0048] In some embodiments of the present disclosure, the acid concentration in the mixture is related or correlated with the reaction temperature of the self-crosslinking reaction and the concentration of hyaluronic acid or its salt. In some embodiments of the present disclosure, the acid concentration in the mixture can be about 0.58N to about 1.0N at a reaction temperature of about -10°C. The acid concentration in the mixture can be about 0.58N to about 1.5N at a reaction temperature of about -20°C. The acid concentration in the mixture can be about 1.0N to about 1.5N at a reaction temperature of about -30°C. In some embodiments of the present disclosure, the acid concentration in the mixture can be about 0.58N to about 1.0N at a reaction temperature of about -10°C, and the concentration of hyaluronic acid or its salt in the mixture is about 10wt%.

[0049] In some embodiments of the present disclosure, the reaction time for the self-crosslinking reaction can be at least 7 days or 14 days, which varies based on the reaction temperature and the concentration of hyaluronic acid or its salt in the mixture.

[0050] In some embodiments of the present disclosure, the concentration of hyaluronic acid or a salt thereof in the mixture can be about 14.5 wt %, the reaction temperature can be about -10°C, and the reaction time can be about 14 to about 42 days, for example, about 21 to about 28 days.

[0051] In some embodiments of the present disclosure, the concentration of hyaluronic acid or a salt thereof in the mixture can be about 14.5 wt %, the reaction temperature can be about -20°C, and the reaction time can be about 7 to about 35 days, for example, about 14 to about 28 days.

[0052] In some embodiments of the present disclosure, the concentration of hyaluronic acid or a salt thereof in the mixture can be about 14.5 wt %, the reaction temperature can be about -30°C, and the reaction time can be about 14 to about 35 days, for example, about 14 to about 28 days.

[0053] In some embodiments of the present disclosure, the concentration of hyaluronic acid or a salt thereof in the mixture can be about 10 wt %, the reaction temperature can be about -10°C, and the reaction time can be about 7 to about 28 days, for example, about 14 to about 28 days.

[0054] In some embodiments of the present disclosure, the concentration of hyaluronic acid or a salt thereof in the mixture can be about 10 wt %, the reaction temperature can be about -20°C, and the reaction time can be about 7 to about 28 days, for example, about 14 to about 28 days.

[0055] In some embodiments of the present disclosure, the concentration of hyaluronic acid or a salt thereof in the mixture can be about 10 wt %, the reaction temperature can be about -30°C, and the reaction time can be about 14 to about 35 days, for example, about 14 to about 28 days.

[0056] In some embodiments of the present disclosure, the method includes carrying out the self-crosslinking reaction only once continuously at about -10°C to about -30°C. That is, after mixing, the mixture is kept at about -10°C to about -30°C throughout the entire reaction time of the self-crosslinking reaction. In some embodiments of the present disclosure, the reaction temperature can be substantially constant throughout the entire self-crosslinking reaction.

[0057] In some embodiments of the present disclosure, prior to step (c), the method further comprises homogenizing and / or washing the product of step (b).In some embodiments of the present disclosure, prior to (c), the method further comprises homogenizing and washing the product of step (b), thus forming the self-crosslinked hyaluronic acid particles.

[0058] In some embodiments of the present disclosure, the product of step (b) is homogenized before the washing step. In some embodiments of the present disclosure, the homogenizing step of the present disclosure means reducing the size of the produce of step (b), thus dispersing the produce of step (b) evenly in a liquid, such as the liquid used in the washing step. The homogenizing step of the present disclosure may include a crushing step, which may be achieved by a homogenizer or through a sieve. In some embodiments of the present disclosure, the product of step (b) is homogenized by a homogenizer.

[0059] The washing step may use water, distilled water, deionized water, saline or a buffer solution. The washing step removes acidic components, such as nitric acid, hydrochloric acid or sulfuric acid, from the product of step (b) and neutralizes the resulting product to a neutral or approximately neutral pH.

[0060] Without intending to be bound by any theory, it is believed that the pH of the self-crosslinked hyaluronic acid particles before steaming can affect the rheological properties of the self-crosslinked hyaluronic acid gel after steaming.In some embodiments of the present disclosure, the pH of the self-crosslinked hyaluronic acid particles is adjusted with a buffer solution, a weak acid and / or a weak base before steaming in step (c).Because the suspended aqueous solvent is removed by sieving or filtration in the washing step, the pH of the self-crosslinked hyaluronic acid particles cannot be easily measured, and therefore the pH of the waste solvent of the washing step is measured instead.The pH before steaming can be adjusted to about pH 7.0 to about pH 6.0, for example about pH 6.3 to about 6.8.

[0061] The means of washing and adjusting the pH of the product of step (b) and / or the self-crosslinked hyaluronic acid particles are not limited to the present disclosure, provided that the desired pH of the self-crosslinked hyaluronic acid particles can be achieved. For example, this can be done alone, together with a buffer solution of the target pH, or with the aid of titration with a low concentration of acidic or basic solution. Since the self-crosslinked hyaluronic acid particles tend to hydrolyze quickly in a basic buffer solution, it is important to protect them from hydrolysis due to the high pH of the washing process. In some embodiments of the present disclosure, the product of step (b) is washed with a neutral buffer solution to a neutral or approximately neutral pH, and the suspended aqueous medium is removed. The self-crosslinked hyaluronic acid particles are then washed at least once with a buffer solution of the target pH, and then adjusted to the target pH by titration with a low concentration of acidic or basic solution (e.g., 0.1 N hydrochloric acid or sodium hydroxide solution). For example, after a low-temperature reaction, the reaction product can be first placed at room temperature, then added to a certain amount of 100 mM phosphate buffer solution at pH 7.0, and homogenized by grinding. After removing the buffer solution by filtration, the product is washed several times with fresh 100 mM phosphate buffer solution at pH 7.0 until the solution has a pH of about 7.0. The product is then washed at least once with a buffer solution of a target pH (e.g., pH 7.0, pH 6.8, pH 6.5, pH 6.3, and pH 6.0), and then titrated to the target pH with a weak acid or base solution. Such a process can minimize hydrolysis of the reaction product during washing and / or pH adjustment.

[0062] The components and concentrations of the buffer solution used are not limited to the present disclosure, provided that they are suitable for human pharmaceutical use. In some embodiments of the present disclosure, the buffer solution can be a phosphate buffer solution with a concentration of about 1 mM to about 1000 mM, about 10 mM to about 300 mM, or about 10 mM to about 100 mM. The pH of the buffer solution can be about pH 6.0 to about 7.0. For example, a phosphate buffer solution containing 100 mM sodium or potassium phosphate solution and having a pH of about 6.0 to about 7.0 can be used.

[0063] After homogenization, washing and pH adjustment, the self-crosslinked hyaluronic acid particles will automatically separate from the aqueous phase into a single layer when left to stand for a certain period of time. The self-crosslinked hyaluronic acid gel that does not automatically separate from the aqueous phase or forms a suspension with the aqueous phase cannot maintain the crosslinked state after steam treatment due to its weak crosslinked structure.

[0064] The method of the present disclosure includes step (c), which is treating the product of step (b) with steam, such as at about 110° C. to about 130° C., for about 10 minutes to about 60 minutes to obtain a self-crosslinked hyaluronic acid gel.

[0065] According to the present disclosure, the structure of the obtained self-crosslinked hyaluronic acid gel can be modified by adjusting the temperature and time of steaming, which results in modification of the viscoelastic properties, such as the elastic modulus (G'), viscosity coefficient (G'') and shear viscosity.

[0066] The means of steam treatment is not particularly limited in the present disclosure, provided that it is sufficient to convert the self-crosslinked hyaluronic acid particles into a sterile high-viscosity self-crosslinked hyaluronic acid gel. In some embodiments of the present disclosure, the pH-adjusted self-crosslinked hyaluronic acid particles are filled into a glass or plastic container (e.g., glass or plastic syringe), and then treated with high-temperature steam. In some embodiments of the present disclosure, the temperature of the steam is about 110°C to about 130°C, about 115°C to about 125°C, or about 120°C to about 121°C. The time of steam treatment is set based on the steam temperature and the required viscoelasticity of the gel, provided that the self-crosslinked hyaluronic acid can be converted from a granular form to a sterile gel form while maintaining the crosslinked state. For example, for 121°C steam treatment, the treatment time can be about 10 minutes to about 60 minutes, about 15 minutes to about 60 minutes, or about 15 minutes to about 35 minutes. Different treatment times may result in different viscoelasticity of the self-crosslinked hyaluronic acid gel. The sterility of the self-crosslinked hyaluronic acid gel of the present disclosure may be verified by pharmacopoeial sterility test methods (e.g., European Pharmacopoeia 9.0 edition).

[0067] The volume of a syringe or container used in the steam treatment of the present disclosure can be from about 0.5 mL to about 50 mL, from about 1 mL to about 10 mL, or from about 3 mL to about 10 mL.

[0068] The literature (K. EDSMAN et al., "Gel properties of hyaluronic acid dermal fillers", Dermatol Surg. Vol. 38, pp. 1170-1179 (2012)) states that in the case of linear hyaluronic acid, when measured at low frequencies, its elastic modulus (G') is lower than its viscous modulus (G''); on the other hand, when measured at high frequencies, its elastic modulus (G') is higher than its viscous modulus (G''). In contrast, in the case of crosslinked hyaluronic acid, due to the crosslinked network structure, its elastic modulus (G') is higher than its viscous modulus (G'') regardless of the frequency of measurement. Such properties of hyaluronic acid gels can be used to determine whether they are linear or crosslinked. Therefore, the self-crosslinked hyaluronic acid gel of the present disclosure refers to a hyaluronic acid gel after steam treatment, the elastic modulus (G') of which, measured at 25°C and at each and every frequency from 0.1 Hz to 10.0 Hz, is equal to or greater than the viscous modulus (G'') and has a tan δ of 1 or less. -1 The shear viscosity of the self-crosslinked hyaluronic acid gel of the present disclosure measured at a shear rate of 20 Pa s or more at 25° C. On the other hand, when the hyaluronic acid gel has an elastic modulus (G') lower than the viscous modulus (G'') at any frequency between 0.1 Hz and 10.0 Hz, it is determined to be linear.

[0069] In some embodiments of the present disclosure, the viscoelasticity of the self-crosslinked hyaluronic acid gel can be measured by a rheometer (TA, AR2000ex) with a 20 mm metal plate at 25° C. and 1.0 Hz. The elastic modulus (G′) is about 30 Pa to about 8000 Pa, about 200 Pa to about 1000 Pa, or about 300 Pa to about 800 Pa. The viscous modulus (G″) is about 20 Pa to about 2000 Pa, about 100 Pa to about 600 Pa, or about 150 Pa to about 500 Pa. The elastic modulus and viscosity of the self-crosslinked hyaluronic acid gel decrease as the time of steam treatment increases.

[0070] In some embodiments of the present disclosure, -1 The shear viscosity of the self-crosslinked hyaluronic acid gel measured at a shear rate of 1 sec and at 25° C. is about 20 Pa·s to about 900 Pa·s, about 100 Pa·s to about 800 Pa·s, or about 250 Pa·s to about 700 Pa·s. In some further embodiments, the shear viscosity of the self-crosslinked hyaluronic acid gel measured at a shear rate of 1 sec and at 25° C. is about 20 Pa·s to about 900 Pa·s, about 100 Pa·s to about 800 Pa·s, or about 250 Pa·s to about 700 Pa·s. -1 The shear viscosity of the self-crosslinked hyaluronic acid gel measured at a shear rate of about 38.04 Pa·s to about 896.91 Pa·s, or about 55.69 Pa·s to about 896.91 Pa·s. In some embodiments of the present disclosure, the shear viscosity of the self-crosslinked hyaluronic acid gel measured at a shear rate of about 25° C. and a shear rate of about 50 sec -1 The shear viscosity of the self-crosslinked hyaluronic acid gel measured at a shear rate of about 0.31 Pa·s or more, e.g., about 0.35 Pa·s or more, about 0.4 Pa·s or more, about 0.5 Pa·s or more, about 0.6 Pa·s or more, about 0.8 Pa·s or more, or about 1 Pa·s or more, at 25° C. and at a shear rate of 50 sec. -1 The shear viscosity of the self-crosslinked hyaluronic acid gel measured at a shear rate of from about 1.97 Pa·s to about 35.66 Pa·s, or from about 2.18 Pa·s to about 35.66 Pa·s.

[0071] In some embodiments of the present disclosure, the equilibrium swelling capacity of the self-crosslinked hyaluronic acid particles is determined as follows: A sample of 1 g of self-crosslinked hyaluronic acid particles that has been homogenized, washed and neutralized to about pH 7.0 is immersed in a pH 6.0 buffer solution containing 0.9wt% sodium chloride and 10mM sodium phosphate at 5°C for 24 hours to swell to equilibrium, and then dried to constant weight at 25°C. The self-crosslinked hyaluronic acid particles swollen with the buffer solution are in granular form, so their density cannot be measured. Therefore, the equilibrium swelling capacity in the present disclosure is determined by the following formula: Equilibrium swelling capacity = (wet weight of self-crosslinked hyaluronic acid particles swollen with a buffer solution / dry weight of self-crosslinked hyaluronic acid particles swollen with a buffer solution)

[0072] In some embodiments of the present disclosure, the equilibrium swelling capacity of the self-crosslinked hyaluronic acid particles is greater than 10, such as from about 12 to about 30, from about 12 to about 20, from about 13 to about 18, or from about 13 to about 16.

[0073] Typically, hyaluronic acid with more efficient cross-linking produces a tighter cross-linked network and therefore the colloid or particle may be harder and have a lower swelling ratio (Mohammed Al-Sibani et al., "Study of the effect of mixing approach on cross-linking efficiency of hyaluronic acid-based hydrogel cross-linked with 1,4-butanediol diglycidyl ether," European Journal of Pharmaceutical Sciences, Vol. 91, pp. 131-137 (2016)). Others have also found that higher cross-linking density results in a lower swelling ratio (Noel L. Davison et al., "Degradation of Biomaterials," Tissue Engineering (2nd ed.), Chapter 6, Elsevier Inc., p. 197 (2014)).

[0074] As reaction time increases, the hyaluronic acid concentration of the resulting self-crosslinked hyaluronic acid particles increases, the recovery rate increases, and the equilibrium swelling capacity decreases.As reaction temperature decreases, the hyaluronic acid concentration of the resulting self-crosslinked hyaluronic acid particles decreases, the recovery rate decreases, and the equilibrium swelling capacity increases.In some embodiments of the present disclosure, the recovery rate of hyaluronic acid is about 70% to 100%.

[0075] Furthermore, the equilibrium swelling capacity is substantially the same for self-crosslinked hyaluronic acid particles at various pHs when adjusted to a pH of 6.0 to 7.0 and then directly dried to constant weight at 25° C. The equilibrium swelling capacity of the self-crosslinked hyaluronic acid particles is independent of pH at least in the range of 6.0 to 7.0, and is approximately the same as the aforementioned equilibrium swelling capacity of self-crosslinked hyaluronic acid particles swollen to equilibrium in a pH 6.0 buffer solution containing 0.9 wt % sodium chloride and 10 mM sodium phosphate at 5° C. for 24 hours.

[0076] The average particle size of the self-crosslinked hyaluronic acid particles of the present disclosure before steam treatment is not particularly limited, and can be obtained with different homogenizers, different rotation speeds, and different homogenization times.In some embodiments of the present disclosure, the average particle size of the self-crosslinked hyaluronic acid particles is about 200 μm to about 3000 μm, about 600 μm to about 2500 μm, or about 1500 μm to about 2200 μm.The average particle size is determined by using saline as a suspension medium, and is measured by a particle size measuring instrument (PARTICA LA-960, HORIBA) at 25°C.

[0077] In some embodiments of the present disclosure, the pH of the self-crosslinked hyaluronic acid gel after steam treatment is from about 4.5 to about 6.5, from about 5.0 to about 6.0, or from about 5.1 to about 5.8.

[0078] The hyaluronic acid concentration of the self-crosslinked hyaluronic acid gel of the present disclosure is determined by pharmacopoeia carbazole assay (e.g., European Pharmacopoeia 9.0 edition).The hyaluronic acid concentration of the self-crosslinked hyaluronic acid gel of the present disclosure increases with increasing reaction time and reaction temperature, and the concentration is about 1.5wt% to about 7.5wt%, about 2.0wt% to about 6.0wt%, about 3.0wt% to about 6.0wt%, or about 4.0wt% to about 6.0wt%.In the present disclosure, the hyaluronic acid concentration of the self-crosslinked hyaluronic acid gel after steam treatment is the same as the hyaluronic acid concentration of the self-crosslinked hyaluronic acid particles before steam treatment.

[0079] The self-crosslinked hyaluronic acid gel of the present disclosure can also be obtained by mixing a linear hyaluronic acid solution or an aqueous phase (e.g., distilled water, saline, or buffer solution) with the self-crosslinked hyaluronic acid particles of the present disclosure, and then treating the resulting mixture with steam, provided that the product meets the definition of a self-crosslinked hyaluronic acid gel as defined in the present disclosure and is determined to be within the scope of the present disclosure.

[0080] In some embodiments of the present disclosure, the osmolarity of the self-crosslinked hyaluronic acid gel after steam treatment can be controlled by the osmolarity of the aqueous or buffer solution used in the washing step and / or pH adjustment step, and is therefore not particularly limited, provided that it is suitable for human pharmaceutical use. The osmolarity of the self-crosslinked hyaluronic acid gel of the present disclosure after steam treatment can be about 10 to about 350 mOsm / kg, about 30 to about 200 mOsm / kg, or about 50 to about 150 mOsm / kg.

[0081] The self-crosslinking hyaluronic acid gel of the present disclosure can be used for bone regeneration and treatment of defects. The experimental method involves a cranial bone defect in rats (Patrick P Spicer et al., "Evaluation of bone regeneration using the rat critical size calvarial defect", Nature Protocol, Vol. 7, pp. 1918-1929 (2012)), in which the wound is covered with self-crosslinking hyaluronic acid gel. The rats are sacrificed 3 months after surgery, and the bones are removed and analyzed by micro-computed tomography. The results are compared with a commercial group, a linear hyaluronic acid group, a blank control group (no substance added after surgery), and a positive control group. The method for producing the self-crosslinking hyaluronic acid gel of the present disclosure is simple and does not involve the use of toxic organic solvents or crosslinking agents, and the obtained self-crosslinking hyaluronic acid gel shows favorable bone repair and regeneration effects in animal experiments.

[0082] The present disclosure provides a modified manufacturing method for synthesizing self-crosslinked hyaluronic acid particles under low temperature.After adjusting to a certain pH, the self-crosslinked hyaluronic acid particles can be treated with steam to form a sterile high-viscosity self-crosslinked hyaluronic acid gel, which can be used for medical applications such as bone regeneration and defect repair, postoperative adhesion prevention, subcutaneous filler, and osteoarthritis treatment.

[0083] The following examples are presented for illustrative purposes only and are not intended to limit the scope of the present invention.

[0084] [Example 1] Reaction conditions: hyaluronic acid (HA) concentration 14.5 wt%, 0.58 N HCl, reaction temperature -10°C The water content is 3.65wt%, and the intrinsic viscosity is 1.7m 3 12 grams of hyaluronic acid, having a molecular weight of 983,000 / kg and an average molecular weight of 983,000, was added to 59.57 grams of distilled water and mixed in a mixer (SPAR FOOD MACHINERY MFG. CO., LTD., Model SP-502A) at 253 rpm for 5 minutes to form a water-swollen hyaluronic acid-containing colloid. The colloid was extruded into stripes through a metal sieve with openings of about 0.2 cm in size, and then 8.43 g of 6N hydrochloric acid (density 1.1 g / cm) was added in the mixer. 3) at the same speed for 5 minutes to form hyaluronic acid-containing colloid and hydrochloric acid in a homogenous manner. The resulting colloid was placed in a serum bottle and kept in a refrigerator at low temperature for 14 days for self-crosslinking reaction as shown in Table 1. After the reaction, the product was thawed at room temperature and then added into 600 mL of 100 mM phosphate buffer solution at pH 7.00 and homogenized with a homogenizer (IKA, model T-25) at 10,000 rpm for 1 minute. The buffer solution was then removed by sieving, and the product was then added into another 600 mL of fresh 100 mM phosphate buffer solution at pH 7.0 and homogenized with a high-speed emulsifying homogenizer (Gordon, model HM25) at 6,000 rpm for 5 minutes. The buffer solution was removed by sieving, and the product was washed several times with 3000 mL of fresh buffer solution until the buffer solution had a stable pH of 7.00±0.10. After filtering to remove the buffer solution, the resulting product was weighed, divided into 5 equal parts, and then washed separately at the target pH (pH 7.00, pH 6.80, pH 6.50, pH 6.30, and pH 6.00) with 10 times the weight of the product of 100 mM phosphate buffer solution, and then titrated with an aqueous solution of 0.1 N hydrochloric acid or sodium hydroxide until the solution reached a stabilized pH equal to the target pH (pH 7.00, pH 6.80, pH 6.50, pH 6.30, and pH 6.00). The obtained hyaluronic acid particles were filtered through a sieve to remove the solution, then filled into a 1 mL syringe and treated with steam at 121°C for different fixed times to convert the self-crosslinked hyaluronic acid particles into self-crosslinked hyaluronic acid gel. In the experiments with reaction times of 21, 28, 35 and 42 days, the method steps and reaction conditions were identical to those described, except for the low reaction time set. The obtained self-crosslinked hyaluronic acid gel was incubated for 14 days for microbial sterility testing and confirmed to be free of microbial growth (negative).

[0085] [Table 1] TIFF0007678850000002.tif79165

[0086] [Example 2] Reaction conditions: hyaluronic acid concentration 14.5 wt%, 1.0 N HCl, reaction temperature -10°C The process steps and reaction conditions of Example 2 were the same as those of Example 1, except that the amount of distilled water was 53.47 g, the amount of 6N HCl was 14.53 g, and the reaction times were 14 days, 21 days, and 28 days.

[0087] [Table 2]

[0088] [Example 3] Reaction conditions: hyaluronic acid concentration 14.5 wt%, 0.58 N HCl, reaction temperature -20°C The process steps and reaction conditions of Example 3 were the same as those of Example 1, except that the reaction temperature was −20° C., and the reaction times were 14 days, 21 days, 28 days, and 35 days.

[0089] [Table 3]

[0090] [Example 4] Reaction conditions: hyaluronic acid concentration 14.5 wt%, 1.5 N HCl, reaction temperature -20°C The process steps and reaction conditions of Example 4 were the same as those of Example 1, except that the amount of distilled water was 46.18 g, the amount of 6N HCl was 21.82 g, and the reaction times were 7 days, 14 days, 21 days, and 28 days.

[0091] [Table 4]

[0092] [Example 5] Reaction conditions: hyaluronic acid concentration 14.5 wt%, 1.0 N HCl, reaction temperature -30°C The process steps and reaction conditions of Example 5 were the same as those of Example 2, except that the reaction temperature was −30° C., and the reaction times were 14 days, 21 days, 28 days, and 35 days.

[0093] [Table 5]

[0094] [Example 6] Reaction conditions: hyaluronic acid concentration 14.5 wt%, 1.5 N HCl, reaction temperature -30°C The process steps and reaction conditions of Example 6 were the same as those of Example 4, except that the reaction temperature was −30° C., and the reaction times were 14 days, 21 days, 28 days, and 35 days.

[0095] [Table 6] TIFF0007678850000008.tif30163

[0096] [Example 7] Reaction conditions: hyaluronic acid concentration 10 wt%, 0.58 N HCl, reaction temperature -10°C The method steps and reaction conditions of Example 7 were the same as those of Example 1, except that the amount of hyaluronic acid was 8.3 g, the amount of distilled water was 63.27 g, the amount of 6N HCl was 8.43 g, and the reaction times were 7 days, 14 days, 21 days, and 28 days.

[0097] [Table 7]

[0098] [Example 8] Reaction conditions: hyaluronic acid concentration 10 wt%, 1.0 N HCl, reaction temperature -10°C The process steps and reaction conditions of Example 8 were identical to those of Example 7, except that the amount of distilled water was 57.17 g, the amount of 6N HCl was 14.53 g, and the reaction times were 14 days, 21 days, and 28 days.

[0099] [Table 8]

[0100] [Example 9] Reaction conditions: hyaluronic acid concentration 10 wt%, 0.58 N HCl, reaction temperature -20°C The process steps and reaction conditions of Example 9 were the same as those of Example 7, except that the reaction temperature was −20° C., and the reaction times were 7 days, 14 days, 21 days, and 28 days.

[0101] [Table 9]

[0102] [Example 10] Reaction conditions: hyaluronic acid concentration 10 wt%, 1.0 N HCl, reaction temperature -20°C The process steps and reaction conditions of Example 10 were the same as those of Example 8, except that the reaction temperature was -20°C.

[0103] [Table 10]

[0104] [Example 11] Reaction conditions: hyaluronic acid concentration 10 wt%, 0.58 N HCl, reaction temperature -30°C The process steps and reaction conditions of Example 11 were the same as those of Example 7, except that the reaction temperature was −30° C., and the reaction times were 14 days, 21 days, 28 days, and 35 days.

[0105] [Table 11]

[0106] [Example 12] Reaction conditions: hyaluronic acid concentration 10 wt%, 1.0 N HCl, reaction temperature -30°C The process steps and reaction conditions of Example 12 were the same as those of Example 8, except that the reaction temperature was −30° C., and the reaction times were 14 days, 21 days, and 28 days.

[0107] [Table 12]

[0108] The physical properties of Examples 1 to 12 are shown in Table 13 and Figures 1 to 5.

[0109] [Table 13] TIFF0007678850000016.tif40165

[0110] [Example 13] Sample preparation and animal testing process Male Wistar rats weighing approximately 360g to 390g were quarantined for 7 days. Room temperature was controlled at 24°C, humidity was maintained at 60% to 70%, and the photoperiod was set at 12 hours light / 12 hours dark. Rats were housed in acrylic cages and had free access to food and water. Rats were anesthetized with isoflurane, and the instruments used throughout the surgery were heat sterilized to prevent rats from dying from postoperative infection. All animal experiments were performed in accordance with the American Physiological Society Guidelines for the Care and Use of Animals.

[0111] The animal test model generally followed the method used by Spicer et al. ("Evaluation of bone regeneration using the rat critical size calvarial defect", Nature Protocols, Vol. 7, No. 10, pp. 1918-1929 (2012)) and Chen et al. ("Bone Formation Using Cross-Linked Chitosan Scaffolds in Rat Calvarial Defects", Implant Dent. Vol. 27, No. 6, pp. 1-7 (2017)). First, rats were anesthetized with the gas anesthetic isoflorane, and then shaved. The surgical area was disinfected with povidone-iodine alcohol solution and 70% alcohol, and then injected with ampicillin. The rat's head was then covered with sterile tissue paper with a cutout hole to expose only the surgical area. The skin and periosteum directly above the head were incised to a size of 1.5 centimeters. The skull was separated from the periosteum by using a periosteal peeler. A 5 mm diameter bone defect was created by using a 5 mm diameter trephine burr in the center of the midline sagittal suture (the central bulge of the skull, adjacent to the parietal bone). Then, 0.15 g to 0.2 g of 2.05 wt% linear hyaluronic acid (control group), self-crosslinking hyaluronic acid gels with different hyaluronic acid contents (experimental groups 1 to 4), or CoreBone 1000 bone meal (positive control group) were implanted into the bone defect, and the bone defect was then covered with a Bio-Gide membrane (3M, Geistlich Bio-Oss®, Switzerland). In the blank group, the bone defect was directly covered with the Bio-Gide membrane. If the animals woke up from anesthesia during surgery, anesthesia was maintained by inhaling anesthetic gas from a gas anesthesia machine through a breathing mask. The preparation conditions and properties of the animal test samples are as shown in Table 14.

[0112] [Table 14]

[0113] The rats were sacrificed 3 months after the experiment. After deep anesthesia, the skulls were removed from the rats. The specimens were first immersed in 10% paraformaldehyde for 4 days for fixation, and then sent to Taiwan Mouse Clinic (TMC) for scanning and analysis using a micro-computed tomography (micro-CT) 3D imaging system (SkyScan 1076, Bruker, Antwerp, Belgium). The parameters were set as follows: image pixel size (μm) = 9, power supply voltage (kV) = 70, power supply current (uA) = 200, filter = Al 0.5 mm, exposure (ms) = 453, rotation step (deg) = 0.2, and region of interest (ROI): diameter 5.5 mm × height 0.5 mm. The micro-CT images were analyzed using sagittal tomography and histomorphometry. The data was evaluated by newly formed bone volume / total volume (BV / TV (%)) to quantify the amount of bone regeneration.

[0114] The results are shown in Figure 6. The blank group (No. 1) and the control group (No. 2) had the lowest percentage of newly formed bone, 21% and 40%, respectively, while the percentages of newly formed bone in experimental groups 1 to 4 (Nos. 3 to 6) were 48%, 61%, 49% and 57%, respectively, and the percentage of newly formed bone in the positive control group (No. 7) was 45%. The data showed that the self-crosslinking hyaluronic acid gel of the present disclosure had superior bone regeneration function to the commercial product (positive control group), linear hyaluronic acid gel (control group) and the blank group. The present invention includes the following aspects. [Section 1] 1. A method for producing a self-crosslinked hyaluronic acid gel, comprising: (a) preparing a colloid containing hyaluronic acid or a metal salt thereof; (b) carrying out the self-crosslinking reaction of the colloid containing hyaluronic acid or a metal salt thereof in an acidic environment at about -10°C to about -30°C for about 7 to about 42 days; and (c) treating the product of step (b) with steam to obtain a self-crosslinked hyaluronic acid gel. [Section 2] Item 1, the method according to item 1, which does not include the use of any cross-linking agent and 2-chloro-1-methylpyridinium iodide (CMPI). [Section 3] Item 3. The method according to item 1, wherein step (a) comprises providing a colloid containing hyaluronic acid or a metal salt thereof in the form of granules, stripes or strings. [Section 4] Item 3. The method according to item 1, wherein in step (b), the acidic environment is provided by an acid having a concentration of about 0.5N to about 1.5N, and the acid is selected from the group consisting of hydrochloric acid, nitric acid, and sulfuric acid. [Section 5] Item 2. The method according to item 1, wherein step (b) comprises mixing a colloid containing hyaluronic acid or a metal salt thereof with an acid at room temperature to form a mixture, and allowing the mixture to stand at about -10°C to about -30°C for about 7 to about 42 days. [Section 6] Item 6. The method according to item 5, wherein the concentration of hyaluronic acid or a metal salt thereof in the mixture is about 10 wt% to about 30 wt%. [Section 7] In step (b), The acidic environment is provided with an acid having a concentration of about 0.58N to about 1.0N, and the self-crosslinking reaction is carried out at about -10°C for about 7 to about 42 days. The acidic environment is provided with an acid having a concentration of about 0.58N to about 1.5N, and the self-crosslinking reaction is carried out at about -20°C for about 7 to about 35 days. The acidic environment is provided with an acid having a concentration of about 0.58N to about 1.0N, and the self-crosslinking reaction is carried out at about -20°C for about 7 to about 28 days. An acidic environment is provided with an acid at a concentration of about 1.0N to about 1.5N, and the self-crosslinking reaction is carried out at about -30°C for about 14 to about 35 days; or Item 2. The method according to item 1, wherein the acidic environment is provided by an acid having a concentration of about 0.58N to about 1.0N, and the self-crosslinking reaction is carried out at about -30°C for about 14 to about 35 days. [Section 8] Item 1. The method according to item 1, comprising carrying out the self-crosslinking reaction only once, and carrying out the entire self-crosslinking reaction continuously at about -10°C to about -30°C. [Section 9] The method according to claim 1, further comprising homogenizing and / or washing the product of step (b) prior to step (c). [Section 10] Item 10. The method of item 1, wherein the product of step (b) has an equilibrium swelling capacity of about 12 to about 30. [Section 11] Item 1. The method of item 1, further comprising adjusting the pH of the product of step (b) to about 6.0 to about 7.0 prior to step (c). [Section 12] The method of claim 1, further comprising adjusting the pH of the product of step (b) to about 6.3 to about 6.8 prior to step (c). [Section 13] Item 1. The method of item 1, wherein step (c) comprises treating the product of step (b) with steam at about 110° C. to about 130° C. for about 10 minutes to about 60 minutes. [Section 14] The self-crosslinking hyaluronic acid gel exhibits at least one of the following: The tan δ of the self-crosslinked hyaluronic acid gel at a concentration of about 1.5 wt % to about 7.5 wt %, measured at 25° C. and at each and every frequency from 0.1 Hz to 10.0 Hz, is less than or equal to 1; The elastic modulus of the self-crosslinked hyaluronic acid gel at a concentration of about 1.5 wt % to about 7.5 wt %, measured at 25° C. and 1.0 Hz, is about 30 Pa to about 8000 Pa; The viscosity of the self-crosslinked hyaluronic acid gel at a concentration of about 1.5 wt % to about 7.5 wt %, measured at 25° C. and 1.0 Hz, is about 20 Pa to about 2000 Pa; At 25℃, 1sec -1 Item 2. The method according to item 1, wherein the shear viscosity of the self-crosslinked hyaluronic acid gel at a concentration of about 1.5 wt% to about 7.5 wt% measured at a shear rate of about 20 Pa·s to about 900 Pa·s. [Section 15] A self-crosslinked hyaluronic acid gel having a concentration of about 1.5 wt% to about 7.5 wt%, the tan δ of which is less than or equal to 1 when measured at 25°C and at each and every frequency from 0.1 Hz to 10.0 Hz, and the self-crosslinked hyaluronic acid gel is sterile. [Section 16] Presents with at least one of the following: The elastic modulus of the self-crosslinked hyaluronic acid gel at a concentration of about 1.5 wt % to about 7.5 wt %, measured at 25° C. and 1.0 Hz, is about 30 Pa to about 8000 Pa; The viscosity of the self-crosslinked hyaluronic acid gel at a concentration of about 1.5 wt % to about 7.5 wt %, measured at 25° C. and 1.0 Hz, is about 20 Pa to about 2000 Pa; At 25℃, 1sec-1 The shear viscosity of the self-crosslinked hyaluronic acid gel at a concentration of about 1.5 wt% to about 7.5 wt%, measured at a shear rate of about 20 Pa s to about 900 Pa s, At 25℃, 50sec -1 Item 16. The self-crosslinked hyaluronic acid gel according to item 15, wherein the shear viscosity of the self-crosslinked hyaluronic acid gel at a concentration of about 1.5 wt% to about 7.5 wt% measured at a shear rate of about 0.31 Pa s to about 35.66 Pa s. [Section 17] Item 16. A pharmaceutical composition for promoting bone regeneration, treating bone defects, preventing postoperative adhesions, subcutaneous filling, and / or treating arthropathy, comprising the self-crosslinking hyaluronic acid gel according to item 15.

[0115] While the present disclosure has been described and illustrated with reference to specific embodiments thereof, these descriptions and illustrations are not limiting, and it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present disclosure as defined by the appended claims.

Claims

1. 1. A method for producing a self-crosslinked hyaluronic acid gel, comprising: (a) preparing a colloid containing hyaluronic acid or a metal salt thereof; (b) carrying out the self-crosslinking reaction of the colloid containing hyaluronic acid or a metal salt thereof in an acidic environment at −10° C. to −30° C. for 7 to 42 days; and (c) treating the product of step (b) with steam to obtain a self-crosslinked hyaluronic acid gel.

2. 10. The method of claim 1, which does not include the use of any cross-linking agent and 2-chloro-1-methylpyridinium iodide (CMPI).

3. 2. The method of claim 1, wherein step (a) comprises providing a colloid containing hyaluronic acid or a metal salt thereof in the form of granules, stripes or strings.

4. 2. The method of claim 1, wherein in step (b), the acidic environment is provided by an acid having a concentration of 0.5N to 1.5N, the acid being selected from the group consisting of hydrochloric acid, nitric acid and sulfuric acid.

5. 2. The method of claim 1, wherein step (b) comprises mixing the hyaluronic acid or a colloid containing a metal salt thereof with an acid at room temperature to form a mixture, and allowing the mixture to stand at -10°C to -30°C for 7 to 42 days.

6. The method according to claim 5, wherein the concentration of hyaluronic acid or its metal salt in the mixture is from 10 wt% to 30 wt%.

7. In step (b), An acidic environment is provided with an acid concentration of 0.58N to 1.0N, and the self-crosslinking reaction is carried out at -10°C for 7 to 42 days. An acidic environment is prepared with an acid concentration of 0.58N to 1.5N, and the self-crosslinking reaction is carried out at -20°C for 7 to 35 days. An acidic environment is prepared with an acid concentration of 0.58N to 1.0N, and the self-crosslinking reaction is carried out at -20°C for 7 to 28 days. An acidic environment is provided with an acid concentration of 1.0N to 1.5N, and the self-crosslinking reaction is carried out at -30°C for 14 to 35 days; or 2. The method of claim 1, wherein the acidic environment is provided by an acid with a concentration of 0.58N to 1.0N, and the self-crosslinking reaction is carried out at -30°C for 14 to 35 days.

8. 2. The method according to claim 1, comprising carrying out the self-crosslinking reaction only once, and carrying out the entire self-crosslinking reaction continuously at -10°C to -30°C.

9. 2. The method of claim 1, further comprising homogenizing and / or washing the product of step (b) prior to step (c).

10. 2. The method of claim 1, wherein the product of step (b) has an equilibrium swelling capacity of from 12 to 30.

11. 10. The method of claim 1, further comprising adjusting the pH of the product of step (b) to between 6.0 and 7.0 prior to step (c).

12. 10. The method of claim 1, further comprising adjusting the pH of the product of step (b) to between 6.3 and 6.8 prior to step (c).

13. 2. The method of claim 1, wherein step (c) comprises treating the product of step (b) with steam at 110° C. to 130° C. for 10 minutes to 60 minutes.

14. The self-crosslinking hyaluronic acid gel exhibits at least one of the following: The tan δ of the self-crosslinked hyaluronic acid gel at a concentration of 1.5 wt % to 7.5 wt %, measured at 25° C. and at each and every frequency from 0.1 Hz to 10.0 Hz, is less than or equal to 1; The elastic modulus of the self-crosslinked hyaluronic acid gels at concentrations of 1.5 wt% to 7.5 wt%, measured at 25°C and 1.0 Hz, is between 30 Pa and 8000 Pa; The viscosity of the self-crosslinked hyaluronic acid gels at concentrations of 1.5 wt% to 7.5 wt%, measured at 25°C and 1.0 Hz, is between 20 Pa and 2000 Pa; At 25℃, 1sec -1 2. The method according to claim 1, wherein the shear viscosity of the self-crosslinked hyaluronic acid gel at a concentration of 1.5 wt.% to 7.5 wt.% measured at a shear rate of 20 Pa·s to 900 Pa·s.

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