阅读说明：本技术 一种壳聚糖季铵盐型脱细胞真皮基质材料的制备方法 (Preparation method of chitosan quaternary ammonium salt type acellular dermal matrix material ) 是由 但年华 丁壮 陈一宁 但卫华 于 2020-12-07 设计创作，主要内容包括：本发明公开了一种壳聚糖季铵盐型脱细胞真皮基质材料的制备方法,其特征是将氧化壳聚糖双季铵盐与脱细胞真皮基质交联,利用氧化壳聚糖双季铵盐中醛基的化学活性使壳聚糖双季铵盐接枝到脱细胞真皮基质上,制得壳聚糖季铵盐型脱细胞真皮基质材料。由于所接枝的壳聚糖双季铵盐是壳聚糖的衍生物,具有良好的生物相容性；同时壳聚糖双季铵盐拥有更多的季铵基团,可以赋予脱细胞真皮基质材料优良的抗菌性能。本方法可以广泛应用于抗菌生物材料的制备。(The invention discloses a preparation method of a chitosan quaternary ammonium salt type acellular dermal matrix material, which is characterized in that oxidized chitosan biquaternary ammonium salt is crosslinked with an acellular dermal matrix, and the chemical activity of aldehyde groups in the oxidized chitosan biquaternary ammonium salt is utilized to graft the chitosan biquaternary ammonium salt on the acellular dermal matrix to prepare the chitosan quaternary ammonium salt type acellular dermal matrix material. Because the grafted chitosan biquaternary ammonium salt is a derivative of chitosan, the grafted chitosan biquaternary ammonium salt has good biocompatibility; meanwhile, the chitosan biquaternary ammonium salt has more quaternary ammonium groups, and can endow the acellular dermal matrix material with excellent antibacterial performance. The method can be widely applied to the preparation of the antibacterial biological material.)

1. A preparation method of chitosan quaternary ammonium salt type acellular dermal matrix material is characterized by comprising the following steps: (1) weighing 100 parts by weight of acellular dermal matrix material, adding 100-1000 parts by weight of buffer solution with pH of 3.0-9.6 at 20-47 ℃, and soaking for 10-60 minutes; then adding 1-20 parts by weight of oxidized chitosan biquaternary ammonium salt with the oxidation degree of 5-80%, and treating for 0.5-12 hours; discarding the waste liquid, adding 200-1000 parts by weight of water, and cleaning for 10-30 minutes; discarding the cleaning waste liquid, adding 100-1000 parts by weight of buffer solution with the pH value of 3.0-9.6, adding 0.5-2.0 parts by weight of amino acid, and treating at 20-40 ℃ for 0.5-4.0 hours; discarding the waste liquid, adding 200-1000 parts by weight of water, and cleaning for 20-60 minutes; discarding the waste liquid, adding 0.1-1.0 weight part of lactic acid or sodium lactate, treating at 20-35 ℃ for 30-90 minutes, and adjusting the pH value of the bath liquid to be neutral; discarding the waste liquid, adding 200-1000 parts by weight of injection water at 20-40 ℃, and cleaning for 1-2 hours; discarding the waste liquid, adding 200-1000 parts by weight of injection water at 20-40 ℃, and cleaning for 1-2 hours.

2. The method for preparing a chitosan quaternaries-type acellular dermal matrix material according to claim 1, wherein the oxidized chitosan quaternaries are products of chitosan quaternaries formed by selective oxidation of sodium periodate and containing a plurality of active aldehyde groups in the structure.

3. The method for preparing a chitosan quaternary ammonium salt type acellular dermal matrix material according to claim 1, wherein the pH of the buffer solution is 3.0 to 9.6, and the pH is achieved by using an acetic acid-sodium acetate buffer solution, a phosphate buffer solution, or a sodium bicarbonate-sodium carbonate buffer solution.

4. The method for preparing a chitosan quaternary ammonium salt-type acellular dermal matrix material according to claim 1, wherein the amino acids are lysine, arginine, and histidine.

5. The method for preparing the chitosan quaternary ammonium salt acellular dermal matrix material according to claim 1, wherein the key properties of the prepared chitosan quaternary ammonium salt acellular dermal matrix material meet the following requirements: (1) the bacteriostasis rate to colibacillus, staphylococcus aureus, candida albicans and pseudomonas aeruginosa is more than or equal to 95 percent; (2) the inhibition rate of the total mucor is more than or equal to 95 percent; (3) the cytotoxicity of the leaching liquor is 0-1 grade, and no skin sensitization reaction exists.

6. The method for preparing an acellular dermal matrix material of chitosan quaternaries of claim 1, wherein the method is used for preparing acellular porcine dermal matrix, acellular fetal bovine dermal matrix, acellular fishskin matrix, and acellular sheep skin matrix.

Technical Field

The invention relates to a preparation method of a chitosan quaternary ammonium salt type acellular dermal matrix material, which is applied to the field of biological materials.

Background

The acellular dermal matrix material is a material which adopts a tissue engineering technology and an acellular technology, removes high antigen components in epidermis and dermis in animal skin, retains a natural structure and a basement membrane of collagen and has good structural stability. The composition contains type I collagen and type III collagen, and mainly type I collagen. Rejection generally does not occur due to the lack of immunological activity of cellular components and antigens. The acellular dermal matrix material can promote the growth of epidermis and the healing of wounds, provides support for the growth of epidermal cells, seals a wound surface, avoids the exposure of the wounds, resists the invasion of bacteria and the like, and is widely applied to the field of skin repair. However, since the main component of the acellular dermal matrix material is collagen fibers, which do not have antibacterial properties, they have a drawback of being easily infected with bacteria in the treatment of wounds that are not completely debrided, and thus it is necessary to impart anti-infectious ability to them.

Through cross-linking modification, the problems of pathogen inactivation of the biological material, elimination or reduction of immunogenicity, improvement of physical and mechanical properties, improvement of degradation resistance and better satisfaction of use requirements can be solved. Physical crosslinking methods (such as thermal dehydrogenation) have not been widely used because they are limited to the surface of the material and have a low degree of crosslinking. The source of the synthetic cross-linking agent is wide, a specific molecular structure can be obtained by a molecular design method, and a plurality of chemical active groups are introduced into the molecules of the cross-linking agent, so that the cross-linking is high. However, the synthetic cross-linking agent (such as glutaraldehyde) is liable to cause poor biocompatibility of the cross-linked material due to by-products and insufficient purity. The natural product and the derivative thereof (such as genipin) have better biocompatibility, but often have fewer varieties and higher preparation and purification cost, and are difficult to be applied in large-scale industrialization.

Saccharides are important organic compounds widely distributed in nature, play an important role in the life activity process, and are the main sources of energy required by all living bodies to maintain life activities. The saccharide has the characteristics of greenness, no toxicity, degradability in vivo, multiple types, wide sources, certain reactivity and the like, and can be used for modifying the acellular dermal matrix material. The oxidation of polysaccharides is generally to oxidize primary hydroxyl groups into aldehyde groups, and commonly used oxidation methods include sodium periodate oxidation, sodium hypochlorite oxidation, hydrogen peroxide oxidation and the like. The sodium periodate has the characteristic of selective oxidation, can selectively oxidize the adjacent dihydroxy to prepare aldehyde group with strong reaction activity, and the content of the aldehyde group can be regulated and controlled, and the operation is simple. The oxidized polysaccharide obtained by oxidizing sodium periodate has a plurality of aldehyde groups with strong chemical activity, can be crosslinked with active groups (such as amino) on an acellular dermal matrix material, and endows the acellular dermal matrix material with high stability, physical and mechanical properties, degradation resistance and the like.

The chitosan is a product obtained by deacetylating chitin, has active property, good hemostatic activity, biodegradability and biocompatibility, and is widely applied to industries such as food, medical treatment, chemical industry and the like. The chitosan contains free amino groups, and can only be dissolved in an acid dilute solution due to strong hydrogen bonding among molecules, but is basically insoluble in neutral, alkaline and organic solvents, so the application of the chitosan is greatly limited. The quaternary ammonium group is grafted into the chitosan molecule, so that the water solubility of the chitosan molecule can be greatly improved, and the antibacterial performance of the chitosan molecule is enhanced to a certain degree. The chitosan quaternary ammonium salt can be used as a natural modified cross-linking agent, and has wide attention on good antibacterial performance and biocompatibility. For example, Guo Zhang adopts the quaternization modification of alkylated chitosan to synthesize a series of N-alkylated chitosan quaternary ammonium salts, and researches the bacteriostatic property of the N-alkylated chitosan quaternary ammonium salts on several bacteria. The result shows that the synthesized chitosan quaternary ammonium salt has stronger antibacterial effect than chitosan, and the antibacterial performance of the chitosan quaternary ammonium salt is influenced by quaternary ammonium groups (Guo is heroic. the substituent groups and amino electropositivity of the water-soluble chitosan derivative influence the antibacterial activity); the bird plum red adopts chitosan quaternary ammonium salt to treat the blend fiber of alginate, so as to prepare the antibacterial wound dressing with higher antibacterial performance (bird plum red, Durem, Zhangbaozhong, and the like, alginate/chitosan derivative composite antibacterial fiber). The chitosan biquaternary ammonium salt is a product obtained by simultaneously grafting two quaternary ammonium salt groups on a chitosan unit, and most of the natural structure of the chitosan is kept, so that the chitosan biquaternary ammonium salt has the excellent properties of both chitosan and quaternary ammonium salt. The chitosan biquaternary ammonium salt has excellent flocculation property, moisturizing hydrophilicity and biocompatibility, and is widely applied to biomedical materials, water treatment, paper making industry and the like. The chitosan biquaternary ammonium salt has excellent water solubility and antibacterial property, but lacks chemical reaction activity; the aldehyde group can be obtained by oxidizing sodium periodate, so that the novel cross-linking agent has chemical reaction activity and antibacterial property, is used for cross-linking the acellular dermal matrix material, can improve the stability and antibacterial property of the acellular dermal matrix material, and can be used for preparing the functional acellular dermal matrix material.

Disclosure of Invention

The invention provides a preparation method for overcoming the defect that the acellular dermal matrix material has no antibacterial property by adopting oxidized chitosan bis-quaternary ammonium salt to crosslink the acellular dermal matrix material. As shown in figure 1, the chitosan quaternary ammonium salt is originally in-NH after being selectively oxidized by sodium periodate₂Loss of quaternary ammonium groups on; and the quaternary ammonium group of the biquaternary ammonium salt of the chitosan on-OH still exists. The double quaternary ammonium salt of the oxidized chitosan can lead the quaternary ammonium group to be grafted on the acellular dermal matrix material, compared with the common single quaternary ammonium salt, the method can introduce more quaternary ammonium groups, thereby compensating the quaternary ammonium groups lost after the oxidative ring opening of the chitosan unit and keeping good antibacterial function.

The purpose of the invention is realized by the following technical measures:

1. a preparation method of chitosan quaternary ammonium salt type acellular dermal matrix material is characterized by comprising the following steps: (1) weighing 100 parts by weight of acellular dermal matrix material, adding 100-1000 parts by weight of buffer solution with pH of 3.0-9.6 at 20-47 ℃, and soaking for 10-60 minutes; then adding 1-20 parts by weight of oxidized chitosan biquaternary ammonium salt with the oxidation degree of 5-80%, and treating for 0.5-12 hours; discarding the waste liquid, adding 200-1000 parts by weight of water, and cleaning for 10-30 minutes; discarding the cleaning waste liquid, adding 100-1000 parts by weight of buffer solution with the pH value of 3.0-9.6, adding 0.5-2.0 parts by weight of amino acid, and treating at 20-40 ℃ for 0.5-4.0 hours; discarding the waste liquid, adding 200-1000 parts by weight of water, and cleaning for 20-60 minutes; discarding the waste liquid, adding 0.1-1.0 weight part of lactic acid or sodium lactate, treating at 20-35 ℃ for 30-90 minutes, and adjusting the pH value of the bath liquid to be neutral; discarding the waste liquid, adding 200-1000 parts by weight of injection water at 20-40 ℃, and cleaning for 1-2 hours; discarding the waste liquid, adding 200-1000 parts by weight of injection water at 20-40 ℃, and cleaning for 1-2 hours.

2. The method for preparing a chitosan quaternaries-type acellular dermal matrix material according to claim 1, wherein the oxidized chitosan quaternaries are products of chitosan quaternaries formed by selective oxidation of sodium periodate and containing a plurality of active aldehyde groups in the structure.

3. The method for preparing a chitosan quaternary ammonium salt type acellular dermal matrix material according to claim 1, wherein the pH of the buffer solution is 3.0 to 9.6, and the pH is achieved by using an acetic acid-sodium acetate buffer solution, a phosphate buffer solution, or a sodium bicarbonate-sodium carbonate buffer solution.

4. The method for preparing a chitosan quaternary ammonium salt-type acellular dermal matrix material according to claim 1, wherein the amino acids are lysine, arginine, and histidine.

5. The method for preparing the chitosan quaternary ammonium salt acellular dermal matrix material according to claim 1, wherein the key properties of the prepared chitosan quaternary ammonium salt acellular dermal matrix material meet the following requirements: (1) the bacteriostasis rate to colibacillus, staphylococcus aureus, candida albicans and pseudomonas aeruginosa is more than or equal to 95 percent; (2) the inhibition rate of the total mucor is more than or equal to 95 percent; (3) the cytotoxicity of the leaching liquor is 0-1 grade, and no skin sensitization reaction exists.

6. The method for preparing an acellular dermal matrix material of chitosan quaternaries of claim 1, wherein the method is used for preparing acellular porcine dermal matrix, acellular fetal bovine dermal matrix, acellular fishskin matrix, and acellular sheep skin matrix.

The invention has the following advantages:

(1) high degree of crosslinking

The oxidized chitosan diquaternary ammonium salt used in the method contains a plurality of aldehyde groups, can form a plurality of binding points with amino groups on the acellular dermal matrix material, and generates high-strength crosslinking, thereby improving the structural stability and the degradation resistance of the acellular dermal matrix material;

(2) good antibacterial and mildewproof performance

According to the method, the chitosan biquaternary ammonium salt is introduced into the acellular dermal matrix material through the aldehyde group, and more quaternary ammonium salt groups are introduced than the common quaternary ammonium salt under the same condition, so that the chitosan biquaternary ammonium salt has better antibacterial and mildew-proof properties. The cross-linked acellular dermal matrix material has a bacteriostasis rate of more than or equal to 95 percent on escherichia coli, staphylococcus aureus, candida albicans and pseudomonas aeruginosa; the inhibition rate of the total mucor is more than or equal to 95 percent;

(3) good biocompatibility

The chitosan is a product obtained by deacetylation of chitin, has active property, good hemostatic activity, biodegradability and biocompatibility, and retains good biocompatibility of the salt. The oxidized chitosan biquaternary ammonium salt is a derivative of chitosan, and inherits the excellent biocompatibility of the chitosan; the residual aldehyde group is subjected to amino acid post-treatment, so that the residual aldehyde group can be removed, and the good biocompatibility of the cross-linking material is ensured. The cytotoxicity of the leaching liquor of the material is 0-1 grade, and no skin sensitization reaction exists;

(4) good controllability

The cross-linking degree and the antibacterial performance can be controlled by controlling the oxidation degree and the dosage of the cross-linking agent;

(5) is biodegradable

Because the chitosan biquaternary ammonium salt has biodegradability, the biodegradability of the cross-linked material can be still maintained after the chitosan biquaternary ammonium salt is connected to the biological material, and the degradation performance of the cross-linked material can be adjusted through the cross-linking degree;

(6) can endow the biological material with good hydrophilic and moisture-keeping properties

The biological material is endowed with the hydrophilic and moisturizing performances of the chitosan biquaternary ammonium salt by introducing the chitosan biquaternary ammonium salt into the biological material, so that the biological material has good hydrophilic and moisturizing performances.

In summary, the invention takes the oxidized chitosan biquaternary ammonium salt as the raw material, and the oxidized chitosan biquaternary ammonium salt is applied to the crosslinking of the acellular dermal matrix material, so that the prepared chitosan quaternary ammonium salt type acellular dermal matrix material has stronger antibacterial performance and better hydrophilic performance, is a preparation method of a novel functional acellular dermal matrix material, and can be widely applied to the preparation of antibacterial biological materials.

Drawings

FIG. 1 is a comparison of the oxidation reaction of chitosan mono-quaternary ammonium salt and chitosan sugar bis-quaternary ammonium salt.

Detailed Description

While the invention has been described in detail in the following for the purpose of illustration, it is to be understood that this invention is not limited thereto but is intended to cover various modifications and changes therein which may be made by those skilled in the art without departing from the spirit and scope of the invention.

Application example 1

(1) Weighing 1kg of acellular pig dermal matrix, and adding the acellular pig dermal matrix into a reactor;

(2) adding 3kg of sodium bicarbonate-sodium carbonate buffer solution with pH of 9.6, and rotating for 30 minutes;

(3) weighing 0.1kg of oxidized chitosan biquaternary ammonium salt with the oxidation degree of 40%, and dissolving the chitosan biquaternary ammonium salt in 0.2kg of sodium bicarbonate-sodium carbonate buffer solution with the pH value of 9.6;

(4) pouring the dissolved oxidized chitosan diquaternary ammonium salt solution into a reactor, heating to 37 ℃, reacting for 6 hours, and pouring out waste liquid;

(5) adding 6kg of deionized water, cleaning for 20 minutes, and pouring out waste liquid;

(6) adding 6kg of sodium bicarbonate-sodium carbonate buffer solution with pH of 9.6, adding 0.01kg of lysine, rotating for 120 min, and pouring out waste liquid;

(7) adding 6kg of deionized water, cleaning for 30 minutes, and pouring out waste liquid;

(8) adding 6kg of deionized water, adding 0.01kg of lactic acid, treating at 30 deg.C for 60 min, and discarding the waste liquid;

(9) adding 6kg of injection water with the temperature of 30 ℃, and cleaning for 1.5 hours; the cleaning solution was poured off, and 6kg of water for injection was added thereto and cleaned for 1.5 hours.

Application example 2

(1) Weighing 100 g of acellular fetal calf dermal matrix, and adding the acellular fetal calf dermal matrix into a reactor;

(2) adding 1000 g of phosphate buffer solution with the pH value of 7.0, and rotating for 10 minutes;

(3) weighing 1 g of oxidized chitosan biquaternary ammonium salt with the oxidation degree of 80%, and dissolving in 20 g of phosphate buffer solution with the pH value of 7.0;

(4) pouring the dissolved oxidized chitosan diquaternary ammonium salt solution into a reactor, heating to 47 ℃, reacting for 30 minutes, and pouring out waste liquid;

(5) adding 1000 g of phosphate buffer solution with the pH value of 7.0, adding 0.5 g of arginine, rotating for 30 minutes, and pouring out waste liquid;

(6) adding 1000 g of deionized water, cleaning for 20 minutes, and pouring out waste liquid;

(7) adding 1000 g of deionized water, adding 0.1 g of sodium lactate, treating for 30 minutes, and discarding the waste liquid;

(8) adding 1000 g of deionized water, cleaning for 20 minutes, and pouring out waste liquid;

(9) adding 1000 g of injection water with the temperature of 20 ℃, cleaning for 1 hour, and pouring out the cleaning solution;

(10) 1000 weight of water for injection was added and washed for 1 hour.

Application example 3

(1) Weighing 100kg of acellular sheep dermal matrix, and adding the acellular sheep dermal matrix into a reactor;

(2) adding 100kg of acetic acid-sodium acetate buffer solution with pH of 3.0, and rotating for 60 minutes;

(3) weighing 20kg of oxidized chitosan biquaternary ammonium salt with the oxidation degree of 5%, adding the weighed oxidized chitosan biquaternary ammonium salt into a reactor, reacting for 12 hours at the temperature of 20 ℃, and pouring out waste liquid;

(5) adding 100kg of deionized water, cleaning for 60 minutes, and pouring out waste liquid;

(6) adding 100kg of acetic acid-sodium acetate buffer solution with pH of 3.0, adding 2kg of histidine, rotating for 60 min, and pouring off the waste liquid;

(7) adding 100kg of deionized water, cleaning for 60 minutes, and pouring out waste liquid;

(8) adding 100kg of deionized water, adding 1.0kg of sodium lactate, treating for 90 min, and discarding the waste liquid;

(9) adding 200kg of injection water at 37 ℃, cleaning for 2 hours, and pouring out the cleaning solution;

(10) 200kg of water for injection was added thereto, and the mixture was washed for 2 hours.