CN106215239A - A kind of preparation method of crosslinked antimicrobial type acellular matrix material - Google Patents

Abstract

The invention discloses the preparation method of a kind of antimicrobial form acellular matrix material.The present invention uses oxidation chitosan quaternary ammonium salt crosslinking acellular matrix material, utilize and form crosslinking between multiple aldehyde radicals and the active group on acellular matrix on oxidation chitosan quaternary ammonium salt, improve the structural stability of material, chitosan quaternary ammonium salt is grafted in acellular matrix material simultaneously, thus gives acellular matrix material high antibacterial bacteriostatic performance.The crosslinking of material can be united two into one by this method with endowing antibacterial, is the preparation method of a kind of novel functional acellular matrix material, can be widely applied to technical field of biological material.

Description

A preparation method of cross-linked antibacterial acellular matrix material

technical field

The invention relates to a preparation method of an antibacterial decellularized matrix material, which is applied in the field of biological materials.

Background technique

The decellularized matrix material is obtained by treating the matrix material with physical, chemical and biological methods, performing decellularization treatment, and removing related antigens. It removes the cellular components in natural materials, retains matrix components, effectively reduces the immunogenicity of natural materials, and at the same time maintains the basic structure and properties of matrix materials (such as macroscopic weaving structure, microscopic pore structure, physical and mechanical strength, etc.) . Common acellular matrix materials include acellular dermal matrix, acellular corneal stroma, amniotic membrane acellular matrix, porcine aortic acellular matrix, bovine (or porcine) pericardial acellular matrix, porcine small intestinal submucosal acellular matrix, and urethral acellular matrix Matrix and decellularized cartilage can be used for repair or tissue filling of skin, blood vessels, valves, urinary tract, etc., and have been widely used in the medical field (Zhang Chen, Wang Zhijun, Gao Jingheng. Research progress of decellularized tissue matrix. Journal of Dalian University, 2007 , 28(3):57-60).

Cross-linking can eliminate or reduce immunogenicity, improve physical and mechanical properties, increase resistance to degradation, and better meet the needs of use. Physical crosslinking methods (such as thermal dehydrogenation) have not been widely used because they are limited to the surface of the material and the degree of crosslinking is low. Synthetic cross-linking agents have a wide range of sources, and specific molecular structures can be obtained by molecular design methods, and multiple chemically active groups are introduced into the cross-linking agent molecules, so the cross-linking is high; however, synthetic cross-linking agents (such as glutaraldehyde) are due to The reasons such as by-products and insufficient purity easily lead to poor biocompatibility of the cross-linked material. Natural products and their derivative cross-linking agents (such as genipin) have relatively good biocompatibility, but there are often fewer varieties, and the cost of preparation and purification is relatively high, so they cannot be used in large-scale industrial applications.

In recent years, with the advancement of science, cross-linking agents of natural product derivatives represented by oxidized natural polysaccharides have become a research hotspot. This type of cross-linking agent uses natural sugars as the matrix, and forms multiple aldehyde groups through oxidation, so that the sugars have cross-linking properties. Due to the good biocompatibility of the polysaccharide, after being introduced into the biomaterial, good biocompatibility of the crosslinked material can still be ensured. For example, oxidized sodium alginate prepared by oxidizing alginate is used as a cross-linking agent for biomedical materials and has achieved good results (Hu, Y, Lju, L, Gu ZP, Dan, WH et al. Modification of collagen with a natural derived cross-linker, alginate dialdehyde[J]. CARBOHYDRATEPOLYMERS, 2014, 102: 324-332.) This kind of cross-linking agent has a higher degree of cross-linking, which overcomes the disadvantage of low degree of physical cross-linking; Good biocompatibility overcomes the disadvantage of poor biocompatibility of synthetic crosslinkers. Because polysaccharides are relatively cheap and easy to obtain, and the preparation cost is relatively low, they have broad application prospects and are ideal biological crosslinking agents.

While biomaterials treat diseases, there are also some problems. It has been found that biomaterials, whether long-term or short-term indwelling in the human body, often cause various bacterial infections. According to reports, there are 50,000 to 200,000 cases of intravascular catheter infection in the United States within a year, and the mortality rate is as high as 20%. Moreover, once these infections occur, even the application of hundreds of times the normal dose of drugs cannot be effectively treated, and the body's own immune system cannot clear these bacteria. According to statistics, 45% of nosocomial infections are related to the use of biological materials (Luo Jianbin. Research progress of antibacterial biomaterials [J]. Polymer Bulletin, 2009, 3:58-60). Infection is also one of the most common complications for biomaterials used to treat body surface wounds and burns. It will cause a large amount of exudate in the wound, decompose extracellular matrix proteins and various growth factors, and hinder epidermal regeneration and wound closure. , thus significantly affecting the recovery process, and may even lead to the death of burn patients (Zhou Ying, Xu Ling. Research progress in antibacterial dressings. Chinese Journal of Injury and Repair, 2012, 7 (3): 307-310). It can be seen that whether it is applied to biomaterials on the body surface or in the body, antibacterial and antibacterial functions are the advanced functions that biomaterials need to possess. The improvement of antibacterial properties of biomaterials has become one of the key technologies to ensure the performance of biomaterials.

Currently widely used antibacterial materials include antibiotics, metal ions, quaternary ammonium salts, natural antibacterial materials, etc. The use of antibiotics is prone to drug resistance; metal ions (such as silver) may have various problems such as cell deterioration, unstable performance, and high cost; common quaternary ammonium salts have good broad-spectrum antibacterial properties, but are often biocompatible Poor; natural antibacterial materials (such as chitosan) have excellent biocompatibility and good antibacterial performance, but their solubility is poor, which limits their application.

Chitosan quaternary ammonium salt is a product obtained by converting the amino group of chitosan into a quaternary ammonium salt group or grafting a low molecular weight quaternary ammonium salt. It belongs to polycationic compound, which has the characteristics of good water solubility, flocculation, biocompatibility, moisture absorption and better antibacterial properties, so its application range is quite wide. (Zhong Jing, Hong Yan, Chen Yong. The latest research progress of chitosan quaternary ammonium salt. China Tissue Engineering Research and Clinical Rehabilitation, 2008, 12 (6): 1115-1118).

Adding chitosan quaternary ammonium salt to biological materials by physical blending can endow biological materials with antibacterial and antibacterial functions, which is a commonly used method at present. However, chitosan quaternary ammonium salt lacks chemically reactive groups, making it difficult to form a strong covalent bond with biological materials. The chitosan quaternary ammonium salt added by physical blending is prone to migration and loss from biological materials, and cannot produce long-term antibacterial and antibacterial properties, and it is difficult to achieve the expected effect.

It can be seen from the above that, on the one hand, the current biomaterials need to be cross-linked with a cross-linking agent with excellent biocompatibility, and at the same time, it is necessary to endow the biomaterials with good antibacterial and antibacterial functions. Current methods often only satisfy one of them. Can crosslinking and antibacterial be combined into one, and at the same time of crosslinking, biomaterials can be endowed with antibacterial properties? In our long-term research on cross-linking of biomaterials, we proposed a new concept of "functionalized scale cross-linking": that is, while cross-linking, biomaterials are endowed with other functions. This point of view provides a new idea for us to find and prepare a new generation of functional crosslinkers. The use of a cross-linking agent with both cross-linking and antibacterial properties can not only meet the needs of cross-linking, but also endow the material with antibacterial properties.

Oxidized chitosan quaternary ammonium salt, whose parent is chitosan, has good biocompatibility; after selective oxidation, chitosan quaternary ammonium salt produces multiple chemically active aldehyde groups, so it can be compatible with biological materials Form a strong cross-linking between them to improve the degradation resistance of the material; at the same time, the chitosan quaternary ammonium salt structure is inserted into the biological material, so it has excellent antibacterial and antibacterial properties. Valence bonds, thus effectively avoiding the migration and loss of antibacterial agents, overcoming the shortcomings of previous methods. This method combines the cross-linking of biological materials and the endowment of antibacterial properties into one, and simultaneously achieves the dual purposes of producing cross-linking effects and endowing antibacterial properties. It is a new preparation method for functional acellular matrix materials and can be widely used. in the field of biomaterials.

Contents of the invention

The object of the present invention is to provide a preparation method of an antibacterial acellular matrix material for the deficiencies of the prior art, which is realized by the following technical measures.

A method for preparing an antibacterial acellular matrix material, characterized in that: Weigh 100 parts by weight of the acellular matrix material, and add 100 to 1000 parts by weight of a buffer solution with a pH value of 3.0 to 10.5 at 20 to 65°C , treated for 10-60min; then add 1-20 parts by weight of oxidized chitosan quaternary ammonium salt with an oxidation degree of 1-98%, and treat for 0.5-24.0 hours; discard the reaction waste liquid, and add 200-1000 parts by weight of water , wash for 10 to 30 minutes; discard the cleaning waste liquid, add 100 to 1000 parts by weight of a buffer solution with a pH value of 3.0 to 10.5, add 0.5 to 2.0 parts by weight of amino acids, and treat 0.5 to 4.0 parts at 20 to 65 ° C hours; discard the waste liquid, add 200-1000 parts by weight of water, and wash for 20-60 minutes; discard the waste liquid, add 100-300 parts by weight of water, add 0.1-1.0 parts by weight of lactic acid or sodium lactate, and At 35°C, treat for 30-90 minutes, adjust the pH of the bath liquid to neutral; discard the waste liquid, add 200-1000 parts by weight of water for injection at 30-42°C, and wash for 1-4 hours; discard the waste liquid, add 200-1000 parts by weight of water for injection at 30-42°C, wash for 1-4 hours.

The preparation method of antibacterial type acellular matrix material according to claim 1, wherein said oxidized chitosan quaternary ammonium salt refers to the structure formed by chitosan quaternary ammonium salt after selective oxidation containing multiple aldehyde groups chitosan quaternary ammonium salt.

The preparation method of antibacterial acellular matrix material according to claim 1, wherein said amino acid refers to glycine, arginine, lysine and histidine.

The preparation method of antibacterial acellular matrix material according to claim 1, wherein said buffer solution with a pH value of 3.0 to 10.5 is obtained by using acetic acid-hydrochloric acid buffer solution, phosphate buffer solution, boric acid-borax Buffer solution, NaHCO ₃ -NaOH buffer solution.

The preparation method of an antibacterial acellular matrix material according to claim 1 is characterized in that the method can be used for acellular dermal matrix, acellular corneal matrix, amniotic membrane acellular matrix, porcine aortic acellular matrix, pericardial acellular matrix , In the preparation of porcine small intestinal submucosal acellular matrix, urethral acellular matrix, and acellular cartilage.

The quaternary ammonium salt of oxidized chitooligosaccharide used in the present invention is of pharmaceutical grade, and other materials are analytically pure or biological reagents.

This cross-linking method has the following advantages:

(1) High degree of cross-linking: Since the oxidized chitosan quaternary ammonium salt molecule contains multiple aldehyde groups, it can combine with amino groups in the acellular matrix material at multiple points to form a higher degree of cross-linking. Can reduce the antigenicity of acellular matrix materials, improve physical and mechanical properties and degradation resistance;

(2) Good antibacterial and antibacterial performance: due to the strong positive charge of chitosan quaternary ammonium salt, it has broad-spectrum antibacterial properties and excellent antibacterial performance. Chitosan quaternary ammonium salt is introduced into the cross-linked acellular matrix material, Therefore, the acellular matrix material is endowed with good antibacterial and antibacterial functions;

(3) Long-lasting antibacterial and antibacterial properties: strong covalent bonds are formed by oxidizing the aldehyde groups in the chitosan quaternary ammonium salt and the amino groups in the acellular matrix material, with large bond energy and high stability, thus effectively avoiding physical damage. The defect of migration and loss of chitosan quaternary ammonium salt, which is easy to be produced by the blending method, can maintain its antibacterial and antibacterial properties for a long time;

(4) Good biocompatibility: After the aldehyde group reaction in the oxidized chitosan quaternary ammonium salt, the chitosan quaternary ammonium salt group remains in the structure, and the chitosan quaternary ammonium salt is derived from chitosan, thus inheriting the Chitosan has good biocompatibility. Amino acid post-treatment is used to remove possible residual aldehyde groups, ensuring good biocompatibility of cross-linked materials;

(5) Good controllability: the degree of cross-linking and antibacterial properties can be controlled by selecting the type of oxidized chitosan, controlling the degree of oxidation, and controlling the amount of cross-linking agent;

(6) Biodegradable: Since the chitosan quaternary ammonium salt is biodegradable, it can still maintain the biodegradability of the cross-linked material after being inserted into the biological material, and its degradation performance can be adjusted by the degree of cross-linking;

(7) Endow biological materials with good hydrophilic and moisturizing properties: chitosan quaternary ammonium salts are introduced into biological materials, and the moisturizing properties of chitosan quaternary ammonium salts are endowed to biological materials, so that the crosslinked materials have Good hydrophilic and moisturizing properties;

(8) Light color: the collagen cross-linked by oxidized chitosan quaternary ammonium salt can keep white, without the dark yellow after cross-linking with glutaraldehyde, and without the dark blue after cross-linking with genipin;

(9) The price of raw materials is low, and it can be industrialized on a large scale: compared with the excellent crosslinking agent of genipal, oxidized chitosan quaternary ammonium salt is cheaper. Therefore, this method can be applied to large-scale industrial production and is widely used in field of biomaterials.

In summary, the present invention uses oxidized chitosan quaternary ammonium salt as a raw material and applies it to the crosslinking of acellular matrix materials. On the one hand, it can reduce the antigenicity of the material and improve the structural stability and degradation resistance of the material; At the same time, it can endow the material with good antibacterial and antibacterial functions, and is a novel preparation method of functional decellularized matrix material, which can be widely used in the field of biological materials.

detailed description

The present invention is specifically described below by examples, it is necessary to point out that this example is only used for further illustration of the present invention, and can not be interpreted as the restriction to protection scope of the present invention, those skilled in the art can according to above-mentioned invention Some non-essential improvements and adjustments have been made to the content.

Example 1

(1) Weighing 100 parts by weight of acellular porcine dermal matrix and adding it to the reactor;

(2) Add 130 parts by weight of boric acid-borax buffer solution with a pH value of 8.5, and rotate for 30 minutes;

(3) Take by weighing 8 parts by weight of oxidized N-hydroxypropyltrimethylammonium chloride chitosan with a degree of oxidation of 8%, and dissolve it in 20 parts by weight of distilled water;

(4) Add the aqueous solution of oxidized chitosan quaternary ammonium salt into the reactor, raise the temperature to 37°C, react for 16 hours, and pour off the reaction waste liquid;

(5) Add 300 parts by weight of pure water, wash for 20 minutes, and pour off the waste liquid;

(6) Add 100 parts by weight of boric acid-borax buffer solution with a pH value of 8.5 at 37°C, add 1.5 parts by weight of lysine, rotate for 2 hours, and pour off the waste liquid;

(7) Add 1000 parts by weight of pure water, wash for 30 minutes, and pour off the waste liquid;

(8) Add 200 parts by weight of pure water, add 0.5 parts by weight of lactic acid, treat for 1.5 hours, and discard the waste liquid;

(9) Add 500 parts by weight of water for injection at 37°C and wash for 1 hour; pour off the cleaning solution, then add 1000 parts by weight of water for injection at 37°C and wash for 3 hours.

Example 2

(1) Weighing 100 parts by weight of bovine pericardium decellularized matrix and adding it to the reactor;

(2) Add 200 parts by weight of acetic acid-hydrochloric acid buffer solution with a pH value of 4.8, and rotate for 60 minutes;

(3) Weigh 4 parts by weight of oxidized N, N, N-trimethyl chitosan quaternary ammonium salt with an oxidation degree of 80%, and dissolve it in 50 parts by weight of acetic acid-hydrochloric acid buffer solution with a pH value of 4.8;

(4) Add the solution of oxidized chitosan quaternary ammonium salt into the reactor, raise the temperature to 42°C, rotate for 8 hours, and pour off the reaction waste liquid;

(5) Add 500 parts by weight of pure water, rotate for 20 minutes, and pour off the waste liquid;

(6) Add 150 parts by weight of acetic acid-hydrochloric acid buffer solution with a pH value of 4.8, add 0.5 parts by weight of arginine, rotate for 4 hours at 42°C, and pour off the waste liquid;

(7) Add 800 parts by weight of pure water, rotate for 20 minutes, and pour off the waste liquid;

(8) Add 100 parts by weight of pure water, add 1.0 parts by weight of sodium lactate, rotate for 90 minutes, and pour out the waste liquid;

(9) Add 1000 parts by weight of water for injection at 42°C, and wash for 2 hours; pour off the cleaning solution, and then add 500 parts by weight of water for injection at 42°C, and wash for 4 hours.

Example 3

(1) Weighing 100 parts by weight of amniotic membrane decellularized matrix and adding it to the reactor;

(2) Add 100 parts by weight of NaHCO ₃ -NaOH buffer solution with a pH value of 9.4, and rotate for 20 minutes;

(3) Weigh 16 parts by weight of oxidized O-hydroxypropyltrimethylammonium chloride N-trimethyl chitosan with a degree of oxidation of 5%, and dissolve it in 50 parts by weight of pure water;

(4) Add the solution of oxidized chitosan quaternary ammonium salt into the reactor, raise the temperature to 32°C, rotate for 24 hours, and pour off the reaction waste liquid;

(5) Add 800 parts by weight of pure water, rotate for 40 minutes, and pour off the waste liquid;

(6) Add 150 parts by weight of NaHCO ₃ -NaOH buffer solution with a pH value of 9.4, add 2.0 parts by weight of glycine, react at 32°C for 1 hour, and pour off the waste liquid;

(7) Add 300 parts by weight of pure water, rotate for 15 minutes, and discard the waste liquid;

(8) Add 300 parts by weight of pure water, add 1.0 parts by weight of lactic acid, rotate for 40 minutes, and discard the waste liquid;

(9) Add 600 parts by weight of water for injection at 37°C, wash for 2 hours; pour off the cleaning solution, then add 1000 parts by weight of water for injection at 32°C, and wash for 4 hours;

(10) Take out the amniotic membrane decellularized matrix and freeze-dry it.

Claims (5)

1. a preparation method for antimicrobial form acellular matrix material, is characterized in that: weigh the acellular matrix material of 100 weight portions Material, at 20～65 DEG C, adds the buffer that pH value is 3.0～10.5 of 100～1000 weight portions, processes 10～60min；So The oxidation chitosan quaternary ammonium salt that oxidizability is 1～98% of rear addition 1～20 weight portion, processes 0.5～24.0 hour；Discard anti- Answer waste liquid, add the water of 200～1000 weight portions, clean 10～30 minutes；Discard cleaning waste liquid, add 100～1000 weight Part the buffer that pH value is 3.0～10.5, add 0.5～2.0 weight portions aminoacid, at 20～65 DEG C, process 0.5～ 4.0 hour；Discard waste liquid, add the water of 200～1000 weight portions, clean 20～60 minutes；Discard waste liquid, add 100～300 The water of weight portion, adds lactic acid or the sodium lactate of 0.1～1.0 weight portions, at 20～35 DEG C, processes 30～90 minutes, will bath Liquid pH regulator is to neutral；Discard waste liquid, the injection water of 30～42 DEG C of addition 200～1000 weight portions, clean 1～4h；Discard Waste liquid, the injection water of 30～42 DEG C of addition 200～1000 weight portions, cleans 1～4h.

The preparation method of antimicrobial form acellular matrix material the most according to claim 1, wherein said oxidation chitosan The structure that quaternary ammonium salt is formed after referring to the oxidation of chitosan quaternary ammonium salt chosen property contains the chitosan quaternary ammonium salt of multiple aldehyde radicals.

The preparation method of antimicrobial form acellular matrix material the most according to claim 1, wherein said aminoacid, refer to Glycine, arginine, lysine and histidine.

The preparation method of antimicrobial form acellular matrix material the most according to claim 1, wherein said pH value be 3.0～ The buffer of 10.5, this pH is by using acetate hydrochloride buffer solution, phosphate buffered solution, boric acid Borax buffering molten Liquid, NaHCO₃-NaOH buffer solution reaches.

5. the preparation method of a kind of antimicrobial form acellular matrix material described in claim 1, it is characterised in that the method can be used for Acellular dermal matrix, cell-eliminating coanea matrix, human acellular amniotic membrane matrix, porcine aorta acellular matrix, pericardium take off cell base Under matter, intestinal mucosa in the preparation of acellular matrix, urethra acellular matrix, de-cellular cartilage.