A collagen hydrogel crosslinked by oxidized sodium hyaluronate, and a preparation method and application thereof

Abstract

The application discloses a preparation method of collagen hydrogel crosslinked by oxidized sodium hyaluronate, and comprises the following steps: collagen pretreatment; preparation of collagen homogenate liquid by high-pressure homogenization; sodium hyaluronate is modified by oxidation with sodium periodate; crosslinking reaction of the oxidized sodium hyaluronate and the collagen homogenate liquid to form solidified hydrogel, and after composite with sulfonated chitosan and coating with trehalose, the collagen hydrogel is obtained. The collagen is pretreated and high-pressure homogenized, so that the collagen fiber particle size is reduced on the basis of guaranteeing the triple helix structure of the collagen, the steric hindrance of the crosslinking reaction is reduced, the crosslinking reaction is more efficient, controllable and uniform. The triple water hydration network is designed, the HA hydrophilic group is introduced by aldehyde group-amino crosslinking, the proportion of bound water is increased by anchoring water molecules through ionic bonds of sulfonic acid groups, after coating with trehalose, a glassy hydration layer is formed, the water molecule migration rate is reduced, and the dehydration and falling back problem of traditional collagen implant within 1-2 weeks is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, in particular to a collagen hydrogel cross-linked by oxidized sodium hyaluronate and a preparation method and application thereof. BACKGROUND

[0002] Collagen is a naturally occurring protein in the human body, with the advantages of triple helix structure of repeating amino acid sequence (Gly-X-Y), low immunogenicity, complete degradability, and the ability to promote cell proliferation, adhesion and differentiation as a cell scaffold. It is an ideal soft tissue filling material. At present, there are many collagen implants on the market at home and abroad, mainly non-cross-linked and cross-linked implants. Non-cross-linked collagen implants, such as Chinese patents CN115572328A and CN116103776A, have short filling and maintaining effects. It is generally believed that the main reason for the effect is the decomposition and metabolism of collagenase in the human body, so researchers have improved the enzyme resistance of collagen through various cross-linking methods. For example, the commonly used chemical cross-linking agent glutaraldehyde can combine the aldehyde group (-CHO) of the peptide chain with the free amino group (-NH2) of the collagen protein to form intramolecular or intermolecular Schiff base covalent cross-linking. This cross-linking significantly enhances the enzyme resistance, thermal stability and mechanical strength of collagen. For example, Chinese patent CN115747993A discloses a collagen fiber with high biocompatibility and stability, a preparation method and application thereof, which uses chemical reagent NHS-SA to cross-link with collagen. However, its anti-enzymatic effect is not good, and the improvement of filling and maintaining effect is limited.

[0003] In the prior art, although the basic research of oxidized hyaluronic acid as a cross-linking agent has made some progress, such as Chinese patent application CN111019162A discloses a chitosan polypeptide derivative self-cross-linked hydrogel using oxidized hyaluronic acid as a cross-linking agent, and Chinese patent application CN120305450A also forms a hydrogel by cross-linking collagen and oxidized hyaluronic acid. However, this hydrogel can only be used for external wound repair and cannot be used as an implant. The existing cross-linked collagen still has technical gaps in safety and precise control of residual amount. The residual free aldehyde group may cause cytotoxicity or immune response risk, and even non-specific binding with other biological molecules in the body, leading to local tissue inflammation or decreased material stability.

[0004] At present, both non-cross-linked and cross-linked collagen implants have good immediate filling effect, but the filling effect falls after 1-2 weeks. The main reason is not only the decomposition and metabolism of collagen by collagenase, but also the dehydration and cohesion of the implant after being implanted into the body. The physiological saline in the implant is quickly absorbed by the human body, resulting in a decrease in the filling volume of the implant, and often requiring additional injection to achieve the patient's satisfaction. SUMMARY

[0005] To this end, the present application provides a collagen hydrogel crosslinked by oxidized sodium hyaluronate and a preparation method and application thereof to solve the problems in the prior art.

[0006] To achieve the above-mentioned object, the present application provides the following technical solutions.

[0007] According to one aspect of the present application, a preparation method of a collagen hydrogel crosslinked by oxidized sodium hyaluronate is provided, comprising the following steps:

[0008] Step one, collagen pretreatment

[0009] The collagen solution is adjusted to the isoelectric point, stirred and precipitated, centrifuged to obtain a precipitate, repeatedly washed by centrifugation with a buffer solution, and the collagen crude fiber precipitate is redissolved with an acetic acid solution, and a pepsin solution is added for reaction, after which the pretreated collagen is obtained after purification.

[0010] Step two, preparation of collagen homogenate

[0011] The pretreated collagen is dissolved with a buffer solution and subjected to high-pressure homogenization with a high-pressure homogenizer to obtain a collagen homogenate.

[0012] Step three, preparation of oxidized sodium hyaluronate

[0013] The sodium hyaluronate solution is subjected to an aldehyde group reaction with sodium periodate, and after the reaction is terminated by adding ethylene glycol, the oxidized sodium hyaluronate is obtained by ultrafiltration concentration, freeze-drying.

[0014] Step four, preparation of hydrogel

[0015] The oxidized sodium hyaluronate is dissolved with a buffer solution to obtain an oxidized sodium hyaluronate solution, and the collagen homogenate is added for crosslinking reaction to form a solidified hydrogel, and the residual unreacted oxidized sodium hyaluronate is removed by washing to obtain an initial hydrogel; after the initial hydrogel is subjected to sulfonated chitosan compounding and trehalose coating, a hydrogel block is obtained, which is the collagen hydrogel.

[0016] Further, the collagen used in the present application is derived from animals. In the step one, the collagen is extracted from animal Achilles tendons or animal skin. The collagen includes but is not limited to collagen extracted from pig Achilles tendons, pig skin, cow Achilles tendons, or cow skin, etc. The collagen of the present application can be obtained by purchase or self-preparation.

[0017] Further, in the step one, the concentration of the collagen solution is 1-6 mg / mL.

[0018] The adjusting pH to the isoelectric point is adjusting pH to 4.5-6.0; the pretreatment method of the application utilizes collagen isoelectric point precipitation method, which can remove acid-soluble impurities (albumin, glycoprotein, etc.) and concentrate collagen; at the collagen isoelectric point (pI≈4.5-6.0), the net charge of collagen molecule surface is zero, and the solubility is the lowest and the collagen precipitates, while the impurities are still dissolved in the liquid phase. Preferably, the stirring and precipitation method is stirring for 4-6 h, and standing at 4℃ for 1.5-2.5 h.

[0019] The pH regulator of the application is one or more of sodium hydroxide, disodium hydrogen phosphate, sodium citrate and citric acid.

[0020] Further, the step of pretreating collagen protein includes: adjusting pH of the collagen protein solution to 4.5-6.0, stirring and precipitating, obtaining the precipitate by centrifugation, repeatedly washing the precipitate by centrifugation with a buffer solution, and obtaining collagen crude fiber precipitate; redissolving the collagen crude fiber precipitate with acetic acid solution, stirring at 3-5℃, then adjusting pH to 2-3, adding pepsin solution, and performing constant temperature reaction, after the reaction is completed, inactivating pepsin by adjusting pH to terminate the reaction, and obtaining pretreated collagen protein by tangential flow ultrafiltration purification; wherein the centrifugal speed is 6000-10000 rpm, and the centrifugal time is 15-20 min.

[0021] The collagen protein of the application is pretreated by pepsin, and the pepsin can selectively remove the terminal peptide of collagen crude fiber (pepsin specifically hydrolyzes aromatic amino acid (Phe / Tyr) carboxyl terminal peptide bond at pH 2-3), and exposes the active site of collagen.

[0022] As an example, the pepsin pretreatment method is: redissolving collagen crude fiber precipitate with 0.3M acetic acid, stirring at 4℃ for 12 h, adding 2M HCl drop by drop, adjusting to pH 2.5±0.1 under magnetic stirring, adding pepsin solution (prepared by precooling 0.1M HCl), the final concentration is 0.8% (w / w, enzyme / collagen), oscillating at 4℃ and 50 rpm for 24 h, after the reaction is completed, adding 1M sodium hydroxide solution to adjust pH to 7 (inactivating pepsin) to terminate the reaction, and obtaining pepsin pretreated collagen protein by tangential flow ultrafiltration purification.

[0023] Further, the step of tangential flow ultrafiltration purification includes: redissolving the precipitate obtained by centrifugation of the reaction liquid with 0.05M HCl solution, repeatedly replacing the liquid with 0.05M HCl solution under the conditions of 100 kDa MWCO, 4℃, transmembrane pressure 0.8 bar and flow rate 5 L / min for 5-6 times, finally adjusting pH to 7 with 1M sodium hydroxide solution, and obtaining pepsin pretreated collagen protein by centrifugation after collagen protein is precipitated.

[0024] Further, in the step two, the high pressure homogenization is performed in three stages, the first stage is at a pressure of 4000±100 psi to break up large aggregates, the second stage is at a pressure of 8000±100 psi to dissociate fiber bundles, and the third stage is at a pressure of 12000±100 psi to obtain uniform nanofibers.

[0025] Further, in the step two, the high pressure homogenization is performed in three stages, the first stage is at a pressure of 4000±100 psi to break up large aggregates, the second stage is at a pressure of 8000±100 psi to dissociate fiber bundles, and the third stage is at a pressure of 12000±100 psi to obtain uniform nanofibers.

[0026] Further, in the step two, the concentration of the collagen homogenate is 50-70 mg / mL.

[0027] Further, in the step three, the sodium hyaluronate solution has a mass concentration of 0.5-5%, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or a range between any of the above values; preferably, the mass concentration of the sodium hyaluronate solution is 1%-4%.

[0028] Further, the mass ratio of sodium hyaluronate to sodium periodate is 1:0.5-1:2.

[0029] Further, in the step two, the sodium hyaluronate has a molecular weight range of 400-2600 kDa, and preferably, the sodium hyaluronate has a molecular weight greater than 1200 kDa.

[0030] Further, in the step three, the mass concentration of the oxidized sodium hyaluronate solution is 3%, and the mass ratio of the collagen homogenate to the oxidized sodium hyaluronate solution is 1:1-5:1, preferably 3:1. The cross-linking reaction temperature is 37°C, and the cross-linking reaction time is 1-3 h.

[0031] Further, in the step three, the washing step includes: dividing the solidified hydrogel into small gel pieces with a volume of 0.1-1 cm 3 , immersing them in a PBS solution, stirring for 2-4 h, centrifuging to remove the supernatant, and repeating the washing operation 3-5 times to obtain an initial hydrogel.

[0032] Further, in the step three, the sulfonated chitosan compounding step includes immersing the initial hydrogel in a sulfonated chitosan solution to obtain a sulfonated chitosan composite hydrogel by vacuum negative pressure infiltration; and the trehalose coating step includes placing the sulfonated chitosan composite hydrogel in a trehalose solution and treating it by vacuum negative pressure to obtain a hydrogel block.

[0033] Further, the sulfonated chitosan compounding step comprises: soaking the initial hydrogel block in PBS buffer at 3-5 DEG C, then treating with 0.1% EDTA solution, and then adding to 30-50 g / L sulfonated chitosan solution, soaking at a vacuum degree of -0.08+ / -0.01 MPa and a temperature of 3-5 DEG C for 1-3 h, then increasing the temperature to 25-27 DEG C and controlling the pH at 6.0+ / -0.1, constant temperature reaction, after the reaction, washing with PBS buffer to remove unbound sulfonated chitosan, and obtaining sulfonated chitosan compounded hydrogel.

[0034] As an example, the sulfonated chitosan compounding step comprises: soaking the initial hydrogel block in PBS buffer at 4 DEG C for 30 min, then treating with 0.1% EDTA solution for 5 min, then adding to 40 g / L sulfonated chitosan solution, soaking at a vacuum degree of -0.08+ / -0.01 MPa and a temperature of 4 DEG C for 2 h, then increasing the temperature to 26 DEG C and controlling the pH at 6.0+ / -0.1, constant temperature reaction for 12 h, after the reaction, washing with PBS buffer to remove unbound sulfonated chitosan, and obtaining sulfonated chitosan compounded hydrogel.

[0035] Further, the trehalose coating step comprises: placing the sulfonated chitosan compounded hydrogel in a mixed solution containing 25-35 g / L trehalose, 0.1% hydroxypropyl methyl cellulose and 0.1M PBS, controlling the temperature at 3-5 DEG C, treating under negative pressure for 2-4 h, and then standing at normal pressure for 1-3 h, and obtaining the hydrogel block.

[0036] As an example, the trehalose coating step comprises: placing the sulfonated chitosan compounded hydrogel in a mixed solution containing 30 g / L trehalose, 0.1% hydroxypropyl methyl cellulose and 0.1M PBS, controlling the temperature at 4 DEG C, treating under negative pressure of -0.1 MPa for 3 h, and then standing at normal pressure (4 DEG C) for 2 h, and obtaining the hydrogel block.

[0037] Further, the buffer is 0.9% sodium chloride or PBS buffer.

[0038] As an example, the hydrogel block obtained by the preparation method of the present application can be freeze-dried or subjected to microporous extrusion granulation to obtain gel microparticles with uniform particle size, and different products can be applied.

[0039] According to another aspect of the present application, the hydrogel prepared by the preparation method of the above-mentioned collagen hydrogel crosslinked by oxidized sodium hyaluronate is used in the preparation of long-acting implant preparations.

[0040] The hydrogel obtained by the present application is mainly used for cosmetic products, including but not limited to long-acting implant preparations for long-acting volume filling of facial tissues such as forehead, tear groove, apple muscle, nasolabial groove and mandible.

[0041] The present application has the following advantages:

[0042] The present application can reduce the particle size of collagen fibers, reduce the steric hindrance of cross-linking reaction, and make the cross-linking reaction more efficient, controllable and uniform by pretreating and high-pressure homogenizing collagen.

[0043] The present application solves the problem of dehydration and falling back of traditional collagen implant within 1-2 weeks by designing a triple hydration network, introducing HA hydrophilic groups through aldehyde-amino cross-linking, anchoring water molecules through ionic bonds of sulfonic acid groups, increasing the proportion of bound water, and forming a glassy hydration layer after coating with trehalose, thereby reducing the mobility of water molecules.

[0044] The present application can wash away free sodium hyaluronate that does not participate in cross-linking reaction by dynamic soaking in buffer solution and centrifugal cleaning, thereby avoiding the unexpected safety risk of free aldehyde groups.

[0045] The cross-linked collagen hydrogel prepared by the present application has the advantages of high support, resistance to collagenase degradation and high hydration, which avoids rapid dehydration and cohesion of collagen protein suspension, maintains good immediate filling effect, and also has long-term maintenance function. DETAILED DESCRIPTION

[0046] The embodiments of the present application are described below by specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the disclosure. Obviously, the described embodiments are part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0047] Collagen solution: Zhejiang Chongshan Biological Products Co., Ltd., 3mg / mL, containing telopeptide collagen solution (type I collagen);

[0048] Pepsin: Sigma-Aldrich, enzyme activity ≥500U / mg;

[0049] Sodium hyaluronate: Shandong Liyang Biological Products Technology Co., Ltd., molecular weight 1.2 million Da;

[0050] Sulfonated chitosan (SCS) solution: Sulfonated chitosan (Taian Jiangzhou Biological Technology Co., Ltd., JZ-20250402) is added to PBS and stirred magnetically for 2h to obtain 40g / L sulfonated chitosan solution;

[0051] Carboxymethyl chitosan solution: Carboxymethyl chitosan (Shanghai Yuanye Biotechnology Co., Ltd., Catalog No. S30948) was added to PBS and stirred magnetically for 2 h to obtain a 40 g / L carboxymethyl chitosan solution until it was transparent;

[0052] Trehalose: CAS 99-20-7;

[0053] Other reagents are commercially available on the market.

[0054] Example 1

[0055] The present embodiment provides a method for preparing a collagen hydrogel crosslinked by oxidized sodium hyaluronate:

[0056] (1) Collagen pretreatment

[0057] A 3 mg / ml collagen solution was added to a 0.5 M Na2HPO4 solution to adjust the pH to 4.5-6.0, stirred and precipitated for 6 h, and then placed at 4°C for 1.5 h. The mixture was centrifuged at 8000 rpm for 15 min, and the precipitate was washed with PBS solution 3 times to obtain a collagen fiber precipitate. The collagen fiber precipitate was resuspended in 0.3 M acetic acid, stirred at 4°C for 12 h, and then 2 M HCl was added dropwise. The pH was adjusted to 2.5±0.1 under magnetic stirring, and a pepsin solution (pre-cooled 0.1 M HCl) was added to a final concentration of 0.8% (w / w, enzyme / collagen). The mixture was shaken at 50 rpm at 4°C for 24 h. After the reaction was completed, 1 M NaOH solution was added to adjust the pH to 7 (to inactivate pepsin), and the reaction was terminated. The precipitate was obtained by centrifugation, resuspended in 0.05 M HCl solution, and then subjected to tangential flow ultrafiltration. The 0.05 M HCl solution was replaced 5-6 times under the following conditions: 100 kDa MWCO, 4°C, transmembrane pressure 0.8 bar, and flow rate 5 L / min. Finally, the pH was adjusted to 7 using 1 M NaOH solution, and the collagen was precipitated by centrifugation to obtain pepsin-pretreated collagen.

[0058] (2) Preparation of collagen homogenate

[0059] The pretreated collagen was diluted with PBS solution to a collagen solution of 60 mg / ml, stirred and mixed, and then subjected to high-pressure homogenization using a high-pressure homogenizer. The high-pressure homogenization was performed in three stages: the first stage pressure was 4000±100 psi, the second stage pressure was 8000±100 psi, and the third stage pressure was 12000±100 psi. The temperature was controlled at 4°C to obtain a collagen homogenate.

[0060] (3) Preparation of oxidized sodium hyaluronate

[0061] Sodium hyaluronate of 120 million Da was dissolved in purified water to prepare a 1.5% sodium hyaluronate solution. Sodium periodate was added according to a mass ratio of sodium hyaluronate to sodium periodate of 1:1.5. The solution was reacted at room temperature for 4 hours in the dark, followed by the addition of an equal volume of ethylene glycol to quench the reaction. The solution was stirred at room temperature for 1 hour. The reacted solution was concentrated by ultrafiltration to remove residual small molecular impurities. The concentrated sodium hyaluronate oxidized solution after ultrafiltration was frozen at -20°C for 4 hours. After vacuum freeze-drying, the freeze-dried product was stored at -20°C after being sealed.

[0062] (4) Crosslinking and gelation of sodium hyaluronate oxidized

[0063] The freeze-dried product of sodium hyaluronate oxidized was dissolved in a PBS solution to prepare a 3% sodium hyaluronate oxidized solution. Collagen homogenate was added according to a mass ratio of collagen homogenate to sodium hyaluronate oxidized solution of 3:1. After stirring uniformly, the solution was incubated at 37°C for 2 hours to form a solidified hydrogel.

[0064] The solidified hydrogel was divided into small pieces with a volume of 0.5 cm 3 The small pieces of hydrogel were soaked in a PBS solution and slowly stirred for 2 hours. The supernatant was discarded by centrifugation, and the washing operation was repeated three times to obtain the initial hydrogel pieces.

[0065] The initial hydrogel pieces were soaked in a PBS buffer at 4°C for 30 minutes, then treated with a 0.1% EDTA solution for 5 minutes, and then added to a 40 g / L sulfonated chitosan solution. The solution was soaked at a vacuum degree of -0.08±0.01 MPa and a temperature of 4°C for 2 hours, then the temperature was increased to 26°C and the pH was controlled at 6.0±0.1. The solution was oscillated (50 rpm) for 12 hours of constant temperature reaction. After the reaction was completed, the solution was washed with a PBS buffer for three times to remove the unbound sulfonated chitosan, and a sulfonated chitosan composite hydrogel was obtained.

[0066] The sulfonated chitosan composite hydrogel was placed in a mixed solution containing 30 g / L trehalose, 0.1% hydroxypropyl methyl cellulose, and 0.1M PBS. The temperature was controlled at 4°C, and the solution was treated at a negative pressure of -0.1 MPa for 3 hours. After standing at normal pressure for 2 hours, a hydrogel piece was obtained, which was the collagen hydrogel.

[0067] (5) Preparation of implant preparation finished product

[0068] The hydrogel piece was extruded and granulated by a microparticle granulator with a micropore size of 200µm. The piece was filled into a pre-filled needle tube at 1 g / branch. After filling, a rubber piston was added for sealing, and an implant preparation finished product was obtained.

[0069] Example 2

[0070] The present embodiment provides a method for preparing a collagen hydrogel crosslinked by sodium hyaluronate oxidized:

[0071] Adjust the concentration of the sodium hyaluronate solution in step (4): dissolve the sodium hyaluronate lyophilizate with PBS solution to prepare a 1% sodium hyaluronate solution, add the collagen homogenate solution according to the mass ratio of collagen homogenate solution:sodium hyaluronate solution 3:1, stir uniformly, and then incubate at 37°C for 2h to form a solidified hydrogel.

[0072] The remaining steps are the same as in Example 1.

[0073] Example 3

[0074] This example provides a method for preparing a collagen hydrogel crosslinked by sodium hyaluronate:

[0075] Adjust the concentration of the sodium hyaluronate solution in step (4): dissolve the sodium hyaluronate lyophilizate with PBS solution to prepare a 5% sodium hyaluronate solution, add the collagen homogenate solution according to the mass ratio of collagen homogenate solution:sodium hyaluronate solution 3:1, stir uniformly, and then incubate at 37°C for 2h to form a solidified hydrogel.

[0076] The remaining steps are the same as in Example 1.

[0077] Example 4

[0078] This example provides a method for preparing a collagen hydrogel crosslinked by sodium hyaluronate:

[0079] Adjust the preparation method of the implant preparation product in step (5): after extrusion granulation, do not perform pre-canned needle filling but use a conventional lyophilization method (trehalose as a lyophilization protective agent, freezing conditions: -80°C rapid freezing, -40°C / 0.1mbar), to obtain a lyophilized hydrogel, with a pore size distribution of 10-50μm micropores accounting for 70% and >100μm macropores accounting for 30%, which can be reconstituted for use.

[0080] The remaining steps are the same as in Example 1.

[0081] Comparative Example 1

[0082] This comparative example provides a method for preparing a collagen hydrogel crosslinked by sodium hyaluronate:

[0083] Step (1) does not perform pepsin pretreatment, and the remaining steps are the same as in Example 1.

[0084] Comparative Example 2

[0085] This comparative example provides a method for preparing a collagen hydrogel crosslinked by sodium hyaluronate:

[0086] Step (2) without high pressure homogenization, and the rest is completely identical with Example 1.

[0087] Comparative Example 3

[0088] This comparative example provides a method for preparing a collagen hydrogel crosslinked by oxidized sodium hyaluronate:

[0089] Step (3) without oxidizing sodium hyaluronate, and the rest is completely identical with Example 1.

[0090] Comparative Example 4

[0091] This comparative example provides a method for preparing a collagen hydrogel crosslinked by oxidized sodium hyaluronate:

[0092] Step (4) without sulfonated chitosan complex treatment, and the rest is completely identical with Example 1.

[0093] Comparative Example 5

[0094] This comparative example provides a method for preparing a collagen hydrogel crosslinked by oxidized sodium hyaluronate:

[0095] Step (4) without trehalose coating, and the rest is completely identical with Example 1.

[0096] Comparative Example 6

[0097] This comparative example provides a method for preparing a collagen hydrogel crosslinked by oxidized sodium hyaluronate:

[0098] Step (4) with carboxymethyl chitosan complex treatment, and the rest is completely identical with Example 1.

[0099] Experimental Example 1

[0100] The relevant parameters of the examples and comparative examples are tested:

[0101] 1. Collagen content: The biuret method is used to determine the collagen content. 1.0 g of hydrogel is precisely weighed and added into 0.5 M acetic acid solution to dissolve and dilute to 10 ml. After centrifugation, the supernatant is taken as the test solution. Bovine serum albumin is taken as the control group, and 10 mg is precisely weighed and dissolved in water to prepare a standard control solution. 0.0 ml, 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, and 1.0 ml of the control solution are precisely measured and placed in a stoppered test tube, and each is added with water to 1.0 ml. Then, 4.0 ml of biuret reagent is added, and immediately mixed, and placed at room temperature for 30 minutes. The absorbance is measured at a wavelength of 540 nm on an ultraviolet spectrophotometer. In addition, 1.0 ml of the test solution is precisely measured and operated in the same manner. The protein concentration in the test solution is calculated from the linear regression equation, and multiplied by the dilution factor to obtain the result.

[0102] 2. Cross-linking degree: The cross-linking degree was tested by the method of trinitrobenzenesulfonic acid. 0.25 g of cross-linked collagen hydrogel was weighed and supplemented with purified water to 0.5 g. The mixture was dissolved in 1 mL of 4% Na2CO3 and 1 mL of 0.5% 2,4,6-trinitrobenzenesulfonic acid (TNBS) solution. The mixture was stirred at 40°C at a speed of 220 r / min for 4 h. 0.5 g of purified water and 0.25 g of uncross-linked collagen hydrogel were subjected to the same operation as the blank control group and the uncross-linked control group, respectively. 3 mL of 6 mol / L hydrochloric acid solution was added and placed in a hot pressure sterilizer at 120°C for 1 h. The obtained solution was diluted with 5 mL of water and extracted with ether 3 times, each time with 20 mL. The ether layer was discarded, and 5 mL of the water phase was taken. The water bath was heated for 15 min, cooled to room temperature, diluted with 15 mL of water, and shaken well. The absorbance was measured at a wavelength of 346 nm.

[0103] ;

[0104] In the formula, W0 is the absorbance of the blank control group; W1 is the absorbance of the uncross-linked collagen hydrogel; and W2 is the absorbance of the cross-linked collagen hydrogel.

[0105] 3. Oxidized sodium hyaluronate residue detection: The carbazole color reaction method was used. 0.1 g of hydrogel was taken and immersed in 9 g of PBS solution at 37°C for 24 hours. After centrifugation, 1 mL of the supernatant was taken as the test solution. 10 mg of glucuronic acid was dissolved in water to make 100 mL as the standard control solution. 0.0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the control solution were accurately measured and placed in a stoppered test tube, each added with water to 1.0 mL. The standard liquid tubes and sample tubes were placed in an ice water bath, and 0.025 mol / L sodium tetraborate sulfate 5 mL was slowly added to each tube with an acid burette. After mixing, it was placed in a boiling water bath for 20 min and then taken out and cooled to room temperature. 0.2 mL of carbazole ethanol solution was added to each test tube, shaken well, and then placed at room temperature for 2 h. The absorbance of each standard tube and sample tube was measured at 550 nm by ultraviolet spectrophotometry. The content of glucuronic acid in the test sample was calculated from the linear regression equation.

[0106] ;

[0107] ρ - the content of glucuronic acid in the sample tube, in units of micrograms per milliliter (µg / mL);

[0108] m2 - the total mass of collagen hydrogel and PBS solution, in units of milligrams (mg);

[0109] m1 - the mass of collagen hydrogel, in units of micrograms (µg).

[0110] 4. Thermal denaturation temperature Tm value: 5.0 mg of the collagen hydrogel was precisely weighed into a test aluminum pan, and the temperature was raised at a rate of 2 ℃ / min under a nitrogen atmosphere. The temperature value corresponding to the absorption peak of the sample was recorded at a temperature range of 25-100 ℃ at a temperature rise rate of 5 ℃ / min.

[0111] 5. Rheological properties: Dynamic rheological measurements were performed using a rheometer using a strain amplitude sweep mode, with a fixed frequency of 10 rad / s and a strain range of 0.1-1000%. The hydrogel was measured in the linear viscoelastic region at a frequency range of 1-100 rad / s, a constant strain of 1%, and a temperature of 25 ℃. The storage modulus (G') and loss modulus (G'') were recorded.

[0112] 6. Hydrogel volume loss rate experiment: After measuring the volume of the collagen hydrogel, it was placed in a 37 ℃ PBS phosphate buffer for 7 days, and the volume shrinkage rate of the hydrogel was calculated.

[0113] 7. Cytotoxicity experiment: The collagen hydrogel was extracted with 0.9% normal saline as the test solution, and the CCK-8 method was used to determine the effect of the extract on cell proliferation rate.

[0114] 8. In vitro degradation experiment: The collagenase was used as the degradation enzyme to perform the in vitro degradation test according to the methods of YY / T0473 and GB / T16886.13.

[0115] The specific results are shown in Tables 1 and 2.

[0116] Table 1

[0117]

[0118] Analysis of experimental results:

[0119] The cross-linking degree of the collagen hydrogel is positively correlated with the concentration of sodium oxidized hyaluronic acid. An increase in cross-linking degree helps the collagen fibers to cross-link to form a more stable network structure, improving the enzyme resistance of collagen protein and thus improving the maintenance effect of soft tissue filling. However, with further increase in the concentration of sodium oxidized hyaluronic acid solution, sodium oxidized hyaluronic acid residues may appear in the collagen protein hydrogel, causing difficulty in removal in the actual production process and the risk of free aldehyde group residues, so the concentration of sodium oxidized hyaluronic acid solution is preferably 1%-4%. The collagen homogenate after high-pressure homogenization has a small particle size and low steric hindrance, which is beneficial to the occurrence of cross-linking reaction. The sulfonated chitosan complexation and trehalose coating are beneficial to enhancing the thermal stability of the hydrogel through ionic bonds and hydrogen bonds.

[0120] Table 2

[0121]

[0122] Experimental result analysis:

[0123] The mechanical strength (storage modulus G' and loss modulus G''), thermal stability and enzyme resistance of the collagen hydrogel crosslinked by oxidized sodium hyaluronate were greatly improved compared with the uncrosslinked collagen hydrogel. By comparing Example 1 with Comparative Example 4 and Comparative Example 5, the hydrogel further solidified by sulfonated chitosan and trehalose enhanced the intermolecular force of the collagen hydrogel, improved its hydration capacity, and was beneficial to reduce water absorption and loss of the collagen hydrogel in the physiological environment after implantation. By comparing Example 1 with Comparative Example 6, sulfonated chitosan had stronger ionic bonding capacity than carboxymethyl chitosan, could more effectively anchor water molecules through ionic bonds, increase the proportion of bound water, and form a more dense network structure, thereby providing better mechanical support and anti-dehydration capacity, and was beneficial to reduce water absorption and loss of the collagen hydrogel in the physiological environment after implantation.

[0124] Experimental Example 2

[0125] In vivo injection experiment of rats:

[0126] After normal feeding of SD rats for one week, the in vivo injection experiment of rats was carried out. After the rats were anesthetized with chloral hydrate, the back was shaved and disinfected with 75% alcohol, and 4 injection points were selected symmetrically along the middle of the back, and 1 mL of collagen hydrogel was injected at each injection point. After injection, the rats were normally fed, and the rat back was observed for swelling. The dissection observation time points of implantation for 1 week, implantation for 1 month, implantation for 3 months and implantation for 6 months were set, and the rats were anesthetized and sacrificed by cervical dislocation for dissection, and the average volume of collagen hydrogel residue was recorded, and the results are shown in Table 3.

[0127] Table 3

[0128]

[0129] Experimental result analysis:

[0130] According to the degradation results in rats, the collagen hydrogel crosslinked by the oxidized sodium hyaluronate of the application can obviously improve the phenomenon of dehydration and cohesion of the collagen hydrogel implanted for 1 week, and the crosslinked collagen hydrogel has a longer maintenance time in rats, which is consistent with the trend of improved enzyme resistance after crosslinking. In addition, through comparative analysis, it can be known that the particle size of the collagen hydrogel is also an important factor affecting its mechanical strength and maintenance time, and the gel with large particle size has better support and maintenance effect. The phenomenon of slight skin redness occurred in rats implanted in early stage in Example 3, and according to the results in Table 2, the cytotoxicity occurred in Example 3 due to the residual free aldehyde group, and the skin has certain irritability. The phenomenon of slight skin redness occurred in rats implanted in early stage in Comparative Example 1, and the collagen hydrogel in Comparative Example 1 is not treated by pepsin, and the end peptide at both ends of the collagen peptide chain has certain immunogenicity, and the skin as a sensitive part will have an allergic reaction, so the collagen hydrogel after the end peptide is removed by pepsin is more preferred.

[0131] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application claimed.

Claims

1. A method for preparing a collagen hydrogel crosslinked with oxidized sodium hyaluronate, characterized by, It comprises the following steps: Step one, collagen pretreatment The collagen solution is adjusted to the isoelectric point, stirred and precipitated, and the precipitate is obtained by centrifugation, and the buffer is repeatedly washed by centrifugation to obtain the collagen crude fiber precipitate; The collagen crude fiber precipitate is redissolved with acetic acid solution, and pepsin solution is added for reaction, and after the reaction is completed, it is purified to obtain pretreated collagen; Step two, preparation of collagen homogenate The pretreated collagen is dissolved in buffer and subjected to high pressure homogenization with a high pressure homogenizer to obtain collagen homogenate; Step three, preparation of oxidized sodium hyaluronate The sodium hyaluronate solution is subjected to aldehyde group reaction with sodium periodate, and after the reaction is terminated by adding ethylene glycol, it is concentrated by ultrafiltration, freeze-dried to obtain oxidized sodium hyaluronate; Step four, preparation of hydrogel The oxidized sodium hyaluronate is dissolved in buffer to obtain an oxidized sodium hyaluronate solution, and the collagen homogenate is added for cross-linking reaction to form a solidified hydrogel, and the residual unreacted oxidized sodium hyaluronate is removed by washing to obtain an initial hydrogel; After the initial hydrogel is compounded with sulfonated chitosan and coated with trehalose, a hydrogel block is obtained, which is the collagen hydrogel.

2. A process for the preparation of a collagen hydrogel crosslinked with sodium hyaluronate according to claim 1, characterized in that, In step one, the collagen is extracted from animal Achilles tendon or animal skin; the concentration of the collagen solution is 1-6 mg / mL; the pH is adjusted to 4.5-6.0; the pH adjuster is one or more of sodium hydroxide, disodium hydrogen phosphate, sodium citrate, and citric acid.

3. The process for preparing a collagen hydrogel crosslinked with sodium hyaluronate according to claim 1, characterized by, The collagen pretreatment step comprises adjusting the pH of the collagen solution to 4.5-6.0, stirring and precipitating, obtaining the precipitate by centrifugation, and repeatedly washing the precipitate with buffer to obtain the collagen crude fiber precipitate; the collagen crude fiber precipitate is redissolved with acetic acid solution, stirred at 3-5℃, then the pH is adjusted to 2-3, pepsin solution is added, and constant temperature reaction is carried out; after the reaction is completed, the reaction is terminated by adjusting the pH to inactivate the pepsin; the pretreated collagen is obtained by tangential flow ultrafiltration purification; wherein the centrifugal speed is 6000-10000 rpm, and the centrifugal time is 15-20 min.

4. The process for preparing a collagen hydrogel crosslinked with sodium hyaluronate according to claim 1, characterized by, In step two, the high pressure homogenization is carried out in three stages, the first stage pressure is 4000±100 psi; the second stage pressure is 8000±100 psi; the third stage pressure is 12000±100 psi; the high pressure homogenization temperature is controlled at 2-8℃; the concentration of the collagen homogenate is 50-70 mg / mL.

5. The process for preparing a collagen hydrogel crosslinked with sodium hyaluronate according to claim 1, characterized by, In step three, the mass concentration of the sodium hyaluronate solution is 0.5-5%; the mass ratio of sodium hyaluronate to sodium periodate is 1:0.5-1:

2.

6. The process for preparing a collagen hydrogel crosslinked with sodium hyaluronate according to claim 1, characterized by, In step four, the mass concentration of the oxidized sodium hyaluronate solution is 3%; the mass ratio of the collagen homogenate to the oxidized sodium hyaluronate solution is 1:1-5:1; the cross-linking reaction temperature is 37℃, and the cross-linking reaction time is 1-3 h.

7. The process for preparing a collagen hydrogel crosslinked with sodium hyaluronate according to claim 1, characterized by, In the fourth step, the washing step comprises: dividing the solidified hydrogel into small pieces with a volume of 0.1-1 cm 3 , soaking in PBS solution, stirring for 2-4 h, centrifuging to remove supernatant, and repeating the washing operation 3-5 times to obtain the initial hydrogel.

8. The process for preparing a collagen hydrogel crosslinked with sodium hyaluronate according to claim 1, characterized by, In the fourth step, the step of sulfonated chitosan compounding comprises immersing the initial hydrogel into a sulfonated chitosan solution to obtain a sulfonated chitosan compounded hydrogel by vacuum negative pressure infiltration; and the step of trehalose coating comprises placing the sulfonated chitosan compounded hydrogel into a trehalose solution and treating by vacuum negative pressure to obtain a hydrogel block.

9. The method for preparing a collagen hydrogel crosslinked with oxidized sodium hyaluronate according to claim 8, characterized in that, In the step of sulfonated chitosan compounding, the initial hydrogel block is soaked in a PBS buffer at 3-5 ℃, then treated with a 0.1% EDTA solution, and then added into a 30-50 g / L sulfonated chitosan solution, and soaked at a vacuum degree of -0.08±0.01 MPa and a temperature of 3-5 ℃ for 1-3 h, then the temperature is increased to 25-27 ℃ and the pH is controlled at 6.0±0.1 for constant temperature reaction, and after the reaction is completed, the PBS buffer is used for rinsing to remove the unbound sulfonated chitosan, thereby obtaining a sulfonated chitosan compounded hydrogel; and in the step of trehalose coating, the sulfonated chitosan compounded hydrogel is placed into a mixed solution containing 25-35 g / L trehalose, 0.1% hydroxypropyl methyl cellulose and 0.1M PBS, the temperature is controlled at 3-5 ℃, and after being treated under negative pressure for 2-4 h, it is placed at normal pressure for 1-3 h, thereby obtaining a hydrogel block.

10. A collagen hydrogel crosslinked with sodium hyaluronate oxidized, characterized by, The hydrogel block is prepared by the preparation method of any one of claims 1-9.

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