阅读说明：本技术 一种用于创面修复富含干细胞的可降解膜的制备方法 (Preparation method of stem cell-rich degradable membrane for wound repair ) 是由 向兴力 于 2020-04-13 设计创作，主要内容包括：本发明公开了一种用于创面修复富含干细胞的可降解膜的制备方法,其特征在于,包括如下步骤：步骤S1、芦荟新甙D/D-烯丙基甘氨酸/柠檬烯-1 2-环氧化物共聚物的制备；步骤S2、可降解膜材料的制备；步骤S3、辐射交联。本发明还提供了根据所述一种用于创面修复富含干细胞的可降解膜的制备方法制备得到的用于创面修复富含干细胞的可降解膜。本发明公开的用于创面修复富含干细胞的可降解膜对创面修复效果好,可降解和生物相容性佳,机械强度大,抗感染抗炎性能优异。(The invention discloses a preparation method of a degradable membrane rich in stem cells for wound repair, which is characterized by comprising the following steps: step S1, preparing an barbaloin D/D-allyl glycine/limonene-12-epoxide copolymer; step S2, preparing a degradable membrane material; and step S3, radiation crosslinking. The invention also provides the degradable membrane rich in stem cells for wound repair, which is prepared by the preparation method of the degradable membrane rich in stem cells for wound repair. The degradable membrane rich in stem cells and used for wound repair disclosed by the invention has the advantages of good wound repair effect, good degradability and biocompatibility, high mechanical strength and excellent anti-infection and anti-inflammatory properties.)

1. A preparation method of a degradable membrane rich in stem cells for wound repair is characterized by comprising the following steps:

step S1, preparation of barbaloin D/D-allylglycine/limonene-12-epoxide copolymer: adding barbaloin D, D-allyl glycine, limonene-12-epoxide and initiator into high boiling point solvent, stirring and reacting at 70-80 deg.C under nitrogen or inert gas atmosphere for 3-5 hr, removing high boiling point solvent by rotary evaporation, dissolving the product with water, filtering to remove insoluble substances, removing water by rotary evaporation, drying at 80-90 deg.C in vacuum drying oven to constant weight to obtain barbaloin D/D-allyl glycine/limonene-12-epoxide copolymer;

step S2, preparation of degradable membrane material: adding the barbaloin D/D-allyl glycine/limonene-12-epoxide copolymer prepared in the step S1, sulfobutyl sodium betacyclodextrin, chitosan quaternary ammonium salt, amino modified glucan, 2- [ oxy-bis (2, 1-ethyleneoxymethylene) ] dioxirane, allyl cyclodextrin and stem cells into a mixed solvent, stirring for 2-4 hours at the temperature of 60-80 ℃, then carrying out ultrasonic treatment for 15-30 minutes, then pouring into a film, and then carrying out freeze drying to obtain a degradable film material;

step S3, radiation crosslinking: and (4) placing the degradable membrane material prepared in the step S2 into a radiation field in the nitrogen atmosphere, and radiating for 30-40 minutes by adopting a cobalt 60-gamma radiation method to obtain the degradable membrane rich in stem cells for wound repair.

2. The method of claim 1, wherein the mass ratio of the aloin D, D-allyl glycine, the limonene-12-epoxide, the initiator and the high boiling point solvent in step S1 is 2:1 (0.2-0.3): 0.03-0.05): 10-16.

3. The method for preparing the stem cell-rich degradable membrane for wound healing according to claim 1, wherein the initiator is at least one of azobisisobutyronitrile and azobisisoheptonitrile.

4. The method for preparing the stem cell-rich degradable membrane for wound repair according to claim 1, wherein the high boiling point solvent is at least one of dimethylsulfoxide, N-dimethylformamide, N-dimethylacetamide and N-methylpyrrolidone.

5. The method for preparing the stem cell-rich degradable membrane for wound repair of claim 1, wherein the inert gas is one of helium, neon and argon.

6. The method for preparing a degradable membrane rich in stem cells for wound healing as claimed in claim 1, wherein the mass ratio of the barbaloin D/D-allylglycine/limonene-12-epoxide copolymer, the sulfobutylbetacyclodextrin sodium, the chitosan quaternary ammonium salt, the amino modified dextran, the 2,2- [ oxybis (2, 1-ethyleneoxymethylene) ] dioxirane, the allylcyclodextrin, the stem cells and the mixed solvent in step S2 is 1:0.3:0.2:0.6:0.1:0.6 (0.01-0.03): 9-15.

7. The method for preparing the stem cell-rich degradable membrane for wound healing according to claim 1, wherein the stem cell is any one of adipose mesenchymal stem cell, bone marrow mesenchymal stem cell, umbilical cord mesenchymal stem cell, placenta mesenchymal stem cell and heart stem cell.

8. The preparation method of the stem cell-rich degradable membrane for wound healing according to claim 1, wherein the mixed solvent is a mixture of N, N-dimethylformamide and water in a mass ratio of 1 (3-5).

9. The stem cell-rich degradable membrane for wound healing, which is prepared according to the preparation method of the stem cell-rich degradable membrane for wound healing of any one of claims 1 to 8.

Technical Field

The invention relates to the technical field of biomedical materials, in particular to a preparation method of a degradable membrane rich in stem cells for wound repair.

Background

The skin is an important tissue organ that covers and protects the body surface. Skin defects and abnormalities are often caused by inflammation, ulcer, trauma, burn, tumor after operation and congenital malformation, clinically, tissue inflammation and ulcer are easily caused by skin damage, and amputation and life risks are caused by severe cases. Therefore, it is necessary to care the skin of the wound with a biological membrane material to achieve the effects of promoting wound repair, preventing wound infection and causing other complications.

Gauze has traditionally been the most used for exposed skin wounds. The gauze is mostly processed by cotton, linen and linen, belongs to inert materials, and has no direct promotion effect on the healing of wound surfaces. With the progress of wound healing research, people recognize that not only wound coverage is required, but also wound healing is required to be assisted. The current clinical conventional treatment means are autologous middle-thick skin, full-thick skin transplantation and allogeneic acellular dermal matrix composite autologous epidermal sheet transplantation. Skin grafting can reconstruct the skin structure of the defect site and restore its appearance and function, but this method lacks sufficient skin resources, is expensive, and also causes pain to the patient, and is difficult to popularize.

Stem cells are a type of pluripotent cells that have the ability to self-replicate. Under certain conditions, it can differentiate into a variety of functional cells. The stem cells are divided into embryonic stem cells and adult stem cells according to the development stage of the stem cells, the cells have the functions of immunoregulation and anti-inflammation besides multidirectional differentiation potential, and the stem cells are added into a bracket material to prepare a biological membrane for treating damaged skin, can effectively promote wound healing and are good products for wound repair.

At present, the scaffold materials used in tissue engineering are mainly divided into two types: chemically synthesized materials and biologically natural materials. Both types of materials have been reported in a number of studies and clinical trials. Wherein the chemical synthetic material comprises polyglycolic acid, polylactic acid, copolymer of polylactic acid and polyglycolic acid, polyethylene glycol and the like; the biological natural material comprises collagen, silk fibroin, alginate, chitosan, hyaluronic acid and the like. The chemical synthetic material has strong mechanical property, but the material is difficult to degrade, the biocompatibility is poor, and the rejection and the surname invasion reaction can be caused by the implantation in vivo. The biological natural material has the characteristics of being close to natural extracellular matrix, has excellent biocompatibility, is nontoxic and harmless as a degradation product, can be absorbed and utilized by organisms, and is favored by researchers, however, the biological natural material is too soft, has poor mechanical property, is easy to tear, and brings much inconvenience to operation and nursing operation; in addition, it is difficult to achieve a degradation rate consistent with tissue regeneration rate for such materials, often resulting in graft and repair failure due to excessive degradation of the material.

The invention discloses a degradable biological membrane rich in stem cell extract for wound repair, which is prepared by separating and culturing mesenchymal stem cells, taking a fibrin membrane as a scaffold material, combining the mesenchymal stem cells cultured in vitro with the fibrin membrane, and transplanting the combined stem cells to a damaged part of cartilage to achieve the effect of repairing the damaged cartilage. However, fibrin films are not strong enough, degrade too quickly, and are expensive, limiting their widespread use.

Therefore, the development of the degradable membrane which has good mechanical strength, strong degradability and good wound repair effect and is used for wound repair and rich in stem cells meets the market demand, has wide market value and application prospect, and has very important significance for promoting the treatment of damaged skin.

Disclosure of Invention

The invention aims to solve the problems and provides a degradable membrane rich in stem cells for wound repair, which has good wound repair effect, good degradability and biocompatibility, high mechanical strength and excellent anti-infection and anti-inflammatory properties. Meanwhile, the invention also provides a preparation method of the degradable membrane rich in stem cells for wound repair, the preparation method is simple and easy to implement, has low requirements on reaction conditions and equipment, has high preparation efficiency, is suitable for continuous large-scale production, and has higher economic value, social value and ecological value.

In order to achieve the above object, the present invention provides the following technical solution, a method for preparing a degradable membrane rich in stem cells for wound repair, comprising the following steps:

step S1, preparation of barbaloin D/D-allylglycine/limonene-12-epoxide copolymer: adding barbaloin D, D-allyl glycine, limonene-12-epoxide and initiator into high boiling point solvent, stirring and reacting at 70-80 deg.C under nitrogen or inert gas atmosphere for 3-5 hr, removing high boiling point solvent by rotary evaporation, dissolving the product with water, filtering to remove insoluble substances, removing water by rotary evaporation, drying at 80-90 deg.C in vacuum drying oven to constant weight to obtain barbaloin D/D-allyl glycine/limonene-12-epoxide copolymer;

step S2, preparation of degradable membrane material: adding the barbaloin D/D-allyl glycine/limonene-12-epoxide copolymer prepared in the step S1, sulfobutyl sodium betacyclodextrin, chitosan quaternary ammonium salt, amino modified glucan, 2- [ oxy-bis (2, 1-ethyleneoxymethylene) ] dioxirane, allyl cyclodextrin and stem cells into a mixed solvent, stirring for 2-4 hours at the temperature of 60-80 ℃, then carrying out ultrasonic treatment for 15-30 minutes, then pouring into a film, and then carrying out freeze drying to obtain a degradable film material;

step S3, radiation crosslinking: and (4) placing the degradable membrane material prepared in the step S2 into a radiation field in the nitrogen atmosphere, and radiating for 30-40 minutes by adopting a cobalt 60-gamma radiation method to obtain the degradable membrane rich in stem cells for wound repair.

Further, in step S1, the mass ratio of the barbaloin D, D-allyl glycine, the limonene-12-epoxide, the initiator and the high boiling point solvent is 2:1 (0.2-0.3): 0.03-0.05): 10-16.

Preferably, the initiator is at least one of azobisisobutyronitrile and azobisisoheptonitrile.

Preferably, the high boiling point solvent is at least one of dimethyl sulfoxide, N-dimethylformamide, N-dimethylacetamide and N-methylpyrrolidone.

Preferably, the inert gas is one of helium, neon and argon.

Further, in step S2, the mass ratio of the barbaloin D/D-allylglycine/limonene-12-epoxide copolymer, the sulfobutylbetacyclodextrin sodium, the chitosan quaternary ammonium salt, the amino-modified dextran, the 2,2- [ oxybis (2, 1-ethyleneoxymethylene) ] dioxirane, the allylcyclodextrin, the stem cells and the mixed solvent is 1:0.3:0.2:0.6:0.1:0.6 (0.01-0.03): 9-15.

Preferably, the stem cell is any one of adipose mesenchymal stem cell, bone marrow mesenchymal stem cell, umbilical cord mesenchymal stem cell, placenta mesenchymal stem cell and heart stem cell.

Preferably, the mixed solvent is formed by mixing N, N-dimethylformamide and water according to the mass ratio of 1 (3-5).

Preferably, the sulfobutylbetacyclodextrin is purchased from noontima biotechnology limited, shandong, under the accession number of excipients: f20190000285; the average molecular weight of the chitosan quaternary ammonium salt is 20 ten thousand, and the substitution degree is 35%; the amino modified glucan has a mass average molecular weight of 7 ten thousand and is purchased from Xian Ruixi Biotechnology Ltd; the allylcyclodextrins were purchased from Linyi Ezilas Biotech, Inc.

The invention also aims to provide the degradable membrane which is prepared according to the preparation method of the degradable membrane for wound repair and rich in stem cells and is used for wound repair and rich in stem cells.

Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:

(1) the degradable membrane rich in stem cells for wound repair provided by the invention is simple and feasible in preparation method, low in requirements on reaction conditions and equipment, high in preparation efficiency, suitable for continuous large-scale production, and high in economic value, social value and ecological value.

(2) The degradable membrane rich in stem cells for wound repair provided by the invention overcomes the defects that the traditional wound repair material is more or less too soft, has poor mechanical property and is easy to tear, and brings much inconvenience to operation and nursing operation; the method has the advantages of difficult realization of the defect that the degradation speed is consistent with the tissue regeneration speed, often causes the failure of transplantation and repair due to too fast material degradation, good wound repair effect, good degradability and biocompatibility, high mechanical strength and excellent anti-infection and anti-inflammatory performance.

(3) According to the degradable membrane rich in stem cells for wound repair, provided by the invention, the copolymer contains the barbaloin D structure, so that free radicals can be eliminated, the functions of resisting aging and keeping skin whitening are achieved, the epoxy group provides a reaction site for the subsequent cross-linking of the membrane, and the glycine structure provides nutrition for the repair of the wound and the growth of new tissues; sulfobutyl-beta-cyclodextrin sodium and chitosan quaternary ammonium salt are connected together through ionic bonds through ion exchange reaction, the amino group on the amino-modified glucan reacts with the epoxy group on the 2,2- [ oxybis (2, 1-ethyleneoxymethylene) ] diepoxy ethane for chemical reaction and crosslinking, and the epoxy group on the copolymer also reacts with the amino-modified glucan; the allyl cyclodextrin, the barbaloin D/D-allyl glycine/limonene-12-epoxide copolymer can be grafted in the radiation stage, and the allyl cyclodextrin and the sulfobutyl betacyclodextrin sodium both contain cyclodextrin structures, so that the components form a unified whole, and the comprehensive performance of the material is effectively improved; the materials are nontoxic and harmless, have no rejection reaction to human tissues, have good biocompatibility, and can resist bacteria and infection, promote the growth of new tissues and the repair of wound surfaces and provide a proper environment for the repair of the wound surfaces under the synergistic effect of the components; the addition of the stem cells can further improve the biocompatibility and the anti-inflammatory and anti-infection performance of the membrane and can further improve the wound repair effect.

Detailed Description

In order to make the technical solution of the present invention better understood by those skilled in the art, the following provides a detailed description of the product of the present invention with reference to the examples.

The raw materials required in this example were purchased from national pharmaceutical group chemical reagents, ltd; the sulfobutyl betacyclodextrin is purchased from Zhiyuan Biotech limited, Shandong Binzhou, and has an auxiliary material registration number: f20190000285; the average molecular weight of the chitosan quaternary ammonium salt is 20 ten thousand, and the substitution degree is 35%; the amino modified glucan has a mass average molecular weight of 7 ten thousand and is purchased from Xian Ruixi Biotechnology Ltd; the allylcyclodextrins were purchased from Linyi Ezilas Biotech, Inc.