Dialdehyde polysaccharide biological cross-linking agent, collagen implant and preparation method of collagen implant

Abstract

The invention relates to the technical field of biomedical materials, and provides a dialdehyde polysaccharide biological cross-linking agent and a collagen cross-linking method thereof. The preparation method of the dialdehyde polysaccharide biological cross-linking agent comprises the following steps: S1, adding polysaccharide into molten imidazole, and heating and stirring to obtain a polysaccharide solution; adding acetylcysteine into the polysaccharide solution, stirring and mixing, and heating and reacting at 30-50 DEG C for 3-7 hours; s2, after the reaction is finished, adding ethanol to precipitate a product, then filtering and washing to remove unreacted substances, and then drying to obtain acetylcysteine modified polysaccharide; and S3, dispersing the acetylcysteine modified polysaccharide in a sodium periodate aqueous solution, uniformly stirring, reacting for 5-7 hours in a dark place at 30-50 DEG C, carrying out suction filtration after the reaction is finished to obtain a precipitation product, filtering, washing, and drying to obtain the acetylcysteine modified dialdehyde polysaccharide. The multifunctional group of acetylcysteine can react with multiple groups in polysaccharide molecules to form a uniform cross-linked network. The uniform cross-linked structure is beneficial to improving the physical properties and compatibility of the hydrogel.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a dialdehyde polysaccharide biocrosslinking agent, a collagen implant and a preparation method thereof. Background Art

[0002] Fillers are medical devices sold for cosmetic and plastic surgery (reconstruction, etc.) purposes and are used by injecting them into the skin tissue at wrinkles and depressions. Most fillers are injected into the skin tissue to improve wrinkles or depressions by increasing the volume of the substance itself and thereby increasing the volume of the skin tissue.

[0003] Collagen is an important structural protein in organisms and is widely used in the biomedical field. Its hydrolyzate (gelatin) has better biodegradability, biocompatibility, weak antigenicity and unique fiber formation properties compared to other biopolymers. However, untreated collagen gels usually show poor material properties. In order to improve the performance of collagen, researchers have tried to enhance its stability and mechanical properties by physical or chemical cross-linking methods, such as using glutaraldehyde and oxidized polysaccharides as cross-linking agents. However, the defect of existing cross-linking agents is that due to rapid cross-linking reactions, electrostatic interactions and hydrogen bonding, collagen will inevitably flocculate locally once it comes into contact with the cross-linking agent. This flocculation phenomenon affects the uniform spatial network structure inside the collagen hydrogel, thereby affecting the physicochemical properties of the hydrogel. To this end, a method for cross-linking collagen with high mechanical strength and biocompatibility is needed. Summary of the invention

[0004] In view of this, the present invention proposes a dialdehyde polysaccharide biocrosslinking agent, a collagen implant and a preparation method thereof, aiming to solve the problem in the prior art that the collagen stability and mechanical properties are insufficient, resulting in unsatisfactory filling effects.

[0005] The technical solution of the present invention is achieved as follows: On the one hand, the present invention provides a method for preparing a dialdehyde polysaccharide biocrosslinking agent, comprising the following steps:

[0006] S1, adding polysaccharide to molten imidazole, heating and stirring to obtain a polysaccharide solution; adding acetylcysteine ​​to the polysaccharide solution, stirring and mixing, and heating at 30-50° C. for reaction for 3-7 hours;

[0007] S2, after the reaction is completed, ethanol is added to precipitate the product, and then unreacted substances are removed by filtration and washing, followed by drying to obtain acetylcysteine-modified polysaccharides;

[0008] S3, dispersing the polysaccharide modified with acetylcysteine ​​in a sodium periodate aqueous solution, stirring evenly, reacting at 30-50°C in the dark for 5-7 hours, and filtering to obtain a precipitated product after the reaction is completed, and then filtering, washing, and drying to obtain the dialdehyde polysaccharide modified with acetylcysteine.

[0009] The multifunctional groups of acetylcysteine ​​can react with various groups in polysaccharide molecules to form a uniform cross-linked network. This uniform cross-linked structure helps to improve the physical properties and compatibility of the hydrogel. Specifically:

[0010] The carboxyl group in acetylcysteine ​​can form hydrogen bonds and ionic bonds with hydroxyl or amino groups in polysaccharide molecules. The carboxyl group in acetylcysteine ​​is introduced during the polysaccharide modification process, which makes the dialdehyde polysaccharide have better hydrophilicity and biocompatibility, making it easier to interact with cells and tissues in the biological environment. In addition, the carboxyl group can also form hydrogen bonds and ionic bonds with the amino group in collagen, promote the interaction between the material and biological tissues, and reduce immune rejection reactions. The introduction of acetyl groups can reduce the polarity of polysaccharides, making them more stable under physiological conditions, reducing nonspecific interactions with other biological molecules, and thus improving the compatibility of materials. The thiol group in acetylcysteine ​​can form covalent bonds with reactive groups (such as aldehyde groups) in polysaccharides, especially by forming disulfide bonds or forming Schiff bases with aldehyde groups. This cross-linking reaction increases the cross-linking density of polysaccharides, significantly enhances the mechanical strength and stability of collagen hydrogels, and makes them more resilient and elastic under external forces.

[0011] In addition, the antioxidant properties of acetylcysteine ​​help protect collagen and polysaccharide structures from damage by oxidative stress, improve the durability and stability of the material, and slow down its degradation rate. Due to the moderate cross-linking reaction rate of dialdehyde polysaccharides, a uniform spatial network structure can be formed in the collagen hydrogel, avoiding the local flocculation phenomenon caused by traditional cross-linking agents. This uniform cross-linking structure helps improve the physical properties of the hydrogel.

[0012] On the basis of the above technical solution, preferably, in step S1, acetylcysteine, EDC and NHS are added to the polysaccharide solution.

[0013] Specifically, EDC (N-hydroxysuccinimide) can activate the carboxyl groups on polysaccharides to form active esters, making them more easily react with the amino groups on acetylcysteine. NHS (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) is used together with EDC. NHS can stabilize the intermediates, make the reaction more efficient and faster, and reduce side reactions. The combination of EDC and NHS can enhance the modification effect of acetylcysteine ​​on polysaccharides, thereby obtaining better material performance in subsequent applications.

[0014] On the basis of the above technical solution, preferably, the mass ratio of EDC:NHS:polysaccharide is 0.5-1.2:0.5-1.2:1.

[0015] On the basis of the above technical solution, preferably, the mass ratio of acetylcysteine ​​to polysaccharide is 0.025-0.075:1.

[0016] On the basis of the above technical solution, preferably, the mass ratio of periodic acid to polysaccharide is 0.5-1.5:1.

[0017] On the basis of the above technical solution, preferably, the polysaccharide is one or a mixture of sodium alginate, xanthan gum, pullulan, dextran, chitosan, guar gum and carrageenan.

[0018] On the other hand, the present invention also provides a method for preparing a collagen implant using a dialdehyde polysaccharide biocrosslinking agent, comprising the following steps:

[0019] S1, dissolving the dialdehyde polysaccharide modified with acetylcysteine ​​in a DPBS buffer solution to obtain a 4-20 mg / mL dialdehyde polysaccharide solution; dissolving collagen in an acetic acid solution to obtain a 30-60 mg / mL collagen solution, and then mixing the collagen solution with the dialdehyde polysaccharide solution to obtain a collagen-dialdehyde polysaccharide mixed solution;

[0020] S2, taking sodium bicarbonate and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), dissolving them in sodium hydroxide solution to obtain a neutralized solution;

[0021] S3, inject the collagen-dialdehyde polysaccharide mixed solution into the injection tube A of the double syringe, mix the neutralization solution and DPBS buffer and inject them into the injection tube B, and push the injection 25-35 times at the same time to finally obtain the cross-linked collagen implant.

[0022] On the basis of the above technical solution, preferably, in step S1, insulin-like growth factor and epidermal growth factor are added to the collagen solution.

[0023] Insulin-like growth factor (IGF) can enhance cell growth and tissue regeneration, and can shorten healing time. In addition, by promoting cell activity and tissue integration, IGF can also significantly improve the biological properties of collagen implants, improve their integration and effect in the body, and improve skin elasticity. Epidermal growth factor (EGF) promotes the proliferation and migration of keratinocytes and other epidermal cells by binding to its receptors, accelerating epidermal renewal and regeneration.

[0024] Insulin-like growth factor and epidermal growth factor have synergistic effects, jointly promoting cell proliferation, migration and tissue remodeling. Insulin-like growth factor mainly acts on deep tissues and matrix, while epidermal growth factor is more focused on promoting epithelial cells and superficial cells. Adding these two growth factors can improve the biocompatibility and functional performance of implants, shorten healing time, and improve repair quality, and is suitable for various trauma and regenerative medical fields.

[0025] On the basis of the above technical solution, preferably, the dosage of insulin-like growth factor and epidermal growth factor is 0.01%-1% of the mass of the collagen solution.

[0026] On the basis of the above technical scheme, preferably, in step S1, the mass ratio of the dialdehyde polysaccharide solution: collagen solution is 5-10:1; in step S2, the concentration of sodium bicarbonate is 1.0%-2.5%, the concentration of HEPES is 100-200mmol / L, and the concentration of sodium hydroxide is 0.5-1.0mol / L; in step S3, the neutralization solution and DPBS buffer are mixed in a volume ratio of 1-1.5:1.

[0027] The dialdehyde polysaccharide biocrosslinking agent, collagen implant and preparation method thereof of the present invention have the following beneficial effects compared with the prior art:

[0028] (1) The multifunctional groups (carboxyl, acetyl, and thiol) of acetylcysteine ​​can react with various groups in polysaccharide molecules to form a uniform cross-linked network. This uniform cross-linked structure helps to improve the physical properties and compatibility of collagen hydrogels.

[0029] (2) Adding EDC during the preparation of dialdehyde polysaccharide biocrosslinkers can activate the carboxyl groups on the polysaccharide to form active esters, which are easier to react with the amino groups on acetylcysteine. When NHS is used together with EDC, NHS can stabilize the intermediates, making the reaction more efficient and rapid, and reducing side reactions. The combination of EDC and NHS can enhance the modification effect of acetylcysteine ​​on polysaccharides, thereby obtaining better material properties in subsequent applications.

[0030] (3) Adding insulin-like growth factor and epidermal growth factor during the preparation of collagen implants can promote cell proliferation, migration and tissue remodeling, and can also improve the biocompatibility and functional performance of the implants, shorten the healing time, and improve the quality of repair. It is suitable for various trauma and regenerative medicine fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 This is a physical picture of the collagen implant prepared by the present invention. DETAILED DESCRIPTION

[0033] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1

[0035] The preparation method of the dialdehyde polysaccharide biocrosslinking agent of this embodiment comprises the following steps:

[0036] S1, adding 10 g of sodium alginate to molten imidazole of 10 times the mass (100 g), heating and stirring at 100° C. to obtain a polysaccharide solution; then adding 0.5 g of acetylcysteine ​​to the sodium alginate solution, the mass ratio of acetylcysteine ​​to sodium alginate being 0.05:1, stirring and mixing, heating at 40° C. to react for 6 h;

[0037] S2, after the reaction is completed, adding excess ethanol to precipitate the product, then filtering to obtain the precipitate, washing and filtering 5 times to remove unreacted substances, and then drying at 40°C to obtain acetylcysteine-modified polysaccharide;

[0038] S3, dispersing the polysaccharide modified with acetylcysteine ​​prepared in step S2 in a sodium periodate aqueous solution with a mass concentration of 10%, wherein the mass ratio of periodate to sodium alginate is 1:1, stirring evenly, reacting at 40° C. in the dark for 6 hours, and filtering to obtain a precipitated product after the reaction is completed, and then washing and filtering 5 times and drying to obtain the dialdehyde polysaccharide modified with acetylcysteine.

[0039] The method for preparing a collagen implant using a dialdehyde polysaccharide biocrosslinking agent comprises the following steps:

[0040] S1, dissolving the prepared acetylcysteine-modified dialdehyde polysaccharide in 10×DPBS buffer solution to obtain 50 mL of a 20 mg / mL dialdehyde polysaccharide solution; dissolving collagen in 1 mmol / L acetic acid solution to obtain 50 mL of a 50 mg / mL collagen solution, and then mixing the dialdehyde polysaccharide solution and the collagen solution in a mass ratio of 8:1 to obtain a collagen-dialdehyde polysaccharide mixed solution;

[0041] S2, take appropriate amount of sodium bicarbonate and HEPES, dissolve them in 0.8 mol / L sodium hydroxide solution, and prepare 30 mL of neutralization solution; wherein the mass concentration of sodium bicarbonate is 2.2%, and the concentration of HEPES is 180 mmol / L

[0042] S3, inject the collagen-dialdehyde polysaccharide mixed solution into the injection tube A of the double syringe with a specification of 10 ml, mix the neutralization solution and DPBS buffer in a volume ratio of 1:1 and inject into the injection tube B of the double syringe, and push injection 30 times at the same time to finally obtain the cross-linked collagen implant.

[0043] Example 2

[0044] Compared with Example 1, Example 2 differs in that EDC and NHS are added to the polysaccharide solution, and the specific steps are as follows:

[0045] The preparation method of the dialdehyde polysaccharide biocrosslinking agent of this embodiment comprises the following steps:

[0046] S1, add 10g of sodium alginate to 10 times the mass (100g) of molten imidazole, heat and stir at 100°C to obtain a polysaccharide solution; then add 0.5g of acetylcysteine, 8g of EDC and 8g of NHS to the sodium alginate solution, the mass ratio of acetylcysteine: EDC: NHS: sodium alginate is 0.05:0.8:0.8:1, stir and mix, and heat to react at 40°C for 6h.

[0047] Steps S2, S3, and the method for preparing the collagen implant using the dialdehyde polysaccharide biocrosslinking agent are the same as those in Example 1.

[0048] Example 3

[0049] The difference between this embodiment and embodiment 2 is that insulin-like growth factor and epidermal growth factor are added when preparing the collagen implant using dialdehyde polysaccharide biocrosslinking agent. The specific method is as follows:

[0050] The preparation method of the dialdehyde polysaccharide biocrosslinking agent is the same as that in Example 2.

[0051] The method for preparing a collagen implant using a dialdehyde polysaccharide biocrosslinking agent comprises the following steps:

[0052] S1, dissolving the prepared acetylcysteine-modified dialdehyde polysaccharide in 10×DPBS buffer solution to obtain 50 mL of a 20 mg / mL dialdehyde polysaccharide solution; dissolving collagen in 1 mmol / L acetic acid solution to obtain 50 mL of a 50 mg / mL collagen solution, adding 0.08% of insulin-like growth factor and 0.08% of epidermal growth factor by mass of the collagen solution, stirring evenly to obtain a collagen solution mixed with growth factors; then mixing the dialdehyde polysaccharide solution and the collagen solution in a mass ratio of 8:1 to obtain a collagen-dialdehyde polysaccharide mixed solution;

[0053] S2 and S3 are the same as in Example 2.

[0054] Example 4

[0055] The preparation method of the dialdehyde polysaccharide biocrosslinking agent of this embodiment comprises the following steps:

[0056] S1, add 10g chitosan to molten imidazole with a mass of 10 times (100g), heat and stir at 100°C to obtain a polysaccharide solution; then add 0.25g acetylcysteine, 5g EDC and 5g NHS to the chitosan solution, the mass ratio of acetylcysteine: EDC: NHS: chitosan is 0.025:0.5:0.5:1, stir and mix, and heat at 30°C to react for 7h.

[0057] S2, after the reaction is completed, excess ethanol is added to precipitate the product, and then the precipitate is filtered to obtain the precipitate, which is washed and filtered 5 times to remove the unreacted substances, and then dried at 40°C to obtain the acetylcysteine-modified polysaccharide.

[0058] S3, dispersing the polysaccharide modified with acetylcysteine ​​prepared in step S2 in a sodium periodate aqueous solution with a mass concentration of 10%, wherein the mass ratio of periodate to chitosan is 0.5:1, stirring evenly, reacting at 30° C. in the dark for 5 hours, and filtering to obtain a precipitated product after the reaction is completed, and then washing and filtering 5 times and drying to obtain the dialdehyde polysaccharide modified with acetylcysteine.

[0059] The method for preparing a collagen implant using a dialdehyde polysaccharide biocrosslinking agent comprises the following steps:

[0060] S1, dissolving the prepared acetylcysteine-modified dialdehyde polysaccharide in 10×DPBS buffer solution to obtain 50 mL of a 4 mg / mL dialdehyde polysaccharide solution; dissolving collagen in 1 mmol / L acetic acid solution to obtain 50 mL of a 30 mg / mL collagen solution, adding 0.01% of insulin-like growth factor and 0.01% of epidermal growth factor by mass of the collagen solution, stirring evenly to obtain a collagen solution mixed with growth factors; then mixing the dialdehyde polysaccharide solution and the collagen solution in a mass ratio of 5:1 to obtain a collagen-dialdehyde polysaccharide mixed solution;

[0061] S2, take appropriate amounts of sodium bicarbonate and HEPES, dissolve them in 0.5 mol / L sodium hydroxide solution, and prepare 30 mL of neutralization solution; wherein the mass concentration of sodium bicarbonate is 1%, and the concentration of HEPES is 100 mmol / L;

[0062] S3, inject the collagen-dialdehyde polysaccharide mixed solution into the injection tube A of the double syringe with a specification of 10 ml, mix the neutralization solution and DPBS buffer in a volume ratio of 1.5:1 and inject into the injection tube B of the double syringe, and push injection 25-35 times at the same time to finally obtain the cross-linked collagen implant.

[0063] Example 5

[0064] The preparation method of the dialdehyde polysaccharide biocrosslinking agent of this embodiment comprises the following steps:

[0065] S1, add 10g of dextran to molten imidazole with a mass of 10 times (100g), heat and stir at 100°C to obtain a polysaccharide solution; then add 0.75g of acetylcysteine, 12g of EDC and 12g of NHS to the dextran solution, the mass ratio of acetylcysteine: EDC: NHS: dextran is 0.075: 1.2: 1.2: 1, stir and mix, and heat to react at 50°C for 3h.

[0066] S2, after the reaction is completed, excess ethanol is added to precipitate the product, and then the precipitate is filtered to obtain the precipitate, which is washed and filtered 5 times to remove the unreacted substances, and then dried at 40°C to obtain the acetylcysteine-modified polysaccharide.

[0067] S3, dispersing the polysaccharide modified with acetylcysteine ​​prepared in step S2 in a sodium periodate aqueous solution with a mass concentration of 10%, with a mass ratio of periodate to dextran being 1.5:1, stirring evenly, reacting at 50° C. in the dark for 7 hours, and filtering to obtain a precipitated product after the reaction is completed, then washing and filtering 5 times and drying to obtain the dialdehyde polysaccharide modified with acetylcysteine.

[0068] The method for preparing a collagen implant using a dialdehyde polysaccharide biocrosslinking agent comprises the following steps:

[0069] S1, dissolving the prepared acetylcysteine-modified dialdehyde polysaccharide in 10×DPBS buffer solution to obtain 50 mL of a 20 mg / mL dialdehyde polysaccharide solution; dissolving collagen in 1 mmol / L acetic acid solution to obtain 50 mL of a 60 mg / mL collagen solution, adding 1% of the mass of the collagen solution to insulin-like growth factor and 1% of the epidermal growth factor, stirring evenly to obtain a collagen solution mixed with growth factors; then mixing the dialdehyde polysaccharide solution and the collagen solution in a mass ratio of 10:1 to obtain a collagen-dialdehyde polysaccharide mixed solution;

[0070] S2, take appropriate amounts of sodium bicarbonate and HEPES, dissolve them in 1 mol / L sodium hydroxide solution, and prepare 30 mL of neutralization solution; wherein the mass concentration of sodium bicarbonate is 2.5%, and the concentration of HEPES is 200 mmol / L;

[0071] S3, inject the collagen-dialdehyde polysaccharide mixed solution into the injection tube A of the double syringe with a specification of 10 ml, mix the neutralization solution and DPBS buffer in a volume ratio of 1.3:1 and inject into the injection tube B of the double syringe, and push injection 25-35 times at the same time to finally obtain a cross-linked collagen implant.

[0072] Example 6

[0073] The preparation method of the dialdehyde polysaccharide biocrosslinking agent of this embodiment comprises the following steps:

[0074] S1, add 10g xanthan gum to molten imidazole with a mass of 10 times (100g), heat and stir at 100°C to obtain a polysaccharide solution; then add 0.6g acetylcysteine, 15g EDC and 15g NHS to the xanthan gum solution, the mass ratio of acetylcysteine: EDC: NHS: xanthan gum is 0.06:1.5:1. After stirring and mixing, heat and react at 50°C for 5h.

[0075] S2, after the reaction is completed, excess ethanol is added to precipitate the product, and then the precipitate is filtered to obtain the precipitate, which is washed and filtered 5 times to remove the unreacted substances, and then dried at 40°C to obtain the acetylcysteine-modified polysaccharide.

[0076] S3, dispersing the acetylcysteine-modified polysaccharide prepared in step S2 in a sodium periodate aqueous solution with a mass concentration of 10%, with a mass ratio of periodate to xanthan gum of 1.2:1, stirring evenly, reacting at 35°C in the dark for 6 hours, and filtering to obtain a precipitated product after the reaction is completed, washing and filtering 5 times, and drying to obtain the acetylcysteine-modified dialdehyde polysaccharide.

[0077] The method for preparing a collagen implant using a dialdehyde polysaccharide biocrosslinking agent comprises the following steps:

[0078] S1, dissolving the prepared acetylcysteine-modified dialdehyde polysaccharide in 10×DPBS buffer solution to obtain 50 mL of a 10 mg / mL dialdehyde polysaccharide solution; dissolving collagen in 1 mmol / L acetic acid solution to obtain 50 mL of a 40 mg / mL collagen solution, adding 0.05% of insulin-like growth factor and 0.05% of epidermal growth factor by mass of the collagen solution, stirring evenly to obtain a collagen solution mixed with growth factors; then mixing the dialdehyde polysaccharide solution and the collagen solution in a mass ratio of 6:1 to obtain a collagen-dialdehyde polysaccharide mixed solution;

[0079] S2, take sodium bicarbonate and HEPES, dissolve them in 0.6 mol / L sodium hydroxide solution to prepare 30 mL of neutralization solution; the concentration of sodium bicarbonate is 1.5%, and the concentration of HEPES is 150 mmol / L;

[0080] S3, inject the collagen-dialdehyde polysaccharide mixed solution into the injection tube A of the double syringe with a specification of 10 ml, mix the neutralization solution and DPBS buffer in a volume ratio of 1:1 and inject into the injection tube B of the double syringe, and push injection 30 times at the same time to finally obtain the cross-linked collagen implant.

[0081] Example 7

[0082] The difference between Example 7 and Example 3 is that the polysaccharide is pullulan, and the rest of the contents are the same as Example 3.

[0083] Example 8

[0084] The difference between Example 8 and Example 3 is that the polysaccharide is guar gum, and the rest of the contents are the same as Example 3.

[0085] Example 9

[0086] The difference between Example 9 and Example 3 is that the polysaccharide is carrageenan, and the rest of the contents are the same as Example 3.

[0087] Comparative Example 1

[0088] Compared with Example 1, Comparative Example 1 directly uses sodium alginate and periodic acid to react to produce dialdehyde polysaccharide, which is then used to prepare collagen implants.

[0089] The preparation method of dialdehyde polysaccharide is as follows: 10g of sodium alginate is dispersed in a sodium periodate aqueous solution with a mass concentration of 10%, the mass ratio of periodate to polysaccharide is 1:1, and stirred for 5-10min. After stirring evenly, the mixture is reacted at 40°C in the dark for 6h, and after the reaction is completed, the precipitated product is filtered out to obtain the precipitated product, which is then washed and filtered 5 times and dried to obtain the dialdehyde polysaccharide.

[0090] Comparative Example 2

[0091] Compared with Example 1, in Comparative Example 2, the mass ratio of acetylcysteine ​​to polysaccharide exceeds the specified range, specifically 0.1:1, and the rest of the contents are the same.

[0092] Comparative Example 3

[0093] Compared with Example 1, in Comparative Example 3, the mass ratio of the dialdehyde polysaccharide solution to the collagen solution exceeds the specified range, specifically 4:1, and the rest of the contents are the same.

[0094] The collagen implants prepared in the examples and comparative examples were placed at 37° C. and incubated for 24 hours to form collagen hydrogels. The mechanical strength and compression failure strength of the gels were tested. The results are shown in Table 1.

[0095] Table 1 Strength of hydrogel

[0096] Compression failure strength / kPa Example 1 72.35 Example 2 78.49 Example 3 78.51 Example 4 78.43 Example 5 78.52 Example 6 78.45 Example 7 78.12 Example 8 78.37 Example 9 78.19 Comparative Example 1 47.42 Comparative Example 2 53.17 Comparative Example 3 55.19

[0097] As shown in Table 1, the strength of the collagen hydrogel prepared by the present invention is as high as 72 kPa or more, which is significantly higher than the strength of the comparative example, indicating that acetylcysteine ​​reacts with various groups of polysaccharide molecules to form a uniform cross-linked structure, thereby improving the strength of the collagen hydrogel. As can be seen from Example 2, the addition of EDC and NHS improves the modification effect of acetylcysteine ​​on polysaccharides, thereby obtaining better mechanical properties in subsequent applications.

[0098] Comparative Example 1 shows that the dialdehyde polysaccharide cross-linked collagen directly generated by sodium alginate and periodic acid reaction has a lower final hydrogel strength. Comparative Example 2 shows that the excessive use of acetylcysteine ​​can cause too many sulfhydryl groups to react with aldehyde groups, forming a softer and more fragile network. This is because the crosslinking between disulfide bonds and sulfhydryl groups (except normal aldehyde groups and amino groups) is relatively weak, and excessive amounts may cause an imbalance in the internal structure of the material. Too much acetylcysteine ​​can seize the strong bond that originally needs to be formed between the collagen chain, resulting in insufficient crosslinking. Comparative Example 3 shows that the reduction of dialdehyde polysaccharide content directly reduces the number of crosslinking points that can be formed with collagen reaction, making the crosslinked network not dense enough. This can significantly reduce the tensile strength and elastic modulus of the material. In addition, insufficient crosslinking may cause the internal structure of the material to be loose, and it is impossible to effectively resist external stress, which can make the material more easily broken and deformed.

[0099] Toxicity test: The MTT method was used to determine the cytotoxicity of the collagen implants in Example 1-3 to rat fibroblasts. The specific operation was as follows: RPMI1640 culture medium containing 10% serum was used as the extraction medium. 2 / mL is the extraction ratio, and the test is carried out after 24h of extraction at 37°C. The cell suspension is added to a 96-well plate, 100 μL per well, and cultured at 37°C, 5% CO2 for 24h. After the culture is completed, the culture medium in the culture plate is discarded, and each plate is divided into groups and the extract (Example 1-3), negative control (RPMI1640 culture medium containing 10% serum), blank control (RPMI1640 culture medium) and positive control (RPMI1640 culture medium containing 0.5% phenol) are added, 5 wells per group, and cultured in a 37°C, 5% CO2 saturated water vapor incubator for 3h. After the culture is completed, the culture medium in the culture plate is discarded, 100 μL of isopropanol is added to each well, oscillated and mixed, and placed in an ELISA reader, and read at 570nm. For each dose group and positive control group, the relative cell proliferation rate is calculated by the OD value of the blank control group, the blank control group is 100%, and the cytotoxicity (%) = absorbance of cells in the test group / absorbance of cells in the blank group × 100%, and the experimental results are shown in Table 2.

[0100] Table 2 Cytotoxicity test results

[0101]

[0102] As shown in Table 2, under the experimental conditions, the relative cell proliferation rates of the extracts of Examples 1-3, negative and positive control groups after contact for 24 hours were 115.66%, 120.69%, 132.18%, 107.2%, 100.0%, and 30.9%, respectively. The negative control group and the positive control group were in line with expectations. The results of this experiment suggest that the samples of Examples 1-3 are not only non-cytotoxic, but also can promote the proliferation of rat fibroblasts, indicating that the hydrogel prepared by the present invention has good biocompatibility, can promote cell proliferation, and improve cell viability.

[0103] Degradability of collagen hydrogel: The collagen hydrogel prepared in the examples and comparative examples was gelled in a test tube and then cut into gel blocks with a length, width and height of 5 mm, with 10 blocks taken in each group. All gel blocks were dried to constant weight, weighed, and the weight before degradation was recorded. Then 10,000 units / mL of lysozyme phosphate buffer was prepared, and the gel blocks of 9 groups were placed in 9 different beakers, 100 mL of lysozyme phosphate buffer was added to each beaker, and then placed in a constant temperature box at 37°C for degradation test. Take 2 gel blocks at 10 days, 20 days, 30 days, and 60 days respectively, dry to constant weight, and record. The results are shown in Table 3.

[0104] Table 3 Hydrogel degradation rate

[0105]

[0106]

[0107] As shown in Table 3, the 60d degradation rate of the hydrogel in the embodiment of the present invention is 45%-55%, which is significantly lower than that in comparative examples 1-3, indicating that the collagen hydrogel prepared in the present application can delay enzymatic hydrolysis and has a strong lasting effect.

[0108] As shown in Comparative Example 1, common dialdehyde polysaccharide is used, and degradation rate is faster, indicating that acetylcysteine ​​is modified polysaccharide, and more dense multiple cross-linked network is obtained, except Schiff base (imine bond) formed by aldehyde group and amino group, the sulfhydryl of acetylcysteine ​​can also form disulfide bond (SS), further stabilize cross-linked network, thereby improve the strength and stability of collagen hydrogel, reduce degradation rate. As shown in Comparative Example 2, increasing the consumption of acetylcysteine, accelerates degradation on the contrary, this is because the excessive use of acetylcysteine ​​can cause too much sulfhydryl modification on polysaccharide molecule, but reduces the effective cross-linking between aldehyde group and collagen amino group. Too much sulfhydryl may compete with aldehyde group to react, causing the formation efficiency of cross-linked network to decrease. Therefore, the consumption of acetylcysteine ​​needs to be accurately controlled, to ensure the optimal stability of cross-linked network and the biocompatibility of material. As shown in Comparative Example 3, the ratio of dialdehyde polysaccharide is too low, and cross-linking reaction is insufficient, causing insufficient cross-linking density. This can make the structure of hydrogel loose, mechanical strength is reduced, and it is easily hydrolyzed and enzymolyzed. Therefore, maintaining the mass ratio of dialdehyde polysaccharide to collagen within a reasonable range is an important factor in ensuring the stability and degradation rate of hydrogels.

[0109] Collagen implantation experiment: The experimental animals were female mice weighing 20-25 g, with 12 mice in each group. The mice were anesthetized and their skins were disinfected according to conventional methods, and the collagen prepared in the embodiments and comparative examples was injected subcutaneously to ensure that the muscles were not pierced. Three mice in each group were killed on the 7th, 15th and 30th days, respectively, and the skin and materials at the injection site were removed together, observed by projection electron microscopy, and pathological analysis was performed.

[0110] Inflammation grading was performed using a semiquantitative scoring system:

[0111] 0 points - no inflammatory response.

[0112] 1 point - mild inflammatory reaction, cells, a small number of lymphocytes and eosinophils can be seen.

[0113] 2 points - moderate inflammatory reaction, with increased numbers of giant cells, lymphocytes, plasma cells, eosinophils, and neutrophils.

[0114] 3 points - severe inflammatory reaction with the presence of microabscesses.

[0115] Table 4 Inflammatory response scores at different observation periods after collagen implantation

[0116] Grouping Example 1 Example 2 Example 3 Comparative Example 1 Day 7 1 0.67 0.33 1.67 Day 15 0.67 0.33 0 1.33 Day 30 0.33 0 0 1 Day 45 0 0 0 0.67

[0117] Note: The values ​​of the examples and comparative examples are all average values.

[0118] As shown in Table 4, after the collagen prepared by the present invention is implanted in the body, the inflammatory period is short and the biocompatibility is good, so it is an ideal repair material.

[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a dialdehyde polysaccharide biocrosslinking agent, characterized in that: The steps include: S1, adding polysaccharide to molten imidazole, heating and stirring to obtain a polysaccharide solution; adding acetylcysteine ​​to the polysaccharide solution, stirring and mixing, and heating at 30-50° C. for reaction for 3-7 hours; S2, after the reaction is completed, ethanol is added to precipitate the product, and then unreacted substances are removed by filtration and washing, followed by drying to obtain acetylcysteine-modified polysaccharides; S3, dispersing the polysaccharide modified with acetylcysteine ​​in a sodium periodate aqueous solution, stirring evenly, reacting at 30-50°C in the dark for 5-7 hours, and filtering to obtain a precipitated product after the reaction is completed, and then filtering, washing, and drying to obtain the dialdehyde polysaccharide modified with acetylcysteine.

2. The method for preparing a dialdehyde polysaccharide biocrosslinking agent according to claim 1, characterized in that: In step S1, acetylcysteine, EDC and NHS are added to the polysaccharide solution.

3. The method for preparing a dialdehyde polysaccharide biocrosslinking agent according to claim 2, characterized in that: The mass ratio of EDC:NHS:polysaccharide is 0.5-1.2:0.5-1.2:

1.

4. The method for preparing a dialdehyde polysaccharide biocrosslinking agent according to claim 1, characterized in that: The mass ratio of acetylcysteine ​​to polysaccharide is 0.025-0.075:

1.

5. The method for preparing a dialdehyde polysaccharide biocrosslinking agent according to claim 1, characterized in that: The mass ratio of periodic acid to polysaccharide is 0.5-1.5:

1.

6. The method for preparing a dialdehyde polysaccharide biocrosslinking agent according to claim 1, characterized in that: The polysaccharide is one or a mixture of sodium alginate, xanthan gum, pullulan, dextran, chitosan, guar gum and carrageenan.

7. A method for preparing a collagen implant using the dialdehyde polysaccharide biocrosslinking agent according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1, dissolving the dialdehyde polysaccharide modified with acetylcysteine ​​in a DPBS buffer solution to obtain a 4-20 mg / mL dialdehyde polysaccharide solution; dissolving collagen in an acetic acid solution to obtain a 30-60 mg / mL collagen solution, and then mixing the collagen solution with the dialdehyde polysaccharide solution to obtain a collagen-dialdehyde polysaccharide mixed solution; S2, taking sodium bicarbonate and HEPES, dissolving them in 0.5-1.0 mol / L sodium hydroxide solution to obtain a neutralized solution; S3, inject the collagen-dialdehyde polysaccharide mixed solution into the injection tube A of the double syringe, mix the neutralization solution and DPBS buffer and inject them into the injection tube B, and push the injection 25-35 times at the same time to finally obtain the cross-linked collagen implant.

8. The method for preparing collagen implants using a dialdehyde polysaccharide biocrosslinking agent as claimed in claim 7, characterized in that: In step S1, insulin-like growth factor and epidermal growth factor are added to the collagen solution.

9. The method for preparing collagen implants using a dialdehyde polysaccharide biocrosslinking agent as claimed in claim 8, characterized in that: The dosage of insulin-like growth factor and epidermal growth factor is 0.01%-1% of the mass of the collagen solution.

10. The method for preparing collagen implants using a dialdehyde polysaccharide biocrosslinking agent according to claim 7, characterized in that: In step S1, the mass ratio of the dialdehyde polysaccharide solution to the collagen solution is 5-10:1; in step S2, the mass concentration of sodium bicarbonate is 1.0% to 2.5%, the concentration of HEPES is 100-200 mmol / L, and the concentration of sodium hydroxide is 0.5-1.0 mol / L; in step S3, the neutralization solution and the DPBS buffer are mixed in a volume ratio of 1-1.5:1.

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